Mechanisms of Resistance in Maize Grain to the Maize Weevil and the Larger Grain Borer - Insect Resistant Maize Recent Advances and Utilization

José-Antonio  Serratos-Hernández

The mechanism of resistance in maize to the stored product insects such as the maize weevil (MW), Sitophilus zeamais Motsch and the larger grain borer (LGB), Prostephanus truncatus Horn has been investigated in relation to secondary chemistry and other biochemical and physical characteristics of maize genotypes. Performance parameters of weevils (number of eggs laid, number of progeny, Dobie index, grain consumption) were negatively and significantly correlated (r = -0.8, P = 0.05) to the most abundant phenolic of grain, E-ferulic acid. With P. truncatus, the weight loss of grain also showed a negative correlation with E-ferulic acid while percent damage of kernels by insects was negatively correlated to p-coumaric acid. These phenolic acids were found in highest concentration in the pericarp and cell walls of the endosperm by fluorescence microscopy. Phenolic acid content was also found to correlate strongly with hardness of the grain, which may be related to the mechanical contributions of phenolic dimers to cereal cell wall strength. In the aleurone layer phenolic acid amines have been detected that have toxic effects on insects.

Insect Resistant Maize Recent Advances and Utilization Proceedings of an International Symposium held at CIMMYT J ohn A. M ihm CIMMYT World Development i Insect Resistant Maize Recent Advances and Utilization Proceedings of an International Symposium held at the International Maize and Wheat Improvement Center (CIMMYT) 27 November - 3 December, 1994 T e c hnic a l Edit or: J ohn A. M ihm CIMMYT World Development ii CIMMYT is an internationally funded, nonprofit scientific research and training organization. Headquartered in Mexico, the Center works with agricultural research institutions worldwide to improve the productivity and sustainability of maize and wheat systems for poor farmers in developing countries. It is one of 16 similar centers supported by the Consultative Group on International Agricultural Research (CGIAR). The CGIAR comprises over 50 partner countries, international and regional organizations, and private foundations. It is co-sponsored by the Food and Agriculture Organization (FAO) of the United Nations, the International Bank for Reconstruction and Development (World Bank), the United Nations Development Programme (UNDP), and the United Nations Environment Programme (UNEP). Financial support for CIMMYT’s research agenda currently comes from many sources, including the governments of Australia, Austria, Belgium, Canada, China, Denmark, the European Union, the Ford Foundation, France, Germany, India, the Inter-American Development Bank, Iran, Italy, Japan, the Kellogg Foundation, the Republic of Korea, Mexico, the Netherlands, Norway, the OPEC Fund for International Development, the Philippines, the Rockefeller Foundation, the Sasakawa Africa Association, Spain, Switzerland, the United Kingdom, UNDP, the USA, and the World Bank Correct citation: Mihm, J.A. (ed.). 1997. Insect Resistant Maize: Recent Advances and Utilization; Proceedings of an International Symposium held at the International Maize and Wheat Improvement Center (CIMMYT) 27 November-3 December, 1994. Mexico, D.F.: CIMMYT. Abstract: This publication reports advances in worldwide research on the mechanisms and bases of insect resistance in maize; the genetics of resistance; on the biotechnological manipulation of resistance; on techniques for the mass rearing of pests, for scoring damage, for conducting bioassays, and for detecting resistance mechanisms; and on the verification and use of resistance. It also describes maize insect pests and related research in specific countries and regions. AGROVOC Descriptors: Maize; Zea mays; Hybrids; Plant breeding; Pest resistance; Chilo; Diatraea; Sitophilus; Lepidoptera; Root eating insects; Stem eating insects; Leaf eating insects; Pest control; Biological control organisms; Research projects. Agris Category Codes: F30, H10 Dewey Decimal Classification: 633.153 ISBN: 968-6923-79-9 Printed in Mexico iii Cont e nt s vii viii Acknowledgments Foreword: Future Opportunities for Host Plant Resistance Research in the CIMMYT Maize Program D.C. Hess Mechanisms and bases of resistance 1 13 21 25 32 46 55 57 62 70 82 91 96 101 106 112 117 An Overview of the Mechanisms and Bases of Insect Resistance in Maize C.M. Smith The Effect of DIMBOA Concentration in Leaf Tissue at Various Plant Growth Stages on Resistance to Asian Corn Borer in Maize C.T. Tseng Impact of Mechanisms of Resistance on European Corn Borer Resistance in Selected Maize Hybrids B.D. Barry and L.L. Darrah Mechanisms and Bases of Resistance in Maize to Southwestern Corn Borer and Fall Armyworm W.P. Williams and F.M. Davis Chemicals Associated with Maize Resistance to Corn Earworm and Fall Armyworm M.E. Snook, B.R. Wiseman, N.W. Widstrom, and R.L. Wilson Mechanisms of Maize Resistance to Corn Earworm and Fall Armyworm B.R. Wiseman Mechanisms of Resistance in Maize to Southwestern Corn Borer and Sugarcane Borer H. Kumar and J.A. Mihm Variability for Maysin in Maize Germplasm Developed for Insect Resistance C. Welcker, G. Febvay, and D. Clavel A Review of Entomological Techniques and Methods Used to Determine Mechanisms and Bases of Stem Borer Resistance in Maize Z.R. Khan An Overview of Research on Mechanisms of Resistance in Maize to Spotted Stem Borer H. Kumar Phytochemical Basis for Multiple Borer Resistance in Maize D.J. Bergvinson, J.T. Arnason, J.A. Mihm, and D.C. Jewell Mechanisms of Resistance in Maize Grain to the Maize Weevil and the Larger Grain Borer J.T. Arnason, B. Conilh de Beyssac, B.J.R. Philogene, D. Bergvinson, J.A. Serratos, and J.A. Mihm Mechanisms of Resistance in Maize to Western Corn Rootworm J.T. Arnason, J. Larsen, R. Assabgui, Y. Xie, J. Atkinson, B.J.R. Philogene, and R.I. Hamilton Mechanisms and Bases of Resistance in Maize to Mites T.L. Archer, F.B. Peairs, and J.A. Mihm Mechanisms and Bases of Resistance in Maize to Chilo Partellus S.S. Sekhon and U. Kanta Maize Resistance to the Lesser Cornstalk Borer and Fall Armyworm In Brazil P.A. Viana and P.E.O. Guimarães Windows of Maize Resistance D.J. Bergvinson iv The genetics of resistance 127 132 139 143 Genetic Basis of Silk Resistance (Antibiosis) to the Corn Earworm in Six Crosses of Maize Lines: Statistical Methodology K. Bondari and B.R. Wiseman Genetics of Maize Grain Resistance to Maize Weevil J.A. Serratos, J.T. Arnason, A. Blanco-Labra, and J.A. Mihm Improving Two Tropical Maize Populations for Resistance to Stunt Complex R. Urbina Response to Selection for Resistance to Leaf Feeding by Fall Armyworm in PopG, a Guadeloupe Maize Population C. Welcker, J.D. Gilet, D. Clavel, and I. Guinet Biotechnological manipulation of resistance 148 155 159 163 172 175 178 Location and Effect of Quantitative Trait Loci for Southwestern Corn Borer and Sugarcane Borer Resistance In Tropical Maize M. Khairallah, M. Bohn, D.C. Jewell, J.A. Deutsch, J. Mihm, D. Hoisington, A. Melchinger and D. González-de-León Developing Insect Resistant Germplasm Using RFLP Aided Breeding Techniques D.L. Benson Construction of a Bioinsecticidal Strain of Pseudomonas fluorescens Active Against the Sugarcane Borer G. Herrera, S.J. Snyman and J.A. Thomson Developing Maize with Resistance to European Corn Borer J. Sagers, D. Mies, M. Edwards, B. Bolan, A. Wang, I. Mettler, L. Barrett, and C. Garrett The Expression of a Synthetic CryIA(b) Gene in Transgenic Maize Confers Resistance to European Corn Borer J.J. Estruch, N.B. Carozzi, N. Desai, G.W. Warren, N.B. Duck, and M.G. Koziel Sustaining Host Plant Resistance Derived Through Conventional and Biotechnological Means K.M. Maredia Insect Resistant Maize: A New Paradigm for Conducting Research J.E. Foster and S. Ramnath Advances in techniques (rearing, rating bioassays, mechanism detection) 184 189 195 203 211 Improved Technologies for Rearing Lepidopterous Pests for Plant Resistance Research F.M. Davis A New Technique for Evaluating Southwestern Corn Borer Damage to Post-Anthesis Maize F.M. Davis and W.P. Williams Assessing Damage by Second-Generation Southwestern Corn Borer and Sugarcane Borer and Development of Sources of Resistance in Tropical and Subtropical Maize H. Kumar and J.A. Mihm Advances in Rating and Phytochemical Screening for Corn Rootworm Resistance D.J. Moellenbeck, D.J. Bergvinson, B.D. Barry and L.L. Darrah Factors Affecting a Laboratory Bioassay for Antibiosis: Influences of Maize Silks on the Corn Earworm and Fall Armyworm Larvae B.R. Wiseman Resistance verification and utilization 217 Development of Germplasm with Resistance to the European Corn Borer B.D. Barry and L.L. Darrah v 221 226 230 234 241 246 255 261 266 Variability for Resistance to Fall Armyworm in Guadeloupe among Maize Populations Improved for Resistance to Various Insects C. Welcker, D. Clavel, J.D. Gilet, F. Felicite, and I. Guinet Maize Germplasm with Resistance to Southwestern Corn Borer and Fall Armyworm W.P. Williams and F.M. Davis Maintenance of, and Requests for, Maize Germplasm Having Resistance to Insect Pests R.L. Wilson Recent Advances in the Development of Sources of Resistance to Pink Stalk Borer and African Sugarcane Borer N.A. Bosque-Pérez, J.G. Kling, and S.I. Odubiyi The Importance of Institutional Linkages for the Development of Multiple Borer Resistant Maize Hybrids J.L. Overman Evaluation and Development of Maize Germplasm for Resistance to Spotted Stem Borer U. Kanta, B.S. Dhillon and S.S. Sekhon Verification and Pre-Commercial Testing of European Corn Borer and Gibberella Ear Rot Resistant Varieties R.I. Hamilton, L.M. Reid, and F. Meloche Introducing Unadapted, Insect–Resistant Maize Germplasm in Three–Way Hybrid Combinations for Resistance to the Maize Stalk Borer J.B.J. van Rensburg European Corn Borer Resistance: Evaluation of Commercial Maize Hybrids and Transgenic Maize Cultivars B.D. Barry and L.L. Darrah Country reports 271 276 279 283 287 291 293 297 Use of CIMMYT’s Multiple Borer Resistance Population for Developing Asian Corn Borer Resistance and Inbreds in China K. He, D. Zhou, and Y. Song Corn Borers Affecting Maize in Egypt M. Soliman Search for Multiple Resistance in Maize to Stem-Borers Under Natural Infestation in Midaltitude Intermediate Maturity Areas in Kenya M. Gethi Developing Rootworm Resistant Maize in México J.F. Pérez Domínguez, J.B. Maya Lozano, and J.A. Mihm Selection Methodology for Resistance to Dalbulus maidis and Fine Stripe Virus Disease in Maize in Peru P.H. Injante Silva, and J. Lescano Muñoz Mass Rearing of Helicoverpa zeae in Peru P.H. Injante Silva Progress of Host Plant Resistance Research to the Asiatic Corn Borer in the Philippines E.C. Fernandez, and D.M. Legacion Two Experimental Maize Varieties Selected for Resistance to Fall Armyworm and Sugarcane Borer in Tabasco, Mexico O.L. Segura-León Conclusion: 301 Host Plant Resistance — Alleviating Poverty and Improving Environmental Stability D.L. Winkelmann 303 Participants and Contact Information vi Ac k now le dgm e nt s I would like to thank UNDP for its long-standing financial support to host plant resistance research at CIMMYT and for funding the symposium and the proceedings. The efforts of editors Mike Listman and David Hodson in preparing the symposium materials for publication are greatly appreciated, as well as the professional design of Eliot Sánchez and Miguel Mellado and the layout of Juan José Joven, Marcelo Ortíz and Antonio Luna. Finally, the symposium and proceedings are especially dedicated to Professor Huai C. Chiang, the Happy Entomologist, in recognition of his pioneering efforts and success in international collaboration to enhance host plant resistance and integrated pest management, of his many contributions to agriculture and to knowledge on maize pest biology, and of his work as a dedicated and inspiring teacher. John A. Mihm vii Fore w ord D.C. Hess, Director, CIMMYT Maize Program Fut ure Opport unit ie s for H ost Pla nt Re sist a nc e Re se a rc h in t he CI M M Y T M a ize Progra m First, let me say that I have personally enjoyed the past four days listening to the some 65 presentations concerning the various aspects of host plant resistance. There is no question but that there has been more experts and expertise on maize host plant resistance here at this conference than ever before in a similar gathering. It is obvious that since the last similar conference held here in 1987, many scientific disciplines have become involved in team efforts to understand the mechanisms and intensify the efforts in increasing the effectiveness of host plant resistance. T he I m port a nc e of H ost Pla nt Re sist a nc e I would like to address the question of why insect host plant resistance is important to the CIMMYT Maize Program. Let me remind you that the mission of the Maize Program is “to help the poor of developing countries by increasing the productivity of resources committed to maize while protecting natural resources.” Maize that can be grown by resource poor farmers without being vulnerable to attacks by insects and without needing the application of usually scarce, expensive and often dangerous insecticides help these farmers increase their production of an essential food product, while protecting the environment. Another reason host plant resistance is important to the CIMMYT Maize Program is because it is a complex trait. National programs of the developing countries often find it beyond their capability to effectively manage this trait, although I hasten to say that there are some programs that have quite successful HPR programs. The trait is also not one that fits private seed companies very well, as they are often required to apply their resources on more short-term research projects. Smaller local seed companies usually find such complicated traits well beyond their very limited resources. It is also interesting to note that the areas of the developing world that need maize resistant to tropical insects are often the areas the multinational seed companies find less attractive markets. Since the trait is complex, it lends itself to the application of more advanced scientific techniques such as marker assisted selection. As we have heard several times during the week, host plant resistance is an important component of integrated pest management (IPM). In fact some would contend that it is by far the most important component of integrated pest management programs. Some of you are aware that there is an effort to establish an IPM facility which will be a collaborative effort by important funders to insure that IPM activities are emphasized and well supported throughout the world. The organizations behind this movement are the World Bank, UNDP, UNCED and USAID. This initiative should certainly boost the IPM efforts and along with them the strengthening of host plant resistance work. I hope that you all agree with me that HPR is an important component of IPM and will be influential whenever possible in assuring that HPR is included in IPM projects. viii Ge t t ing H PR M a ize t o Fa rm e rs I know you have seen about as many slides and overheads this week as you can stand; however, I would like to show just one more: Re a c hing De ve loping Count ry Fa rm e rs w it h I nse c t Re sist a nt M a ize Agronomic Proven improvement / • NARSs • OPVs sources of incorporation of • Inbreds resistance various traits • Hybrids (+ biotech) • National seed Farmers in companies developing • Multi-national countries seed companies • NGOs The diagram represents a series of steps that must occur in order for us to fulfill the objective of making insect resistant maize available to farmers; in CIMMYT’s case, to farmers of the developing world. Beginning at the left side of the diagram, I believe significant progress has been made in this area, largely due to the efforts of individuals in this room, especially those of John Mihm, Frank Davis, and Bill Wiseman. Through what we would now call conventional methods, these and other scientists have proven beyond a doubt that effective insect resistance maize is available and with enough effort the trait can be transferred to all types or genotypes of maize. This is not to say we are done with this part of the equation; there is certainly more work to do in this area and we do not yet know the limit that can be reached with host plant resistance. As we move to the next step of improving the insect resistant lines or varieties for agronomic and other traits, we are not so far along, at least at CIMMYT. A tremendous amount of work will be required to accomplish this task and, even after resistance is available in more productive and acceptable genotypes, they will have to be tested in new open pollinated varieties, inbreds and hybrids. We at CIMMYT will be making a concentrated effort to move the present level of resistance into the mainline breeding programs to help with this step. The next step is to deliver the products to those that can be effective in further research and evaluations, developing and recommending specific products for specific ecologies. These include national agricultural research programs, national and local seed companies as well as multinational seed companies, and non governmental organizations. And of course the final test is that of the farmers themselves. Unfortunately, at this time satisfactory insect resistant products have not been made available to farmers in any significant manner, especially in CIMMYT’s target areas in developing countries. The above challenges are far too large to be accomplished by a single organization but will require the efforts and linkages of all of our organizations. We think it is so important that we are contemplating developing a global special project that would enable us, working with others, to enhance the possibilities of success in accomplishing these goals. Certainly we at CIMMYT consider host plant resistance work one of our primary objectives. We believe that the time is right for host plant resistance to make significant impacts on the developing world , since the needs are so clear and the benefits of insect resistant maize are so great, for both productivity and the environment. We look forward to joining all of you in working on these very important tasks. 1 An Ove rvie w of t he M e c ha nism s a nd Ba se s of I nse c t Re sist a nc e in M a ize C. M. Smith, Department of Entomology, Kansas State University Abst ra c t Many insect resistant maize varieties have been developed during the past 50 years, due to the development of highly efficient techniques for maize insect pest rearing, artificial infestation and damage evaluation. Through the efforts of an international working group of scientists, maize genotypes developed primarily from the Antigua Group 2 gene bank and selected from it at CIMMYT have been shown to be resistant to many of the major lepidopterous pests of maize in the world. In several resistant varieties, resistance is controlled by different allelochemicals. The cyclic hydroxamic acid DIMBOA, and its decomposition product, 6-MBOA, occur in the foliage of some resistance sources. The flavone glycoside maysin and its related luteolin c-glucosides occur in the silks of other resistant varieties. These allelochemicals kill or impair the growth of many of the major insect pests of maize. Several morphological factors, including increased leaf fiber content, increased silica content, increased vascular bundle density, increased husk tightness and decreased leaf trichome density also contribute to some sources of resistance that do not have high levels of DIMBOA or maysin. Insect resistant maize greatly increases farming efficiency since insect control is available for the cost of only the seed. In addition, research on developing resistant varieties provides 100- to 300-fold greater returns on investment than research to develop insecticides. During the past 20 years, insect resistant maize in the United States has helped prevent the application of several million tons of insecticides onto croplands, reduced insecticide rates and applications, and encouraged the use of biological and cultural insect control practices in integrated maize insect pest management programs. Several examples demonstrate how insect resistant maize varieties act synergistically with both biological and chemical insect control tactics. National agricultural program staffs in many countries should work jointly with scientists located at centers that are members of the Consultative Group for International Agricultural Research to train farmers about the benefits of insect resistant maize varieties in insect pest management and incorporate insect resistance genes into locally adapted varieties which possess grain quality and yield desirable to specific localized conditions. I nt roduc t ion Studies of insect resistant maize began borer(SCB) Diatraea saccharalis (F.) in in the early 1900’s, when Hinds (1914) Caribbean and Mexican maize Though there are few written accounts, demonstrated the value of maize husk populations, respectively. Peairs (1977) early farmers in Africa, the Americas tightness and thickness for corn also identified resistance to the fall and Asia probably selected edible earworm (CEW), Helicoperva zea armyworm(FAW), Spodoptera frugiperda plants resistant to insect pests and (Boddie), resistance and Gernert (1917) (J.E. Smith) in tropical Mexican maize saved seed of these plants to continue demonstrated that corn leaf aphid populations. growing them in successive years. (CLA), Rhopalosiphum maidis (Fitch), Crops with insect resistant properties resistance existed in teosinte x yellow Over 300 varieties of insect resistant have helped United States agriculture dent corn hybrids. The first maize alfalfa, corn, sorghum, and wheat are for over 200 years. Wheat varieties with varieties with resistance to the grown presently in Africa, Asia, Europe resistance to the Hessian fly (HF), European corn borer(ECB), Ostrinia and the United States. Of these, over Mayetiola destructor (Say), were grown nubilalis (Hubner) were studied by one-half are cereal grains and many in New York around the beginning of Huber et al. (1928). In research at were developed by scientists at the the 1800’s. CIMMYT, Elias (1970) and Peairs (1977) International Maize and Wheat identified resistance to the sugarcane Improvement Center (CIMMYT) or 2 C. M. SMITH scientists around the world cooperating device, the bazooka or plant inoculator, or because there are no organized with CIMMYT researchers. In Missouri, that is used to infest plants with systems of pesticide distribution. a major U. S. maize producing state, neonate lepidoptera larvae mixed in cob Potential human health hazards are over 75% of all varieties grown possess grits (Davis and Williams 1980; Mihm et high with insecticide use, due to limited some resistance to whorl, leaf and al. 1978; Wiseman and Widstrom 1980; farmer training about insecticide sheath collar feeding of the ECB (Barry Wiseman et al. 1980 ) has greatly application methods and often and Darrah 1991). Today, entomologists improved the efficiency and accuracy of inadequate water supplies. The need and maize breeders continue to make many insect resistance plant breeding for insect resistant maize varieties is global progress toward the release and programs and tremendously also high in the tropics, since pest production of multiple insect resistant accelerated the rate of progress of incidence is greater than in temperate maize varieties. Through the efforts of identifying sources of resistance in regions, due to rapid pest population an international working group of maize to many foliage feeding increases, which lead to several scientists, maize genotypes derived Lepidoptera. continuous pest generations each year. (Smith et al. 1989). primarily from the CIMMYT Antigua Group 2 germplasm have been shown In this paper, I will provide some to be resistant to several major working definitions on plant resistance The effects of plant resistance to insects lepidopterous pests of maize in Africa, to insects, discuss the advantages to the are cumulative over time, and the Asia, Latin America and North America use of insect resistant maize, and review longer resistance is employed and (Ampofo et al. 1986; Dabrowski 1990; the allelochemical and morphological effective, the greater the benefits of its Dabrowski and Nyrangiri 1983; Davis mechanisms of insect resistance in use. Panda (1979) demonstrated an and Williams 1986; Davis et al. 1988; maize. average 12-fold population reduction among 25 different insect pests Mihm 1985; Smith et al. 1989). Ec onom ic Adva nt a ge s damaging 10 food and fiber crops. In a 10 year study of rice insect pest related- In the first textbook on insect resistance in crop plants, Painter (1951) described There is a major economic advantage to crop losses in the Philippines, Waibel methods to measure plant resistance to the use of insect resistant varieties by (1987) determined that the 10-year insects. Since then, gas and high farmers. Insect resistant crops greatly average yield losses of insect resistant pressure liquid chromatography, x-ray increase farming efficiency by reducing rice varieties were approximately one- crystallography and mass spectral or eliminating the costs of insecticides half (14%) of the losses in susceptible analysis have become routinely used to and reduce or eliminate the risk of yield rice varieties (26%). quantify allelochemicals involved in losses from insect damage. When insect maize resistance to insects. resistant varieties are planted, insect Plant resistance research provides a control is available for little more than substantially greater return (as much as Transmission and scanning electron the cost of the crop seed, and there is 120-fold greater) on each research microscopy also permit the study of the often no need or in many cases, a dollar invested, compared to research cellular as well as the whole structure greatly reduced need to purchase on the development of insecticides. morphological bases of insect resistant insecticides or the equipment to apply Since the late 1960s, wheat varieties maize. them for pest control. The advantages with HF resistance have been proven to to the use of insect resistant varieties are return approximately $600 per research Artificial diets and rearing methods especially important in developing dollar invested, compared to a $5 have been developed for many of the countries, where farmers can rarely return per dollar spent on insecticide major maize pest insects of the world afford to purchase insecticides for crop development (Painter 1968). (Mihm 1983a,b,c; Ortega et al. 1980). protection. In this setting, they provide These accomplishments have greatly practical and economical ways to Insect resistant cultivars of alfalfa, corn, increased the rate at which new sources minimize losses to insect pests (Mihm barley and wheat have been proven to of insect resistance have been 1989). Many of these farmers have a have marked economic advantages in identified. The invention and widely limited access to insecticides, because United States agriculture (Luginbill accepted use of a very simple plastic they lack the income to purchase them 1969; Maxwell et al. 1972; Painter 1968). AN OVERVIEW OF INSECT RESISTANCE IN MAIZE 3 Based on reductions in the costs of Isenhour and Wiseman (1987) found a Wiseman (1986) confirmed the insecticide applications and reduced synergistic interaction between existence of a synergistic interaction insect damage, the value of insect genotypes of maize resistant to FAW between maize varieties resistant to leaf resistant cultivars of these crops during and its parasite, Campoletis sonorensis feeding by the FAW and the nuclear the 1970’s was nearly $500 million each (Cameron). Parasitism results in further polyhedrosis virus (NPV). The year (Schalk and Ratcliffe 1976). reductions in FAW larval weights over protozoan parasite, Nosema pyrausta Though insect resistant crops are sound those caused by FAW consumption of and maize varieties resistant to leaf and economic investments for the resistant foliage alone and has no sheath-collar feeding by the ECB, agricultural economy of any country, adverse effects on parasite interact to significantly reduce ECB United States crop production using development. populations (Lynch and Lewis 1976; Lewis and Lynch 1976). insect and mite resistant alfalfa, barley, corn, sorghum, and wheat cultivars In research with Cotesia marginiventris currently returns an economic benefit of (Cresson), a naturally occurring parasite Moderately insect-resistant crop over $1.4 billion each year. of FAW, Riggin et al. (1992, 1994) varieties are normally compatible with demonstrated in laboratory and field different types of biological control. studies that FAW-resistant maize However, some resistant varieties that varieties have no negative effect on the possess high levels of toxic plant rate of FAW parasitism and that FAW allelohemicals or dense levels of leaf or Com pa t ibilit y w it h I nt e gra t e d Pe st M a na ge m e nt larvae feeding on resistant plants are stem trichomes have been shown to Insect resistant maize varieties more heavily parasitized than those have negative effects on beneficial generally compliment integrated pest feeding on susceptible plants. insects (Campbell and Duffey 1979; Obrycki et al. 1983). Similarly, management (IPM) tactics such as chemical and biological insect control Wiseman et al. (1976) demonstrated that allelochemicals mediating insect (Table 1.). Improved maize varieties higher levels of the predator Orious resistance in plants may adversely resistant to the CEW require much less insidiosus Say, are found on maize affect the synergism of resistance with insecticide (in some cases, as much as hybrids tolerant to CEW during and NPV (Felton et al. 1987). 28-fold less ) than susceptible varieties after silking. This interaction to achieve equivalent control (Wiseman contributes to a greater suppression of Plant breeding goals, however, et al. 1975). Insecticides applied to CEW larval populations on the resistant normally strive to incorporate moderate maize varieties with intermediate and hybrid than on susceptible hybrids. levels of insect resistance in varieties with yield, processing and cooking high levels of resistance to the ECB are of little benefit in reducing borer The interactions of viruses and fungi qualities acceptable to farmers and damage in the field (Robinson et al. with insect resistant maize varieties are consumers. Such varieties also guard 1978). not well known. However, Hamm and against the development of resistancebreaking insect biotypes and insure a Table 1. Examples of synergistic interaction of insect resistant maize with various integrated pest management tactics. IPM Tactic Insect affected Reference(s) Insecticidal Corn earworm European corn borer Wiseman et al. 1975 Robinson et al. 1978 Biological Archytus marmoratus and Ichneumon promissorius Campoletis sonorensis Cotesia marginiventralis Nosema pyrausta Nuclear polyhedrosis virus Orious insidiosus longer useful life of resistant varieties that work synergistically with natural enemies. The numerous advantages of the compatibility of maize resistance to pests with other IPM tactics are sufficient to indicate that varieties produced by all maize improvement programs should possess some level of Corn earworm Fall armyworm Fall armyworm European corn borer Fall armyworm Corn earworm Mannion et al. 1994 Isenhour and Wiseman 1987 Riggin et al. 1994 Lewis and Lynch 1976 Hamm and Wiseman 1986; Wiseman and Hamm 1993 Wiseman et al. 1976 insect resistance. Unfortunately, many current maize varieties have limited, if any, insect resistance. 4 C. M. SMITH Environm e nt a l a nd Soc ia l Adva nt a ge s metabolites (Pearce et al. 1991). Induced categorization of phenomena and the resistance may last from a few to basic study of the causative factors or several days. processes.” In his discussion of the different types of plant resistance to In addition to being compatible with IPM tactics and economically Ca t e gorie s of Re sist a nc e insects, Horber (1980) chose to describe the three elements of the resistance advantageous to farmers, insect resistant crop varieties, including some In addition to the types of resistance triad as functional categories of maize varieties, improve the quality of described above, three categories have resistance. Smith (1989) termed these the environment and the general health been referred to since their description categories functional modalities of of agricultural producers and by Painter (1951). Antibiosis and resistance. consumers. By reducing the amount of nonpreference resistance describe the insecticides applied in maize reaction of an insect to a plant, while According to Webster’s 7th New production, as shown above, insect tolerance resistance describes the Collegiate Dictionary, a “category” is a resistant maize varieties increase the reaction of a plant to insect infestation general class or group, and a safety of food produced for animal and and damage. In antibiosis resistance, “modality” is a classification or form. human consumption, protect water the biology of the pest insect is Conversely, a “mechanism” is a supplies from insecticide contamination adversely affected after feeding on the fundamental physical or chemical and help improve the general quality of plant. With nonpreference resistance process involved in or responsible for water resources. (now referred to by many researchers an action, reaction or other natural as antixenosis ( Kogan and Ortman phenomenon. The term “basis” refers 1978 )), the plant is as a poor host and to the foundation or principal the pest insect then selects an alternate component of anything. Thus, the “Plant resistance to insects” is the host. Plant tolerance describes the terms category and modality refer to genetically inherited qualities that inherent genetic vigor or growth the way a group of items are classified, result in a plant of one variety or capacity of a resistant plant that gives it while the terms basis and mechanism species being less damaged than a the ability to withstand or recover from denote the principal process governing susceptible plant lacking these insect damage that a susceptible plant a natural phenomenon. qualities. Resistance is a relative cannot survive. De finit ions In applying these terms to the study of property, based on the comparative reaction of resistant and susceptible In describing his attempts to classify plant resistance to insects, many plants, grown under similar conditions, causes of plant resistance to insects, examples exist to show that insects are to the pest insect. “Pseudo”- or “false Painter (1951) stated “I have attempted affected by resistant plants in ways we resistance” may occur in susceptible to work out a classification of those categorize or classify as antibiosis or plants due to earlier than normal items suggested as ‘cause(s)’ of antixenosis, while plants themselves planting, low levels of insect resistance so as to emphasize the insect- demonstrate tolerance as a third type of infestation, or variations in plant interrelations that are a feature of resistance. In contrast to Painter’s use temperature, day length, soil chemistry insect resistance.” Painter then of the term, I propose that the term and plant or soil water content. presented the now classic diagram of “mechanisms” be used to describe the “Associational resistance” refers to a the three-fold basis of field plant underlying chemical or morphological normally susceptible plant growing in resistance to insects, consisting of what plant processes that, where known, are association with a resistant plant, and he termed the three bases or responsible for the (negative) reaction deriving protection from insect mechanisms of resistance. However, in of insects to resistant plants. To predation. “Induced resistance”, the the legend explaining the diagram, he describe the outcome of insect-plant enhancement of a plant’s pest defense referred to these as mechanisms of interactions, I propose the use of the system in response to external physical resistance classifications. term “categories” to refer to antibiosis, antixenosis and other as of yet or chemical stimuli, (Kogan and Paxton 1983) occurs in many crops due to the Horber (1980) referred to Painter’s triad undefined types of plant-insect elicitation of endogenous plant of resistance as a ... “workable interactions, observed as responses of compromise between mere AN OVERVIEW OF INSECT RESISTANCE IN MAIZE 5 insects to plant resistance mechanisms. actual contributions of plant factors to toxins, or, the absence of feeding or I will use these definitions throughout each category of resistance. From a oviposition stimulants. In one instance, the remainder of this manuscript. practical standpoint, the absolute the lack of nutrients has been shown to contribution of a given category may affect insect resistance in maize. Penny Often, antibiosis and antixenosis never need to be fully understood et al. (1967) determined that maize resistance overlap because of the before a resistant variety is released. resistant to ECB larvae had an ascorbic acid content that was inadequate to difficulty involved in designing experiments to delineate between the From an ecological and environmental support normal ECB larval growth. two. Horber (1980) stated that “all three standpoint, tolerance has many Resistance may also be a result of the categories, while workable, are advantages, since it does not adversely density of external or internal plant arbitrary and vaguely delineated,” affect beneficial insects or exert structural features that either alter since not all resistance can be assigned sufficient selection pressure on pest insect behavior or reduce insect into one of these categories. An insect insect populations to develop biotypes digestion. In some maize varieties, the confined to a resistant plant may fail to as does antibiosis alone. Often content of silica containing cells is high gain weight at the rate it normally does however, agricultural producers tend enough to adversely affect ECB larval on a susceptible plant, due presumably to prefer varieties with antibiosis and feeding and impart some resistance to to the presence of antibiotic properties antixenosis resistance that reduce pest ECB (Rojanaridpiched et al. 1984). in the plant. However, reduced weight abundance. We, as conscientious gain may also be due to the presence of agricultural researchers also often The lethal effects from both an antixenotic physical or chemical screen for antibiosis and antixenosis in allelochemical and morphological feeding deterrent that causes aberrant developing maize varieties. However, factors may be acute, often affecting behavior in the test insect, resulting in a tolerance in maize to the northern corn young larvae, or chronic, and lead to weakened physiological condition. rootworm (NCRW), Diabrotica barberi mortality in older larvae, prepupae, Smith and Lawrence, the western corn pupae, and adults, where larvae and Antibiosis exists in maize to the aphid rootworm (WCRW), Diabrotica virgifera pupae fail to pupate and eclose, Metopolophium dirhodum (Walker) virgifera LeConte, the CEW, the maize respectively. Individuals surviving the (Argandona et al. 1980); the CLA (Long borer, Chilo partellus (Swinhoe) and direct effects of these plant defenses et al. 1977); the CEW (Waiss et al. 1979; ECB, are well documented (Dabrowski may exhibit the debilitating effects of Wiseman et al. 1992a,b) the ECB (Klun and Nyangiri 1983; Mollenbeck et al. reduced body size and weight, et al. 1970; Robinson et al. 1982b); the 1994; Ortman et al. 1968; Wiseman and prolonged periods of development in FAW ( Hershey 1978; Wiseman et al. Widstrom 1992; Wiseman et al. 1972; the immature stages, and reduced 1981) and the southwestern corn Zuber et al. 1971). At CIMMYT, fecundity as surviving adults. borer(SWCB), Diatraea grandiosella Dyar Hershey (1978) identified several (Davis et al. 1989). Antixenosis exists in progeny from three tropical maize maize to the CEW (Wiseman et al. populations with tolerance to the FAW 1977), the ECB (Robinson et al. 1978), and Smith (1982) developed moderate Organic acids were some of the first the FAW (Wiseman et al. 1981), the levels of FAW tolerance in selected allelochemicals found to mediate maize weevil (MW), Sitotroga zeamais lines of Tuxpeno germplasm. antibiosis to insects in several maize Motchulsky, the rice weevil (RW), Sitophilus oryzae (L.) (Singh et al. 1972; Wiseman et al 1974) and the SWCB (Davis et al. 1989). Smith (1982) Pla nt Alle loc he m ic a ls varieties. An aglucone in maize foliage, Alle loc he m ic a l a nd M orphologic a l M e c ha nism s of Re sist a nc e identified both antibiosis and 2,4-dihydroxy-7-methoxy-2H-1, 4benzoxazin-3(4H)-one, (DIMBOA) is one of the more widely studied plant allelochemicals affecting crop antixenosis resistance to FAW in Both chemical and morphological resistance to arthropods. When normal, certain Caribbean maize germplasm. maize defenses mediate resistance to healthy maize foliage is mechanically insect pests. Resistance may be due to damaged, the glucoside, 2-0-glucosyl-4- Very detailed sets of experiments are the presence of olfactory repellents, hydroxy-1, 4-benzoxazin-3-one, is normally required to delineate the feeding or oviposition deterrents, and enzymatically converted to DIMBOA 6 C. M. SMITH (Fig. 1 ) (Loomis et al. 1957; Smissman Feng et al. (1990, 1992) demonstrated toxic or deterrent to several insects, et al. 1957; Wahlroos and Virtanen that ingestion of DIMBOA and MBOA Bjostad and Hibbard (1992) found that 1959). DIMBOA and its decomposition by ECB greatly increases the levels of MBOA functions as a volatile attractant product, MBOA have antibiotic effects activity of several detoxification to WCRW in combination with carbon on the ECB (Barry et al. 1994; Campos enzymes, including cytochrome b5 , dioxide. Related research (Aboufakhr et et al. 1988; Klun and Brindley 1966; NADH oxidase, NADH cytochrome c al. 1994) has demonstrated that MBOA Klun et al. 1967; 1970, Robinson et al. reductase and o-demethylase. is non-toxic to WCRW larvae. Other major foliage or stem feeding 1982b), and limited antibiotic effects on the SWCB and the FAW (Nicollier et al. Xie et al. (1990, 1992) demonstrated that lepidopterous pests of maize do not 1982). Robinson et al. (1982a) developed CIMMYT maize lines developed by suffer significant adverse effects from an accurate, efficient thin layer Agriculture Canada with high DIMBOA or MBOA. chromatography (TLC) technique to DIMBOA root content negatively affect identify maize lines with high the emergence of WCRW adults and The flavone glycoside maysin (Fig. 2), is concentrations of MBOA for ECB that one high DIMBOA line is an allelochemical contained in the silks resistance. Barry et al. (1994) surveyed significantly less damaged by CRW of maize varieties resistant to CEW and ECB leaf feeding resistance and larvae than a low DIMBOA line. FAW (Waiss et al. 1979; Ellinger et al. DIMBOA content in progeny of crosses Although MBOA has been shown to be 1980; Wiseman et al. 1992a). Increasing of resistant and susceptible maize varieties and found the two traits to be positively correlated. Their results and CH3 O O Glucose O CH3 O O OH CH3 O O those of Sullivan et al. (1974) however, O indicate that some maize germplasm that resists ECB leaf feeding does so N without a high DIMBOA content. O N OH N O H OH Glucoside DIMBOA The CLA and the aphid Metopolophium CH3 O dirhodum (Walker) are also adversely O OH N O MBOA affected by DIMBOA (Argandona et al. 1980; Long et al. 1977). CLA population levels sustained on various maize HMBOA varieties are strongly correlated to the DIMBOA concentration of each variety (Beck et al. 1983). HMBOA (Fig. 1), another intermediate degradation product of DIMBOA (Feng et al. 1992; OH Figure 1. Production of DIMBOA (2,4-dihydroxy-8-methoxy-2H-1, 4-benzoxazin3(4H)-one), MBOA (6-methoxyben-zoxazolinone) and HMBOA (2-hydroxy-7methoxy-1,4-benzoxazin-3-one) by enzymatic hydrolysis of a glucoside of mechanically damaged maize foliage (from Campos et al. 1988; Feng et al. 1992; and Klun et al. 1967). Kumar et al. 1994) may also have toxic effects on ECB. N-O-ME DIMBOA (2hydroxy-4, 7-dimethoxy-1,4- (a) OH benzoxazin-3-one), yet another related compound, exists in higher concentrations than DIMBOA or MBOA in the surface waxes of some OH HOOC HO 1993). Total surface wax content of these varieties is higher than in susceptible varieties. H H OH OH O HO O SWCB-resistant maize varieties derived from CIMMYT germplasm (Hedin et al. R1 (b) H CH3 O O H OH H OH H R2 O O α-Rha Figure 2. Chlorogenic acid (a) and the related flavonoid glycosides (b) maysin (R1 = OH, R2 = OH), apimaysin (R1 = CH3, R2 = CH3) and 3'-methoxymaysin (R1 = CH3, R2 = OH) from foliage of insect resistant maize cultivars which inhibit growth of the corn earworm, Helicoverpa zea Boddie and fall armyworm, Spodoptera frugiperda (J. E. Smith) (Gueldner et al. 1991; Wiseman et al. 1992a). AN OVERVIEW OF INSECT RESISTANCE IN MAIZE 7 the concentration of maysin in artificial Widstrom 1992; Wiseman et al. 1977), negatively correlated with SWCB larval diets inhibits the growth of these the MW, (Wiseman et al. 1974) and the feeding damage. Ng (1988) found that insects proportionally (Wiseman et al. RW (Singh et al. 1972). Maize varieties Mp701 has more vascular bundles, 1992a). The related luteolin c-glycosides with reduced trichome density and thicker cuticle and a thicker outer chlorogenic acid, apimaysin (the delayed development of pubescence epidermal cell wall than susceptible apigenin analogue of maysin) and 3' - have been shown to be less preferred inbred lines. Recent results by Davis et methoxymaysin (Fig. 2) may also for oviposition by CEW and are al. (1995) with Mp496, 704, 706 and 708 contribute to the resistance of maize to resistant to larval feeding (Wiseman et confirm these findings and also the CEW and the FAW (Gueldner et al. al. 1976; Widstrom et al. 1979). At demonstrate that inner whorl leaves of 1991,1992; Wiseman et al. 1992b). CIMMYT, screening and breeding these Mp inbred lines have thicker maize for oviposition nonpreference is leaves and thicker upper and lower leaf Growth inhibition in insects feeding on avoided, since moth oviposition epidermal cell walls than susceptible resistant maize may also be related to behavior can evolve to overcome the inbred lines. Leaf feeding damage by altered nutrient levels. Early research oviposition resistance of germplasm SWCB and FAW larvae is highly conducted by Penny et al. (1967), and because soil and environmental correlated with epidermal cell wall determined that maize resistant to ECB factors interact to make adult thickness. In research with another Mp larvae had an ascorbic acid content oviposition behavior measurements resistance source, MpSWCB-4, Yang et inadequate for larval growth. difficult to reliably predict (Mihm 1989). al. (1991, 1993) determined that Pla nt M orphology Several maize inbred (Mp) lines whorl leaves removes resistance to removal of leaf cuticular lipids from developed jointly by scientists at the FAW larval feeding. Gel Several types of morphological USDA Crop Science Research electrophoresis of the total leaf protein defenses in maize varieties deter insect Laboratory at Mississippi State, extracts from field grown tissues of feeding and oviposition (Table 2). As Mississippi (including Mp496, 701, 704, Mp496, 701, 707, and 708 has identified previously mentioned, increased leaf 706, and 708) have morphological polypeptides which predict SWCB and and stem silica content contribute to defenses related to their resistance to FAW resistance (Callahan et al. 1992). ECB resistance in some maize varieties the CEW, the ECB, the FAW, the SWCB (Rojanaridpiched et al. 1984). Tight- and the SCB (Davis et al. 1988). Hedin Many of these Mp lines have been used husked maize ears, a character also et al. (1984) demonstrated that Mp701 as resistance components to develop mentioned previously, continue to and Mp496 have higher hemicellulose the CIMMYT multiple borer resistant contribute the resistance of current and crude fiber content than susceptible (MBR) maize population 590 (Benson varieties to the CEW (Wiseman and inbred lines, and that crude fiber is 1986). Bergvinson (1993) found significant correlations between the leaf Table 2. Morphological defenses of insect resistant maize. fiber content and cell wall dehydrodiferulic acid content of MBR Defense Insect(s) affected Reference(s) Dense surface waxes Southwestern corn borer Fall armyworm Hedin et al. 1993 Yang et al. 1991,1993 High fiber, dense vascular bundles, high hemicellulose, thick cuticle European corn borer Fall armyworm Southwestern corn borer Sugarcane borer Bergvinson 1993, Davis et al. 1995, Hedin et al. 1984, Ng 1988 Low trichome density Corn earworm Widstrom et al. 1979, Wiseman et al. 1976 New discoveries in crop plant rapidly, and maize insect resistance Silica European corn borer Rojanaridpiched et al. 1984 Tight husks Corn earworm Wiseman et al. 1977, Wiseman and Widstrom 1992 Wiseman et al. 1974 Singh et al. 1972 lines with ECB leaf feeding damage, and that leaf toughness was inversely related to leaf feeding damage. Ge ne t ic a lly T ra nsform e d M a ize molecular genetics are occurring research is currently moving molecular Maize weevil Rice weevil biology into maize production and protection (Koziel et al. 1993). Within 8 C. M. SMITH the next five years, hybrid maize be the selection of well-defined, somaclonal variants indicated above containing transgenic insect resistance functional IPM systems in which to test did not prove to be highly resistant to will be sold commercially in the United different release strategies. FAW in field trials at CIMMYT (Mihm et al. unpublished manuscript). States. The resistance factor(s) in these hybrids is derived from the HD-1-delta- I nduc e d Re sist a nc e Sum m a ry a nd Conc lusions endotoxin gene that encodes plant DNA to produce a crystal protein from New discoveries in the area of induced the bacteria Bacillus thuringiensis (B.t.). plant resistance to arthropods indicate During the past thirty years, numerous The protein is toxic to insects but not to that this physiological process is likely sources of multiple insect resistant mammals. Research during the next a part of a general maize plant maize germplasm have been decade will attempt to develop gene protection mechanism against insect developed, and a detailed release strategies that maximize the life damage. Guiterrez et al. (1988) understanding of the allelochemical span of different B.t. genes for insect demonstrated that in a maize variety and morphological mechanisms of resistance in maize and other crops. with high DIMBOA content and some of this germplasm has begun to CIMMYT’s varietal release strategy is to resistance to the maize borer (MB), be understood. The production of pyramid B.t. genes into maize Sesamia nonagrioides, and in a variety maize varieties with genetically- populations with existing multigenic with low DIMBOA content and expressed pest resistance has improved pest resistance, in order to enhance susceptibility to MB, both varieties farming profitability and both the levels and durability of plant contained significantly increased leaf environmental safety in many resistance to maize pests. DIMBOA content within 3 days of MB developed countries. Techniques infestation. Thus, the existence of the invented by maize researchers in There is a real need for varietal release same physiological phenomenon in developing these varieties have also strategies that avoid promoting the both insect-resistant and susceptible provided many benefits to global development of resistance-breaking maize varieties indicates the possibility agricultural research and production. insect biotypes similar to those that of using the inherent induced response These have all been truly remarkable have developed resistance to of all maize genotypes to develop types developments. insecticides. Such strategies are of insect resistance to complement necessary because of the high potential previously identified allelochemical However, these accomplishments are that exists for the selection of B.t.- and morphological based sources of yet to result in a corresponding resistant pest populations when seed of maize resistance to insects. increase in the use of insect resistant maize by farmers in many developing transgenic crops are marketed for production. Gene release strategies are Ca llus T issue Cult ure countries of the world. In the next five years, Africa’s population will grow at especially necessary for highly polyphagous pest insects, such as The callus tissues of some maize a rate of 3% annually, but food migratory Lepidoptera that will be varieties exhibit resistance to the FAW, production will increase by only 2% exposed to the B.t. toxin in maize and SWCB and CEW that closely resembles each year, computing to an annual other crops in the same agroecosystem. damage to whole plant foliage African food production shortage of (Williams and Davis 1985; Williams et about 250 million tons by the end of the The development of successful gene al. 1985, 1987a, 1987b; Isenhour and century (Anonymous 1992). African release strategies will depend on the Wiseman 1988). Isenhour and Wiseman food production capabilities have ability of researchers in government, (1991) isolated somaclonal variant steadily eroded over the past 20 years, industry and universities to plants regenerated from callus tissues but there is limited use of IPM or insect cooperatively conduct field of maize genotypes resistant to FAW resistant crop varieties in most of experiments that test several different that have greater levels of FAW African agriculture. types of gene release techniques. An resistance than non-regenerated lines. additional factor that will directly affect The use of regenerated lines in a With increasing demands for an the success of the development of breeding program for enhanced insect abundant and safe world food supply, transgenic plant release strategies will resistance should proceed with caution, there are many countries where insect however, as field screening of the resistant maize can make an important AN OVERVIEW OF INSECT RESISTANCE IN MAIZE difference. What will the strategies be varieties is recognized as one of the for the 21st century to ensure most highly productive areas of deployment of insect resistant maize modern agricultural research. Genes for varieties? I believe a real challenge now resistance to most of the major maize exists for International Agricultural insect pests have been identified and Research Centers to work with National incorporated into maize breeding Agricultural Research Staffs (NARS) to programs in many countries, and the deploy insect resistant maize varieties future is bright for continuing success into the field in the same way that in many other parts of the world. New genes have been deployed around the and emerging genetic technologies also world to be screened for resistance. In promise to enhance the types and order for this cooperative effort to numbers of insect resistance genes work, NARS will need to actively available for placement into maize provide funding and personnel in this varieties. There is also a solid process. NARS, agricultural understanding or the major plant economists, rural sociologists and pest chemical and physical factors management workers must help mediating maize resistance to certain farmers realize the benefits and major insect pests. With all of these limitations of insect resistant maize factors in place, there are really no varieties in their fields. Farmers must major reasons why varieties with be assisted to understand that insect resistance to all major insect pests of resistant maize can lower yield losses maize cannot be developed and from insect damage and increase their cultivated. A key to this harvests and market profits. accomplishment will be to mesh the IPM needs of maize farmers at the local What will the research agenda for level with the sociological needs of maize insect resistance be in the next farmers in each maize growing location century? International research teams (Peairs 1989). When this is such as those mentioned in this paper accomplished, varieties with the must continue to develop and refine necessary combinations of insect accurate and efficient maize insect pest resistance, high yield and good grain bioassay techniques, continue to quality can be “tailored” to fit the needs discover the functional categories and of farmers in specific geographic underlying mechanisms mediating conditions. resistance, and continue to develop and refine microanalytical techniques to Re fe re nc e s determine resistance mechanisms. Although knowledge continues to accumulate at a rapid rate concerning the allelochemical and morphological bases of insect resistance in maize plants, in only a few cases such as DIMBOA, is the specific site of activity of a plant allelochemical on insect metabolism actually known. The science of identifying, quantifying and developing insect resistant maize Aboufakhr E.M., B.E. Hibbard, and L.B. Bjostad. 1994. 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Smith) and sugarcane borer (Diatraea saccharalis F.): Evaluation techniques and potential for genetic improvement. Ph.D. Thesis, Cornell Univ., Ithaca, NY, USA. Hinds, W.E. 1914. Reducing insect injury in stored corn J. Econ. Entomol. 7: 203211. Horber, E. 1980. Types and classification of resistance. In F.G. Maxwell and P.R. Jennings (eds.). Breeding Plants Resistant to Insects. John Wiley and Sons, New York. 15-21. Huber, L.L., C.R. Neiswander, and R.M. Salter. 1928. The European corn borer and its environment. Ohio Agr. Exp. Sta. Bull. Isenhour, D.J., and B.R. Wiseman. 1987. Foliage consumption and development of the fall armyworm (Lepidoptera: Noctuidae) as affected by the interactions of a parasitoid, Campoletis sonorensis (Hymenoptera: Ichneumonidae), and resistant corn genotypes. Environ. Entomol. 16: 11811184. ibid. 1988. Incorporation of callus tissue into artificial diet as a means of screening corn genotypes for resistance to the fall armyworm and the corn earworm (Lepidoptera: Noctuidae).. Soc J. Kansas Entomol. 61: 303-307. Ibid. 1991. Fall armyworm resistance in progeny of maize plants regenerated via tissue culture. Fla. Entomol. 74: 221228. Klun, J.A., and T.A. Brindley. 1966. Role of 6-methoxybenzoazolinone in inbred resistance of host plant (maize) to firstbrood larvae of the European corn borer. J. Econ. Entomol. 59: 711-718. Klun, J.A., C.L. Tipton and T.A. Brindley. 1967. 2,4-dihydroxy-7-methoxy-1, 4benzoxazin-3-one (DIMBOA), an active agent in the resistance of maize to the European corn borer. J. Econ. Entomol. 60: 1529-1533. Klun, J.A., W.D. Guthrie, A.R. Hallauer, and W.A. Russell. 1970. Genetic nature of the concentration of 2,4-dihydroxy 7-methoxy 2H-1,4 benzoxazin-3 (4H)one and resistance to the European corn borer in a diallell set of eleven maize inbreds. Crop Sci. 10: 87-90. Kogan, M., and E.E. Ortman. 1978. Antixenosis-a new term proposed to replace Painter’s “Non-preference” modality of resistance. Bull. Entomol. Soc. Am. 24: 175-176. Kogan, M., and J. Paxton. 1983. Natural inducers of plant resistance to insects. In P.A. Hedin (ed.), Plant Resistance to Insects. Am. Chem. Soc. Symp. Series 208, American Chemical Society, Washington, DC. 153-171. Koziel, M.G., G.L. Beland, C. Bowman, N.B. Carozzi, R. Crenshaw, L. Crossland, J. Dawson, N. Desai, M. Hill, S. Kadwell, K. Launis, K. Lewis, D. Maddox, K. McPherson, M.R. Meghji, E. Merlin, R. Rhodes, G.W. Warren, M. Wright, and S.V. Evola. 1993. Field performance of elite transgenic plants expressing an insecticidal protein derived from Bacillus thuringiensis. Bio/Technol. 11: 194-200. Kumar, P., D.E. Moreland, and W.S. Chilton. 1994. H-1, 4-benzoxazin3(4H)-one, an intermediate in the biosynthesis of cyclic hydroxamic acids in maize. Phytochem. 36: 893-898. AN OVERVIEW OF INSECT RESISTANCE IN MAIZE Lewis, L. C. and R.E. Lynch. 1976. Influence on the European corn borer of Nosema pyrausta and resistance in maize to leaf feeding. Environ. Entomol. 5: 139-142. Long, B.J., G.M. Dunn, J.S. Bowman, and D.G. Routley. 1977. Relationship of hydroxamic acid content in corn and resistance to the corn leaf aphid. Crop Sci. 17: 55-58. Loomis, R.S., S.D. Beck and J.F. Stauffer. 1957. The European corn borer, Pyrausta nubilalis (Hubn), and its principal host plant. V. A chemical study of host plant resistance. Plant Physiol. 32: 379-385. Luginbill, P., Jr. 1969. Developing resistant plants — an ideal method of controlling insects. USDA, ARS. Prod. Res. Rpt. III. 14pp. Lynch, R.E., and L.C. Lewis. 1976. Influence on the European corn borer of Nosema pyrausta and resistance in maize to sheath-collar feeding. Environ. Entomol. 5: 143-146. Mannion, C.M., J.E. Carpenter, B.R. Wiswman, and H.R. Gross. 1994. 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SMITH Smith, M.E. 1982. Studies on fall armyworm resistance in Tuxpeno and Antigua maize populations. Ph.D. Thesis, Cornell University, Ithaca, NY, USA. Smith, M.E., J.A. Mihm, and D.C. Jewell. 1989. Breeding for multiple resistance to temperate, subtropical and tropical maize insect pests at CIMMYT. In Toward insect resistant Maize for the Third World: Proceedings of the International Symposium on Methodologies for Developing Host Plant Resistance to Maize Insects. 222-234. Mexico, D. F.:CIMMYT. Sullivan, S.L., V.E. Gracen, and A. Ortega. 1974. Resistance of exotic maize varieties to the European corn borer, Ostrinia nubilalis (Hubner). Environ. Entomol. 3: 718-720. Wahlroos, V., and A.I. Virtanen. 1959. The precursors of 6- MBOA in maize and wheat plants, their isolations and some of their properties. Acta Chem. Scan. 13: 1906-1908. Waibel, H. 1987. The Economics of Integrated Pest Control in Irrigated Rice. A Case Study from the Philippines. SpringerVerlag. Berlin. Waiss, A.C, Jr., B.G. Chan, C.A. Elliger, B.R. Wiseman, W.W. McMillian, N.W. Widstrom, M.S. Zuber, and A.J. Keaster. 1979. Maysin, a flavone glycoside from corn silks with antibiotic activity toward corn earworm. J. Econ. Entomol. 72: 256-258. Widstrom, N.W., W.W.McMillian, and B.R. Wiseman. 1979. Ovipositional preference of the corn earworm and the development of trichomes on two exotic corn selections. Environ. Entomol. 8: 833-839. Williams, W.P., and F.M. Davis. 1985. Southwestern corn borer larval growth on corn callus and its relationship with leaf feeding resistance. Crop Sci. 25: 317319. Williams, W.P., P.M. Buckley, and F.M. Davis. 1985. Larval growth and behavior of the fall armyworm (Lepidoptera: Noctuidae) on callus initiated from susceptible and resistant corn hybrids. J. Econ. Entomol. 78: 951954. ibid. 1987a. Tissue culture and its use in investigations of insect resistance in maize. Agric. Ecosyst. Environ. 18: 185190. ibid. 1987b. Feeding response of corn earworm (Lepidoptera: Noctuidae) to callus and extracts of corn in the laboratory. Environ. Entomol. 16: 532534. Wiseman, B.R., and J.J. Hamm. 1993. Nuclear polyhedrosis virus and resistant corn silks enhance mortality of corn earworm (Lepidoptera, Noctuidae) larvae. Biol. Control. 3(4): 337-342 Wiseman, B.R. and N.W. Widstrom. 1980. Comparison of methods of infesting whorl-stage corn with fall armyworm larvae. J. Econ. Entomol. 73: 440-442. ibid. 1992. Resistance of corn populations to larvae of the corn earworm (Lepidoptera: Noctuidae). J. Econ. Entomol. 85: 601-605. Wiseman, B.R., F.M. Davis, and J.E. Campbell. 1980. Mechanical infestation device used in fall armyworm plant resistance programs. Fla. Entomol. 63: 426-432. Wiseman, B.R., E.A. Harrell, and W.W. McMillian. 1975. Continuation of tests of resistant sweet corn hybrid plus insecticides to reduce losses from corn earworm. Environ. Entomol. 2: 919-920. Wiseman, B.R., W.W. McMillian, and N.W. Widstrom. 1972. Tolerance as a mechanism of resistance in corn to the corn earworm. J. Econ. Entomol. 65: 835837. ibid. 1974. Techniques, accomplishments, and future potential of breeding for resistance in corn to the corn earworm, fall armyworm and maize weevil, and in sorghum to the sorghum midge. In F.G. Maxwell, and F.M. Harris (eds.) Proc. Summer Inst. Biol. Control Plant Insects Dis. Univ. Mississippi Press, Jackson, MS. 381-393. ibid. 1976. Feeding of corn earworm in the laboratory on excised silks of selected corn entries with notes on Orius insidiosus. Fla. Entomol. 59: 305-308. ibid. 1977. Ear characteristics and mechanisms of resistance among selected corns to corn earworm. Fla. Entomol. 60: 97-103. Wiseman, B.R., M.E. Snook, D.J. Isenhour, J.A. Mihm, and N.W. Widstrom. 1992a. Relationship between growth of corn earworm and fall armyworm larvae (Lepidoptera: Noctuidae) and maysin concentration in corn silks. J. Econ. Entomol. 85: 2473-2477. Wiseman, B.R., M.E. Snook, R.L. Wilson, and D.J. Isenhour. 1992b. Allelochemical content of selected popcorn silks - Effects on growth of corn earworm larvae (Lepidoptera, Noctuidae). J. Econ. Entomol. 85: 2500-2504. Wiseman, B.R., W.P. Williams, and F.M. Davis. 1981. Fall armyworm: resistance mechanisms in selected corn genotypes. J. Econ. Entomol. 74: 622-624. Xie, Y.S., J.T. Arnason, B.J.R. Philogene, and J.D.H. Lambert. 1990. Role of 2,4dihydroxy-7-methoxy-1, 4-benzoxazin3-one (DIMBOA) in the resistance of maize to western corn rootworm, Diabrotica virgifera virgifera (LeConte) (Coleoptera: Chrysomelidae). Can. Ent. 122: 1177-1186. Xie, Y.S,. J.T. Arnason, B.J.R. Philogene, J. Atkinson, and P. Morand. 1992. Behavioral responses of western corn rootworm larvae to naturally occurring and synthetic hydroxamic acids. J. Chem. Ecol. 18: 945-957. Yang, G., K.E. Espelie, B.R. Wiseman, and D.J. Isenhour. 1991. Activity of maize leaf cuticular lipids in resistance to leaffeeding by the fall armyworm. Fla Entomol 74: 229-236. Yang, G., B.R. Wiseman, D.J. Isenhour, and K.E. Espelie. 1993. Chemical and ultrastructural analysis of corn cuticular lipids and their effect on feeding by fall armyworm larvae. J. Chem. Ecol. 19: 2055-2074. Zuber, M.S., G.J. Musick, and M.L. Fairchild. 1971. A method of evaluating corn strains for tolerance to the western corn rootworm. J. Econ. Entomol. 64: 1514-1518. 13 T he Effe c t of DI M BOA Conc e nt ra t ion in Le a f T issue a t V a rious Pla nt Grow t h St a ge s on Re sist a nc e t o Asia n Corn Bore r in M a ize C.T. Tseng, Corn Research Center, Tainan Dais Abst ra c t The chemical analytical values obtained for MBOA (6-methoxy-2-benzoxazolinone) were related to the labile cyclic hydroxamic acid precursors DIMBOA (2,4-dihydroxy-7-methoxy-(2H)-1,4-benzoxazin-3(4H)-one) which is formed enzymatically from its glucosides when the leaf tissues are crushed or placed at high temperature. The results of chemical analysis revealed that the MBOA concentrations in leaf tissue decreased as the plants grew towards maturity, inversely the TLC plate ratings increased as the plants grew older. This showed that there were higher MBOA concentrations in the leaf tissue of earlier stage plants than in those of later ones. Leaf-feeding damage ratings caused by artificial infestation with Asian corn borer (ACB), Ostrinia furnacalis (Guenée) egg masses and number of surviving borer larvae per plant increased as the plants grew older, indicating that younger plants were more resistant than older ones to cornborer feeding. Of 11 inbred lines tested JT 30-1-1-1-15-3 and CI31A had lower leaf-feeding ratings, lower number of surviving larvae per plant and higher MBOA concentrations than any other lines at various stages of plant development. This implies that these 2 lines possess a remarkable degree of resistance to leaf-feeding by corn borer. The correlation coefficients of MBOA concentrations with leaf-feeding ratings at the 4th, 6th, 8th, 10th and 12th leaf stages were as follows: -0.85, -0.84, -0.86, -0.82, and -0.84 respectively, while the correlation coefficients of MBOA concentrations with number of surviving larvae at the same leaf stages in order were as follows: -0.88, -0.83, -0.82, -0.78 and -0.80 respectively. The negative correlations of MBOA concentrations with leaf-feeding ratings and number of surviving borer larvae per plant were highly significant. This means that the higher the MBOA concentrations in leaf tissue, the lower the leaf-feeding ratings and number of surviving borer larvae per plant. The results prove that DIMBOA is an important chemical factor responsible for resistance in maize to the Asian corn borer. I nt roduc t ion concentration in leaf tissue was used as the plant, but the overall concentration one of the indicators for selecting maize in the whole plant decreased as the DIMBOA (2,4-dihydroxy-7-methoxy- inbreds resistant to leaf-feeding by the plant matured (Klun and Robinson. (2H)-1,4-benzoxazin-3(4H)- one) was ECB (Klun and Robinson 1969; Sullivan 1969). The high concentration of first associated with insect resistance in et al. 1974; Russell et al. 1975). DIMBOA in seedling corn may explain the apparent resistance of young corn crop plants when Klun et al (1967) isolated it from corn seedlings and The concentration of DIMBOA in maize to the ECB (Klun and Robinson 1969; bioassayed it in artificial diets for the was found to vary between different Guthrie 1974). European corn borer (ECB), Ostrinia plant tissues. Concentrations were nubilalis (Hubner). They found that this generally highest in the root and then in The precursor to DIMBOA occurs as a compound inhibited larval decreasing order of concentration; the glucoside in intact maize tissue. When development and caused 25% stalk, whole plant and leaf (Klun and plant tissues are crushed, the glucoside mortality. These results, and associated Robinson 1969). Moreover, the is hydrolyzed by a plant enzyme to the experimental evidence, revealed that concentrations in the various tissues aglucone, 2,4-dihydroxy-7-methoxy-1, the compound is a chemical factor in were different for each inbred. 4- benzoxazin-3(4H)-one (DIMBOA) the resistance of maize to first brood Biosynthesis of the benzoxazinone took (Klun and Robinson. 1969). DIMBOA is ECB. As a result, DIMBOA place throughout the development of chemically labile and slowly 14 C.T. TSENG decomposes to 6-methoxy-2- M a t e ria ls a nd M e t hods replicates with a split-plot design — main plots : inbreds; subplots : plant benzoxazolinone (MBOA), which is chemically stable (Fig. 1). Thus, The 11 dent corn inbreds chosen in this growth stages; sub-subplots : infesting DIMBOA concentration in plant tissue study and their origins are listed in artificial ACB egg masses inside the could be estimated by analyzing for Table 1. The experiments were whorl leaves (Tseng and Twu 1974) MBOA. The MBOA analytical value is conducted at the Corn Research Center, (Fig. 2) and cutting the whorl leaf for interpreted as a stoichiometric measure Tainan DAIS, Potzu, Chiayi, using four chemical analysis of DIMBOA of DIMBOA formed as the result of enzymatic cleavage of its glucoside precursor (Klun et al. 1967). CH3O O O Glucose CH3O O O OH CH3O O Klun et al. (1970) used a diallel set of 11 N maize inbreds (55 single cross hybrids) OH to study the concentration of DIMBOA in whorl leaf tissue and the resistance to leaf-feeding by first-generation ECB. The correlation between concentration of DIMBOA in plant tissue and level of O N Crushing O (Aglucone) DIMBOA Glucoside N Heating H 6 MBOA Figure 1. Formation of DIMBOA (Aglucone) and MBOA from a glucoside occurring in maize tissue. resistance was highly significant for the inbreds (r=-0.89) and the single crosses Table 1. Eleven dent corn inbreds used in the study and their origins. (r=-0.74). Genetic effects due to general Inbred Derivation Origin JT 30-1-1-1-15-3 YT 148-2-1-1-2-1 JWL. 305 x Tainan DMR #2 Yellow hard endosperm x Tainan DMR #2 South African Yellow x Tainan DMR #2 Cogollero x Tainan DMR #2 (Amber x (B 57 x B 37) x Akbar) x Tainan DMR #2 Pendu x Tainan DMR #2 Antigua Gr. x Tainan DMR #2 Iowa 2 ear syn. Midland “A” O. P. Midland Wilson Farm Reid CIMMYT CIMMYT and specific combining ability were highly significant for both traits, but general combining ability accounted for 84% of the variation in the resistance ratings and for 91% of the variation in the concentration of DIMBOA. These results provided further evidence that DIMBOA is a chemical factor in the resistance of maize to the ECB. However, most chemicals exhibit their specific properties only in host plant resistance (HPR) to insects (Beck 1965; Guthrie 1974). Hence, further studies were needed to determine whether the maize inbreds with high DIMBOA concentration would exhibit similar levels of resistance to the Asian corn borer (ACB) Ostrinia furnacalis (Guenée). This study was carried out to determine the changes of DIMBOA concentration in all stages of maize development and to evaluate any relationship between DIMBOA and resistance to leaf-feeding by ACB. Figure 2. Infesting artificial ACB egg masses inside the whorl leaves. ST 153-1-3-2-2-1 CT 139-5-1-1-1-1 ANMT 55-1-3-2-2-1 PT 169-1-1-4-1-1 ANT 176-1-3-5-13-3 B 49 CI31A B 52 WF 9 CIMMYT CIMMYT CIMMYT CIMMYT CIMMYT Iowa State USDA Iowa State Indiana State THE EFFECT OF DIMBOA CONCENTRATION IN LEAF TISSUE AT VARIOUS PLANT GROWTH STAGES Leaf Whorl Leaf 15 concentration (Fig. 3). Samples Five rows in each plot were infested analyzed were taken at the 4th, 6th, with ACB egg masses at the 4th, 6th, 8th, 10th, and 12th leaf stages, defined 8th, 10th and 12th leaf stages of plant according to the uppermost leaf whose development, respectively. Infestations collar was visible (Ritchie and Hanway were made in 3 applications of 3 egg 1982). masses (Ca. 450 eggs/plant), each spaced 1 day apart. Leaf-feeding Stalk Root Figure 3. Samples of plant tissues taken for chemical analysis of DIMBOA concentration (after Klun and Robinson 1969). The 11 inbreds were planted in 10-row damage was rated on a plot basis, 21 plots (24 hills of two seeds/hill and days after egg hatching, using a scale of thinned to one plant/hill) in 1985 and 1 to 9 (1 = no damage, 9 = extremely 1986. The distance between rows was damaged) (Guthrie et al. 1960) (Fig. 4 75 cm and between hills within row and Fig. 5). After rating, 10 plants from distance was 25 cm. each row were dissected to count the number of surviving borer larvae per plant (Fig. 6). The other 5 rows in each plot were used for DIMBOA analysis. Whorl leaves from 10 plants in each row were collected at the 4th, 6th, 8th, 10th and 12th leaf stages of plant development, respectively. The whorl leaves collected were placed in plastic bags and stored Figure 5. Susceptible inbred line of dent maize rated 9 according to the visual rating system (Guthrie et al. 1960). Figure 4. Resistant inbred line of dent maize rated 1 according to the visual rating system (Guthrie et al. 1960). Figure 6. Dissecting the infested stalks to count the surviving Asian corn borer larvae 16 C.T. TSENG at -23ºC prior to analysis. The frozen 0.5g of dried ground leaf tissue; after commercial MBOA (Calbiochem- leaf tissue was then thawed, dried in an shaking vigorously for 1 min, this Behring Corp., P. O. Box 12087, San oven at 45ºC, and ground into a fine solution was poured into a Buchner Diego, California), as a control, were powder. The chemical determinations funnel (lined with filter paper), and an spotted on an individual plate. Each carried out on the ground tissue were aspirator vacuum (Fig. 7) filtered the sample of the 4 replications was actually for MBOA, expressed as mg filtrate into a 500 ml flask. The filtrate repeated twice. The 7 spots were placed MBOA/g of plant tissue (Brendenberg was then poured into a 100 ml beaker along one edge of the plate (Fig. 9). et al. 1962; Klun and Robinson; 1969; and allowed to cool (the leaf residue Klun et al. 1970; Klun, 1970; Tseng et al. was discarded). Four drops of After spotting, the chromatogram was 1984). concentrated hydrochloric acid were developed with chloroform: ethyl- added to acidify the filtrate (pH 1.0). acetate: cyclohexane (4:4:2 vol./vol.). The procedures used to obtain The acidified filtrate was poured into a After development, the plates were quantitative measurement of MBOA in separating funnel (Fig. 8) and 40 ml of removed from the solution, dried, and leaf tissue were modified from those diethyl ether were added. After then redeveloped in the same direction used by Klun and Robinson (1969). For vigorously shaking the funnel, the with cyclohexane : isobutanol (85 : 15 each sample, 20 ml of boiling water water and ether were allowed to vol./vol.). The chromatogram was then were added to a 70 ml jar containing separate, then each layer was drained air-dried and two observers visually into 100 ml beakers; the aqueous phase rated, under short wave uv light (254 was then poured back into the nm), the intensity of each MBAO spot separating funnel. To wash MBOA from the extracts in classes of 1 to 5 from the aqueous phase as completely (1=highest intensity, 5=lowest as possible, the procedure involving intensity) as described by Robinson et the separating funnel was repeated al. (1982). Beuchner funnel Filter paper Sample twice, then the aqueous phase was Filtrate Aspirator vacuum Figure 7. Buchner funnel and aspirator vacuum. discarded. Anhydrous calcium chloride When the intensity ratings were was added to the ether layer to remove completed, the area of the silica gel any water left in the ether. The ether corresponding to the reference MBOA was allowed to evaporate under a fume spot was scraped from the hood, and the ether soluble residue was chromatogram and transferred to a dissolved in 1 ml ethyl acetate: benzene disposable pasteur pipette plugged solution (1:1 vol./vol.). with glass wool (Fig. 10). MBOA was then eluted from the silica gel with 6 ml A 100 µl aliquot of this solution was of 95% ethanol and the uv absorbance then spotted on a 20 x 20 cm glass plate of this solution was measured at 231 covered with a thin layer of silica gel nm with a Beckman Model DB (GF 254 Brinkmann Instruments , Spectrophotometer. The uv Westbury, NY). Six samples plus spectrophoto-metric percent transmission (T %) was read twice for Silica gel Ether layer Glass wool Water layer 6 Figure 8. Separating funnel. 5 4 MBOA 3 2 1 Figure 9. Thin layer chromatography (TLC) plate. Figure 10. Pasteur pipette plugged with glass wool. 17 THE EFFECT OF DIMBOA CONCENTRATION IN LEAF TISSUE AT VARIOUS PLANT GROWTH STAGES Leaf-feeding ratings and larval survival Among all the inbreds tested JT 30-1-1- (mg MBOA/g dried leaf tissue) was then calculated from a MBOA standard Leaf-feeding rating, after artificial leaf-feeding rating and number of curve. infestation with ACB egg masses, and surviving borer larvae per plant in all each sample. The MBOA concentration 1-15-3 and CI31A had both the lowest the number of surviving larvae per leaf stages. CT139-5-1-1-1-1 and WF9 Thus, we used two methods for plant both increased as the plants had the highest leaf-feeding ratings and measuring DIMBOA concentrations in matured (Table 3), indicating that numbers of surviving borer larvae per maize leaf tissue: 1) Chemical analysis young plants were more resistant to plant in all growth stages of plant for mg MBOA/g of maize leaf tissue leaf-feeding by ACB than older ones. development. and 2) thin layer chromatography (TLC) to rate differences visually in the concentration of MBOA (TLC plate rating). Table 2. Mean concentrations of MBOA in leaf tissue and TLC plate ratings at various leaf stages. Leaf stage Data on leaf-feeding ratings, number of surviving larvae per plant, mg MBOA/ Inbred 4 6 8 10 12 mg MBOA/g dry weight 4 6 8 10 12 TLC plate ratings g dried leaf tissue and TLC plate ratings collected from above experiments were statistically analyzed to elucidate significant differences between experimental results (Steel and Torrie 1960). Re sult s MBOA leaf tissue content and TLC readings The results of chemical analysis for MBOA concentration and TLC plate JT 30-1-1-1-15-3 YT 148-2-1-1-2-1 ST 153-1-3-2-2-1 CT 139-5-1-1-1-1 ANMT 55-1-3-2-2-1 PT 169-1-1-4-1-1 ANT 176-1-3-5-13-3 B 49 CI31A B 52 WF 9 3.60 2.70 2.51 1.52 2.01 1.81 1.68 2.90 3.56 2.31 1.28 3.25 2.15 1.81 1.21 1.80 1.23 1.70 2.20 2.90 1.80 0.92 2.85 1.80 1.65 1.10 1.41 0.90 1.15 1.75 2.60 1.45 0.75 2.50 1.40 1.10 0.95 1.20 0.80 0.96 1.40 2.45 1.30 0.66 2.15 1.20 0.85 0.66 0.95 0.70 0.80 1.40 2.10 1.25 0.48 1.7 2.5 3.5 4.5 4.0 4.5 4.5 2.0 1.7 3.5 4.5 1.8 2.8 4.0 4.5 4.5 4.5 4.5 2.5 2.0 3.5 4.6 2.2 3.0 4.5 4.5 4.5 4.5 4.5 3.0 2.5 4.0 5.0 3.0 3.5 4.5 4.7 4.8 4.7 4.6 3.5 3.0 4.5 5.0 3.5 4.0 4.5 5.0 5.0 5.0 5.0 4.0 3.5 4.5 5.0 LSD (0.05) Any two means of MBOA concentrations between leaf stages for the same inbred is 0.64 Any two means of MBOA concentrations between inbreds for the same leaf stage is 0.85 Any two means of TLC plate ratings between leaf stages for the same inbred is 0.45 Any two means of TLC plate ratings between inbreds for the same leaf stage is 0.55 rating for MBOA spot intensity revealed that the highest MBOA concentrations were at the 4th leaf stage Table 3. Mean leaf-feeding ratings after artificial infestation with ACB egg masses and number of surviving larvae per plant at various leaf stages. and the lowest were at the 12th leaf stage for all inbreds (Table 2). Of all Leaf stages Inbred 4 inbreds tested JT 30-1-1-1-15-3 and CI31A had the highest and WF 9 had the lowest MBOA concentration in all plant growth stages. The former possessed about three times more MBOA than the latter. The TLC plate ratings showed just the inverse, as the highest ratings were at the 12th leaf stage and the lowest were at the 4th leaf stage. JT 30-1-1-1-15-3 and CI31A had the lowest rating throughout all growth stages and amongst all inbreds. JT 30-1-1-1-15-3 YT 148-2-1-1-2-1 ST 153-1-3-2-2-1 CT 139-5-1-1-1-1 ANMT 55-1-3-2-2-1 PT 169-1-1-4-1-1 ANT 176-1-3-5-13-3 B 49 CI31A B 52 WF 9 1.5 2.5 3.0 5.0 3.5 4.0 4.0 2.5 1.5 3.0 5.0 6 8 10 12 Leaf-feeding ratings 1.5 3.0 3.5 4.5 4.0 4.5 4.5 3.0 2.0 3.5 5.5 2.0 3.6 5.0 6.0 5.0 6.0 6.0 4.0 2.5 4.5 6.5 2.5 4.5 6.0 6.5 6.0 6.0 6.0 5.0 2.5 5.0 7.5 3.0 5.5 6.5 7.5 6.5 7.5 6.5 5.5 3.0 5.5 8.5 4 6 8 10 12 No. of surviving larvae 1.2 2.2 2.8 5.6 4.0 4.4 4.8 2.8 1.5 3.1 6.5 1.6 3.0 3.5 6.5 4.3 6.2 5.0 3.5 1.3 3.5 8.0 2.4 5.0 6.5 7.0 5.5 7.6 6.5 5.0 2.6 5.5 8.5 2.8 7.5 7.5 8.5 6.5 8.4 7.3 5.5 3.0 4.5 9.8 2.5 6.5 8.0 8.8 7.0 8.6 6.8 4.5 2.4 5.4 10.5 LSD (0.05) Any two means of leaf-feeding ratings between leaf stages for the same inbred is 1.0 Any two means of leaf-feeding ratings between inbreds for the same leaf stage is 1.5 Any two means of numbers of surviving larvae per plant between leaf stages for the same inbred is 1.1 Any two means of numvers of surviving larvae per plant between inbreds for the same leaf stage is 1.9. 18 C.T. TSENG Correlation of MBOA leaf tissue content with leaf-feeding ratings at various leaf stages of 11 inbreds at the 4th, 6th, 8th, 10th materials in stock or locally available. and 12th leaf stages, were -0.88, -0.83, - However, since ACB resistance 0.82, -0.78 and -0.80 respectively (Fig. mechanisms in maize are unclear, it is The correlation coefficients of MBOA 12). The correlation of MBOA difficult to know where to collect or concentrations in leaf tissue with leaf- concentration with number of how to identify the resistant germplasm feeding ratings of 11 inbreds at the 4th, surviving larvae per plant was also (Beck 1965; Dahms 1972). The breeding 6th, 8th, 10th and 12th leaf stages, were highly significant in all growth stages of maize varieties resistant to ACB -0.85, -0.84, -0.86, -0.82 and -0.84, of plant development. would be more efficient, if we knew more about the resistance mechanisms. respectively (Fig. 11). The correlation of MBOA concentration with leaf-feeding Disc ussion In this study we used 11 inbreds to determine the relationship of MBOA rating was highly significant throughout all growth stages of plant It is imperative to have resistant concentration in leaf tissue with development. germplasm available for breeding resistance to ACB at various growth insect resistant crop varieties; this holds stages of plant development, and also Correlation of MBOA leaf tissue content with larval survival at various leaf stages true in developing maize that possesses to provide more information to identify resistance to ACB. Resistant germplasm resistant inbreds. The results from leaf- can be obtained through introductions feeding ratings after artificial The correlation coefficients of MBOA or exchanges with foreign or domestic infestation with ACB egg masses at the concentrations in leaf tissue with the research institutes and through 4th, 6th, 8th, 10th and 12th leaf stages number of surviving larvae per plant, identifying resistance sources in (Table 3) indicated that the lowest leaf- Leaf-feeding rating Leaf-feeding rating feeding ratings and number of 8 8 A 6 Y=8.07 - 2.01X r= -0.85 (p 0.05) B 6 Y=7.61 - 2.16X r= -0.84 (p 0.05) surviving borer larvae per plant were at the 4th leaf stage for all inbreds tested. However, leaf-feeding ratings and 4 4 2 2 0 0 number of surviving larvae per plant increased as plants matured. This indicated that the young plants were more resistant to ACB than the older 0 0.6 1.2 1.8 2.4 3.0 3.6 MBOA concentration (mg/g dry weight) 0 0.6 1.2 1.8 2.4 3.0 3.6 MBOA concentration (mg/g dry weight) 8 8 C 6 Y=8.96 - 2.85X r= -0.36 (p 0.05) 4 4 2 2 0 0 maintain the leaf-feeding rating and number of surviving larvae per plant at D 6 ones. Therefore, if the inbreds could Y=9.24 - 3.05X r= -0.32 (p 0.05) a low level throughout all growth stages of plant development, they would possess the high resistance level to ACB. Among all inbreds tested, JT301-1-1-15-2 and CI31A had the lowest leaf- feeding ratings and numbers of 0 0.6 1.2 1.8 2.4 3.0 3.6 MBOA concentration (mg/g dry weight) 0 0.6 1.2 1.8 2.4 3.0 3.6 MBOA concentration (mg/g dry weight) surviving larvae per plant at all leaf stages. This showed that JT30-1-1-1-15-3 and CI31A were more resistant to ACB than other inbreds. Leaf-feeding rating 8 E 6 Y=9.87 - 3.69X r= -0.84 (p 0.05) 4 Figure 11. Correlation coefficients of MBOA concentration in leaf tissue with leaf feeding ratings at the 4th (A), 0 th th th th 0 0.6 1.2 1.8 2.4 3.0 3.6 6 (B), 8 (C), 10 (D) and 12 (E) leaf stages, respectively. MBOA concentration (mg/g dry weight) 2 DIMBOA (2, 4-dihydroxy-7-methoxy-1, 4-benzoxazin-3(4H)-one) is chemically labile and slowly decomposes to 6methoxy-2- benzoxazolinone (MBOA), which is chemically stable (Brendenberg et al. 1970; Klun, 1970). THE EFFECT OF DIMBOA CONCENTRATION IN LEAF TISSUE AT VARIOUS PLANT GROWTH STAGES 19 with leaf-feeding ratings at the 4th, 6th, means that the inbreds with greater determined by chemical analysis of 8th, 10th and 12th leaf stages were - MBOA concentrations will possess dried plant tissue for MBOA (Klun and 0.85, -0.84, -0.86, -0.82 and -0.84 greater resistance to ACB. The Robinson, 1969; Klun et al. 1970; Klun, respectively (Fig. 11), whereas the experimental data proved that 1970). The results of chemical analysis correlation coefficients of MBOA DIMBOA was a significant biochemical of MBOA concentrations and TLC plate concentrations with numbers of factor in maize responsible for ACB ratings (Table 2) revealed that MBOA surviving borer larvae per plant at the resistance. concentration decreased as plants grew same leaf stages were -0.88, -0.83, -0.82, toward maturity. Inversely, TLC plate -0.78 and -0.80 respectively (Fig. 12). rating increased as plants grew older. These results clearly indicate that the This showed that young plants relationship between MBOA contained a higher MBOA concentration and both leaf-feeding concentration than older ones. Among rating and number of surviving borer 11 inbreds tested, JT 30-1-1-1-15 -3 and larvae per plant was highly significant. CI31A had the highest MBOA In other words, the higher the MBOA concentrations and the lowest TLC concentration in leaf tissue, the lower plant ratings. The correlation the leaf-feeding rating and the number coefficients of MBOA concentrations of surviving larvae per plant. This No. of surviving larvae/plant Thus, DIMBOA concentration can be 8 8 A 6 Y=9.49 - 3.40X r= -0.88 (p 0.05) 4 4 2 2 0 0.6 1.2 1.8 2.4 3.0 3.6 MBOA concentration (mg/g dry weight) 10 10 No. of surviving larvae/plant Y=10.58 - 3.43X r= -0.83 (p 0.05) 0 0 0 0.6 1.2 1.8 2.4 3.0 3.6 MBOA concentration (mg/g dry weight) No. of surviving larvae/plant B 6 C 8 Y=10.78 - 3.40X r= -0.82 (p 0.05) D 8 6 6 4 4 2 2 Y=11.41 - 3.66X r= -0.78 (p 0.05) 0 0 0 0.6 1.2 1.8 2.4 3.0 3.6 MBOA concentration (mg/g dry weight) 0 0.6 1.2 1.8 2.4 3.0 3.6 MBOA concentration (mg/g dry weight) 10 E 8 Y=11.66 - 4.59X r= -0.80 (p 0.05) 6 4 2 0 0 0.6 1.2 1.8 2.4 3.0 3.6 MBOA concentration (mg/g dry weight) Figure 12. Correlation coefficients of MBOA concentration in leaf tissue with number of surviving borer larvae per plant at the 4th (A), 6th (B), th th th 8 (C), 10 D) and 12 (E) leaf stages, respectively. Re fe re nc e s Beck, S.D. 1965. Resistance of plants to insects. Annu. Rev. Entomol. 1 : 207-232. Brendenberg, J.B-Son, E. Honkanen, and A.I. Virtanen. 1962. The kinetics and mechanism of decomposition of 2,4dihydroxy-1,4-benzoxazine-3-one. Acta. Chem. Scand. 16 : 135-141. Dahms, R.G. 1972. Techniques in the evaluation and development of host plant resistance. J. Environ. Qual. 1 : 254-259. Guthrie, W.D., F.F. Dicke, and C.R. Neiswander. 1960. Leaf and sheath feeding resistance to the European corn borer in eight inbred lines of dent corn. Ohio Agr. Exp. Sta. Res. Bull. 860. Guthrie, W.D.1974. Techniques, accomplishments and future potential of breeding for resistance to European corn borer in corn. In F.G. Maxwell. and E.F. Harris, (eds.), Biological Control of Plants, Insects, and diseases. Jackson, Miss, Univ. Press. 359-380 Klun, J.A., C.L. Tipton, and T.A. Brindley. 1967. 2, 4-dihydroxy-7-methoxy-1, 4benzoxazin-3-one (DIMBOA), an active agent in the resistance of maize to the European corn. borer. J. Econ. Entomol. 60 : 1529-1533. Klun, J.A., and J.F. Robinson. 1969. Concentration of two 1,4benzoxazinones in dent corn at various stages of development of the plant and its relation to resistance of the host plant to the European corn borer. J. Econ. Entomol. 62 : 214-220. Klun, J.A. 1970. Relation of chemical analysis for DIMBOA and visual resistance rating for first-brood European corn borer. Proc. Annu. Cornsorghum Res. Conf. 24 : 61-67. Klun, J.A., W.D. Guthrie, A.R. Hallauer, and W.A. Russell. 1970. Genetic nature of the concentration of 2,4-dihydroxy-7methoxy-2H-1, 4-benzoxazin-3(4H)-one and resistance to the European corn borer in a diallel set of eleven maize inbreds. Crop Sci. 10 : 87-90. Ritchie, S.W., and J.J. Hanway. 1982. How a corn plant develops.Special Rep. No. 48. ISU, Coop. Exten. Ser. Ames, Iowa. 20 C.T. TSENG Robinson, J.F., J.A. Klun, W.D. Guthrie, and T.A. Brindley. 1982. European corn borer leaf-feeding resistance : A simplified technique for determining relative differences in concentrations of 6-methoxy bonzoxazolinone (Lepidoptera : Pyralidae). J. Kans. Entomol. soc. 55 : 297-301. Russel, W.A., W.D. Guthrie, J.A. Klun, and R. Grindeland. 1975. Selection for resistance in maize to first-brood European corn borer by using leaffeeding damage of the insect and chemical analysis for DIMBOA in the plant. J. Econ. Entomol 68 : 31-34. Sullivan, S.L., V.E. Gracen, and A. Ortega. 1974. Resistance of exotic maize varieties to the European corn borer, Ostrinia nubilalis (Hubner), Environ. Entomol. 3 : 718-720. Steel, R.G.D., and J.H. Torrie. 1960. Principles and procedures of statistics. Mc Graw-Hill Book Co., New York. Tseng, C.T. and C.J. Twu. 1974. Studies on mass production of the Asian corn borer with artificial medium. Rep. Corn Res. 10 : 34-39. Tseng, C.T., W.D. Guthrie, W.A. Russell, J.C. Robbins, J.R. Costa, and J.J. Tolleffson. 1984. Evaluation of two procedures to select for resistance to the European corn borer in a synthetic cultivar of maize. Crop Sci. 24 : 11291133. 21 I m pa c t of M e c ha nism s of Re sist a nc e on Europe a n Corn Bore r Re sist a nc e in Se le c t e d M a ize H ybrids B.D. Barry and L.L. Darrah University of Missouri, Columbia, U.S.A. Abst ra c t Four commercial maize hybrids plus a susceptible and a resistant check were compared in experiments to determine which mechanism(s) of resistance — i.e., 1) preference, 2) antibiosis and/or 3) tolerance to first and second generation European corn borer (ECB) — contributed to overall resistance. Preference evaluations were made under natural ECB infestations in an area where ECB is endemic using six replications and counting shot holes for the first generation and number of egg masses and tunnel length (stalk splitting) for the second generation. Antibiosis was determined by manual infestations and no infestation and using Guthrie’s (1960) scale for first generation ECB. second generation ECB antibiosis was determined by splitting stalks of manually infested and non-infested hybrids and estimating the amount of tunneling. Tolerance was measured with leaf-feeding ratings and the amount of tunneling and all hybrids were infested with 0, 30, 120, and 240 larvae per plant. In all six experiments, yields were measured to relate the effects of resistance mechanisms and infestation levels. I nt roduc t ion Our report concerns the effects of mechanisms of resistance on European corn borer (ECB), Ostrinia nubilalis (Hübner), resistance in selected maize, Zea mays (L.), hybrids. The ECB is an Old World insect and before maize was introduced into Europe, the ECB was of limited economic importance except in hemp, Cannabis sativa (L.), and hops, Humulus lupulus (L.). This borer probably arrived in the United States about 1914 in a shipment of hemp and was first described as a pest of maize (Fig. 1, photograph taken in 1918 by B. E. Hodgson, Medford, MA) in 1917 (Vinal 1917). Estimated losses in the Corn Belt due to this insect during 1981 Figure 1. European corn borer damage to maize in 1918, four years after ECB was introduced into the United States (photograph by B. E. Hodgson, Medford, MA). Table 1. Estimated economic losses due to second generation ECB damage in field corn harvested for grain in 1982. were 190 million dollars (Table 1), but the most frequently quoted figures for average annual losses are between 200 Location continues to monitor ECB populations Missouri Iowa Illinois United States and their data suggest losses in Illinois † and 500 million dollars. The Illinois Entomology Extension Service ‡ Area (000 ha) Yield (t/ha) ECB per 100 plants 797 5,322 4,605 29,604 6.53 7.60 8.42 7.21 141 51 26 ‡ 31 1982 price of corn averaged $2.45. Calculated from average statistics for the above three states. † Losses (US$000’000) 21.23 59.64 29.14 191.35 22 B.D. BARRY AND L.L. DARRAH for 1991, 1992, and 1993 of 324, 33, and Huber et al. (1928) proposed host-plant several lepidopteran pests of maize. Of 101 million dollars, respectively. The resistance (HPR) as a means of control. course, the father of HPR was R.H. ECB population in Illinois in 1991 was L.H. Patch worked with field maize; Painter from Kansas State University the highest since 1949 (personal and his colleague, M. Schlosberg, (Fig. 4). Painter began promoting HPR communication, Mike Gray, Illinois another entomologist, worked with as early as 1923 and published his first Extension Service, 1994). If we sweet maize (personal communication, book on the subject in 1951 (Painter extrapolate from the Illinois data for the Orlo Vance, retired, 1994). These 1951). He defined resistance and the acreage of maize for the U.S. Corn Belt, scientists made many contributions mechanisms of resistance: antibiosis, losses would have been 2,197; 236; and toward our current HPR programs for non-preference, and tolerance: 706 million dollars for 1991, 1992, and ECB resistance. Ideas and techniques 1993, respectively (Table 2). for manually infesting plants for • “Resistance” refers to the heritable screening, laboratory rearing, and a qualities of the host which allow The ECB has been the most studied damage rating scale began with these infested (with insects) cultivars to economic pest of maize in the United scientists. Guthrie et al. (1960) produce more than similar cultivars States and the majority of studies have developed a rating scale for ECB (Figs. dealt with control. As early as the 1920s, 2 and 3) that is currently used for without these qualities. • “Antibiosis” refers to adverse biological consequences to the life history of an insect due to the Table 2. Statistics for maize and estimated ECB damage in Illinois from 19911993 and extrapolations for the United States. Illinois Hectares planted (millions) Yield (t/ha) Price ($/t in Iowa) Plants infested with ECB (%) Average number of borers/plant Total tons produced (millions) Loss due to ECB (millions of tons) Loss in dollars (millions) United States Hectares planted (millions) Loss in dollars (millions, extrapolated) † feeding on a resistant host. The effects may be death, small size, low ` 1991 Year 1992 1993 4.5 8.16 96.85 † 91.4 3.3 36.98 3.35 324.0 4.5 9.36 87.40 30.9 0.3 42.39 0.38 33.3 4.2 6.72 107.48 50.3 1.1 28.54 0.94 101.2 weight, reduced fecundity, extended life cycle, and/or abnormal 30.6 2,197.1 32.1 235.9 29.7 706.5 Second highest recorded infestation (highest was during the 1940s). Entomologists in Illinois surveyed 10 fields in 35 counties by examining samples of 25 plants in each field. behavior. • “Non-preference” refers to the lack of attractiveness of a host for food and shelter for an insect. The nonpreference attribute of a host could Class 1. No visible leaf injury or a small amount of pin or fine shot-hole type of injury on a few leaves. Class 2. Small amount of shot-hole type lesions on a few leaves. Class 3. Shot-hole injury common on several leaves. Class 4. Several leaves with shot-hole and elongated lesions. Class 5. Several leaves with elongated lesions. Class 6. Several leaves with elongated lesions (about 2.5 cm). Class 7. Long lesions common on about one-half of the leaves. Class 8. Long lesions common on about two-thirds of the leaves. Class 9. Most leaves with long lesions. Figure 2. Leaf damage corresponding to Guthrie et al.’s 1-9 rating scale for first generation ECB damage. Figure 3. Description for leaf damage corresponding to Guthrie et al.’s 1-9 rating scale for first generation ECB damage. IMPACT OF MECHANISMS OF RESISTANCE ON EUROPEAN CORN BORER also discourage continued habitation M a t e ria ls a nd M e t hods fertilization, herbicide treatment, and other cultural practices for the 1994 even though it had served as shelter • 23 and/or as an oviposition site. Four widely-grown commercial maize evaluation were those used by farmers “Tolerance” is the ability of the host hybrids (one from each of four in the area. Experiments were planted plant to support a certain population companies) plus two hybrid checks on 18 May 1994 in a field fertilized with level of insects due to plant vigor, or were selected primarily for tolerance 168 kg/ha N, 67 kg/ha P2O5, and 78 kg the ability to repair the damaged and/or antibiosis for ECB. Each hybrid /ha K2O. Atrazine and metolachlor tissue without loss of quality or was entered twice, once for manual or were applied at rates of 1.8 and 2.2 kg yield. This mechanism of resistance natural infestation and once as a control a.i./ha, respectively, for weed control. may be rendered ineffective, protected from insects. A research site, No insecticides were applied at however, if the pest population is too approximately 1 km north east of planting. All plots were four rows wide large. Grand Pass, Missouri, was chosen (0.91 m [36"] between rows) and 4.9 m because of having an endogenous (16' long). Antibiosis and tolerance population of ECB. Planting, experiments had four replications and Research focused towards insect resistance has primarily been concerned with antibiosis, although some scientists have studied adult nonpreference and tolerance for a number of insects. Wiseman and his colleagues at the U.S. Department of Agriculture, Agricultural Research Service, Insect Biology and Population Management Research Laboratory, Tifton, GA, have been the most prolific researchers in this work (Chang et al. 1985; Waiss et al. 1979; Widstrom et al. 1979; Wilson et al. 1984; Wiseman 1985; Wiseman and Bondari 1992; Wiseman and McMillian 1980; Wiseman and Widstrom 1986; Wiseman et al. 1967, 1972, 1977, 1981, and 1983) and they have demonstrated both non-preference and tolerance in maize for corn earworm, Helicoverpa zea Figure 4. R.H. Painter and colleagues: R.H. Painter, E.C. Ortman, and E.L. Sorenson (left to right). (Boddie), and fall armyworm Spodoptera frugiperda (J.E. Smith). Barry and Darrah (1988) have shown that adult nonpreference and antibiosis resistance for Table 3. Comparisons of means of first and second generation ECB activities on three maize cultivars as they relate to host plant resistance in Missouri (Barry and Darrah 1988). 1984-1985† ECB can exist within a single cultivar (Table 3). Insect activity objective was to evaluate the First generation Egg masses/plant‡ Larvae/plant Larvae/eggmass Second generation Egg masses/plant‡ Tunnel (cm)/plant Tunnel (cm)/egg mass mechanisms of resistance for their † We designed six experiments to study the three mechanisms of resistance for both generations of ECB by using manual and natural infestations. Our contribution to overall ECB resistance in selected maize hybrids. ‡ 1986† Mo-2 ECB MFA 5802 Mo-2 ECB Wf9 x W182E 0.7a 0.3a 0.6a 0.9b 0.6b 1.1b 8.3a 3.2a 0.4a 11.5b 12.8b 1.2b 0.4a 0.9a 6.0a 1.5b 2.7b 7.0a 7.9a 12.0a 1.6a 4.3b 29.0b 9.6b Means for insect activity (horizontally between two cultivars) followed by the same letter are not significantly different according to Duncan’s Multiple Range Test at the 0.05 probability level. Data for the 1984-1985 study were derived from observations of 150 plants and from 50 plants for the 1986 study. Each egg mass contained 20-25 eggs. 24 B.D. BARRY AND L.L. DARRAH • the non-preference experiments had six live, neonate ECB larvae. Before replications because we were harvest, but at least 60 days after Second generation ECB tolerance depending upon natural infestation in infestation, five stalks from each plot During anthesis, hybrids were the latter. were randomly selected and split manually infested with 0, 30, 100, from the node above the ear to and 240 neonate ECB larvae for the ground level, and centimeters of infested treatment. Larvae were tunneling were estimated. applied in the leaf axils near the ear Experiment #3: zone. As in Experiment #2, five thuringiensis (Bt) (Dipel, Abbott Lab., First generation ECB nonpreference stalks were randomly selected from North Chicago, IL., or Bio-bit, E.I. This experiment was dependent on the center two rows of each plot, then split to measure ECB tunneling. Data were collected from the center two rows of each plot. In each experiment, control plots were treated with Bacillus DE1) • Experiment #6: on a 7-10 natural ECB populations to infest day schedule beginning at about the plants. Six replications were used. eight-leaf stage and continuing to one About 10 days after the first egg interval beyond anthesis. Plots infested masses from overwintering ECB with ECB for first generation studies were observed on plants in ECB- The results of our field evaluations of received Bt treatments beginning about treatment plots , one center row of four commercial maize hybrids and 21 days after manual infestations were each plot was checked for the two hybrid checks to determine which made, and the second generation plots number of plants exhibiting shot mechanisms of resistance are holes (Fig. 5). responsible for first and second Experiment #4: Second generation ECB resistance are generation ECB non-preference presented in Tables 4 to 6. In these Natural populations of ECB were tables, letter subscripts are used to Experiment #1: depended upon for infestation. Four indicate significant differences in the First generation ECB antibiosis days after moths were seen and the vertical direction, and superscripts are All plants in the center two rows of first egg masses were found in the used to indicate significant differences the four-row plots for the infested plots during anthesis, egg masses in the horizontal direction. The yields treatment were manually infested by were counted by examining using a bazooka (Mihm, 1983a, 10 plants selected at random 1983b) during the whorl stage of from the two center rows of plant development with each plot. Measurement of approximately 100 live, neonate ECB the amount of stalk larvae. Twenty-one days after tunneling was done as in Dupont, Wilmington, were treated from about the eight-leaf until mid-whorl stage of plant • development. The experiments were: • Experiment #2. infestation the plots were rated for ECB leaf damage using Guthrie et • Re sult s a nd Disc ussion • Experiment #5: First al.’s (1960) scale of 1 to 9 (1 = no generation ECB tolerance damage and 9 = severe damage; Figs. The center two rows of each 2 and 3). plot for the infested Experiment #2: treatment were all manually Second generation ECB antibiosis infested during the whorl All plants of the center two rows of stage of plant development the four-row plots for the infested with 0, 30, 100, and 240 treatment were manually infested neonate ECB larva and leaf during anthesis in the leaf axils, damage was rated as in within one leaf above or below the Experiment #1. top ear zone, with approximately 100 1 Mention of a trademark or proprietary product does not constitute a guarantee, warranty, or recommendation of the product by the U.S. Department of Agriculture or the University of Missouri and does not imply its approval to the exclusion of other products that may also be suitable. Figure 5. Shot holes; taken from Patch (1943). IMPACT OF MECHANISMS OF RESISTANCE ON EUROPEAN CORN BORER 25 among hybrids and within treatments Experiment #2 There were no significant differences for all experiments were highest for This experiment was done to evaluate between infested and non-infested Pioneer Brand 3184 (resistant check) antibiosis to the second generation ECB treatments within any hybrid. No and ICI Seeds 8326, with more than 12.1 (Table 4). The amount of tunneling was conclusions were made about antibiosis t/ha. DeKalb Genetics 623 and Ciba very low, which indicated a poor for second generation ECB in these 4666 yields were 10.9 to 12.1 t/ha. survival following infestation, hybrids. Pioneer Brand 3471 (unadapted in particularly because the susceptible Missouri) yielded in the range of 9.1 to check hybrid did not have much Experiment #3 10.6 t/ha, where as the susceptible tunneling. There were some significant This experiment was done to determine check hybrid yielded slightly over 6.0 differences among hybrids within the if non-preference was a mechanism for t/ha. Although manual insect non-infested treatment, but the first generation ECB resistance (Table infestations were made to enhance amounts of tunneling were small. Stalk 5). For comparison, naturally infested insect damage, they were not as tunneling differences observed in this vs. protected treatments were used and successful as we had anticipated (this experiment had no biological meaning. number of shot holes were counted as was not unusual in ECB research plots in the Midwest in 1994). Experiment #1 Table 4. Evaluation of four commercial hybrids plus two check hybrids for antibiosis by the first and second generation of ECB in Missouri in 1994. This experiment was done to evaluate First generation antibiosis to first generation ECB (Table 4). Leaf-feeding ratings among hybrids within the infested plots were the same except for Pioneer Brand 3471, which was significantly better (lower rating). Several significant differences in leaffeeding damage for the non-infested hybrids were found, although the ratings were too low to have biological meaning. DeKalb Genetics 623 and Pioneer Brand 3184 had the lowest Infested Hybrid ICI Seeds 8326 DeKalb Genetics 623 Ciba 4666 Pioneer Brand 3471 Pioneer Brand 3184 (Check) Wf9 x W182E (Check) Average rating LSD 0.05 = 0.5 Average yield LSD 0.05 = 0.47 † Rating (1-9) 3.5aa‡ a 2.8 a 3.0aa a 1.8 b 2.8aa a 3.0 a a 2.8 infested and non-infested treatments within hybrids, all were significantly different except for Pioneer Brand 3471, which indicated antibiosis resistance for first generation ECB. There were no significant differences in yield between infestation treatments within hybrids. Even though this experiment was not designed to look at tolerance, all in the infested plots. Yield (t/ha) 12.36ab a 11.61 b 11.79ab a 8.82 c 14.05ad a 6.34 d 1.4 Infested Hybrid ICI Seeds 8326 DeKalb Genetics 623 Ciba 4666 Pioneer Brand 3471 Pioneer Brand 3184 (Check) Wf9 x W182E (Check) Average tunnel length/plant LSD 0.05 = 0.38 Average yield LSD 0.05 = 0.49 † ‡ tolerance, particularly since survival was also a trend noted for lower yields 1.2bbc b 1.0 c 1.8bab a 2.0 a 1.0bc b 1.2 bc b 10.83a Second generation hybrids appeared to have some and damage were low this year. There † Rating (1-9) 10.46a and Ciba 4666 had significantly higher leaf-feeding damage ratings between Yield (t/ha) 12.41ab a 11.21 c 11.11ac a 8.73 d 13.61aa a 5.81 e ratings, where as Pioneer Brand 3471 ratings. However, when comparing Non-infested § ¶ § Non-infested § Tunnel (cm) Yield (t/ha) Tunnel (cm) Yield (t/ha) 0.25aa¶ a 0.51 a 0.64aa a 0.38 a 0.25aa a 0.25 a a 12.99aa a 10.73 c 12.00ab a 10.25 c 12.76aab a 8.73d 0.51ab a 1.40 a 0.38aa a 0.76 b 0.13ab a 0.25 b a 12.59aa a 11.14 b 12.47aa a 9.84c 12.52aa a 9.85c 0.38 0.57 11.25a 11.40a Rating is according to Guthrie’s (1960) scale of 1-9 (1 = no damage, 9 = severe damage). For first generation ECB data, superscript letters indicate significance horizontally (LSDs 0.05, rating = 1.1; yield = 1.16) and subscript letters indicate significance vertically (LSDs 0.05, rating = 0.8; yield = 0.82) in the table. If letters are different, the numerical values are significantly different. Average tunnel length/plant. For second generation ECB data, superscript letters indicate significance horizontally (LSDs 0.05, tunnel = 0.91; yield = 1.20) and subscript letters indicate significance vertically (LSDs 0.05, tunnel = 0.64; yield = 0.85) in the table. If letters are different, the numerical values are significantly different. 26 B.D. BARRY AND L.L. DARRAH an indicator of attractiveness. There Experiment #4 traits observed. A few significant were no significant differences between This experiment determined whether differences were noted among hybrids treatments for either yield or number of non-preference was a mechanism for within treatments, but they were not plants having shot holes. The natural second generation ECB resistance consistent. Again, natural ECB populations of ECB did not develop in (Table 5). Naturally infested vs. populations did not develop in good synchrony with the maize hybrids. protected plots were used as the synchrony with the crop. However, there was a trend for treatments; and egg mass counts, increased yield for hybrids with tunneling, and yield were observed. Experiment #5 protected treatment, except for Pioneer There was no significant difference This experiment determined whether Brand 3184. between the treatments for any of the tolerance was important for first generation ECB resistance by observing ECB leaf-feeding damage and yield (Table 6). A significant difference in Table 5. Evaluation of four commercial hybrids plus two check hybrids for preference by the first and second generation of ECB in Missouri in 1994. ECB leaf-feeding damage was observed for every hybrid when no infestation First generation Natural † Hybrid was compared with the infestation rate Protected † of 240 larvae per plant. There were Shot holes (no. plants) Yield (t/ha) Shot holes (no. plants) Yield (t/ha) generally no significant differences in 1.6ab‡ a 6.5a 1.8bb a 3.0 b 1.8ab a 1.2 b 2.6a 12.61aa a 11.15 b 12.14aa a 9.47 c 12.73aa a 6.85 d 2.0ab a 3.2 b 6.5aa a 2.7 b 2.3ab a 1.8 b 3.1a 12.90aa a 11.41 b 12.34aa a 10.19 c 12.57aa a 7.46 d indicating good tolerance. Additionally, 11.14a Experiment #6 ICI Seeds 8326 DeKalb Genetics 623 Ciba 4666 Pioneer Brand 3471 Pioneer Brand 3184 (Check) Wf9 x W182E (Check) Average number of plants/plot with shot holes LSD 0.05 = 1.7 Average yield LSD 0.05 = 0.39 10.83a yields across treatments within hybrids, these results suggested that we should have used 240 larvae per plant for our first generation ECB infestations in Missouri in 1994. This experiment determined the Second generation Natural Hybrid ICI Seeds 8326 DeKalb Genetics 623 Ciba 4666 Pioneer Brand 3471 Pioneer Brand 3184 (Check) Wf9 x W182E (Check) Average no. of egg masses/ plant LSD 0.05 = 1.4 Average tunnel length/plant LSD 0.05 = 0.33 Average yield LSD 0.05 = 0.54 † ‡ § ¶ # Egg § ¶ masses Tunnel (no.) (cm) 3.2a# ab a 3.3ab 5.0aa 5.2aa 1.6ab 4.0aa 3.7a a 0.51ab a 0.76ab a 0.51ab 0.25ab 0.84aa a 0.69ab importance of tolerance to second Protected Yield (t/ha) 12.10aa 11.16ab a 11.18ab 9.99acd 10.46bbc 9.07ad 0.59a Egg § ¶ masses Tunnel (no.) (cm) 2.8aa 1.8aa 3.7aa 2.8aa 3.7aa 3.7aa 3.1a a 0.69ab 0.91aa 0.33ab 0.33ab 0.94aa a 0.76ab generation ECB resistance by observing Yield (t/ha) 12.53aa 11.18ac 11.45abc 9.71ad a 12.17ab 9.45ad 10.66 the manual infestations were not effective and no conclusions about tolerance for second generation ECB could be made. Our experiments indicated that Pioneer Brand 3471 has antibiosis as a resistance mechanism for first generation ECB 0.66a a tunneling and yield (Table 9). However, when manually infested with 120 a 11.08 Average number of plants/plot with shot holes. For first generation ECB data, superscript letters indicate significance horizontally (LSDs 0.05, shot holes = 4.03; yield = 0.97) and subscript letters indicate significance vertically (LSDs 0.05, shot holes = 2.85; yield = 0.68) in the table. If letters are different, the numerical values are significantly different. Average number of egg masses/plant. Average tunnel length/plant. For second generation ECB data, superscript letters indicate significance horizontally (LSDs 0.05, egg masses = 3.5, tunnel = 0.81; yield = 1.34) and subscript letters indicate significance vertically (LSDs 0.05, egg masses = 2.5, tunnel = 0.56; yield = 0.94) in the table. If letters are different, the numerical values are significantly different. larvae per plant. Because of very low second generation ratings (including our susceptible check), no significant antibiosis was determined. The preference studies were dependent upon natural infestations of ECB and since these populations were very low, no preference or non-preference was found for either generation. In tests for IMPACT OF MECHANISMS OF RESISTANCE ON EUROPEAN CORN BORER tolerance, no significant differences check) were resistant when using were found for yield. The infestation Guthrie et al.’s 1 to 9 scale (Guthrie et rate of 240 larvae per plant, however, al., 1960). During the 1994 growing showed significant differences in leaf season, in our plots, 100 or fewer larvae feeding (antibiosis) for all hybrids. per plant were inadequate for making Those hybrids with higher leaf-feeding good evaluations. Chang, N.T., B.R. Wiseman, R.E. Lynch, and D.H. Habeck. 1985. Fall armyworm: Expression of antibiosis in selected grasses. J. Entomol. Sci. 20: 179188. Guthrie, W.D., F.F. Dicke, and C.R. Neiswander. 1960. Leaf and sheath feeding resistance to the European corn borer in eight inbred lines of dent corn. Ohio Agr. Exp. Sta. Res. Bull. 860. Huber, L.L., C.R. Neiswander, and R.M. Salter. 1928. The European corn borer and its environment. Ohio Agr. Expt. Sta. Bull. No. 429. ratings [ICI Seeds 8326 (4.0 rating), Re fe re nc e s DeKalb Genetics 623 (3.8 rating), and Ciba 4666 (5.0 rating)] would be Barry, Dean, and L.L. Darrah. 1988. Nonpreference resistance to European corn borer (Lepidoptera: Pyralidae) in the Mo-2 ECB maize cultivar. J. Kansas Entom. Soc. 61: 72-75. considered intermediate in resistance, and the check Wf9xW182E (6.0 rating) was susceptible. Pioneer Brand 3471 and Pioneer Brand 3184 (resistant 27 Table 6. Evaluation of four commercial hybrids plus two check hybrids for tolerance by the first and second generation of ECB in Missouri in 1994. First generation Noninfested † Hybrid ICI Seeds 8326 DeKalb Genetics 623 Ciba 4666 Pioneer Brand 3471 Pioneer Brand 3184 (Check) Wf9 x W182E (Check) Average rating LSD 0.05 = 0.4 Average yield LSD 0.05 = 0.51 Rating (1-9) 1.0c‡ b 1.0cb b 2.2 a b 1.2 b 1.2bb 2.2ca 10.99 30 larvae per plant 100 larvae per plant Rating (1-9) Yield (t/ha) Rating (1-9) Yield (t/ha) Rating† (1-9) Yield (t/ha) 12.96aa 11.16ba a 12.60a a 9.39 c 13.04aa 6.79ab d 1.9c 1.8bc b 1.0cc b 2.8a ab 1.5 bc 1.5ab bc 3.0ca 12.79aa 11.83ab ab 12.43 ab a 9.27 c a 12.26ab b 6.16d 1.9c 2.2bbc 2.8bb b 2.5bc ab 2.0 c 2.0ab c 4.5ba 13.05aa 12.18ab b 11.20c a 9.34 d 13.03aa 7.43ae 2.7b 4.0ac 3.8ac a 5.0b a 2.8d 2.2ae 6.0aa 13.15aa 11.51ab 12.89a a 9.54c 11.98ab 7.35ab d 4.0a 10.79 a † 240 larvae per plant Yield (t/ha) a † 11.04 a a 11.07 Second generation Noninfested § Hybrid ICI Seeds 8326 DeKalb Genetics 623 Ciba 4666 Pioneer Brand 3471 Pioneer Brand 3184 (Check) Wf9 x W182E (Check) Average tunnel length/plant LSD 0.05 = 0.30 Average yield LSD 0.05 = 0.70 † ‡ § ¶ 30 larvae per plant § 100 larvae per plant § Tunnel (cm) Yield (t/ha) Tunnel (cm) Yield (t/ha) Tunnel (cm) Yield (t/ha) 0.38a¶ cd 1.27aa 0.76ab a 0.13 d a 0.64 bc a 0.64 bc 12.38aab 11.76ab 13.09aa a 9.59 c a 12.06 b a 9.41 c 0.64a 0.51aab 0.13bc 0.25abc a 0.25 bc a 0.38 bc a 0.76a 12.90aa 10.81ac 11.91ab a 10.49 c a 12.18ab a 8.33 d 0.38a 0.13ab 0.51ba 0.64aa a 0.13 b b 0.00b a 0.64a 12.41aab 11.53acd 13.13aa a 10.70 d a 12.06 bc a 9.16 e 0.34b 11.38a 11.10a 11.50a 240 larvae per plant § Tunnel (cm) 0.51aa 0.76ab a 0.13ab a 0.00 b a 0.51a a 0.76a Yield (t/ha) 12.72aab 11.51ac 13.07aa a 10.26 d a 11.75 bc a 9.36 e 0.44a 11.38a Rating is according to Guthrie’s (1960) scale of 1-9 (1 = no damage, 9 = severe damage). For first generation ECB data, superscript letters indicate significance horizontally (LSDs 0.05, rating = 1.0; yield = 1.25) and subscript letters indicate significance vertically (LSDs 0.05, rating = 0.5; yield = 0.62) in the table. If letters are different, the numerical values are significantly different. Average tunnel length/plant. For second generation ECB data, superscript letters indicate significance horizontally (LSDs 0.05, tunnel = 0.76; yield = 1.70) and subscript letters indicate significance vertically (LSDs 0.05, tunnel = 0.56; yield = 1.20) in the table. If letters are different, the numerical values are significantly different. 28 B.D. BARRY AND L.L. DARRAH Mihm, J.A. 1983a. Efficient mass rearing and infestation techniques to screen for host plant resistance to maize stem borers, Diatraea spp. Centro International de Mejoramiento de Maiz y Trigo. El Batán, Mexico. Technical Bulletin. Mihm, J.A. 1983b. Efficient mass-rearing and infestation techniques to screen for host plant resistance to fall armyworm, Spodoptera frugiperda. Centro International de Mejoramiento de Maiz y Trigo. El Batán, Mexico. Technical Bulletin. Painter, R.H. 1951. Insect resistance in crop plants. The MacMillan Co., New York. Patch, L.H. 1943. Survival, weight, and location of European corn borers’ feeding on resistant and susceptible field corn. J. Agric. Res. 66: 7-19. Vinal, S.C. 1917. The European corn borer, Pyrausta nubilalis Hübner, a recently established pest in Massachusetts. Massachusetts Agric. Exp. Stn. Bull. 178. Waiss, A.C., B.G. Chan, C.A. Elliger, B.R. Wiseman, W.W. McMillian, N.W. Widstrom, M.S. Zuber, and A.J. Keaster. 1979. Maysin, a flavone glycoside from corn silks with antibiotic activity toward corn earworm. J. Econ. Entomol. 72: 256-258. Widstrom, N.W., W.W. McMillian, and B.R. Wiseman. 1979. Oviposition preference of the corn earworm and the development of trichomes on two exotic corn selections. Environ. Entomol. 8: 833-839. Wilson, R.L., B.R. Wiseman, and N.W. Widstrom. 1984. Growth response of corn earworm (Lepidoptera: Noctuidae) larvae on meridic diets containing fresh and lyophilized corn silk.. J. Econ. Entomol. 77: 1159-1162. Wiseman, B. 1985. Types and mechanisms of host plant resistance to insect attack. Insect Sci. Applic. 6: 239-242. Wiseman, B.R., and K. Bondari. 1992. Genetics of antibiotic resistance in corn silks to the corn earworm (Lepidoptera: Noctuidae) larvae. J. Econ. Entomol. 85: 293-298. Wiseman, B.R., F.M. Davis, and W.P. Williams. 1983. Fall armyworm: Larval density and movement as an indication of nonpreference in resistant corn. Prot. Ecol. 5: 135-141. Wiseman, B.R., and W.W. McMillian. 1980. Feeding preferences of Heliothis zea larvae preconditioned to several host crops. J. Georgia Entomol. 15: 449-453. Wiseman, B.R., W.W. McMillian, and N.W. Widstrom. 1972 Tolerance as a mechanism of resistance in corn to the corn earworm. J. Econ. Entomol. 65: 835837. Wiseman, B.R., R.H. Painter, and C.E. Wassom. 1967. Preference of first-instar fall armyworm larvae for corn compared with Tripsacum dactyloides. J. Econ. Entomol. 60: 1738-1742. Wiseman, B.R., and N.W. Widstrom. 1986. Mechanisms of resistance in ‘Zapalote Chico’ corn silks to fall armyworm (Lepidoptera: Noctuidae) larvae. J. Econ. Entomol. 79: 1390-1393. Wiseman, B.R., N.W. Widstrom, and W.W. McMillian. 1977. Ear characteristics and mechanisms of resistance among selected corns to corn earworm. Fla. Entomol. 60: 97-103. Wiseman, B.R., W.P. Williams, and F.M. Davis. 1981. Fall armyworm: Resistance mechanisms in selected corn. J. Econ. Entomol. 74: 622-624. 29 M e c ha nism s a nd Ba se s of Re sist a nc e in M a ize t o Sout hw e st e rn Corn Bore r a nd Fa ll Arm yw orm W.P. Williams and F.M. Davis, USDA-ARS, Mississippi State. Abst ra c t Maize, Zea mays L., germplasm lines with resistance to leaf feeding by the southwestern corn borer (SWCB), Diatraea grandiosella Dyar, and fall armyworm (FAW), Spodoptera frugiperda (J.E. Smith), have been developed and released. A series of experiments were conducted to determine the mechanisms and bases of this resistance. Field experiments have shown that antibiosis is a mechanism of resistance to both insects. When resistant and susceptible maize genotypes were infested with either SWCB or FAW neonates, the larvae that were recovered 10 to 14 days later from susceptible genotypes weighed twice as much as those recovered from resistant genotypes. Laboratory experiments using excised leaf tissue and liquid pressed from leaves demonstrated that larval non-preference is also a mechanism of resistance of these germplasm lines. When experiments were conducted using callus tissue of susceptible and resistant maize genotypes, both SWCB and FAW larvae preferred to feed on callus of susceptible genotypes. Larvae that fed on the susceptible calli weighed twice as much as those that fed on resistant calli. Similar differences in size were observed when larvae were fed on lyophilized leaf tissue of susceptible and resistant genotypes. Factors responsible for these differences in growth are not fully known; however, leaf tissue from the inner whorls of resistant genotypes tends to be tougher than that from susceptible genotypes. The cuticle and epidermal cell wall of resistant genotypes is generally thicker. Leaves of resistant plants have a higher fiber content and lower total protein content. A 33kD polypeptide found in callus tissue appears to be associated with resistant genotypes. Electrophoretic analysis of whorl leaf tissue also indicated a possible association of resistance to SWCB and FAW with 21kD and 36kD polypeptides. I nt roduc t ion In our work with SWCB and FAW resistance in maize, we have not only Fie ld a nd Gre e nhouse Ex pe rim e nt s Identifying germplasm with resistance posed these questions, but have to a pest is critical to the success of any undertaken research to try to answer One of the first experiments conducted plant resistance research program. them. Scott and Davis (1981) released to determine mechanisms of resistance Therefore, developing methods of the first germplasm with resistance to operating in germplasm released from evaluating damage to the pest and SWCB and FAW in 1974. Soon our program compared MpSWCB-4 locating suitable germplasm to thereafter, attempts to determine how and Antigua 2D-118 (FAW resistant evaluate receive a high priority at the the resistant plants differed from germplasm identified at Tifton, GA) inception of a new program. However, susceptible plants and to compare the with susceptible genotypes (Wiseman once germplasm with resistance has responses of larvae to resistant and et al. 1981). Choice tests to determine been identified, other questions quickly susceptible plants began. As we larval preference were conducted by arise. Why is the germplasm resistant? developed and released additional randomly placing leaf sections of 5 What mechanisms of resistance are germplasm lines, we have continued genotypes along the outer edge of operating? How is the resistance our investigations in these areas using 25.4-cm-diam. dishes, then 200 first inherited? How effective is the field and greenhouse experiments, instar larvae were placed in the center. resistance in reducing yield losses? chemical analyses, microscopy, and The dishes were maintained in laboratory bioassays. darkness for 18 hours; larvae on or 30 W.P. WILLIAMS AND F.M. DAVIS under each leaf section were then Several field experiments have also growth with 30 neonates per plant. counted. Fewer larvae were found on indicated that SWCB survival and Larvae were counted and weighed 14 the resistant genotypes than the growth are reduced on plants of leaf- days later. The significantly lower susceptible genotypes (Table 1). Tests feeding resistant genotypes (Davis and weights of larvae recovered from the were also conducted to compare Williams 1986; Davis et al. 1991; resistant hybrids provide evidence for growth of FAW larvae on leaf sections Williams et al. 1989). Data from one of antibiosis as a mechanism of resistance of the same genotypes (Table 1). After 8 these (Davis et al. 1991) are given in in these hybrids. Differences in larval days, larvae fed on leaf tissue of Table 3. In this experiment, plants were survival between resistant and MpSWCB-4 were significantly smaller infested in the mid-whorl stage of susceptible hybrids were less distinct, than those fed on any other genotype. Larvae fed on Cacahuacintle X tissue consumed 72.4 cm2 leaf tissue while those fed on MpSWCB-4 consumed Table 1. Number of FAW larvae present on leaf sections of different maize genotypes after 18 hours in a choice test, and mean weights of larvae fed for 8 days on the same genotypes in a no-choice test (Wiseman et al. 1981). only 21.5 cm2 tissue. MpSWCB-4 showed the highest level of resistance with both antibiosis and non-preference expressed. The resistance of Antigua 2D-118 appeared to be primarily nonpreference. Genotype Cacahuacintle X s Ab24E x Mp305 Mp4008 Antigua 2D-118 MpSWCB-4 Further evidence of the high degree of a non-preference of Antigua 2D-118 was b provided by a field cage test designed c to determine if FAW larvae were No. of larvaeb Wt. of larvae (mg)c (18 hr) choice (8 days) no-choice Field ratinga S S R R R 17.7 a 13.0 b 8.5 c 5.8 cd 2.1 d 333.5 a 263.3 b 193.3 c 229.6 bc 151.8 d S, susceptible; R, resistant. Means (based on 30 replicates) followed by the same letter do not differ significantly (P = 0.05, Duncan’s Multiple Range Test). Means (based on 50 replicates) followed by the same letter do not differ significantly (P 0.05, Duncan’s Multiple Range Test). crawling off resistant plants (Wiseman et al. 1983). Test plants of 3 genotypes were planted approximately 120 cm apart and each was surrounded by Table 2. Mean number of FAW larvae moving from test maize genotypes surrounded by susceptible trap plants at various time intervals after infestation (Wiseman et al. 1983). plants of a susceptible hybrid. The test plants were infested with 10, 20, or 40 newly hatched larvae per plant. At 3, 5, Genotype larvae that had moved from test plants Antigua 2D-118 MpSWCB-4 Cacahuacintle X s were counted (Table 2). Significantly a 7, and 11 days after infestation, the more larvae crawled from Antigua 2D- b 118 than from MpSWCB-4 or Cacahuacintle Xs, which did not differ. An additional investigation was conducted to determine survival and 3 R R S 0.6 a 0.1 b 0.2 b genotypes (Williams et al. 1983b). Again, larval weights and survival were lower on Antigua 2D-118 and MpSWCB-4 than on Ab24E x Mp305 and Cacahuacintle Xs, indicating antibiosis and possibly non-preference as resistance mechanisms in the 2 resistant types of germplasm. Days after infestation 5 7 5.6 a 2.1 b 2.1 b 5.9 a 3.7 b 3.3 b 11 8.0 a 5.0 b 4.5 b R, resistant; S, susceptible. Means within a column followed by the same letter do not differ (P = 0.05, Duncan’s Multiple Range Test. Table 3. Number and weights of SWCB larvae 14 days after infestation of hybrids with 30 larvae/plant at Mississippi State, MS (Davis et al. 1991). No. larvae growth of FAW larvae under field conditions using some of the same b Field a classification Hybrid Ab24E x Va35 T202 x Va35 Ab24E x Tx601 Mp496 x Mp701 Mp701 x Mp705 Mp703 x Mp704 Mp704 x Mp707 LSD (0.05) a Larval wt (mg) Classificationa 1988 1989 1988 1989 SxS SxS SxS RxR RxR RxR RxR 4.1 2.7 3.4 2.1 2.8 1.8 2.5 1.7 4.9 4.7 5.6 2.2 1.6 1.3 0.6 1.6 60.1 62.1 53.3 15.6 13.2 11.3 10.0 7.8 55.5 57.1 47.7 7.0 8.4 10.2 7.6 10.5 S, susceptible; and R, resistant, to SWCB leaf feeding. MECHANISMS AND BASES OF RESISTANCE IN MAIZE TO SOUTHWESTERN CORN BORER 31 especially in 1988. The differences in program (Davis 1989). The diet is based correlated with SWCB and FAW leaf number of larvae recovered from on wheatgerm, casein, sucrose, feeding damage ratings (Table 4). resistant and susceptible hybrids could vitamins, salts, agar, and antimicrobial Larval weights were negatively be attributed to either non-preference or agents. Newly hatched SWCB larvae correlated with thickness. In a second antibiosis. were fed on the test diets for 5 days and part of this investigation, the pressure then weighed. The extracts from both required to split whorl tissue of the the susceptible and resistant tissue same lines was determined. Although caused only limited inhibition of larval the required pressure differed between growth that did not appear to be years and there was a significant biologically significant (Hedin et al. interaction with years, greater pressure While field and greenhouse 1984). Analyses to determine the was generally required to split the experiments yielded information on the composition of the residue did not whorl tissue of resistant genotypes. mechanisms of resistance operating in yield any information that suggested a this germplasm, it became obvious to us basis of resistance. We concluded that that other types of experiments would the celluloses and hemicelluloses be necessary for a more thorough making up the higher fiber content of At about the same time the anatomical understanding of what factors are the resistant genotypes could studies commenced, we undertook responsible for the resistance. This led contribute to leaf toughness, investigations to determine whether the us into the area of chemical analyses indigestibility, and intractability to reductions in larval weight expressed and laboratory bioassays. Whorl tissue metabolism by the insect and this might on plants of resistant genotypes in the from resistant and susceptible be at least a part of the basis of field would also be expressed if larvae genotypes was analyzed. The tissue resistance. were fed undifferentiated maize callus Che m ic a l Ana lyse s, Bioa ssa ys, a nd Ana t om ic a l Obse rva t ions T issue Cult ure tissue. Initially, we conducted from the resistant genotype was at least 25% higher for crude fiber, acid Because of the ambiguity of our results experiments to determine whether detergent fiber, lactic acid, calcium, and at this point, we were somewhat SWCB larvae would feed and develop glutamate-oxalacetate transaminase. discouraged and were unsure as to on callus tissue and whether larval The susceptible genotype was at least whether we should look for an growth was affected by callus genotype 25% higher for crude protein, crude anatomical or biochemical basis of (Williams et al. 1983a). We found that lipid, ash, stearic and oleic acids, and resistance. Investigations were begun to the larvae did indeed feed and develop silica (Hedin et al. 1984). It appeared determine whether anatomical on callus tissue and those fed on callus that components associated with differences exist between a leaf-feeding of resistant maize genotypes were nutrition, such as protein, minerals, and resistant line, Mp704, and a susceptible generally smaller. lipids, were higher in the susceptible line, Ab24E. Ng (1988) found that the genotype, whereas fiber was higher in number of vascular bundles per unit the resistant genotype. Subsequent area was greater in Mp704 whorl leaf analyses of tissue from additional tissue than in Ab24E whorl leaf tissue. genotypes generally supported this The cuticle and outer cell wall of the conclusion. epidermis on both the upper and lower Table 4. Correlation coefficients (r) between anatomical characteristics and SWCB and FAW damage ratings and larval weights for susceptible and resistant maize lines (Davis et al. 1994). leaf surfaces of Mp704 leaves were In another facet of this investigation, Cell wall complex thickness found to be thicker. whorl tissue was freeze-dried, ground, and extracted by Soxhlet with In a follow-up study, 4 resistant and 6 cyclohexane/ethyl acetate/acetic acid, susceptible lines were included (Davis 500/500/1 (CHEA). The tissue was et al. 1994). Again, both the upper and subsequently extracted at boiling reflux lower cell wall complexes of the with methanol/water, 7/3 (mw). The resistant lines were thicker. The extracts were incorporated into the thickness of the upper and lower cell artificial diet used in our rearing wall complexes were also highly Insect Upper Lower Southwestern corn borer Damage score 0.92** Larval weight - 0.85** 0.92** - 0.85** Fall armyworm Damage score Larval weight 0.91** - 0.71* 0.91** - 0.71* * Significant at P <0.05. ** Significant at P <0.01. 32 W.P. WILLIAMS AND F.M. DAVIS Encouraged by the results of our initial susceptible hybrids (Table 6) (Williams were placed in complete darkness for tests, we designed experiments to et al. 1985). As with SWCB, antibiosis 24 hours; larvae present on each measure both SWCB and FAW growth was apparently operating as a portion of callus were then counted on resistant and susceptible genotypes. mechanism of resistance to FAW. (Williams et al. 1985). Twice as many larvae were attracted to the callus of We evaluated a diallel cross for leaf feeding by SWCB in the field and for Additional investigations were susceptible hybrids (Table 7), indicating larval growth on callus in the conducted to determine whether non- non-preference for the callus of laboratory (Williams and Davis 1985). preference might also be operating as a resistant hybrids. Both the leaf feeding ratings and the mechanism of resistance. To determine larval weights clearly delineated whether FAW larvae, upon hatching, A similar experiment was conducted to resistant and susceptible hybrids (Table fed preferentially on callus of different determine whether SWCB larvae 5). The differences in larval growth maize hybrids, approximately 500 mg exhibited a preference for callus of indicated that antibiosis was acting as a of callus of 4 hybrids was placed in the some genotypes (Williams et al. 1987a). mechanism of resistance. Significant corners of six plastic containers (130 x In this experiment, callus of 4 hybrids differences in larval weight were also 130 x 55 mm). Approximately 50 was equally spaced around the expressed when FAW larvae were fed blackhead-stage eggs were placed in perimeter of Petri plates (150 mm for 7 days on callus of resistant and the center of each container. Containers diameter) and 50 eggs, just prior to hatch, were placed in the center of the plate. Larvae were counted after 24 Table 5. Mean ratings of SWCB leaf-feeding damage in the field and weights of larvae grown on callus initiated from resistant and susceptible maize hybrids (Williams and Davis 1985). a Hybrid Classification Ab24E x Tx601 Ab24E x GT112 Tx601 x GT112 Mp496 x Mp704 Mp496 x Mp78:518 Mp704 x Mp78:518 LSD (0.05) a b c 7.1 6.9 6.7 5.5 4.9 4.8 0.5 17.8 19.2 16.5 11.1 10.3 11.0 2.3 Classificationa Pioneer Brand 3369A Ab24E x Va35 Mp496 x Mp704 Mp703 x Mp704 S S R R No. of larvaeb 52 a 48 a 34 b 25 b S indicates susceptible; R, resistant to leaf feeding in field tests. Means not followed by the same letter differ at the P = 0.05 level of significance (Student-Newman-Keuls test). a b S S R R We have also conducted similar our leaf feeding resistant and susceptible lines (Williams et al. 1987b). The results were similar to those obtained with SWCB and FAW. No. of Classificationa larvaeb Pioneer Brand 3369A Ab24E x Va35 Mp496 x Mp704 Mp703 x Mp704 feeding susceptible hybrids (Table 8). (CEW), Helicoverpa zea (Boddie), using Table 7. Mean number of FAW larvae present on different maize hybrids 24 hours after infestation with 50 blackhead-stage eggs (Williams et al. 1985). Hybrid strongly preferred callus of the leaf- experiments with corn earworm S indicates susceptibility and R, resistance. Damage was visually rated 14 days after infestation with 30 larvae per plant on a scale of 0 (no damage) to 9 (heavy damage) in 1982 and 1983. Larvae were weighed after feeding on callus for 7 days. Hybrid b 7-Day larval c wt (mg) SxS SxS SxS RxR RxR RxR Table 6. Weights of FAW larvae fed on callus of resistant and susceptible maize hybrids for 7 days (Williams et al. 1985). a Leaf feeding b damage hours. As with FAW, the SWCB larvae 13 a 15 a 6b 7b S indicates susceptibility to leaf feeding; R indicates resistance. Means not followed by the same letter differ at the P = 0.05 level of probability (Student-Newman-Keuls test). The differences we observed in larval growth and preference associated with maize exhibiting different levels of Table 8. Number of SWCB larvae feeding on callus 24 hours after infestation with 50 blackhead-stage eggs (Williams et al. 1987a). Hybrid Classificationa Ab24E x Va35 SC229 x Tx601 Mp496 x Mp701 Mp704 x Mp706 a b S S R R No. of larvaeb 6.4 a 7.6 a 2.7 b 1.3 b S indicates susceptibility to leaf feeding; R indicates resistance. Means (10 replications) followed by the same letter differ at the P = 0.05 level of significance (Student-Newman-Keuls test). MECHANISMS AND BASES OF RESISTANCE IN MAIZE TO SOUTHWESTERN CORN BORER 33 resistance to FAW and SWCB leaf- the amino acids alanine and valine, but and frozen at -18°C. The whorl tissue feeding in the field renewed our aspartic acid was non-preferred (Hedin was later lyophilized and ground to a interest in trying to find a chemical et al. 1993). fine powder. Diets were prepared by combining 250 ml distilled water, 2400 basis for this resistance. Chemical analyses of callus of susceptible and In other investigations, lyophilized leaf mg agar, 12.5 mg gentamicin sulfate, resistant genotypes indicated that the tissue of corn genotypes with varying 132 mg sorbic acid, and 528 mg ascorbic most obvious difference between the levels of resistance to FAW and SWCB acid. The mixture was heated to 82°C two was a higher amount of aspartic was added to an artificial diet. FAW while stirring, and 11 g lyophilized acid in the resistant callus (Hedin et al. larvae grew well on all diets into which tissue was then added. The mixture 1990). We also found that when choice lyophilized tissue had been was then dispensed in 10-ml aliquots tests were conducted in which a series incorporated, and weights of larvae fed into 30-ml cups. of amino acids were compared in on resistant and susceptible genotypes attractiveness with water, only aspartic did not differ (F. Davis, unpublished). Experiments were carried out by acid elicited less response than water. This indicated to us that the diet was infesting cups with neonates, covering either masking differences that would them with insert paperboard caps, and have been expressed in growth of placing them in an environmental larvae fed on susceptible or resistant chamber maintained at 28°C with a genotypes in the field or lyophilization photoperiod of 12:12 (L:D). FAW larvae Because of the difficulty of producing destroyed genotypic differences among were weighed after 10 days and SWCB callus in the quantities needed by the tissue samples. larvae after 14 days. For both insects, La bora t ory Bioa ssa ys a nd Che m ic a l Ana lyse s the larvae weighed significantly less chemist with whom we were working, we sought other approaches for We, therefore, designed a bioassay when fed on diets containing tissue of investigating the chemical basis of based primarily on lyophilized leaf resistant genotypes (Table 9). The resistance. To determine whether tissue (Buckley et al. 1991; Williams and bioassay was also used successfully larvae discriminated among extracts Buckley 1992; Williams et al. 1990a). For with CEW (Table 10) (Buckley et al. from leaves of different genotypes, these bioassays, whorl leaves were 1991). plant whorls were pressed in a harvested when plants reached the mid- hydraulic press. Filter paper disks were whorl stage of growth. They were This was the first bioassay that we had saturated with extracts from different trimmed to approximately 15 cm in used which appeared to have promise genotypes and randomly placed length, placed in plastic freezer bags, as a way of comparing various fractions of susceptible and resistant maize around the perimeter of 150-mm diameter Petri plates. Forty neonate larvae were placed in the center of the dish, and the dishes were placed in darkness for 4 hours. The number of larvae on each filter paper were then Table 9. Weights of FAW and SWCB larvae reared for 10 and 14 days, respectively, on diets containing lyophilized whorl tissue of various inbred lines (Williams et al. 1990a). counted. We found that larvae were attracted to susceptible genotypes Larval weight (mg) Inbred line a FAW SWCB 172 b 185 b 239 a 150 b 116 b 161 a 112 b 126 b 62 c 86 c 97 c 53 c 65 c 71 c 69 c 46 d twice as often as to resistant genotypes (Williams et al. 1987b). Choice tests conducted following fractionation of leaf extracts indicated that FAW were more strongly attracted to the amino acids of extracts from susceptible genotypes than to those from resistant ones (Hedin et al. 1990). Further experimentation also indicated that SWCB larvae exhibited a preference for Susceptible Ab24E SC229 Tx601 Va35 Resistant Mp701 Mp705 Mp704 Mp707 a Means in a column followed by the same letter do not differ (P <0.05) (StudentNewman-Keuls test). genotypes. In one investigation, we evaluated FAW larval growth, not only on susceptible and resistant inbred Table 10. Weights of CEW larvae reared for 11 days on diets containing only lyophilized whorl tissue of corn inbreds (Buckley et al. 1991). Inbred Ab24E Tx601 Va35 Mp704 Mp707 Mp708 LSD (0.05) a a Classification S S S R R R Larval wt (mg) 223 139 59 11 26 12 28 S indicates susceptibility to leaf feeding; R indicates resistance. 34 W.P. WILLIAMS AND F.M. DAVIS lines, but also on mixtures of tissue on this problem. We do make these from resistant and susceptible lines conclusions about the basis of resistance (Williams and Buckley 1992). We found from our research: I de nt ific a t ion of Prot e ins Assoc ia t e d w it h Re sist a nc e Because we have been unable to generally that larvae fed on mixtures of • Resistant genotypes probably do not definitely identify specific substances contain a highly toxic substance. responsible for resistance in the lines we Such characteristics as leaf toughness have released, we have attempted to may be a part, but not the complete, identify proteins associated with basis of resistance. resistance (Paiva 1988; Callahan et al. Nutritional differences between 1992; Jiang 1994). We assumed that was 238 mg, while those fed on only tissue of resistant and susceptible although proteins per se might not affect resistant tissue weighed 114 mg, and genotypes may be associated with larval growth, it should be possible to resistance. find differences in proteins that were There are likely several factors involved in some way in the synthesis consistent with the presence of reduced responsible for resistance in the lines of those substances that affect larval amounts in resistant genotypes of we have released. growth. This work has involved the tissue of susceptible and resistant genotypes exhibited weight gains less than those of larvae fed on susceptible • tissue alone, but greater than those fed only resistant tissue. The mean weight of those fed on susceptible tissue alone • those fed on a mixture of the two weighed 185 mg. This would be • electrophoretic analyses of proteins substances essential for larval growth. extracted from whorl leaf tissue We have also carried out experiments using methods similar to those described in the previous experiment (Williams and Buckley 1992) except various combinations of water extracts and residues of lyophilized tissue of Table 11. Weights of FAW larvae fed on test diets composed of various combinations of residues and extracts of lyophilized leaf tissue from resistant and susceptible maize genotypes (Williams and Buckley 1992). resistant, Mp708, and susceptible, Ab24E, genotypes replaced the 10 g Larval wt. (mg) Diet composition lyophilized tissue in our usual bioassay occur in the extracts or residues. The results (Table 11) indicated that the a determine whether the extraction process itself affected larval growth and to determine whether the factors causing differences in larval growth on resistant and susceptible genotypes water extracts provided substances (Jiang 1994) of resistant and susceptible maize genotypes. In a comparison of polypeptides present in the leaf-feeding resistant line Mp708 with the lines from which it was developed, Mp704 and Tx601 (Williams et al. 1990b), Callahan et al. (1992) a Lyophilized tissue (S) Lyophilized tissue (R) Water extract (S) + residue (S) Water extract (R) + residue (S) Water extract (S) + residue (R) Water extract (R) + residue (R) Residue (S) Residue (R) LSD (0.05) diet. This was done to help us (Callahan et al. 1992) and callus tissue 165 20 106 106 13 10 11 1 22 found 8 polypeptides present in both Mp708 and its resistant parent, Mp704, which were absent in the susceptible Tx601. The full complement of polypeptides was not present in 1 other resistant line nor completely absent from 3 other susceptible lines (Table 12). S indicates leaf feeding susceptible Ab24E; R indicates leaf feeding resistant Mp708. The combined presence of polypeptides 5(36kD) and 7(21kD) was, however, essential to growth, but the resistant and susceptible genotypes provided these equally well. Larval growth Table 12. Summary of two-dimensional gel data of 8 maize lines with regard to presence (+) or absence (-) of 8 polypeptides (Callahan et al. 1992). indicated that genotypic differences between residues are responsible for differences in growth. We have not yet been able to capitalize on this. In further fractionation of the residue, we apparently have either lost or changed substances essential for growth. We are, however, still working Line a 1 2 3 Mp708 (R) Mp704 (R) Mp707 (R) Mp496 (R) Tx601 (S) Ab24E (S) GT106 (S) SC229 (S) + + - + + + - + + + + - a Polypeptide number 4 5 + + + + + + + + + + + + R indicates resistance to leaf feeding; S indicates susceptibility. 6 7 8 + + + - + + + + + - + + - MECHANISMS AND BASES OF RESISTANCE IN MAIZE TO SOUTHWESTERN CORN BORER specific to the resistant lines. Further with resistance represents one of our research will be needed to reveal the more definitive findings. We are now significance of these findings. attempting to identify the protein and determine its function in the plant. Extensive analyses by Jiang (1994) of the proteins of callus of resistant and From the many experiments we have susceptible maize lines revealed one conducted, we conclude that both protein (33kD) that was consistently antibiosis and non-preference are present in callus of resistant, but not operating as mechanisms of resistance. susceptible, genotypes. The n-terminal We have not found a single factor, such amino acid sequence of the 33kD as a strong toxin, to which resistance protein suggests that it may be a can be attributed. It may well be that cysteine proteinase. In F2 progeny of the resistance we have found is the resistant by susceptible cross, conditioned by several factors, such as Mp704 x Tx601, concentration of the leaf toughness, increased fiber, and 33kD protein and weight of larvae reduced nutritional quality of the feeding on those callus lines were resistant plants. If this is true, it would negatively correlated. explain our difficulties in identifying those factors. One interesting fact was observed during this investigation. Callus of Ac k now le dgm e nt Mp704 is normally not friable, but after culturing for extended periods of time, Contribution of the USDA, ARS Crop it sometimes becomes friable or easily Science Research Laboratory in crumbled. In one insect feeding trial, cooperation with the Mississippi both friable and non-friable Mp704 Agricultural and Forestry Experiment callus was included. The FAW larvae Station, Mississippi State, MS, USA. that fed on friable callus were much Published as Paper no. P-8614 of the heavier than those fed on non-friable Mississippi Agric. and Forestry Exp. callus. After this was observed, an Stn. experiment was designed to compare growth on callus with friable and nonfriable morphology. The results indicated that FAW larvae fed on friable Mp704 callus and friable callus of the hybrid, Mp704 x Tx601, were not only heavier than those fed on non- Table 13. Mean weights of FAW larvae fed nonfriable and friable calli of resistant and susceptible maize lines for 7 days (Jiang 1994). friable callus of the same genotypes, but also heavier than those fed on callus of the susceptible line, Tx601 (Table 13). Analysis of the proteins of the friable and non-friable callus revealed that loss of the 33kD protein accompanied the change in morphology. This provides additional Genotype Mp704 (R)a Mp704 x Tx601 (RxS) Tx601 (S) evidence that this protein may play a LSD role in resistance. The indication that a the 33kD protein may be associated a Callus morphology Larval wt. (mg) Nonfriable Friable 59 130 Nonfriable Friable Nonfriable 93 131 100 (0.05) 16 S indicates susceptibility to leaf feeding; R indicates resistance. 35 Re fe re nc e s Buckley, P.M., F.M. Davis, and W.P. Williams. 1991. Identifying resistance in corn to corn earworm (Lepidoptera: Noctuidae) using a laboratory bioassay. J. Agric. Entomol. 8: 67-70. Callahan, F.E., F.M. Davis, and W.P. Williams. 1992. Steady-state polypeptide profiles of whorl tissue from lepidoptera-resistant and susceptible corn lines. Crop Sci. 32: 1203-1207. Davis, F.M. 1989. Rearing the southwestern corn borer and fall armyworm at Mississippi State. In Toward Insect Resistant Maize for the Third World: Proceedings of the International Symposium on Methodologies for Developing Host Plant Resistance to Maize Insects, 27-36. Mexico, D.F. CIMMYT. Davis, F.M., G.T. Baker, and W.P. Williams. 1994. Anatomical characteristics of maize resistant to leaf feeding by southwestern corn borer (Lepidoptera: Pyralidae) and fall armyworm (Lepidoptera: Noctuidae). J. Agric. Entomol. 12: 55-66. Davis, F.M. and W.P. Williams. 1986. Survival, growth, and development of southwestern corn borer (Lepidoptera: Pyralidae) on resistant and susceptible maize hybrids. J. Econ. Entomol. 79: 847-851. Davis, F.M., W.P. Williams, S.S. Ng, and G.W. Videla. 1991. Growth and survival of southwestern corn borer on whorl and reproductive stage plants of selected corn hybrids. Southwestern Entomol. 16: 144-154. Hedin, P.A., F.M. Davis, W.P. Williams, and M.L. Salin. 1984. Possible factors of leaf feeding resistance in corn to the southwestern corn borer. J. Agric. Food Chem. 32: 262-264. Hedin, P.A., W.P. Williams, F.M. Davis, and P.M. Buckley. 1990. Roles of amino acids, protein, and fiber in leaffeeding resistance of corn to the fall armyworm. J. Chem. Ecol. 16: 19771995. Hedin, P.A., W.P. Williams, P.M. Buckley, and F.M. Davis. 1993. Arrestant responses of southwestern corn borer larvae to free amino acids: structureactivity relationships. J. Chem. Ecol. 19: 301-311. Jiang, B. 1994. Relationship of a 33 kD putative cysteine proteinase with fall armyworm resistance in corn. Ph.D. dissertation, Mississippi State Univ., Mississippi State, MS. 36 W.P. WILLIAMS AND F.M. DAVIS Ng, S.S. 1988. Southwestern corn borer, Diatraea grandiosella Dyar, and the fall armyworm, Spodoptera frugiperda (J.E. Smith): biology and host plant resistance studies. Ph.D. dissertation, Mississippi State Univ., Mississippi State, MS. Paiva, R. 1988. Electrophoretic analysis of proteins from fall armyworm resistant corn and susceptible corn genotypes. M.S. thesis, Mississippi State Univ., Mississippi State, MS. Scott, G.E. and F.M. Davis. 1981. Registration of Mp496 inbred of maize. Crop Sci. 21: 353. Williams, W.P. and P.M. Buckley. 1992. Growth of fall armyworm (Lepidoptera: Noctuidae) larvae on resistant and susceptible corn. J. Econ. Entomol. 85: 2039-2042. Williams, W.P., P.M. Buckley, and F.M. Davis. 1985. Larval growth and behavior of the fall armyworm (Lepidoptera: Noctuidae) on callus initiated from susceptible and resistant corn hybrids. J. Econ. Entomol. 78: 951954. Williams, W.P., P.M. Buckley, and F.M. Davis. 1987a. Tissue culture and its use in investigations of insect resistance of maize. Agric., Ecosystems, and Environ. 18: 185-190. Williams, W.P., P.M. Buckley, and F.M. Davis. 1987b. Feeding response of corn earworm (Lepidoptera: Noctuidae) to callus and extracts of corn in the laboratory. Environ. Entomol. 16: 532534. Williams, W.P., P.M. Buckley, and F.M. Davis. 1989. Combining ability for resistance in corn to fall armyworm and southwestern corn borer. Crop Sci. 29: 913-915. Williams, W.P., P.M. Buckley, P.A. Hedin, and F.M. Davis. 1990a. Laboratory bioassay for resistance in corn to fall armyworm (Lepidoptera: Noctuidae) and southwestern corn borer (Lepidoptera: Pyralidae). J. Econ. Entomol. 83: 1578-1581. Williams, W.P., P.M. Buckley, and V.N. Taylor. 1983a. Southwestern corn borer growth on callus initiated from corn genotypes with different levels of resistance to plant damage. Crop Sci. 23: 1210-1212. Williams, W.P. and F.M. Davis. 1985. Southwestern corn borer larval growth on corn callus and its relationship with leaf feeding resistance. Crop Sci. 25: 317319. Williams, W.P., F.M. Davis, and G.L. Windham. 1990b. Registration of Mp708 germplasm line of maize. Crop Sci. 30: 757. Williams, W.P., F.M. Davis, and B.R. Wiseman. 1983b. Fall armyworm resistance in corn and its suppression of larval growth and survival. Agron. J. 75: 831-832. Wiseman, B.R., F.M. Davis, and W.P. Williams. 1983. Fall armyworm: larval density and movement as an indication of non-preference in resistant corn. Prot. Ecol. 5: 135-141. Wiseman, B.R., W.P. Williams, and F.M. Davis. 1981. Fall armyworm: resistance mechanisms in selected corns. J. Econ. Entomol. 74: 622-624. 37 Che m ic a ls Assoc ia t e d w it h M a ize Re sist a nc e t o Corn Ea rw orm a nd Fa ll Arm yw orm M.E. Snook, USDA-ARS-PRU, Russell Research Center B.R. Wiseman, USDA-ARS-IBPMRL, Tifton N.W. Widstrom, USDA-ARS-IBPMRL, Tifton and R.L. Wilson, USDA-ARS, Plant Introduction Station, Ames Abst ra c t The resistance of certain corn silks to the corn earworm, Helicoverpa zea (Boddie) and fall armyworm, Spodoptera frugiperda, is due to the presence in the silks of one major luteolin-C-glycoside called maysin. A recent HPLC screening of over 1,100 corn inbreds, populations, Plant Introductions, and various unassigned lines for maysin content has resulted in the discovery of a number of lines with high levels of maysin. This screening also led to the discovery of several lines with relatively high levels of flavone-C-glycosides, other than maysin. Laboratory bioassays showed a high correlation between antibiosis activity and flavone content and type. Compounds identified include 3'methoxymaysin, the apigenin-analogue of maysin (apimaysin), and 4"-hydroxy-maysin. Several lines were found to contain large levels of isoorientin, 6-C-glucosylluteolin. Bioassays determined that it was almost as active as maysin, while apimaysin and 3'-methoxymaysin were about half as active in reducing corn earworm growth. Chlorogenic acid was also found in silks and was shown to be almost as active as maysin in the bioassay. Incorporation of these new compounds into corn silks of new germplasm should greatly increase corn earworm and fall armyworm resistance. OH I nt roduc t ion O HO The control of the corn earworm, Helicoverpa zea, (Boddie) (CEW) and fall armyworm, Spodoptera frugiperda (FAW) (J.E. Smith), in corn by H H Me O H O OH H result in higher yields and decreased silks were first reported to possess an antibiosis factor to the CEW by Straub and Fairchild (1970). After several O OH O α-Rha increased natural resistance would H O OH H OH OH OH H Galactoluteolin attempts were made to isolate the al. 1970), Waiss et al. (1979) and Elliger hexos-4-ulosyl)-luteolin] (Fig. 1) that was shown to possess high antibiosis activity. Wiseman et al. (1992) showed O HO et al. (1980a) successfully characterized [2"-O-L-rhamnosyl-6-C-(6-deoxy-xylo- O H O OH H OH De-Rhamnosylmaysin CH3 OH OH 5 OH OH O H H O HO OH H O OH 6 4 H H O OH OH factor (Starks et al. 1965; McMillian et a C-flavone glycoside called maysin CH3 OH H H O O HO OH H H HOH2C OH OH O HO agrochemical expenses. Zapalote Chico R R = OH Maysin A R =H Apimaysin B R = OCH3 3’-Methoxymaysin HO OH 1 3 2 O CH=CH-C—O α-Rha 4”-Hydroxy-maysin Figure 1. Structures of corn silk polyphenols. 3-Caffeoylquinic Acid (Chlorogenic Acid) CO2H 38 M.E. SNOOK, B.R. WISEMAN, N.W. WIDSTROM AND R.L. WILSON that maysin was also active against HPLC analysis column chromatography followed by a FAW. Elliger et al.(1980b) tested a Sufficient numbers of plants were second preparative reversed-phase number of flavonoids for growth sampled to give approximately 30 g of separation. The n-butanol residue inhibition against CEW and silk/sample. The silks were weighed, (dissolved in water) was demonstrated that the presence of placed immediately in 8 oz jars chromatographed on a column packed adjacent hydroxyl groups on the B ring (Teflon-lined cap) and the jars were with Waters PrepPAK 500 C18 of the flavone was essential for activity, filled with 100% MeOH (approx. 180 cartridge material (Millipore Corp., a structural feature exhibited by maysin mL). Samples were stored at 0oC until Milford, MA) and eluted with water (Fig. 1). Maysin can thus be considered analysis. Chrysin was added as internal and 50% methanol/water. The latter a natural insecticide that is target standard. After ultrasonication for 20 fraction was evaporated to dryness and specific (CEW) and is present at the min, aliquots of the solution were submitted to silicic acid (SA) right place (silk) and right time (first analyzed by reversed-phase HPLC, as (Mallinckrodt, 100 mesh, washed with instar) to stop insect infestation. previously described (Snook et al. methanol and activated at 155°C for 1 1989), using an H2O/MeOH linear hr) column chromatography. The We have recently developed a high gradient from 10% to 90% MeOH in 35 column was packed in CH2Cl2 and after performance liquid chromatographic min, a flow rate of 1 mL/min, and applying the sample to the top of the (HPLC) method for the determination detection at 340 nm. Each solvent column (as a SA/sample deposited of maysin in corn silks (Snook et al. contained 0.1% H3PO4. Most analyses mixture), eluted with CH2Cl2 followed 1989). Besides allowing us to monitor were performed with an Altex by ethyl acetate or acetone/ethyl maysin levels accurately, the HPLC has Ultrasphere C18, 5 micron (4.6 X 250 acetate mixtures. Most of the flavonoids provided a more complete profile of the mm, Beckman Instruments, Norcross, of interest were found in the ethyl flavonoid contents of the silks than was GA) column. Additional analyses for acetate eluant. After evaporation to previously possible. To date, we have apimaysin and 3'-methoxymaysin were dryness, the SA separated flavonoids surveyed the maysin content of silks made with a Hypersil Phenyl, 5 micron were dissolved in 40% MeOH/H2O from 1,129 corn inbreds, populations, (4.6 x 250 mm, Alltech Associates, and submitted again to reversed-phase plant introductions (PI), and various Deerfield, IL) column. chromatography using the following unassigned collections. In addition to linear solvent program: 40-60% MeOH/ discovering many new sources of corn Isolation of flavone glycosides H2O in 400 min. 8 mL fractions were with high silk maysin levels, several Typical isolation procedures, following collected and column effluents were lines were identified that contained the methodology of Snook et al. (1993, monitored at 340 nm. high levels of related flavonoids. We 1994) for silk flavone glycosides, were report here the identification of these as follows: Identification. Isolated flavonoids were identified by UV, NMR (1H and 13C), new flavonoids and their biological activity towards CEW in a laboratory Extraction. Silk/methanol extracts bioassay. were filtered, concentrated, extracted and FAB/MS spectrometric methods. with CH2Cl2, followed by extraction Bioassay procedures with n-butanol. The n-butanol was Silk extract bioassay. Silk/methanol evaporated to dryness (a small amount extracts were bioassayed by the method Plants of water, added at the end of the of Wiseman et al. (1992) and Snook et Plants were grown between 1989 and evaporation, facilitated the removal of al. (1994). A small aliquot of the extracts 1994 at the Coastal Plain Experiment the last traces of n-butanol). The was analyzed for maysin content and Station, Tifton, GA, under standard residue was dissolved in 40% MeOH/ the remaining solution filtered into a 1 cultural practices of fertilizer and weed H2O and submitted to preparative L roundbottom flask, 5 g of celufil (US control. Silks were covered to prevent reversed-phase column Biochemical, Cleveland, OH) was pollination and were sampled when 3-5 chromatography. added and the solvent was evaporated M a t e ria ls a nd M e t hods to deposit the extract onto the celufil. days old. Isolation. Isolation was mainly by The dried celufil/extract mixture was preparative reversed-phase, silicic acid then added to 100 g of diluted pinto CHEMICALS ASSOCIATED WITH MAIZE RESISTANCE TO CORN EARWORM 39 bean diet (3 mL diet:2 mL water), 10 mL reduction reached a plateau at maysin activity (<0.2% fresh wt.). Fully 50% of were dispensed into plastic diet cups levels >0.4%. From these data, 0.2% has the inbreds were completely devoid of and one neonate CEW added. After 8 been deduced as the minimum level maysin or only possessed trace days the weights of the worms were needed for resistance, with levels amounts of maysin (Table 4). In this recorded. Appropriate MeOH/celufil >0.3% most desirable, because of study, we found a number of corn blanks were used. The experiment was possible yearly variation. inbreds and populations with high silk maysin levels above the 0.2% fresh arranged in a randomized complete block design with 15 replications. These results have prompted us to weight threshold, considered survey other corn inbreds, populations significant for CEW antibiosis. Model compound bioassay and plant introductions (PIs) for Approximately 1/5 of both the inbreds (Microbioassay method). Isolated maysin content. Other high maysin and populations were found to have flavonoids or commercially available lines may have more desirable maysin levels >0.2%. Most of these fall compounds (chlorogenic acid) were agronomic characteristics than in the 0.2-0.5% range. Fully 1/3 of the deposited onto celufil as above. Zapalote Chico for development of high inbred maysin lines contained Concentrations for each compound new, stable corn inbreds with a silk-maysin, at a greater concentration were 240, 120, 60, and 30 mg/2 g celufil. sufficient level of maysin for resistance. than Z. Chico, based on the amount of Each compound/celufil mixture was Flavonoid analyses were performed on maysin per quantity of silk. Only 5% of added to 25 g of diluted pinto bean diet. the methanol extracts of the silks of 497 the populations had maysin levels Detached, disposable plastic pipette inbreds and 295 populations of corn, >0.2%. Relatively few PIs (12%) had bulbs were filled with 2 g of the diet/ selected as representing a broad genetic high maysin levels in their silks. celufil mixture, allowed to solidify, and base. In addition, 337 PIs and However, the silks of PI340856 one neonate CEW was placed on the unassigned germplasm sources of corn averaged 0.743% maysin, over 3 years. diet. The bulbs were placed in diet cups from the North Central Regional PI This PI is a popcorn from the Eldredge and larval weights measured after 8 Station, Ames, Iowa, were also collection. Crosses of PI340856 with a days. There were ten replications for analyzed. The results of these analyses number of inbreds produced high each compound concentration. are given in Tables 1-4 and showed that levels of maysin in the progeny there is a wide range in silk maysin (Wiseman et al. 1992). Prior to our levels, from <0.01% to >0.5% fresh analyses, only Z. Chico was known to weight. contain maysin although several other Re sult s a nd Disc ussion New sources of high maysin germplasm As expected, the majority of lines tested absorptions in their methanol silk Waiss et al. (1979) determined that a (82.6%) contained levels of maysin extracts (Waiss et al. 1979). We have level of maysin of 0.15% (wt/wt of diet) below that considered necessary for now identified almost 200 inbreds, lines possessed maysin type UV in laboratory bioassays reduced CEW larval weights by 50%. We have bioassayed the silk methanol extracts of 100 compared the growth of CEW to the maysin level (Fig. 2). A highly significant negative relationship (r =-0.81) was found between maysin concentration in fresh silks and 8-day larval weights of CEW. This study showed that silk maysin concentration of 0.2% (fresh wt.) reduced larval weights to about 50% and that higher maysin levels (>0.3%) inhibited larval growth by about 80%. Larval growth Percent inhibition of growth 50 different corn lines and crosses and 80 60 40 20 r = -0.81 0 0 0.2 0.4 0.6 0.8 Maysin (% fresh weight) 1 1.2 Figure 2. Percent growth inhibition of CEW versus silk maysin levels. 40 M.E. SNOOK, B.R. WISEMAN, N.W. WIDSTROM AND R.L. WILSON Table 1. Silk maysin levels in inbreds (percent fresh weight). A102 Ab16 Ab416 Ab602 Ab604B Ab608A Ab612A Ab616 AB618 Akd24 Akd26 Akd34 Akd52 B1138T B14(T) 0.307 0.443 0.244 0.463 0.229 0.464 0.778 0.233 0.204 0.305 0.230 0.206 0.239 0.220 0.322 C1-5 C.I.37B C.I.64 C.I.83A C.I.317B E239S E2629P F45 F54 F98 GE37 GE58 GE70 GE74 GE80 0.271 0.368 0.356 0.307 0.314 0.204 0.321 0.270 0.257 0.296 0.897 0.523 0.279 0.302 0.847 A103 Ab20 Ab28E Ab30 Ab44B Ab418 Akd36 Akd38 B64 C103 CK3W C.I.85B C.I.287 C.I.38B ESDJ1 A286Y Ab59 Ab412A Ab424 Akd32 B41 B504 C1-11 C.I.21 C.I.84B D160 0.185 0.194 0.131 0.192 0.173 0.108 0.181 0.181 0.170 0.169 0.155 0.167 0.166 0.172 0.111 0.051 0.050 0.077 0.093 0.085 0.081 0.077 0.095 0.079 0.097 0.099 E263S F2L F44 F47 F101 GE62 GE82 GE86 GE109 GEC8 GEC116B GT107 GT114 GT166 Hy2 (normal) E226S E241S FF8 GA221 GA222 GE76 GE90 GE92 GE291 GE331 GT112 (old) 0.196 0.161 0.140 0.181 0.122 0.130 0.138 0.164 0.158 0.131 0.181 0.154 0.259 0.107 0.166 0.068 0.070 0.097 0.093 0.099 0.060 0.077 0.075 0.076 0.079 0.097 A239 Ab12A Ab18 Ab26 Ab408 Ab454 Ab610 AC454 AC455 AC456 AC543 Akd40 B2 B37 B539 BJ28 BJ30 C.I.82B C.I.88A C.I.90A C.I.91C C.I.121 D113 D187 D287 E199S E2667P SC324 SC333 SC335 SC344 SC359 SC375 SC402 SC403 SC441 SC444 Su4(Red) Syn3B Syn23 Syn49 Syn52 ES1W ES2W ESN F1D F6 FF3 GA152 GA209 GA212 GA215 GA219 GCP9A GE10 GE19 GE25 GE38 GE54 GE68 GE72 GE78 GE88 GE129 GE205 GE275 GE281 GE293 GE297 T11 T101 T111 T125 T127 T129 T133 T135 T137 T141W T143W T202 T206 T210 T216 (Silk M a ysin Le ve ls > 0 .2 % ) 0.299 Mp704 0.392 0.245 Mp707 0.274 0.240 NC24 0.278 0.200 NC45 0.362 0.259 NC64 0.310 0.246 NC264 0.274 0.510 R4 0.223 0.493 SC90 0.233 0.566 SC102 0.301 0.246 SC114 0.380 0.293 SC229 0.205 0.459 SC243 0.263 0.890 SC245A 0.313 0.200 SC249 0.298 0.249 SC249A 0.219 (Silk M a ysin Le ve ls 0 .1 -<0 .2 % ) L601 0.192 SC54 0.138 Lahue0-514W 0.112 SC60 0.169 Lahue9-032A 0.107 SC84E 0.168 M68 0.111 SC144 0.179 May79 0.110 SC229 0.162 Mo15W 0.124 SC229MH 0.186 Mo47 0.112 SC235R3 0.124 Mo102 0.135 SC254 0.138 Mp426 0.192 SC277 0.186 Mp464 0.118 SC343 0.115 NC254 0.177 SC357 0.106 Oh26F 0.154 SC401 0.117 R101 0.179 SC413 0.202 Rec.38-11 0.109 Syn36 0.129 SC46 0.170 T105 0.140 GT3 0.099 Mp337 0.098 H21 0.095 N6 0.093 H30 0.059 NC7 0.058 L708 0.055 NC232 0.063 L765 0.076 Oh7B 0.066 Lahue9-213D 0.069 Oh422 0.059 Lahue9-996 0.089 Pa36 0.085 M14(Mo) 0.068 Pa884P 0.054 Mo12 0.80 R227 0.50 Mo13 0.060 SC16 0.078 Mo426 0.095 SC228A 0.053 (Silk M a ysin Le ve ls < 0 .0 5 % ) GE311 L503 GE317 L578dd CEW GE321 L578 GE325 L605 GE333 L609 GE335 L615 GE337 L617 GE339 L621 GE341 L668 GE440 L678 GE247-205B L690 GEC40 L699 GEC119A L709 GT9 L764 GT11 L814 GT102 M6 GT119 M102 GT150 Miss.Ace996.3 H84 Mo1W JLM1 Mo5 K44 Mo6 K5Y2-3 Mo16W KY21 Mo17NSyn KyWS1 Mo20W L317 Mo45 L317(la) Mo46 L501 Mp1D T222 0-115 T224 0-145 T234 0-159A T331 0-177 Tx44-91 0-190 Va35 0-509 W48FSK 0-530A WPT4 0-538A WT12 0-572A WT34 0-677B WT46 0-708A YT14 0-956A YT23W 0-1032 YT27W 0-1130 YT37 0-1243 GE84 GE295 GEC100 GT106 GT114 GT154 GT169a Gu54-5 H31 H45 L90 L329 Mo10 Mo14W Mp311 SC250A SC265R T226 T238 T315 Tx501 Tzi30 W22 W23 WF-038B WT23 0-835 0-909 0-1480A 0-1566 0.473 0.336 0.523 0.358 0.296 0.215 0.226 1.125 0.260 0.521 0.514 0.328 0.390 0.238 0.206 0-1836 1-1566 2-07A 2-043 2-635 9-96A 9-502B 9-676A 9-928A 49-1201B 57-163 79:295-2 79:301-2 8940C 91201Y 0.325 0.377 0.214 0.278 0.360 0.533 0.290 0.228 0.266 0.284 0.300 0.216 0.267 0.420 0.521 T115 T139 T204D T208 T212 T236 T244 TGY2 Tx501 Tzi1 Tzi8 Tzi15 Tzi24 WF9 WH SC256 SC311A SC346 SC353 SEG SH258 Syn15 T8 T220 T240 T242 0.116 0.130 0.138 0.127 0.185 0.118 0.166 0.168 0.169 0.108 0.114 0.115 0.199 0.174 0.194 0.058 0.050 0.070 0.099 0.091 0.070 0.064 0.057 0.099 0.077 0.050 WT31 WVLFPC1x7 YT14 0-102 0-641 1-1072 2-717 49-1684 9-032A 9-110C 9-201 9-213F 9-238 9-880 0.129 0.181 0.174 0.129 0.151 0.119 0.195 0.198 0.105 0.102 0.197 0.112 0.107 0.125 T246 Tx601 Tx46139 0-514A 0-1325A 0-1432 0-11830 1-40A 2-673 7-104 9-886 0.093 0.099 0.065 0.085 0.091 0.099 0.081 0.061 0.071 0.050 0.053 Mp113 MP303 Mp307 Mp309 Mp313E Mp317 Mp335 MP339 MP410 Mp412 MP420 MP446 Mp448 Mp460 (Miss.66) Mp462 Mp466 MP496 MP708 N20 N101 N104 N106 N132 NC220 NC222 NC224 NC605 0-1290 1-34B 1-222A 1-278 1-759 1-837 1-919 1-977A 2A12 2A44 2-12B 2-535A 3L2 5-666A 8HL6 Oh45B Oh56 SC253R3 SC2-3 Sa4(W) SC15 SC44 SC73 SC91 SC152 SC212 SC213R SC214 SC225 SC233 SC235 SC246C SC257 SC260R SC270RS SC273 SC276R SC278DY SC279-4 SC285 SC301 SC310 8-12A 9-54C 9-218 9-220 9-230B 9-245 9-908A 9-971 9-1028 33-16 48-1166 49-1166 49-1170 49-1550 936-2179 CHEMICALS ASSOCIATED WITH MAIZE RESISTANCE TO CORN EARWORM Table 2. Silk maysin levels in populations (percent fresh weight). AERD (C1) Amar. Salv. X’s Ant.2D-118 ANTB-EP ANTB-EPDS ANTB-EPM ANTB-SIDS ANTB-SIM Azteca X’s Azul B-20# B-70# B-81# B-219# BlueK.M. Catito Limon 0.297 0.243 0.211 0.457 0.564 0.934 0.532 1.031 0.263 0.238 0.241 0.303 0.229 0.200 0.200 0.392 Ant 20xTxp ANTB-SI Arroc. Amao X’s Azul Blandito sonora Bofo Bolita B-116# B-12# B127# B-137# B-18# B-200# B-208# B-220# B-260# B-40# B-46# B-50# B-80# B-94# Camp. Group 365# 408X 2PR 415x 3PR 538x 1T 581x 1T 690x 1T 0.115 0.186 0.170 0.121 0.176 0.161 0.123 0.102 0.122 0.145 0.157 0.190 0.104 0.127 0.105 0.127 0.127 0.105 0.103 0.118 0.120 0.156 0.180 0.161 0.128 0.106 0.174 0.164 Amar. Wh. Flint Antigua 2D-109 Antigua X’s AntiguaGp2(blanco) Argentina Ark CB B-8# B-10# B-16# B-23# B-31# B-60# B-101# B-133# B-160# B166# 0.054 0.054 0.052 0.052 0.074 0.054 0.065 0.056 0.080 0.097 0.088 0.071 0.059 0.072 0.074 0.080 Alapaha Altiplano B-1# B-15# B-25# B-63# B-109# B-120# B-140# B-144# B-155# B-240# B-252# BSP2CI Caribe Salvadoreno OP (Silk Maysin Levels > 0.2%) CB65 0.252 Kyle Late Syn CC-MIO 0.203 Kyle Long Ear Syn Chis Group X’s 0.365 MWSA Coah Group 0.200 Oax. Comp Group Colorado Manfredi GP 0.295 Panama Gpo 84A 1 Cow Corn 0.278 PR 70B 602-604 Cuba III 0.200 RFC-FI(C9) Dial-4 P28 0.263 Salvadoreno Dial-5 P43 0.262 Strawberry Dent (Tex) Ducle Ja 1 0.255 Tabloncillo X’s ETO X’s 0.233 Tbly Syn Florident White 0.217 Trinidad X’s Gourdseed Dent 0.297 Z. Chico (2451)#(P)C3 GT CEW-RSB 0.220 123# Guat. Gp030-1A 0.295 1243x 2PR# Hond. Group 0.344 1299x 1T (Silk Maysin Levels 0.1-<0.2%) Caribbean # 0.183 Granada X’s Chiapas 138 Ear 1... 0.111 Guad X’s Chiapas Gp0 41 Ear... 0.111 Harinoso Sudan Coe G12#in 0.171 Indian Chief Comiteco X’s 0.168 Jamaica X’s Comp (Va) 0.144 MAS (pwnf) Compuesto Am. Caribe 0.102 Mexican ? #1 Costa Rica X’s 0.179 MOM Syn #3 Crillo de Cat. X’s 0.120 Mosby’s Prol Cuba X’s Low Ear Syn 0.125 MWSB Dial 4 Suwan 1 0.133 M-A[MoSQA(S7-H)C12] Dial 5 P22 0.156 Neal’s Pay Dial 8 P63QPM 0.150 N.L. Group Dial 8 P64QPM 0.115 Oloton No. 1 Diallel (Late) 0.165 Pencil Cob (Tex) Dial-4 P24 0.176 Peru X’s Diente de Caballo 0.143 PR69A 42 Duloillo Noroeste 0.108 PR70A 475 FAW-CC [C5] 0.133 Puerto Rico # FLA 767 Syn 0.126 RS 10(C3) Fla Comp 0.155 San Croix X’s FSC 662-25 0.139 SC Syn. 706x 2T 0.113 960x 2PR 713x 2T 0.147 1603# 721x 3PR 0.121 1762x 1T 824x 1T 0.170 1858x 1T 917# 0.179 2110x 1T 933x 4PR 0.157 2300x 1PR (Silk Maysin Levels 0.05-<0.1%) B-178# 0.065 MEX. (VA.) B-181# 0.084 Pepitilla No. 1 B-193# 0.071 PEX (VA) Barbadox X’s 0.077 Robyn Cacahuacintle X’s 0.053 San Vic x’s Canilla 0.074 Seneca Ind. Mix Chiapas Gp0 41... 0.067 Spykepit X’s Chih Group X’s 0.084 Syn TW Clavilla # 0.056 Tuxpan Dom. Rep. 0.050 VMX FLA C62 Syn 0.080 Yellow Hickory King Gobi Yell 0.059 Yellow Jellicorse Golden Beauty 0.098 Yuc. Group GT-MAS: gk 0.087 38x 5PR Homedale 0.052 111A Comp Legg Prol 0.060 500x 1T (Silk Maysin Levels < 0.05%) CB So Long Ear Syn Celaya Mayorbella Chantelpa Chaparro... Mic’s Success Chapalate X’s MPCS-1A Coroico Natal Wh. Horsetooth Douthit Prol OP24# Farmer’s Comp OP60-9# FSH MR San Pedro 1 Gaspe Flint SGP-MIO Guat. Gp013-5 SI1285 Syn A High Guat. Gp021-11 Syn A. High 3rd CYC IK Syn L Jarvis Syn Mdy Jellicorse x South Teko Yellow Knightin 8-Row Yellow Neals Pay 0.299 0.221 0.235 0.565 0.256 0.359 0.564 0.255 0.203 0.343 0.321 0.271 0.350 0.419 0.289 0.337 133# 142# 1439x 4T 1487# 14x 3T# 1520 1889x 2PR 1973x 2T 2280x 1T 37x 7PR 524x 2T 762# 78x 1T 891x 3T# 984x 1PR 998x 1T 0.224 0.342 0.243 0.210 0.306 0.235 0.392 0.215 0.327 0.242 0.240 0.275 0.808 0.243 0.213 0.452 0.131 0.115 0.145 0.101 0.105 0.106 0.200 0.164 0.110 0.137 0.140 0.191 0.100 0.173 0.107 0.118 0.135 0.180 0.114 0.135 0.194 0.120 0.102 0.102 0.194 0.162 0.112 0.118 Shumway’s Goli Snow’s St. Croix St. Crush Syn. A# SWCB Syn X Syn F Syn Kby S. African Syn #1 S.A. Yellow Syn Tamps Gpo #1 Tepecintle X’s Tuxpeno No. 3 Vandeno X’s Ver X’s Z. Chico X’s 10LDD Sel. Rec. 44x 4PR 117# 121# 203# 210# 238# 362# 2302x 5PR 2375x 3PR 2377x 5PR 3146x 1T 3296x 1T 3371x 4PR 0.149 0.118 0.121 0.152 0.124 0.140 0.120 0.135 0.145 0.106 0.105 0.105 0.197 0.137 0.126 0.154 0.161 0.102 0.148 0.103 0.126 0.100 0.106 0.159 0.192 0.125 0.145 0.137 0.063 0.053 0.075 0.051 0.056 0.064 0.053 0.067 0.071 0.054 0.052 0.053 0.065 0.056 0.086 0.054 697x 1T# 958# 1007x 1T 1218x 3PR 1455# 1508# 1515# 1548x 2T 1953 1PRA# 2019x 2PR 2041x 4PR 2745x 2T 3316x 3PR 3457x 3PR 8056# 0.073 0.093 0.076 0.077 0.089 0.082 0.054 0.075 0.081 0.086 0.050 0.096 0.064 0.053 0.097 Y&W-16 Lines Syn Z. Grande X’s 3x 1T# 42x 5PR 126# 135# 234x 1PR 997X 1T 1113x 2PR 1208x 1T 1968x 2PR 2116x 3T 2206x 4T 2370X 1T 41 42 M.E. SNOOK, B.R. WISEMAN, N.W. WIDSTROM AND R.L. WILSON Table 3. Silk maysin levels in plant introductions (PI) (percent fresh weight). PI 172328 PI 194791 PI 208473 PI 213742 PI 217404 PI 217460 PI 219874 0.211 0.972 0.295 0.972 0.374 0.411 0.544 PI 219889 PI 221839 PI 222319 PI 222497 PI 278722 PI 340837 PI 340838 0.520 0.242 0.394 0.251 0.228 0.225 0.297 (Silk Maysin Levels > 0.2%) PI 340840 0.336 PI 340870 0.356 PI 340844 0.276 PI 340872 0.609 PI 340856 0.823 PI 340873 0.531 PI 340859 0.207 PI 438942 0.239 PI 340865 0.581 PI 444142 0.370 PI 340867 0.201 PI 444443 0.324 PI 340869 0.753 PI 445235 0.222 PI 445630 PI 474214 PI 515375 PI 515551 PI 516037 PI 516120 PI 540777 0.258 0.284 0.261 0.320 0.313 0.233 0.229 PI 571793 AMES 10585 AMES 10587 AMES 10589 AMES 10590 AMES 14099 AMES 8177 0.611 0.421 0.914 0.914 0.975 0.238 0.292 PI 165457 PI 180359 PI 184282 PI 193655 PI 194386 PI 197094 PI 219885 PI 220065 PI 224083 PI 227937 PI 245138 PI 257626 PI 257629 PI 331441 0.150 0.105 0.182 0.131 0.109 0.116 0.185 0.142 0.108 0.176 0.155 0.112 0.194 0.118 PI 340863 PI 340866 PI 340871 PI 347252 PI 414182 PI 414184 PI 430456 PI 443442 PI 443762A PI 443859 PI 444010 PI 444042 PI 444217 PI 444331 0.124 0.175 0.132 0.102 0.183 0.198 0.135 0.131 0.110 0.192 0.177 0.126 0.102 0.128 (Silk Maysin Levels 0.1-<0.2%) PI 444364 0.177 PI 503727 0.163 PI 444562 0.100 PI 503728 0.192 PI 444686 0.156 PI 503794 0.180 PI 444785 0.198 PI 503806 0.141 PI 444868 0.118 PI 503832 0.187 PI 444872 0.135 PI 514923 0.103 PI 445002 0.153 PI 515065 0.133 PI 445056 0.118 PI 515076 0.175 PI 445248 0.135 PI 515078 0.101 PI 445377 0.104 PI 515126 0.152 PI 445422 0.152 PI 515213 0.162 PI 445504 0.102 PI 515219 0.185 PI 445514 0.121 PI 515302 0.149 PI 474215 0.102 PI 515326 0.116 PI 515408 PI 515425 PI 515428 PI 515461 PI 515558 PI 516061 PI 516155 PI 532310 PI 532319 PI 532324 PI 540779 PI 571795 PI 571899 AMES 8426 0.138 0.132 0.135 0.157 0.156 0.137 0.197 0.107 0.112 0.126 0.186 0.102 0.104 0.167 AMES 8462 AMES 8473 AMES 8482 AMES 10358 AMES 10501 AMES 10538 AMES 10551 AMES 10579 AMES 10623 AMES 10665 AMES 10672 AMES 15695 0.152 0.119 0.117 0.117 0.141 0.130 0.139 0.191 0.146 0.182 0.106 0.145 PI 162927 PI 181988 PI 183753 PI 218174 PI 221826 PI 257619 PI 331455 PI 331456 PI 331708 PI 340836 PI 340843 PI 347251 PI 357097 PI 357098 PI 357112 PI 357115 0.088 0.080 0.080 0.077 0.071 0.062 0.089 0.078 0.067 0.084 0.073 0.080 0.067 0.055 0.097 0.069 PI 357120 PI 357125 PI 367115 PI 430455 PI 443779 PI 443794 PI 443805 PI 443827 PI 443849 PI 443992 PI 444029A PI 444029B PI 444174 PI 444239 PI 444282 PI 444292 0.050 0.084 0.075 0.071 0.065 0.063 0.058 0.077 0.072 0.074 0.069 0.065 0.098 0.067 0.065 0.075 (Silk Maysin Levels 0.05-<0.1%) PI 444320 0.065 PI 503725 0.064 PI 444607 0.060 PI 503731 0.081 PI 444859 0.082 PI 503764 0.093 PI 444923 0.073 PI 503793 0.073 PI 445299 0.084 PI 503849 0.080 PI 445307 0.064 PI 503863 0.084 PI 445432 0.092 PI 514735 0.073 PI 445585 0.076 PI 514768 0.053 PI 445641 0.075 PI 514848 0.058 PI 474213 0.066 PI 514947 0.096 PI 483495 0.061 PI 514987 0.095 PI 484435 0.063 PI 514995 0.066 PI 484535 0.051 PI 515003 0.064 PI 503667 0.084 PI 515009 0.051 PI 503678 0.086 PI 515064 0.085 PI 503722 0.052 PI 515097 0.077 PI 515106 PI 515107 PI 515112 PI 515114 PI 515115 PI 515134 PI 515205 PI 515355 PI 515464 PI 515997 PI 516039 PI 520631 PI 520691 PI 520693 PI 522309 PI 532312 0.067 0.050 0.063 0.056 0.067 0.067 0.094 0.051 0.065 0.066 0.070 0.060 0.061 0.088 0.076 0.068 PI 532315 PI 571801 PI 572066 AMES 8428 AMES 8477 AMES 8491 AMES 8493 AMES 8497 AMES 8503 AMES 8515 AMES 8521 AMES 10363 AMES 10446 AMES 10465 AMES 10635 AMES 10638 0.097 0.059 0.095 0.061 0.088 0.058 0.098 0.065 0.059 0.089 0.081 0.087 0.091 0.061 0.050 0.066 (Silk Maysin Levels <0.05%) PI 444731 PI 514843 PI 444991 PI 514858 PI 445401 PI 514896 PI 484506 PI 514921 PI 485139 PI 514932 PI 485256 PI 514994 PI 485257 PI 515008 PI 485316 PI 515108 PI 490973 PI 515111 PI 501124 PI 515113 PI 501126 PI 515116 PI 503660 PI 515117 PI 503669 PI 515122 PI 503688 PI 515411 PI 503697 PI 515436 PI 503720 PI 515457 PI 503723 PI 515462 PI 503736 PI 515467 PI 503861 PI 515467 PI 504301 PI 515490 PI 515528 PI 515529 PI 515529 PI 515531 PI 520626 PI 520702 PI 521313 PI 532321 PI 532327 PI 540767 PI 571493 PI 571506 PI 571511 PI 571582 PI 571754 PI 571767 PI 571803 PI 571897 PI 572049 AMES 8225 PI 174416 PI 186221 PI 193653 PI 193658 PI 194390 PI 194741 PI 213796 PI 213807 PI 219871 PI 219886 PI 219888 PI 221825 PI 221831 PI 221844 PI 222307 PI 222309 PI 233007 PI 303850 PI 303851 PI 317330 PI 331440 PI 331442 PI 331443 PI 331452 PI 331709 PI 340853 PI 347253 PI 347254 PI 357094 PI 357101 PI 357121 PI 357122 PI 357129 PI 390837 PI 443931 PI 443997 PI 444000 PI 444125 PI 444139 PI 444223 AMES 8248 AMES 8429 AMES 8488 AMES 8498 AMES 8501 AMES 8573 AMES 8577 AMES 10024 AMES 10042 AMES 10074 AMES 10075 AMES 10076 AMES 10362 AMES 10382 AMES 10436 AMES 10560 AMES 13932 CHEMICALS ASSOCIATED WITH MAIZE RESISTANCE TO CORN EARWORM Table 4. Distribution of maysin in corn germplasm. Maysin levels % fresh weight 43 unassigned line, Ames 1903, contained Inbreds Populations Plant introductions Total % of total > 0.2 > 0.05-<0.2 < 0.05 90 155 252 64 172 59 42 178 117 196 505 428 17.4 44.7 37.9 Total # of lines 497 295 337 1,129 100.0 maysin, apimaysin and 3'methoxymaysin (Fig. 3). Very few lines contained high levels of 3'-methoxymaysin. Inbred Tx501 was the best source of this compound, containing 0.19% (Fig. 4). Other good sources are lines 9-201 (0.297%), and populations, and PIs with high maysin apimaysin (Fig. 3) and 3'- SC144 (0.293%). Populations with high silk levels. These lines form an methoxymaysin (Fig. 4). One line, the 3'-methoxymaysin are 891x 3T# important, new genetic base for inbred NC7, was unique in that it (0.243%), Kyle Late Syn (0.155%), 998x breeding studies to produce produces 0.614% fresh weight 1T# (0.132), and Oloton No.1# (0.109%). agronomically acceptable CEW apimaysin along with only a trace resistant germplasm. amount of maysin. Recently, inbred Of the other maysin analogues Mp416 was found to produce 0.72% identified in corn silks, only two occur Isolation and identification of new corn silk flavones apimaysin and only 0.088% maysin. in amounts to be significant for CEW SC353 is another good source of resistance. One of these compounds is In addition to identifying corn maysin and apimaysin (0.40% and isoorientin (6-C-glucosylpyranosyl- germplasm with high maysin contents, 0.22% respectively). Only one luteolin) (Fig. 1), first found in inbred the survey resulted in the discovery of population line was found to contain T218 (Snook et al. 1994) (Fig. 5). Our several inbreds, populations and PIs high apimaysin (3146x 1T#). An previous report (Snook et al. 1993) on with very high levels of flavone the identification of this compound as glycosides related to maysin. Some of galactoluteolin was based on these lines showed high activity preliminary NMR data. Further studies GT114 Maysin towards CEW and therefore, it was of have shown that the compound is interest to identify the compounds isoorientin (glucosylluteolin). Other responsible. The compounds were lines where isoorientin occurs are T315 isolated by a combination of solvent and Mo6. T218 also contained de- partitioning and column rhamnosylmaysin (Fig. 1), which has lost the ether-bonded rhamnose. and co-workers (Elliger et al. 1980a) previously identified an apigeninanalog of maysin (called apimaysin) and 3'-methoxymaysin (Fig. 1) from Zapalote Chico, in which they occur in minor amounts. Our analysis of Zapalote Chico showed apimaysin and Apimaysin NC7 3’-Methoxymaysin TX501 Apimaysin Pop. 3146x 1T# Maysin 3'-methoxymaysin to be present in only 0.019% and 0.045% fresh weight, while maysin was at the 0.35% level (averaged over 4 years). We have Ames1903 (Argentina) determined that most corn lines with Apimaysin Maysin Maysin Detector response (340 nm) by preparative reversed-phase). Elliger Detector response (340 nm) chromatography (silicic acid followed 3’-Methoxymaysin Population Oloton No. 1# Maysin 3’-Methoxymaysin high maysin levels have minor levels of apimaysin and 3'-methoxymaysin. However, our survey identified several lines that had very high levels of 0 5 10 15 20 25 30 Figure 3. Corn lines with high levels of apimaysin. 0 5 10 15 20 25 30 Figure 4. Corn lines with high levels of 3'-methoxymaysin. 44 M.E. SNOOK, B.R. WISEMAN, N.W. WIDSTROM AND R.L. WILSON However, the level of this compound groups based on the presence of One line, T218, has high levels of was rather variable from year to year. specific flavonoids. The first group is isoorientin while others, such as the The other maysin analogue, which was characterized by lines low levels (>0.05- Eldridge Popcorn Collection PI340853, found in appreciable quantities in only 0.1%), medium levels (>0.1-<0.2%) or contain rhammosylisoorientin. The 3 lines, is 4"-hydroxymaysin (4"-OH- high levels (>0.2%) of maysin. corn line Azul was also found to maysin). Lines containing 4"-OH- Examples of these lines are given in contain this compound, along with maysin in levels sufficient to be Figure 6. They comprise fully 62.1% of maysin. The seventh type of corn considered resistant are A103, ESDJ1 all lines tested (Table 4). It thus appears flavone profile is typified by ESDJ1 and CML131. that maysin is widespread in corn (Fig. 6), where relatively large amounts germplasm, but, as mentioned before, of 4 -hydroxymaysin are found. HPLC characterization of corn silk flavones. only 17% of corn lines have maysin The HPLC analyses of such a large resistant. However, many of the lines Biological activity of maysin and maysin-analogues number of inbreds, populations and between 0.05 and 0.2 have the potential Isolated flavonoids were submitted to PIs revealed that practically all silks for maysin to be increased to >0.2% laboratory bioassays against CEW and could be classified into seven major with a minimum of effort. FAW. As shown in Figure 7, maysin levels high enough to be considered produced larval weights that were only Galactoluteolin T218 De-rhamnosylmaysin The second group of silks is 16% of controls (at 12.6 mM conc.). characterized by low flavonoid FAW was more sensitive to the effects containing lines (<0.05%, which is of maysin, producing larvae weighing equivalent to trace levels) and represent only 6% of controls at only 11.5 mM almost 38% of lines (Fig. 6). The third concentration of maysin. and fourth groups of corn lines are Chlorogenic Acid Detector response (340 nm) T315 Galactoluteolin Maysin those that contain apimaysin and 3'- The isolated corn flavones- maysin, methoxymaysin respectively. Although apimaysin, 3'-methoxymaysin, only 1% of lines contained these isoorientin and 4"-hydroxymaysin, compounds in high levels, they were were tested in the microbioassay found in measurable quantities in 12% method. In this test, maysin reduced of the lines. The fifth and sixth types of the weights of CEW by 92% (15.4 mM) corn flavone profiles are those while isoorientin gave worm weights containing isoorientin flavones (Fig. 6). about 76% of controls at the highest level tested (19.85 mM) (Fig. 8). 4"- Galactoluteolin Mo6 Maysin Maysin Galactoluteolin Chlorogenic Acid Galactoluteolin High Apimaysin Lines High Maysin Lines Detector response (340 nm) PI340853 (Eldridge popcorn collection) Chrysin (ISTD) ISTD High Galactoluteolin Lines Chlorogenic Acid Chlorogenic Acid Low Maysin Lines ISTD Apimaysin Chlorogenic Acid Low Apiaysin Lines Galactoluteolin 3’-Methoxymaysin Maysin Maysin 0 5 10 15 20 Time (min) 25 30 Figure 5. Corn lines with high levels of isoorientin. 0 5 10 15 20 25 30 Time (min) 0 5 10 15 20 25 30 Time (min) 0 5 10 15 20 25 30 Time (min) Figure 6. Characteristic HPLC polyphenolic profiles of major corn silk types. CHEMICALS ASSOCIATED WITH MAIZE RESISTANCE TO CORN EARWORM Hydroxymaysin was found to be just as The bioassay data show that maysin, active as maysin in the test. Apimaysin isoorientin, and chlorogenic acid are and 3'-methoxymaysin both gave about comparable in activity against CEW. 50% inhibition of growth at the Breeding experiments are currently maximum concentrations tested (15.9 underway to incorporate all three and 15.1 mM respectively). Elliger et al. active compounds into one line that, (1980b) reported 3'-methoxymaysin as hopefully, will possess high antibiosis about half as active as maysin based on activity against CEW and FAW and be ED50 concentrations (mM/kg to retard useful for production of naturally growth to 50% of control). Chlorogenic resistant hybrids. acid also has an ortho- Re fe re nc e s dihydroxybenzene structure and is found in small amounts in corn silk. It was found to be active against CEW, resulting in an 80% reduction of growth at 20.5 mM concentration (Fig. 8). % of control Fall armyworm Corn earworm 100 80 60 40 20 0 0 5 10 Maysin concentration (mM) 15 Figure 7. Growth of fall armyworm (FAW) and corn earworm (CEW) versus concentration of maysin. Elliger, C.A., B.G. Chan, A.C. Waiss, Jr., R.E. Lundin and W.F. Haddon. 1980a. C-Glycosylflavones from Zea Mays that inhibit insect development. Phytochemistry 19: 293-297. Elliger, C.A., B.G. Chan, and A.C. Waiss, Jr. 1980b. Flavonoids as larval growth inhibitors. Naturwissenschaften 67: 358-360. McMillian, W.W., B.R. Wiseman, and A.A. Sekul. 1970. Further studies on the response of corn earworm larvae to extracts of corn silks an kernels. Ann. Entomol. Soc. Am. 59: 863-864. Snook, M.E., N.W. Widstrom, and R.C. Gueldner. 1989. Reversed-phase high-performance liquid chromatographic procedure for the determination of maysin in corn silks. J. Chromatogr. 477: 439-447. Snook, M.E., R.C. Gueldner, N.W. Widstrom, B.R. Wiseman, D.S. Himmelsbach, J.S. Harwood, and C.E. Costello. 1993. Levels of maysin analogues in silks of maize germplasm. J. Agric. Food Chem. 41: 1481-1485. % of control Maysin Apimaysin Methoxymaysin Glactoluteolin Chlorogenic acid C-4”-Hydroxymaysin 100 80 60 40 20 0 0 5 10 15 20 Concentration (mM) 25 30 Figure 8. Growth of corn earworm (CEW) versus concentration of corn flavonoids and chlorogenic acid. 45 Snook, M.E., N.W. Widstrom, B.R. Wiseman, R.C. Gueldner, R.L. Wilson, D.S.Himmelsbach, J.S. Harwood, and C.E. Costello. 1994. New flavone Cglycosides from corn (Zea mays L.) for the control of the corn earworm (Helicoverpa zea). In P.A. Hedin (Ed.), Bioregulators for Crop Protection and Pest Control, American Chemical Society, Washington, DC, ACS Symposium Series #557, 122-135. Starks, K.J., W.W. McMillian, A.A. Sekul, and H.C. Cox. 1965. Corn earworm larval feeding responses to corn silk and kernel extracts. Ann. Entomol. Soc. Am. 58: 74-76. Straub, R.W. and M.L. Fairchild. 1970. Laboratory studies of resistance in corn to the corn earworm. J. Econ. Entomol. 63: 1901-1903. Waiss, Jr., A.C., B.G. Chan, C.A. Elliger, B.R. Wiseman, W.W. McMillian, N.W. Widstrom, M.S. Zuber, and A.J. Keaster. 1979. Maysin, a flavone glycoside from corn silks with antibiotic activity toward corn earworm. J. Econ. Entomol. 72: 256-258. Waiss, Jr., A.C.; Chan, B.G.; Elliger, C.A.; Dreyer, D.L.; Binder, R.G.; Gueldner, R.C. 1981. Insect growth inhibitors in crop plants. Bull. Entomol. Soc. Am. 27: 217-221. Wiseman, B.R., M.E. Snook, D.J. Isenhour, J.A. Mihm, and N.W. Widstrom. 1992. Relationship between growth of corn earworm and fall armyworm (Lepidoptera : Noctuidae) and maysin concentration in corn silks. J. Econ. Entomol. 85: 2473-2477. 46 M e c ha nism s of M a ize Re sist a nc e t o Corn Ea rw orm a nd Fa ll Arm yw orm B.R. Wiseman, Research Entomologist, USDA-ARS-IBPMRL, Tifton Abst ra c t Tolerance, non-preference, and antibiosis, the mechanisms of resistance in maize, Zea mays L., to Helicoverpa zea (CEW) (Boddie) and Spodoptera frugiperda (FAW) (J. E. Smith) have been described for some maize cultivars. The behavior of larvae and, to a lesser extent, of adults of these pest insects as it relates to non-preference has been delineated for a few cultivars. CEW moths preferred to oviposit on the adaxial over abaxial surface of young maize leaves of both resistant and susceptible genotypes. Foliage of Antigua 2D-118 is less pubescent and less preferred than Cacahuacintle X’s. FAW larval behavior on both leaf surfaces with and without cuticular lipids was monitored by video camera. Larvae showed more non-acceptance behavior on the untreated foliage than that with cuticular lipids removed. The effects on the insect’s life history of maize cultivars with antibiotic resistance have been shown and include reduced size of larvae, prolonged length of both the larval and pupal cycle, reduced pupal weights, reduced fecundity, increased number of instars, and decreased head capsule size. Tolerance to FAW was shown as a resistance mechanism in some commercial hybrids. The 12-leaf stage tolerated damage by the FAW larvae better than the 8-leaf stage. Yield reduction was 32.4% at the 8-leaf stage compared to 15.4% at the 12-leaf stage. Two predictive models of maize resistant to CEW and FAW illustrate the value and impact of resistance on developing populations of pest insects. I nt roduc t ion cultivars) and absolute (plants or A case in point is the resistance of cultivars not preferred even when certain maize genotypes to the CEW. Maize, Zea mays L., resistance to corn plants or cultivars are grown or tested Painter (1951) described an “unclassified earworm (CEW), Helicoverpa alone) (Owens 1975). Antibiosis resistance mechanism” in which the (=Heliothis) zea (Boddie) and fall denotes adverse biological effects (e.g., importance of long husks of maize was armyworm (FAW) Spodoptera frugiperda larval mortality, extended development discussed in relation to its resistance to (J.E. Smith) may be defined as “the time, etc.) on the insect pest as it uses CEW. This concept of the “unclassified relative amount of heritable qualities the resistant plant for food. On the resistance mechanism” lingered for possessed by the plant which influence other hand, tolerance describes a plant several years. In fact, most of the early the ultimate degree of damage done by or cultivar that is able to yield well works on maize resistance to CEW the insect” (Painter 1951). Painter despite infestations that seriously involved mechanical factors: long, tight further classified resistance into three damage and reduce yield of susceptible husks, and such factors as silk-balling or mechanisms: non-preference, plants. Generally one or more of these husk protection (Luckman, et al. 1964; antibiosis, and tolerance (Painter 1951, three mechanisms may occur in the Wiseman, et al. 1970; McMillian and 1968). Non-preference results when a same resistant cultivar. Researchers Wiseman 1972). Most workers omitted plant does not possess the normal often fail to recognize this possibility studies on the mechanisms of resistance, attractive substances or qualities for because of a lack of ingenuity in instead researching the broad-based oviposition, establishment and/or designing experiments to separate the chemical factors (Walter 1957; Knapp et feeding, or possesses repellent or mechanisms of resistance or to al. 1965, 1967; McMillian and Wiseman deterring substances. There are two understand the importance of the 1972) or correlating CEW resistance in types of non-preference: relative (non- biological phenomena involved with maize with plant physical factors preferred plants or cultivars in the each resistance mechanism. (Widstrom and McMillian 1967; presence of susceptible plants or Widstrom et al. 1970). MECHANISMS OF MAIZE RESISTANCE TO CORN EARWORM AND FALL ARMYWORM Anderson (1944) stated “it is a T ole ra nc e 47 that maintained a high moisture content over the period of development fundamental principle, too often ignored, that before a biological Tolerance resistance is associated with of CEW larvae. In addition, these phenomenon is to be investigated on the plant’s ability to recover and yield tolerant hybrids or cultivars were the mathematical level it must be satisfactorily, despite insect damage. found to have little or no maysin thoroughly analyzed on the biological Tolerance also can mean that the content (Waiss et al. 1979), later found level.” This principle may be applied to resistant plant simply tolerates the pest to be a major factor for the basis of premature studies on the biochemical insect in the presence of a population of antibiosis resistance (Wiseman et al. basis of resistance factors. Knapp et al. insects equal to that which damages a 1992a,b). (1967) and Straub and Fairchild (1970) susceptible plant or cultivar. In 1972, were among the first to study the Wiseman et al. reported that when The establishment of FAW tolerance in mechanisms of resistance in maize to plants were planted early in the maize had not been achieved until the CEW, but their studies delineated only growing season, two resistant maize last 20 years, though many observers antibiosis. The early progress in hybrids, Dixie 18 and 471-U6 X 81-1, have suggested that maize cultivars do identifying FAW resistant maize was supported numbers of CEW larvae on tolerate large numbers of larvae and much slower because of inadequate the ear that were similar to those on damage (Brett and Bastida 1963; rearing and/or infestation procedures. ears of susceptible hybrids but suffered Wiseman and Davis 1979; Ortega et al. However, Wiseman et al. (1966, 1967) much less damage (Table 1). At a later 1980; Mihm 1989). However, Wiseman found resistance in an Antigua race of planting date the number of CEW and Isenhour (1993) did show that maize. With the advent of artificial larvae in the ears of the resistant tolerance existed in some commercial rearing of FAW (Burton 1967; Burton hybrids was greater, yet the damage to hybrids. They showed that the 12-leaf and Perkins 1989) and infestation the ears was significantly less than that stage tolerated damage by the FAW procedures (Mihm 1983; Wiseman et al. on the susceptible hybrids. Thus, the larvae better than the 8-leaf stage. Yield 1980), sources of FAW resistance in resistance of Dixie 18 and 471-U6 X 81-1 reduction was 32.4% at the 8-leaf stage maize have been found, developed and was identified as tolerance. Later compared to 15.4% at the 12-leaf stage. released (Wiseman and Davis 1990). studies, Wiseman et al. (1976, 1981a), where CEW larvae were fed fresh silks N on-pre fe re nc e The basic triad of the resistance of Dixie 18 and 471-U6 X 81-1, mechanisms proposed by Painter (1951) supported these findings. Larvae and Few studies have been conducted to is usually elucidated by specifically percent mortality of larvae that were determine the mechanism of non- designed experiments to demonstrate fed fresh silks of Dixie 18, 471-U6 X 81- preference in maize to either CEW or the independence of the three 1, or silks of susceptible cultivars did FAW. Ovipositional non-preference components; however, resistant not differ for 6- and 10-day weights or against Antigua 2D-118 by CEW was cultivars often possess combinations of % mortality. Ears of tolerant maize reported by Widstrom et al. (1979). these mechanisms, especially non- hybrids were described by Wiseman et CEW moths preferred to oviposit on preference and antibiosis (Wiseman al. (1977) as having tight husks, long the adaxial as compared to abaxial 1990). With this combination of silk channels, and large amounts of silk surface of young maize leaves of both mechanisms, a cultivar that is nonpreferred does not require the same level of antibiotic resistance. Thus, Table 1. Tolerance as a mechanism of resistance in maize to the corn earworm (CEW). different cultivars may possess the CEW injury in indicated plantingsa Larvae per ear in indicated plantinga Hybrid Early Late Early Late mechanisms of resistance of maize to Dixie 18 (R) Asgrow 200 B (S) Ioana (S) 471-U6 X 81-1 (R) 3.6b 6.1d 5.7c 2.9a 2.5a 4.6c 7.3d 3.6b 0.8b 0.8b 0.7a 0.7a 1.7c 1.3b 1.0a 1.4b FAW and CEW. a same levels of resistance with different mechanisms of resistance and/or levels of the resistance components. The remainder of this paper will be devoted to recent elucidations of the Means in columns followed by the same letter are not significantly different (P < 0.01). CEW injury means are the depth of penetration into the ear in cm (Wiseman et al. 1972). 48 B.R. WISEMAN resistant and susceptible genotypes. stage. The data from this test confirmed cuticular lipids on feeding by FAW Antigua 2D-118, which is less that Antigua 2D-118 had a higher level larvae. Larval behavior on the adaxial pubescent than Cacahuacintle X’s, was of non-preference than MpSWCB-4 and abaxial leaf surfaces with and less preferred than Cacahuacintle X’s. (Table 3). Yang et al. (1993a) reported without cuticular lipids was monitored Subsequent studies have shown similar results, as there were fewer by video camera. Larvae showed more progress in selecting within Antigua larvae on resistant genotypes than on non-acceptance behavior on the 2D-118 for a more pubescent type and susceptible ones, indicating the untreated foliage containing cuticular within Cacahuacintle X’s for a less cuticular lipids are involved in lipids than on foliage from which the pubescent type to demonstrate the resistance. cuticular lipids were removed. Larvae traveled greater distances and crawled ovipositional behavioral preferences of Wiseman and Isenhour (1988) faster when they were on upper leaves speculated from studies where they fed rather than lower leaves and when they Non-preference by CEW larvae for silks green or yellow whorl tissue to FAW were on the abaxial leaf surface than on of resistant maize was reported by larvae that the presumed antibiotic the adaxial surface. Yang et al. (1993c) Wiseman et al. (1983a). They found in resistance of ‘Antigua 2D-118’ and found that larvae weighed more and laboratory choice tests that significantly ‘MpSWCB-4’ could actually be developed faster when they were more larvae had fed on silks of behavioral resistance (i.e., non- reared on diet containing maize foliage ‘Stowell’s Evergreen’ sweet maize after preference), due to the fact that larvae from which the cuticular lipids had 4 days than on silks of ‘Zapalote Chico’. fed yellow whorl tissue were smaller been removed than when they were fed But, when larvae were placed on silks than those fed green whorl tissue, untreated foliage. of these two maize cultivars in both regardless of whether plants were choice and no-choice situations in the resistant or susceptible. Yang et al. Resistance of maize silks to FAW larvae laboratory, many larvae had crawled (1991, 1993b,c) performed a chemical was first reported by Wiseman and off the Zapalote Chico silks after 4 days, and ultrastructural analysis of maize Widstrom (1986). This resistance and significantly more larvae were leaves and studied the effect of manifested itself as non-preference and the female CEW (Wiseman et al. 1988). found on Stowell’s Evergreen silks (Table 2). Thus, it was concluded that the resistant feeding responses of CEW Table 2. Mean percent corn earworm (CEW) larvae on silks of Zapalote Chico (ZC) and Stowell’s Evergreen (SEG) after 4 days of laboratory infestation. larvae observed in the field (Wiseman % larvae et al. 1978) could be due in part to nonLarge dish preference. Larvae which fed in the silk channel of Zapalote Chico for 3 to 6 Initial larval placement days girdled the silks to the point Zapalote Chico Stowell’s Evergreen Center where the exposed silk mass was detached from the silk channel. Exposed larvae then faced the behavioral decision of leaving the silk ZC 19.4 11.1 * * Small dish SEG ZC 80.6 88.9 7.5 7.5 5.6 SEG * * * 92.5 92.5 94.4 Mean percent comparing ZC vs SEG with an asterisk between are significantly different (P < 0.01). Large dish = 25.5 cm dia. and small dish = 8 cm dia. (Wiseman et al. 1983a). channel (non-preference) or attempting to penetrate deeper into the silks, which would retard their development (antibiosis). Non-preference by larvae of FAW has been studied using both leaves and silks of the maize plant. Wiseman et al. (1983b) found that significantly more FAW larvae crawled off resistant plants than off susceptible plants in the whorl- Table 3. Mean number of fall armyworm (FAW) larvae moving from resistant or susceptible corn genotypes to surrounding trap plants (common hybrid) at varying intervals after infestation, 1981. Genotype a Antigua 2D-118 MpSWCB-4 Cacahuacintle X’s a b Larval numbers on surrounding trap plants at b days after infestation 3 5 7 11 0.6a 0.1b 0.2b 3.6a 2.1b 2.1b 5.9a 3.7b 3.3b 8.0a 5.0b 4.5b Antigua 2D-118 and MpSWCB-4 = resistant and Cacahuacintle X’s = susceptible. Means within a column followed by the same letter are not significantly different (P < 0.05) (Wiseman et al. 1983b). MECHANISMS OF MAIZE RESISTANCE TO CORN EARWORM AND FALL ARMYWORM 49 antibiosis. Larvae moved off or away stage plants prompts the larvae to et al. (1991) also associated the from Zapalote Chico silks regardless of move about on the resistant plant in production of additional instars and whether larvae had a choice or not. search of a more appropriate feeding reduced head capsules with antibiotic Overall, a 6 to 1 ratio of larvae site. Non-preference such as reported factors in the silks. Waiss et al. (1979) preferred silks of the susceptible entry here could be a valuable tool by itself suggested that a portion of the (83%) to silks of the resistant entry or when used with certain other antibiotic factor in Zapalote Chico was (15%). All of the silks of the susceptible components of pest management that “maysin”, a luteolin-C-glycoside. cultivar were consumed, while only could take advantage of these Henson et al. (1984) found no about 10% of the silks of the resistant characteristics of larval behavior. relationship between maysin concentration in maize silks and ear cultivar were consumed when larvae Ant ibiosis were presented with a choice (Table 4). penetration by CEW larvae. Also, Wiseman et al. (1985) found no Similar differences were found when the larvae were placed initially on the Walter (1957) was one of the first to significant relationship between resistant or susceptible silks (Table 5). demonstrate that the resistance in silks growth of CEW larvae that were fed on However, more silks of the resistant of some maize lines was due to silks and/or silk diets and maysin cultivar (20%) were fed upon when the antibiosis. Straub and Fairchild (1970) concentration of silks. However, larvae were initially placed on the and Wiseman et al. (1976 and 1981a) Wiseman et al. (1992a) later found a susceptible silks compared with those showed that silks of Zapalote Chico significant (P < 0.01) relationship in initially placed on the resistant silks possessed a CEW larval growth four separate tests between reduced (10%). Yet, when the silks of the two inhibitor. Wiseman and Isenhour (1990) growth of CEW and increased maysin cultivars were mixed, about 90% of the found additional adverse biological concentration, whether maysin was fed silks of the susceptible cultivar were characteristics associated with the directly in meridic diets or fed as silk- fed upon as compared with no feeding antibiotic responses when CEW were diets. A biological relationship must be on the resistant silks. fed on resistant silk-diets (such as established between the suspected prolonged developmental time, chemical basis of resistance in the silks The non-preference mechanism of reduced weight of pupae, and reduced and the insect. resistance against both CEW and FAW fecundity reduced by as much as 65% associated with maize silks or whorl- over 4 generations) (Table 6). Wiseman Recently two additional cultivars (GT114 and PI340856; Wilson et al. Table 4. Preference of neonate fall armyworm (FAW) larvae for either silks of Stowell’s Evergreen or Zapalote Chico. Silks Stowell’s Evergreen Zapalote Chico Mean % larvae on % silks consumed % feeding on mixed silks 80a 20b 100a 10b 90a 0b 1991) have been identified with high levels of antibiosis as well as high levels of maysin (Wiseman and Widstrom 1992; Wilson and Wiseman 1988; Wiseman et al. 1992a,b). PI340856 has some of the highest levels of maysin found to date, and is highly Means within a column followed by the same letter are not significantly different (P < 0.05; least significant difference test) (SAS Institute 1982). Mixed silks are a mixture of Stowell’s Evergreen and Z. Chico silks. (Wiseman and Widstrom 1986) resistant, while the resistance of PI340853 is high, but the silks do not contain maysin (Wiseman et al. 1992b). The resistance of PI340856 is governed Table 5. Mean percent of fall armyworm (FAW) found on silks of Zapalote Chico (ZC) or Stowell’s Evergreen (SEG) four days after infestation. Initial placement of larvae Mean % of larvae on ZC SEG % silks consumed ZC SEG Zapalote Chico Stowells’ Evergreen 17.5 10 10 20 * * 80 90 * * 70 90 Percents separated by * are significantly different (P < 0.05; least significant difference test (SAS Institute 1982). About 2.5% of the larvae placed initially on ZC were not accounted for after 4 days. (Wiseman and Widstrom 1986). by a single dominant gene (Wiseman and Bondari 1995), whereas the inheritance of PI340853 silk resistance is not known to date. Antibiosis to FAW was discovered in whorl-stage maize by Wiseman et al. (1981b). They found that FAW larvae 50 B.R. WISEMAN that fed on resistant genotypes were less than consumption on more were fed for 10 days on a complete significantly smaller than those fed on susceptible plants (Table 8). Resistance meridic diet containing fresh silks of susceptible maize genotypes (Table 7), in maize silks has been demonstrated at Zapalote Chico (200 mg/ml diet), their and that consumption of leaves of a much higher level (Wiseman and final weight averaged 4 mg compared to resistant plants was also significantly Widstrom 1986). When FAW larvae 361 mg for larvae fed on the control meridic diet without corn silks (Table 9). Table 6. Mean growth, development time, and egg production of corn earworm (CEW) after having fed on susceptible, low resistance, intermediate-resistance, and high resistance diets over four generations. Treatmenta Generation 1 Lab C Susceptible Bean diet Low-resistant Intermediateresistant High-resistant MSDc Generation 2 Lab C Susceptible Bean diet Low-resistant Intermediateresistant High-resistant MSDc Generation 3 Lab C Susceptible Bean diet Low-resistant Intermediateresistant High-resistant MSDc Generation 4 Lab C Susceptible Bean diet Low-resistant Intermediateresistant High-resistant MSDc 9-day Weight of Adult Relative larval weight Pupation pupae eclosion egg (mg) (d) (mg) (d) production — 399b 494a 148c 26d — 14.3a 14.3a 16.9b 22.5c — 542a 562a 475b 471b — 24.8a 24.9a 27.6b 32.5c — 21a 21a 20a 22a Table 7. Mean weight of fall armyworm (FAW) larvae after 8 days of a no-choice test involving leaf sections of resistant and susceptible maize entries, 1980. Field ratinga Genotype Cacahuacintle X’s Ab24E X Mp305 Antigua 2D-118 Mp4008 MpSWCB-4 a b Mean larval wt. (mg)b S S R R R 333.5a 263.3b 229.6bc 193.3c 151.8d S, Susceptible; R, resistant. Means followed by the same letter are not significantly different at P < 0.05. Means of 50 replications. (Wiseman et al. 1981b). 6d 30 30.0d 0.63 302c 32 39.3d 0.49 10b 3 821a 691b 692b 310c 12.2a 13.0b 12.8b 14.7c 530ab 554a 537ab 503b 22.4a 23.2a 23.3a 25.5b 27a 24b 27a 25ab 20d 11d 42 25.2d 25.6d 0.53 431c 420c 39 35.9c 38.3d 0.96 18c 14d 3 840a 715b 708b 325c 11.3a 12.3b 11.4a 16.3c 565a 526bc 537b 512c 21.8a 22.9a 22.2a 27.6b 26a 23a 29a 23a 74d 7e 39 21.2d 35.1e 0.64 376d 263e 20 31.0c 46.5 0.64 13b 2c 5 781a 673b 609c 400d 12.8a 13.5ab 13.8bc 14.6c 546a 556a 555a 517b 23.4a 24.1b 24.3b 25.9c 25a 24a 19bc 23ab Table 9. Weight of fall armyworm (FAW) larvae after feeding 10 days on silks of maize mixed in meridic diets, 1984. 16c 6d 4 Amount of silks (mg) per ml diet Stowell’s Evergreen Zapalote Chico 0 25 50 100 200 Slope b 357 394 337 23 246 -16 361 271 150 41 4 -43 Table 8. Total leaf consumption, percentage consumption, and mean weight of fall armyworm (FAW) larvae after 8 days of a no-choice feeding test involving leaves of a resistant and a susceptible maize, 1980. Genotype Cacahuacintle X’s MpSWCB-4 Total Mean Field consumption % larval wt. 2 rating (cm ) consumption (mg) S* R 72.4a 21.5b 37.1a 10.9b 294.2a 77.5b Total consumption, percent consumption, and mean larval weight followed by the same letter are not significantly different at P < 0.05. Means of 12 replications. * S, susceptible; R, resistant. (Wiseman et al. 1981b). Mean wt. of larvae (mg) 165e 11f 49 17.3d 37.8e 0.77 502b 249c 21 28.5d 49.1e 0.68 a Lab C, laboratory control larvae from the laboratory culture on bean diet; susceptible, diet of Stowell’s Evergreen sweet corn, 25 mg dry silk/ml of dilute bean diet; bean diet, larvae on pinto bean diet; low resistance, 25 mg dry Zapalote Chico silk/ml of dilute pinto bean diet; intermediate-resistance, 50 mg dry Zapalote Chico silk/ml of dilute pinto bean diet; high resistance, 75 mg dry Zapalote Chico silk/ml of dilute pinto bean diet. Relative egg production was based on the system used by Perkins et al. 1973. (Wiseman and Isenhour 1990). Means within a column for each generation not followed by the same letter are significantly different (P < 0.05, k-ratio = 100; Waller and Duncan 1969). c MSD = Minimum significant difference. a b * * * * * a Cultivar means separated by * are significantly different (P < 0.05; least significant differences) (SAS Institute 1982). Expressed per 25 mg of silk per ml diet. (Wiseman and Widstrom 1986). MECHANISMS OF MAIZE RESISTANCE TO CORN EARWORM AND FALL ARMYWORM Pla nt Re sist a nc e a nd I nt e gra t e d Pe st M a na ge m e nt (I PM ) 51 Communication, J. Coppedge). Large identified in recent years (Wiseman and CEW populations develop on Davis 1990; Wiseman and Isenhour susceptible maize hybrids from May 1990; Wilson et al. 1991; and Wiseman through mid-September in the and Widstrom 1992). Wiseman and Plant resistance to insects in each crop- southern and southeastern United Isenhour (1990) demonstrated the insect relationship should be the hub of States, and through the northern areas effects of antibiosis on several integrated approaches to pest of the USA. Populations of female biological parameters of larvae of CEW. management (Fig. 1). Though the moths emerging from 200,000 ha of They showed that a low level of effects of the resistant cultivar are maize in the southern USA have been resistance reduced CEW larval growth specific, cumulative and persistent, it estimated at 148 to 716 million (74 to and extended its life cycle by ca. 3 days. can be used safely and compatibly in 358 million females/200,000 ha, An intermediate level of antibiosis combination with any one or more of assuming a 50:50 sex ratio) which could reduced the larval growth, extended its the conventional integrated produce economic infestations on 3.0 to life cycle by ca. 8 days and reduced the components that radiate outward from 14.3 million ha of other crops (Raulston fecundity of females by ca. 30 percent. the central hub of insect pest et al. 1992). A high level of antibiosis in the silks caused a drastic reduction in larval management. The effects of the resistant cultivar have been For many years field maize was growth, extended the life cycle by ca. 20 demonstrated over and over again in protected from damage by CEW larvae days and reduced fecundity by ca. 65 crops such wheat, alfalfa, grapes, by growing tolerant hybrids (Wiseman percent. The very high level of sorghum, maize and grasses. Thus, it is et al. 1984). However, the commercial resistance found in the silks of the our responsibility to keep on maize industry and growers changed popcorn, PI340856, resulted in total promoting and demonstrating the from full season hybrids, which gave larval mortality (Wilson et al. 1991 and benefits of plant resistance to insects so the husk protection (mechanical Wiseman et al. 1992b). that the next generation of scientists resistance) or tolerance to CEW larvae, can appreciate its true value and also to the open, short husk hybrids. This Resistance to CEW larvae in silks of have materials and ideas to build on. change by growers, industry and users commercial maize hybrids could is probably the main reason we have reduce CEW populations, keeping them Losses by CEW larvae in field maize seen increased losses in field maize in from developing into huge populations have been estimated at 2.5% annually recent years. which cause tremendous economic losses in not only maize but in cotton, for the USA. Losses in recent years in Georgia have ranged from 1.5 to 16.7%. High levels of antibiosis in the silks of soybeans, peanut, sorghum, and Losses for popcorn and sweet maize for some maize cultivars have been vegetables (Table 10). human consumption can be as high as 50%, but these high losses rarely occur because the crops are protected by as Table 10. Cumulative effects of various levels of resistance in maize silks on numbers of corn earwom (CEW) larvae and generations per year. many as 29 applications of insecticides for the control of insects (Personal Inherited Sterility Chemical Cultural Control Host Plant Resistance Pathogens Predators Parasites Figure 1. Integrated components of pest management that could be used in a sustainable system for agricultural production. Susceptible Low resistance Intermediate resistance High resistance Very high resistance Number of larvae Number of generations 1.6 x 106 3.1 x 105 1.8 x 104 1.7 x 102 0 6 5 5 4 0 Assuming an initial corn earworm population of 100 moths, a 50:50 sex ratio, beginning May 1 with an egg production of 1000 eggs per female moth and each generation egg to adult mortality of 99 percent due to natural causes, 27 days/generation on a susceptible host 30, 35, and 47 days on a host with a low, intermediate, and high level of resistance, and no development on the low, intermediate, high and very high silk resistant maize hybrids. Also based on the findings of Wiseman and Isenhour (1990) of no additional mortality of larvae on the low resistant hybrid, 25 percent additional mortality of larvae on the intermediate, 50 percent additional mortality of larvae on the high resistant silks (Wiseman et al. 1978) and total larval mortality on the silks of the very high resistant hybrid silks (Wiseman et al. 1992b). Wiseman and Isenhour (1990) also showed that the intermediate and high resistant silks could cause a reduction in female fecundity of 30 and 65 percent, respectively. An assumption is made that silking maize was available from May 1 in the south to September 20 in the more northern areas of the U.S. 52 B.R. WISEMAN Over 1.6 million CEW larvae would surviving CEW larvae could be reduced compared to none produced on the survive after 6 generations, as a result to negligible levels. Thus, this safe, highly resistant grasses. Multiple of the constant build-up on a nonpolluting, persistent, specific, and resistance in cultivars of maize, susceptible maize hybrid. With a low cumulative control method is a feasible sorghum, and millet attacked in level of resistance (i.e., one that extends alternative to chemical pesticides and sequence would result in 6.9 times the life cycle by 3 days per generation), can be implemented by farmers. fewer larvae, 3.4 times fewer moths or 6.7 times fewer eggs on resistant 312 thousand larvae would survive after 5 generations — 5 times less than Plant resistance to FAW may be viewed cultivars than on susceptible cultivars the number produced on the as another model system where the by the end of the third generation. susceptible hybrid. An intermediate resistant cultivar is the hub (Fig. 1) for Integration of plant resistance with level of silk resistance that extends each an integrated approach to pest other control tactics would produce an generation by 8 days, increases management (Wiseman 1996). Plant even greater impact on FAW mortality by 25 percent, and reduces resistance alone would have a populations. fecundity 30 percent could reduce tremendous impact on FAW surviving to 17,800 the number of CEW populations (Table 11). There would be Scientists and the general public are larvae after 5 generations, 17.6 times 196.8 thousand times more larvae becoming increasingly aware of the fewer than those surviving on the low- produced in the 6 generations on the need to reduce our reliance on fossil resistance hybrid and 87.8 times fewer susceptible maize than by the 4 fuels and lessen the contamination of than the larvae that result from feeding generations completing their life cycle air, rivers, and lakes associated with on the susceptible hybrid. A high level on the resistant maize. On susceptible applying more and more pesticides to of resistance in the silks of maize sorghum, there would be 13.3 million produce crops. It is now clear that hybrids could reduce the number of times more larvae produced by the end society’s needs can be met using generations per season to 4 and the of the 6 generations compared to the 3 techniques based on ecological number of surviving larvae to 168 — generations completing their life cycle principles, techniques that lie within 106, 1,860, and 9,301 times fewer larvae on resistant sorghum. However, on our grasp and which minimize than those produced on the susceptible millet there would only be detrimental effects on the environment. intermediate, low-resistance or 544 times more larvae produced by the Likewise, current trends in entomology, susceptible hybrids, respectively. The end of the 4 generations as compared to both at the state and federal level, are very high silk resistant hybrid would the number produced at the end of the 3 emphasizing area-wide management of not permit any increase in CEW generations on resistant millet. But there pests, reduced use of pesticides, populations. By integrating high levels would be 24 million times more larvae improved food safety, and more of silk resistance with other forms of produced by the end of the third sustainable systems of agriculture. The pest management, populations of generation on susceptible grass safety and compatibility of resistant cultivars helps reduce pesticide use, Table 11. Cumulative effects of resistance in maize, sorghum, millet, and grass on number of fall armyworm (FAW) and generations per year on each host. Crop Susceptible cultivar Resistant cultivar Maize Sorghum Millet Grass b Sequence 6.1 x 1013 6.1 x 1012 9.8 x 108 2.4 x 107 2.0 x 107 3.1 x 108 4.6 x 105 1.8 x 106 0 2.9 x 106 a b increases food safety, and boosts profits by reducing production costs. Resistant Number of FAW larvae and (generations)a (6) (6) (4) (5) (3) poses no hazard to production workers, (4) (3) (3) (1) (3) Assumptions: 100 moths in the initial infestation; 50:50 sex ratio; 1000 eggs/female (Lynch et al. 1989); 95% natural mortality; additional mortality on resistant maize (50%; Wiseman et al. 1981c), sorghum (66%; Diawara et al. 1991), millet (50% reduction in oviposition, 10% larval mortality; Leuck 1970), and grass (100%; Wiseman et al. 1982; Lynch et al. 1983; Chang et al. 1985); 27 days/generation on a susceptible host and 35 days on a resistant host; and an unlimited food supply. Depicts population increases on a sequence of hosts, i.e., maize, sorghum, and millet. Grass is not included because of the present confusion on host strains. (from Wiseman 1996). cultivars should be the focal point for the area-wide management of insect pests. Ac k now le dgm e nt Dr. Robert E. Lynch is acknowledged for his critical review of this manuscript and for his creative input to the information in Table 10. MECHANISMS OF MAIZE RESISTANCE TO CORN EARWORM AND FALL ARMYWORM Re fe re nc e s Anderson, E. 1944. Homologies of the ear and tassel in Zea mays. Mo. Bot. Gard. Ann. 31: 325-340. Brett, C.H., and R. Bastida. 1963. Resistance of sweet corn varieties to the fall armyworm, Lyphygma frugiperda. J. Econ. Entomol. 56: 162-167. Burton, R.L. 1967. Mass rearing the fall armyworm in the laboratory. United States Department of Agriculture. ARS. Report. ARS-33-117. 12. Burton, R.L., and W.D. Perkins. 1989. Rearing the corn earworm and fall armyworm for maize resistance studies. pp. 37-45. In: Toward insect resistant maize for the third world: Proc. of an international symposium on methodologies for developing host plant resistance to maize insects. Mexico, D.F., CIMMYT. Carpenter, J.E., J.R. Young, H.L. Cromroy, and A.N. Sparks. 1987. Corn earworm (Lepidoptera: Noctuidae): comparison of field survival of larvae from normal and irradiated parents. J. Econ. Entomol. 80: 83-886. Chang, N.T., B.R. Wiseman, R.E. Lynch, and D.H. Habeck. 1985. Fall armyworm: expression of antibiosis in selected grasses. J. Entomol. Sci. 20: 179188. Diawara, M.M., B.R. Wiseman, and D.J. Isenhour. 1991. Mechanism of whorl feeding resistance to fall armyworm among converted sorghum accessions. Entomol. Exp. Appl. 60: 225-231. Henson, A.R., M.S. Zuber, L.L. Darrah, D. Barry, L.B. Robin, and A.C. Waiss. 1984. Evaluation of an antibiotic factor in maize silks as a means of corn earworm (Lepidoptera : Noctuidae) suppression. J. Econ. Entomol. 77: 487-490. Knapp, J.L., P.A. Hedin and W.A. Douglas. 1965. Amino acids and reducing sugars in silks of corn resistant or susceptible to corn earworm. Ann. Entomol. Soc. Amer. 58: 401-402. Knapp, J.L., F.G. Maxwell, and W.A. Douglas. 1967. Possible mechanisms of resistance of dent corn to the corn earworm. J. Econ. Entomol. 60: 33-36. Leuck, D.B. 1970. The role of resistance in pearl millet in control of the fall armyworm. J. Econ. Entomol. 63: 16791680. Luckman, W.H., A.M. Rhodes, and E.V. Wann. 1964. Silk balling and other factors associated with resistance of corn to corn earworm. J. Econ. Entomol. 57: 778-779. Lynch, R.E., W.G. Monson, B.R. Wiseman, and G.W. Burton. 1983. Bermudagrass resistance to the fall armyworm (Lepidoptera: Noctuidae). Environ. Entomol. 12: 1837-1840. Lynch, R.E., K.F. Nwanze, B.R. Wiseman, and W.D. Perkins. 1989. Fall armyworm (Lepidoptera: Noctuidae) development and fecundity when reared as larvae on different meridic diets. J. Agric. Entomol. 6: 101-111. McMillian, W.W., and B.R. Wiseman. 1972. Host plant resistance : A twentieth century look at the relationship between Zea mays L. and Heliothis zea (Boddie). Fla. Agric. Exp. Sta. Monograph Series 2. Mihm, J.A. 1983. Efficient mass rearing and infestation techniques to screen for host plant resistance to maize stem borers, Diatraea sp. Technical Bulletin. International maize and wheat improvement center. El Batan, Mexico. Mihm, J.A. 1989. Evaluating maize for resistance to tropical stem borers, armyworms, and earworms. In Toward Insect Resistant Maize for the Third World: Proceedings of an International Symposium on Methodologies for Developing Host Plant Resistance to Maize Insects, 109-121. Mexico, D.F.: CIMMYT. Ortega, A., S.K. Vasal, J.A. Mihm, and C. Hershey. 1980. Breeding for resistance in maize. In F.G. Maxwell, and P.R. Jennings, eds., Breeding Plants Resistant To Insects, 371-419. New York: John Wiley and Sons. Owens, J.C. 1975. An explanation of terms used in insect resistance to plants. Iowa State J. Res. 49: 513-517. Painter, R.H. 1951. Insect resistance in crop plants. New York: The MacMillan Co. Painter, R.H, 1968. Crops that resist insects provide a way to increase world food supply. Kans. Agric. Expt. Sta. Bull. 520. Perkins, W.D., R.L. Jones, A.N. Sparks, B.R. Wiseman, J.W. Snow, and W.W. McMillian. 1973. Artificial diets for mass rearing the corn earworm. USDAARS Production Research Report 154. Raulston, J.R., S.D. Pair, J. Loera, A.N. Sparks, W.W. Wolf, J.K. Westbrook, G.P. Fitt, and C.E. Rogers. 1992. Helicoverpa zea (Lepidoptera: Noctuidae) pupa production in fruiting corn in Northeast Mexico and South Texas. Environ. Entomol. 21: 1393-1397. SAS Institute. 1982. SAS user’s guide: statistics. Cary, N.C.: The SAS Institute. Straub, R.W. and M.L. Fairchild. 1970. Laboratory studies of resistance in corn to the corn earworm. J. Econ. Entomol. 63: 1901-1903. Waiss Jr., A.C., B.G. Chan, C.A. Elliger, B.R. Wiseman, W.W. McMillian, N.W. Widstrom, M.S. Zuber and A.J. Keaster. 1979. Maysin, a flavone glycoside from corn silks with antibiotic activity toward corn earworm. J. Econ. Entomol. 72: 256-258. 53 Waller, R.A. and D.B. Duncan. 1969. A bayes rule for the symmetric multiple comparison problem. J. Amer. Stat. Assoc. 64: 1484-1499. Walter, E.V. 1957. Corn earworm lethal factor in silks of sweet corn. J. Econ. Entomol. 50: 105-106. Widstrom, N.W. and W.W. McMillian. 1967. Character correlation-importance and use in studies on insect resistance in corn. Agron. Abstr. Amer. Soc. Agron. 1967 : 22. Widstrom, N.W., W.W. McMillian and B.R. Wiseman. 1970. Resistance in corn to the corn earworm, Heliothis zea (Boddie), and fall armyworm, Spodoptera frugiperda (J.E. Smith) (Lepidoptera : Noctuidae) Part IV : Earworm injury among corn inbreds as related to various climate and plant characteristics. J. Econ. Entomol. 63: 803-808. Widstrom, N.W., W.W. McMillian and B.R. Wiseman. 1979. Ovipositional preference of the corn earworm and the development of trichomes on two exotic corn selections. Environ. Entomol. 8: 833-839. Wilson, R.L. and B.R. Wiseman. 1988. Field and laboratory evaluation of selected maize plant introductions for corn earworm responses at two locations. Maydica 33: 179-187. Wilson, R.L., B.R. Wiseman, and G.L. Reed. 1991. Evaluation of J. C. Eldredge popcorn collection for resistance to corn earworm, fall armyworm (Lep.: Noct.), and European corn borer (Lep.: Pyral.). J. Econ. Entomol. 84: 693-698. Wiseman, B.R. 1982. The importance of Heliothis-crop interactions in the management of the pest. In Proc. of the International Workshop on Heliothis Management, ICRISAT, 15-20 November 1981, Patancheru, A. P., India. 209-222. Wiseman, B.R. 1990. Plant resistance to insects in the southeastern United States—an overview. Fla. Entomol. 73: 351-358. Wiseman, B.R. 1996. Plant resistance to the fall armyworm, Spodoptera frugiperda (J. E. Smith). Integrated Pest Management Reviews. (Accepted) Wiseman, B.R., and K. Bondari. 1995. Inheritance of resistance in maize silks to the corn earworm. Entomologia Exp. Appl. 77: 315-321. Wiseman, B.R., and F.M. Davis. 1979. Plant resistance to the fall armyworm. Fla. Entomol. 62: 123-130. Wiseman, B.R., and F.M. Davis. 1990. Plant resistance to insects attacking corn and grain sorghum. Fla. Entomol. 73: 446-458. 54 B.R. WISEMAN Wiseman, B.R., F.M. Davis, and J.E. Campbell. 1980. Mechanical infestation device used in fall armyworm plant resistance programs. Fla. Entomol. 63: 427-432. Wiseman, B.R., F.M. Davis, and W.P. Williams. 1983b. Fall armyworm: Larval density and movement as an indication of nonpreference in resistant corn. Protection Ecology. 5: 135-141. Wiseman, B.R., H.R. Gross, N.W. Widstrom, A.C. Waiss, Jr., and R.L. Jones. 1988. Resistance of corn to Heliothis zea. In G.A. Herzog, S. Ramaswamy, G. Lentz, J.L. Goodenough, and J.J. Hamm, eds., Theory and tactics of Heliothis population management. III-Emerging control tactics and techniques, 21-30. Southern Cooperative Series Bulletin No. 337. Wiseman, B.R., R. Gueldner, and R.E. Lynch. 1982. Resistance in common centipedegrass to the fall armyworm. J. Econ. Entomol. 75: 245-247. Wiseman, B.R., and D.J. Isenhour. 1988. Feeding responses of fall armyworm larvae on excised green and yellow whorl tissue of resistant and susceptible corn. Florida Entomol. 71: 243-249 Wiseman, B.R. and D.J. Isenhour. 1990. Effects of resistant corn silks on corn earworm (Lepidoptera : Noctuidae) biology : A laboratory study. J. Econ. Entomol. 83: 614-617. Wiseman, B.R. and D.J. Isenhour. 1993. Response of four commercial corn hybrids to infestations of fall armyworm and corn earworm (Lepidoptera: Noctuidae). Florida Entomol. 76: 283-292. Wiseman, B.R., D.J. Isenhour, and V.R. Bhagwat. 1991. Stadia, larval-pupal weight, and width of head capsules of corn earworm after feeding on varying resistance levels. J. Entomol. Sci. 26: 303309. Wiseman, B.R., W.W. McMillian, and N.W. Widstrom. 1970. Husk and kernel resistance among maize hybrids to an insect complex. J. Econ. Entomol. 63: 1260-1262. 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Ecol. 19: 2055-2074. 55 M e c ha nism s of Re sist a nc e in M a ize t o Sout hw e st e rn Corn Bore r a nd Suga rc a ne bore r H. Kumar, Faridabad, Haryana, India I nt roduc t ion For studying non-preference and The quadrants created by the antibiosis in maize to insects, the plants intersection of the line for mean foliar Maize is an important cereal crop used were grown in the fields at Tlatizapan damage and the regression line of foliar for food and fodder. CIMMYT has an and Poza Rica and infested with damage vs. yield reduction were used active program that has developed D. grandiosellaand D. saccharalis. At to separate the components of maize germplasm with a desirable level varying intervals, the plants were resistance into tolerance and antibiosis. of resistance to insect pests. The examined for leaf damage and larval objective of this study was to generate survival and growth. In some quantitative information on the experiments, the excised parts of the components of resistance in the single plants were also infested with When the single cross hybrids Ki3 x cross hybrids and their parents to the D. grandiosellain the laboratory and CML131, CML67 x CML135 and stem borers, Diatraea grandiosella (Dyar) lesions on leaves due to larval feeding CML135 x CML139 were infested with and Diatraea saccharalis Fabricius. The were measured using graph paper. For D. grandiosella larvae for 21 days, leaf mechanisms of resistance, non- determining tolerance in hybrids, the feeding ratings on the resistant hybrids preference, antibiosis and tolerance are plants of each of the three hybrids were (67 x135 & 135 x139) were significantly studied in two ways: grown in Tlaltizapan in four row plots, lower than the susceptible hybrid (Fig. • Responses of insects to plants replicated three times. Two rows of 1). Larval survival and weight gained (orientation, feeding, metabolism of each plot were infested with 60 larvae by the surviving larvae on the resistant the ingested food, larval growth and of D. grandiosella while the other two hybrids were also low (Fig. 1). Similar development, fecundity and rows were protected with insecticides. results were obtained when the hybrids oviposition). Data on foliar damage ratings and grain and inbreds were infested with D. Plant characters determining yield loss suffered by each hybrid were saccharalis (Figs. 2 and 3.). In a separate responses of the insects (biochemical used to separate the components of experiment, when the three hybrids and physical). resistance into tolerance and antibiosis. and their inbreds were infested with • M a t e ria l a nd M e t hods Survival, % Foliar ratings hybrids and the five inbreds developed at CIMMYT were used for the present Weight, mg 25 10 The following three single cross Re sult s a nd Disc ussion 60 50 20 8 A A A 6 40 15 study: • Hybrids - Ki3 x CML131 (susceptible), CML67 x CML135 30 4 B B • Inbreds - CML131 (S), CML135 (I), CML139 (R), CML67 (R), and Ki3 (S). B 10 B 20 2 5 (resistant) and CML135 x CML139 (resistant). F=6.35; LDS=22.9 F=7.56; LDS=14.5 F=70.25; LDS=0.75 0 0 Ki3/131 67/135 135/139 10 B B Ki3/131 67/135 135/139 0 Ki3/131 67/135 135/139 Figure 1. Diatraea grandiosella larval damage, survival and weight gained on 3 hybrids in 21 days. 56 H. KUMAR AND J.A. MIHM D. grandiosella for 96 hours, the larval of the resistant and susceptible inbreds the susceptible hybrid Ki3 x CML131 survival on all the hybrids and inbreds (CML67 and CML131) and hybrids (67 fell distinctly into “antibiosis absent/ was the same (Fig. 4). However, 21 x 135 & 135 & 139) were offered to D. tolerance” absent quadrant (Fig.6). For days after infestation, larval survival on grandiosella, larval survival was 100% the hybrid CML67 x CML135 and the resistant hybrids and inbreds was on all genotypes (Fig.5). However, CML135 x CML139, the tolerance and significantly lower than the susceptible larval feeding as indicated by the area antibiosis types of resistance checks (Fig. 4). In the laboratory of the leaves eaten was significantly mechanisms were operating against D. bioassays, when the excised leaf whorls lower on the resistant hybrids and grandiosella because all the points were Survival (%) 20 Ratings 7 6 scattered in “antibiosis absent/ feeding non-preference and tolerance present” and “antibiosis antibiosis were the mechanisms present/tolerance present” quadrants. of resistance in hybrids 15 5 inbreds (Fig. 5). This showed that developed at CIMMYT. Our data show that CIMMYT has 4 developed potentially useful single- 10 3 2 5 CML135 x CML139 CML67x CML135 0 Ki3 x CML131 CML135 x CML139 CML67x CML135 0 Ki3 x CML131 1 A significant correlation between cross hybrids which have adverse the foliar damage caused by D. effects on the larval feeding and grandiosella and the yield growth/development of stem borers. In reduction indicated a possible addition to resistance, the hybrids partitioning of the resistance possess tolerance to stem borers; i.e., components into antibiosis and surviving larvae would not cause any tolerance (Fig.6). The points for yield reduction in the resistant hybrids. Figure 2. Diatraea saccharalis damage and survival on three maize hybrids. Ratings 10 Feeding (area fed, mm2) 500 Survival 12 8 10 400 8 300 6 6 4 200 4 100 CML 139 CML 135 CML 67 0 CML 131 CML 139 0 CML 135 0 CML 67 2 CML 131 2 Figure 3. Diatraea saccharalis damage and survival on five maize inbred lines. CML 131 CML 67 Figure 5. Feeding response of Diatraea grandiosella on 2 inbreds and 2 hybrids in the laboratory for 48 hours. 50 % 60 % 100 50 80 y = 10.6x - 63.8 40 A 60 Antibiosis + BTolerance B - 40 0 5 0 0 CML CML CML CML 131 67 135 139 Antibiosis + Tolerance + -10 20 10 Antibiosis Tolerance + 10 30 20 Tolerance Antibiosis - 30 20 40 Ki 3xCML 131CML 67XCML 135 Ki 3/131 67/135 135/139 Figure 4. Diatraea grandiosella larval survival on 4 inbreds and 3 hybrids at 21 days after infestation. 6 Ki3 x CML131 CML135 x CML139 CML67 x CML135 7 8 Foliar damage ratings 9 10 Figure 6. Correlation between foliar damage and yield reduction on maize hybrids and inbred lines by Diatraea grandiosella. 57 V a ria bilit y for M a ysin c ont e nt in M a ize Ge rm pla sm De ve lope d for I nse c t Re sist a nc e C. Welcker, INRA, Centre Antilles-Guyane, URPV, Guadeloupe, FWI G. Febvay, INRA, INSA Laboratoire de Biologie appliquée, Villeurbanne, France and D. Clavel, CIRAD-CA, Programme maïs, Montpellier, France Abst ra c t First described in the Mexican maize landrace Zapalote chico, the flavone maysin has been identified as a potent factor in antibiosis to corn earworm Helicoverpa zea (Boddie). This study was conducted to determine maysin content in 20 inbreds and populations of maize which were being developed for resistance to insects. This genetic material and checks were evaluated in the field for corn earworm injury and leaf feeding damage by larvae of fall armyworm, Spodoptera frugiperda (J.E. Smith). Maysin levels in silks and young leaves were measured using HPLC. Maysin levels in silks ranged from 0 to 4 mg/g of fresh weight. The main part of the studied material contained maysin below 1.5 mg/g, the concentration considered to be necessary for resistance based on larval toxicity. Several populations reach the resistance level of Zapalote chico, but a few other populations also possess minor levels of the substance. Among other things, maysin level can be used as a selection criterion to increase the diversity of resistance mechanisms in source germplasm. I nt roduc t ion Several maize populations have been constituted from local maize landraces identified, including Zapalote chico, and introduced, resistance source Fall armyworm (FAW), Spodoptera with high antibiosis against CEW germplasm are being improved through frugiperda (J.E. Smith), and corn (Straub and Fairchild 1970; Wiseman et recurrent selection (Welcker, 1993). earworm (CEW), Helicoverpa zea al. 1976; Wiseman et al. 1977). Waiss et (Boddie) (Lepidoptera, Noctuidae), are al. (1979) suggested that a part of the The three major objectives of the the major pests of maize, Zea mays L., in resistance in silks is due to maysin, a present study were to determine 1) the the southeastern United States, Central flavonol-C-glycoside compound. The maysin content in maize populations America and the Caribbean. For FAW, biological relationship between maysin used and developed in Guadeloupe for damage to maize is caused by leaf concentration in the silks and its effect resistance to fall armyworm and corn feeding during the whorl stage on the growth of CEW and FAW is now earworm; 2) the potential interest of (Bunting 1986). The CEW larvae usually clearly demonstrated (Wiseman et al. some populations; 3) the potential feed on the whorl tissue leaves, 1992, Wiseman et al. 1993). usefulness of maysin content as a selection criterion. emerging tassel and silks in the tips of the ears. While feeding on silks, larvae The French National Institute for often mine into the silk channels and Agronomical Research (INRA) and the damage the accompanying kernels Centre for International Cooperation in (Wiseman and Isenhour 1992). Agricultural Research for Materials Development of plant resistance to Developement (CIRAD) carry out in The study included maize lines and insects is one of the most promising Guadeloupe a maize breeding program populations with agronomic methods for controlling insect pests in focused on insect resistance and characteristics related to host plant maize (Wiseman and Davis 1990). adaptation to Caribbean farming resistance and with potential interest conditions. Base populations M a t e ria ls a nd M e t hods 58 C. WELCKER, G. FEBVAY AND D. CLAVEL for our breeding program (Table 1.) At resistance to FAW: MpSWCB4 and 1993 at INRA Domaine Godet (Grande first, we used Zapalote chico, a CEW Antigua and the lines Mp705 and Terre, latitude 16°20”N, 35 masl, resistant population with high maysin CML67, derived from these sources average annual rainfall 1,300 mm, levels, as a positive check for maysin (USDA Mp, CIMMYT). We chose also average temperature 25.8°C). concentration in silks. Zapalote grande, the well adapted, intermediate two populations from the USDA- resistance cultivar, ‘Spectral’, and Silk and leaf extracts were analysed for Georgia (GTDDSA and GTRI4), and at PopG-C1a, issued from the first cycle of maysin following the procedure least one population from Central recurrent selection for FAW resistance described by Snook et al. (1989) using America (Maïa XXIX) were chosen for in a composite of Guadeloupean maize reversed-phase HPLC. The silk their resistance to CEW. The cultivar ecotypes showing good performance analyses were conducted (5 to 25 Stowell Evergreen was used as a under FAW and CEW (Welcker et al., replicates by genotype) on silk mass negative check for maysin these proceedings). removed from the husk channel (3 to 7 g) of individual ears (1 day after silk concentration. The susceptible local population Fond’or and a very Additional sources included TZBR-E3, a emergence). For the leaf analyses (5 to susceptible line (Mo17) were also population with resistance to African 30 replicates by genotype), the plants described. sugarcane stem borer (SSB), Eldana were at mid-whorl stage and the tissue saccharina Walker, introduced from sampling was restricted to the furl Material resistant to FAW was also IITA (Kling and Bosque-Perez, 1995); leaves (4 to 9 g) of individual plants. included in this study because, on the Desirade, a local, early maturing Maysin was identified by its retention one hand, as described by Wiseman et population; KI3, a susceptible check line time, measured with a standard kindly al. (1992), FAW larvae development for borers; B86, a line resistant to supplied by Neil Widstrom (USDA could be affected by maysin and, on the European corn borer, Ostrinia nubilalis GA). Maysin concentrations are other hand, we wanted to detect maysin (Hübner); CI66; and Sure Sweet, a sweet expressed as mg/g of plant tissue, in organs fed on by FAW. Therefore, we corn selected for resistance to insects at fresh weight. studied the two main sources of the Tropical Agricultural Research Station, Mayaguez, Puerto-Rico. Table 1. Maize germplasm studied. Genotype Zapalote chico Zapalote Grande GTDDSA GTRI4 Sure Sweet Maïa XXIX MpSWCB4 PopG-C1a Desirade Fond’or Spectral TZBR-E3 Antigua 2D-118 Mo17 Mp705 Ki3 CML67 B86 Stowell Evergreen Ci66 91-27-63 # 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Origin CIMMYT CIMMYT USDA USDA USDA EMBRAPA USDA INRA INRA INRA INRA IITA CIMMYT USDA USDA CIMMYT CIMMYT USDA USDA USDA Methods FAW R r r R S R S R - R: resistant material S: susceptible material r: population with intermediate level of resistance CEW Resistance to CEW was R r r r r r s S S - Re sult s Corn earworm injury Mean CEW injury ratings for the evaluated in a replicated trial in studied material are given in Table 2. 1993 at INRA Domaine Duclos These results highlight the potential of (Basse Terre, 110 masl, latitude TZBR-E3 and POPG-C1a as new 16°12”N, average annual rainfall sources of resistance to CEW. These are 2,840 mm, average temperature improved populations developed for 24.5°C), an area where frequent resistance to SSB and FAW, and heavy natural CEW respectively. So the indications are that infestations occur. CEW injury they exhibit multiple resistance. For was rated for depth of kernal TZBR-E3, this could be explained by feeding on random sampled the similarity in the feeding behaviour individual ears. of both insects. Sure Sweet is a sweet corn which was improved for Resistance to FAW was visually resistance to CEW at the Tropical rated plot by plot, 14 days after Agricultural Research Station, Puerto- infestation with 25 larvae per Rico. The results prove the plant (at the 5-leaf stage), on a effectiveness of this selection. On the scale of 0 (no damage) to 9 other hand, no information can explain (heavy damage) (Davis et al., the level of resistance observed for KI3. 1992). The trial was carried out in VARIABILITY FOR MAYSIN CONTENT IN MAIZE GERMPLASM 59 Response to leaf-feeding by fall armyworm Of potential interest also are PopG-C1a Maysin content in silks and Spectral, which showed an A wide range of responses was Mean FAW leaf-feeding ratings are intermediate level of resistance to FAW. detected for maysin in silks, indicating given in Table 3. These data show the Other local materials and germplasm the usefulness of maysin high level of resistance to FAW for improved for resistance to CEW measurements. Figure 1 provides an MpSWCB4, Antigua, and derived lines. appeared susceptible. example of contrasting (Zapalote chico and Stowell Evergreen) and Table 2. Mean corn earworm injury rating on maize populations. Genoytpe Mo17 CML67 B86 Stowell evergreen Fond’or MpSWCB4 GTDDSA CI66 GTRI4 Antigua 2D 118 Mp705 Spectral Desirade Z. Chico Z. Grande Ki3 PopG-C1a Maia XXIX Sure Sweet TZBR.E3 a Ratinga Replicates 10.1 4.4 3.8 2.7 2.4 2.3 2.1 2.1 1.9 1.3 1.2 1.1 1.0 1.0 1.0 0.6 0.4 0.3 0.2 0.1 Statistical grouping 19 66 19 3 63 67 35 8 20 41 35 51 67 45 13 24 68 54 56 61 a b b c c cd cd cde cde de de e e ef ef ef ef f f f Mean of individual ratings of depth of kernal feeding (cm) Standards Chrysin Chlorogenic Acid Maysin Table 3. FAW leaf-feeding rating in maize germplasm. Genotype Mo17 Z. Grande Ki3 GTRI4 Maia XXIX TZBR.E3 GTDDSA B86 Z. Chico Desirade Fond’or PopG-C1a Spectral Mp705 Antigua 2D.118 MpSWCB4 CML67 Detector response (340 nm) Maysin 8.5 7.6 7.3 7.1 7.1 7.0 6.8 6.7 6.7 6.6 6.5 6.5 6.5 4.4 4.2 3.8 2.6 a Visually rated on a 0-9 scale (0 = no damage; 9 =heavy damage). SD Line = 0.33 SD Pop. = 0.55 a Maysin levels in silks ranged from 0 to nearly 4 mg/g of plant tissue, fresh weight (Table 4). As expected, maysin concentration in the silks of Zapalote chico were significantly higher than in the other genotypes. Several laboratory bioassays have shown that a maysin concentrations of 1.5 mg/g of fresh weight reduce CEW larvae growth by 50% (Waiss et al. 1979; Wiseman et al. 1992). In this study, we found several maize populations and lines with silk maysin content below this threshold — considered to be significant for CEW antibiosis. Among these low maysin populations and lines were the negative control Stowell Evergreen and two populations Table 4. Maysin concentrations in silks (mg/g of fresh weight). Maysin content Genotype Zapalote chico Mean rating intermediate (PopG-C1a) spectra. Chrysin (IS) PopG-C1a Maysin Stowell Evergreen Chrysin (IS) Chrysin (IS) Maysin Time Figure 1. HPLC spectra of silks. CI66 B86 GT DDSA Fond’or Ki3 Antigua 2D.118 Stowell evergreen CML67 Maia XXIX Desirade Sure Sweet Mp705 PopG-C1a Mo17 Spectral MpSWCB4 Z. Grande TZBR E3 GTRI4 Z. Chico Mean +/- SE Replicates 0.00 0.02 0.08 0.11 0.34 0.36 0.44 0.87 0.93 0.99 1.00 1.04 1.07 1.08 1.11 1.20 1.52 1.60 2.08 3.71 +/- 0.00 +/- 0.01 +/- 0.03 +/- 0.02 +/- 0.11 +/- 0.06 +/- 0.38 +/- 0.21 +/- 0.18 +/- 0.44 +/- 0.26 +/- 0.13 +/- 0.28 +/- 0.13 +/- 0.21 +/- 0.23 +/- 0.10 +/- 0.29 +/- 1.11 +/- 0.44 5 5 7 7 5 24 5 4 15 10 4 15 10 5 11 11 3 10 3 8 Statistical grouping a a a a ab ab abc abcd bcd bcd bcde bcde bcde bcde bcde cde de de e f 60 C. WELCKER, G. FEBVAY AND D. CLAVEL improved for resistance to insects, arbitrary threshold. For such genotypes (provided that samples were GTDDSA and Antigua. However, some we could expect a 30% reduction in representative, in the latter case). Field populations showed a maysin larval growth. resistance to CEW is also present in other populations: PopG-C1a, Maïa concentration above this threshold: Zapalote grande, GTRI4 (known to HPLC spectra of leaves XXIX and, at a lower level, Spectral. have antibiosis) and TZBR-E3 In contrast to the clean HPLC traces These populations possess a minor apparently new germplasm with obtained from silks, many unidentified level of antibiosis which could be of antibiosis. The maysin concentration of peaks were observed in extracts from interest in breeding programs. many entries (MpSWCB4 and Mp705, young leaves (Fig. 2). Hence, maysin Maysin has been found at an Spectral, PopG-C1a, or Maïa) was not measurements were more difficult. The intermediate level in silks of material significantly different from this maximum level of maysin (0.1mg/g improved for FAW resistance plant tissue, fresh weight) was found in (MpSWCB4, PopG and Spectral), Zapalote Chico leaves. suggesting this as a possible selection Standards Chrysin Chlorogenic Acid criterion to maintain variability in Disc ussion resistance mechanisms in source germplasm. Detector response (340 nm) Maysin Correlation between maysin concentration and resistance Chrysin (IS) Zapalote chico Maysin Chrysin (IS) Antigua 2D-118 Maysin No significant correlation was found Plant to plant variation for silk maysin concentration between maysin concentration and the We have reported in Figure 4 the inter- level of resistance to CEW in the population variability of maysin studied material (Fig. 3), suggesting the concentration versus its intra- involvement of other resistance factors population variation. Some genotypes, or mechanisms. For instance, Widstrom in particular guadeloupean materials, et al. (1992) showed that the resistance present an intermediate level with from GTDDSA is primarly tolerance. interesting intra-population variation. However, maysin appears to contribute This new variability would be worth to antibiosis resistance to CEW in studying, assuming it is not simply the Zapalote chico and probably in TZBR- expression of environmental effects. E3, Zapalote grande and GTRI4 Time Figure 2. HPLC spectra of leaves. 2 14 A 6 20 2 0 Desirade 1.5 8 4 4 r = -0.15, p = 0.53 Standard deviation of silk maysin concentration Level of H. zea damages 10 18 B 10 19 3 13 16 17 B Desirade Spectral PopG-C1a 9 1 PopG-C1a A 1 8 7 6 11 0.5 B 7 15 9 11 65 8 4 2 12 0 1 2 3 Silk maysin concentration (mg/g fresh weight) Figure 3. Correlation between silk maysin concentration and damages. 13 16 18 1 0 4 3 10 20 12 Spectral 5 B 17 15 14 2 1 2 3 Silk maysin concentration (mg/g fresh weight) 4 Figure 4. Variability for maysin concentration in silks within and between maize populations. VARIABILITY FOR MAYSIN CONTENT IN MAIZE GERMPLASM Maysin in leaves Maysin concentrations in leaves were 30 times less than those in silks — below the concentration threshold required to significantly reduce FAW growth as reported in the literature (Wiseman et al. 1992). However, the characterization of material improved for resistance to FAW is still of interest, as it could reveal maysin in some populations with multiple insect resistance and could be a way to maintain genetic variability when improving populations. This study reports new available variability for antibiosis resistance to CEW, partly resulting from maysin content. Use of maysin as a criterion could be of special interest for describing initial variability and for breeding genotypes which combine several resistance factors. Re fe re nc e s Bunting, G.D. 1986. A review of plant response to fall armyworm, Spodoptera frugiperda (J.E. Smith), injury in selected field and forage crops. Fla. Entomol. 69: 549-559. Davis, F.M., S.S. Ng, and W.P. Williams. 1992. Visual rating scale for screening whorl-stage corn for resistance to fall armyworm. Mississippi Agricultural and Forestry Experiment Station Technical Bulletin 186. Kling, J.G., and N.A. Bosque-Pérez. 1995. Progress in screening and breeding for resistance to the maize stem borers Eldana saccharina and Sesamia calamistis. In D.C. Jewell, S.R. Waddington, J.K. Ransom, and K.V. Pixley (eds.), Maize Research for Stress Environments: Proceedings of The Fourth Eastern and Southern Africa Regional Maize Conference, Harare, Zimbabwe, 28 March 1 April 1994. Mexico, D.F.: CIMMYT. Snook, M.E., N.W. Widstrom, and R.C. Gueldner. 1989. Reversed-phase highperformance liquid chromatographic procedure for the determination of maysin in corn silks. J. Chromatogr. 477: 439-447. Straub, R.W., and M.L. Fairchild. 1970. Laboratory studies of resistance in corn to the corn earworm. J. Econ. Entomol. 63: 1901-1903. Waiss, A.C., B.G. Chan, C.A. Elliger, B.R. Wiseman, W.W. McMillian, N.W. Widstrom, M.S. Zuber, and A.J. Keaster. 1979. Maysin, a flavone glycosine from corn silks with antibiotic activity toward corn earworm. Pestic. Biochem. Physiol. 72: 256-258. Welcker, C. 1993. Breeding for resistance in maize to fall armyworm in Caribbean region. Plant Resistance to Insects News Letter 20: 19-20. 61 Welcker, C., J.D. Gilet, D. Clavel, and I. Guinet. This proceedings. Response to selection for resistance to leaf feeding by fall armyworm in PopG, Guadeloupean maize population. Widstrom, N.W., W.P. Williams, B.R. Wiseman and F.M. Davis. 1992. Recurrent selection for resistance to leaf feeding by fall armyworm on maize. Crop Sci. 32: 1171-1174 Wiseman, B.R., and F.M. Davis. 1990. Plant resistance to insects attacking corn and grain sorghum. Fla. Entomol. 73: 446-458. Wiseman, B.R., and D.J. Isenhour. 1992. Relationship of planting dates and corn earworm developmental parameters and injury to selected maize entries. Maydica 37: 149-156. Wiseman, B.R., W.W. McMillian, and N.W. Widstrom. 1976. Feeding of corn earworm in the laboratory on excised silks of selected corn entries with notes on Orius insidiosus. Fla. Entomol. 59 (3): 305-308. Wiseman, B.R., M.E. Snook, and D.J. Isenhour. 1993. Maysin content and growth of corn earworm larvae (Lepidoptera, Noctuidae) on silks from first and second ears of corn. J. Econ. Entomol. 86: 939-944. Wiseman, B.R., N.W. Widstrom, and W.W. McMillian. 1977. Ear characteristics and mechanisms of resistance among selected corns to corn earworm. Fla. Entomol. 60: 97-103. Wiseman, B.R., M.E. Snook, and D.J. Isenhour, J.A. Mihm, and N.W. Widstrom. 1992. Relationship between growth of corn earworm and fall armyworm larvae (Lepidoptera, Noctuidae) and maysin concentration in corn silks. J. Econ. Entomol. 85: 24732477. 62 A Re vie w of Ent om ologic a l T e c hnique s a nd M e t hods U se d t o De t e rm ine M e c ha nism s a nd Ba se s of St e m Bore r Re sist a nc e in M a ize Z.R. Khan, International Center of Insect Physiology and Ecology, Mbita Point, Kenya Abst ra c t Among numerous insects which attack maize, Zea mays L., stem borers are ubiquitous and major pests. These lepidopterous insects infest the maize crop throughout its growth from seedling emergence to maturity. Maize in every country and type of crop cultivation is usually infested by one or more stem borer species. The use of borerresistant maize varieties is an ideal method of managing these pests. Breeding for stem borer resistance has become a major research objective in most of the maize growing countries of Africa, Asia, and the Americas. Success in breeding for stem borer resistance depends upon the development of effective and efficient techniques for screening germplasm for sources of resistance. Screening techniques were presented at the first CIMMYT symposium on developing insect resistant maize in 1987. After sources of resistance have been identified and developed to some usable form (i.e., inbreds), the mechanism(s) and bases of resistance should be determined to fully understand the nature of the resistance and how to best use the resistance source in breeding programs and the resistant cultivars in integrated pest management programs. This paper serves as a review of some entomological techniques which have been used to determine mechanism(s) operating in resistant plants and to elucidate the chemical and/or physical factors (bases) responsible for resistance. M e c ha nism s of Re sist a nc e operating in a resistant plant, susceptible control has acquired a experiments must be carefully designed heavy population accumulation. Plants The mechanisms of resistance in plants that prove or disprove the involvement are then evaluated for insect settling to insects have been divided by Painter of each of the three mechanisms (Davis response, feeding damage and/or (1951) into three categories: non- 1985). Different experimental test oviposition. Techniques for measuring preference, antibiosis, and tolerance. procedures are necessary to non-preference in resistant maize are as Antixenosis has been suggested as a differentiate among non-preference, follows. replacement for the term ‘non- antibiosis, and tolerance. Studying non- preference’ (Kogan and Ortman 1978). preference requires testing with insects Larval orientation and settling - The Non-preference reduces the insects’ under choice and no-choice conditions; female moths are usually responsible three major behavioral responses, i.e., for antibiosis, testing must take place for selecting the plants for their larvae oviposition, orientation, and/or under no-choice conditions and, for or progeny to feed upon. However, feeding (Saxena 1985). Antibiosis tolerance, pest infested and uninfested upon emergence the larvae must find a adversely affects the biology of insects conditions (Davis 1985). suitable site to initiate feeding. The larvae do have the option of accepting (e.g., survival, developmental time, and fecundity). Tolerance is the ability of Non-preference the plant as a host or not. Orientation plants to compensate for insect damage Non-preference denotes the presence of and settling responses of an insect to a without adversely affecting the insects’ morphological and/or chemical plant plant are generally measured in choice biology and/or behavior. factors that adversely affect insect tests by observing the number of behavior. To confirm non-preference, individuals which initially orient To determine which mechanism or plants are planted together, infested toward a plant (orientation), and then combination of mechanisms are with test insects, and left until the remain settled for some time for A REVIEW OF ENTOMOLOGICAL TECHNIQUES AND METHODS USED TO DETERMINE MECHANISMS AND BASES OF STEM BORER RESISTANCE IN MAIZE 63 feeding or oviposition. While the cm high) connected with a 10 cm, each test sample. Test insects were orientation response is measured Y-shaped tube. Test materials were placed in the bioassay arena and the within a few minutes to an hour of the kept in one or both of the rearing cups number of E. lignosellus crawling into release of the test insect, settling and the cups were capped. Twenty each tube was recorded after 30 min or responses are generally measured at neonate larvae were placed in the at 5 min intervals for 30 min. longer time intervals. The following entrance of the Y-tube after which the methods have been used to measure tube is sealed with a cork and kept in Choice test - Using a choice test, Davis orientation. darkness. The number of larvae et al. (1989) determined the presence of reaching each rearing cup was recorded non-preference mechanism in selected after 3 h. maize hybrids to Diatraea grandiosella To test orientation of lesser cornstalk Inner whorl leaf tissue disks (2 cm (Swinhoe) to various susceptible and borer, Elasmophalus lignosellus (Zeller) diam.) from the test plants were resistant sorghum cultivars can be used (Fig. 1) to multiple samples, an randomly placed equidistant from each with maize also. Plants of each test eight-sample olfactometer was other on a piece of paper towel in a cultivar were grown in 3.0 m x 2.5 m constructed by Huang et al. (1990). plastic dish containing 2% agar for plots in five rows parallel to the wind Eight sample tubes were connected in a providing moisture to prevent tissue direction. A rectangular tray (40 cm circle equidistantly to the bottom of a drying. Each tissue disk was held flat to long x 25 cm wide), with two longer Petri dish (15 cm diameter). The Petri towel surface by inserting two small sides continuing upward as 10 cm high dish served as a bioassay arena. A small pins. The pins keep the disks from vertical walls, was lined with filter air-inlet pore was made on the outside folding and allows a thigmotoxic paper. The tray was placed 20 cm from of each tube to eliminate the differences condition that is favored by these stem the downwind end of each plot with its in airflow rate. Small holes were drilled borer larvae. 50 to 75 blackhead stage long axis parallel to and in line with the in clusters in the center of the dish to eggs were placed in the center of each central row of plants so that which an exhaust tube was attached dish. The dishes were then held in distance-perceivable stimuli from the and connected to a vacuum pump. All complete darkness to prevent effects of plants could reach the tray. 20 neonate air-inlet pores were enclosed in an light on larval distribution within C. partellus larvae were released across air-inlet chamber formed by gluing chambers. The larvae on each tissue the middle of the tray and the number another Petri dish of the same size, with disk were counted 24 h after egg hatch. of larvae that moved to the two ends of holes for the corresponding tubes and Leaf tissue from resistant hybrids was the tray in 30 min were recorded. The exit tube, to the bottom of the bioassay significantly less preferred by both percentage of larvae reaching the end arena. Air (100 ml/min) passed into the species of stem borers than leaf tissue nearest the plants reflected the level of inlet tubes was distributed in the from susceptible hybrids. larval attraction to the plants. air-inlet chamber, entered each tube, Attraction test - The method used by Dyar and Ostrinia nubilalis (Hübner). Saxena (1990) to determine the attraction of larvae of Chilo partellus passed into the arena, and was To determine whether neonate larvae Olfactometer - The orientational exhausted at the base of the bioassay of stem borers orient and settle responses of neonate larvae to the odor arena. A cotton ball was placed above preferentially on callus initiated from of plants can be studied using various susceptible or resistant plants, larval 9 kinds of olfactometers. The response can be measured in two-choice or multi-choice olfactometers depending used for studying orientational responses of maize stem borers. The olfactometer was constructed from two plastic rearing cups (4 cm diameter, 4.5 methodology of 3 6 7 4 Chang et al. (1985) for fall armyworm, Spodoptera frugiperda (J.E. Smith), can be were measured following the 2 8 upon the number of test samples. A simple Y-shaped olfactometer, used by orientation and settling responses 1 To vacuum pump 5 Overview of the bioassay arena Williams et al. (1987). Five pieces of callus (0.5 g) of each test Air from outside of lab Figure 1. A multi-choice olfactometer. 1, bioassay arenas; 2, air-inlet chamber; 3, sample tube; 4, airflow meter; 5, filter fitted with activated carbon; 6, air-inlet tubes; 7, exit tube; 8, air-inlet pore; 9, insect releasing hole (Huang et al. 1990). 64 Z.R. KHAN cultivar were placed in a circular planted in 3-row plots 5 m long with a The field cage experiment designed by manner equidistantly from the center of spacing of 75 cm and 30 cm between Wiseman et al. (1983) for fall a Petri dish. 50 to 100 blackhead stage and within rows, respectively. The armyworm can also be used to evaluate eggs or freshly hatched larvae were central row was planted with a cultivar stem borers’ movement from transferred carefully to the center of different from the two adjoining rows. susceptible and resistant maize plants. each Petri dish. The Petri dish was kept Twenty days after germination, each A susceptible or resistant plant was in complete darkness and number of plant in the middle row was infested surrounded by susceptible plants larvae present on each callus was with an egg mass. All plants were spaced alternately at 30 cm and 40 cm recorded at 1, 6, 12, 24, and 48 hours dissected 7 days after infestation and from the central test plant (Fig. 2). The after infestation. Williams et al. (1987) the number of larvae recovered from surrounding plants were spaced about reported significantly more each plant was recorded and mapped. 12.5 cm apart. The test plant was D. grandiosella, D. saccharalis, and Dispersal of first-instar larvae increased infested with a known number of O. nubilalislarvae preferred the callus twofold when infested resistant neonate larvae and the number of originating from maize hybrids which cultivar, IC22-CM, was surrounded by larvae present on the surrounding were susceptible to leaf feeding. a susceptible cultivar, and decreased plants 4, 6, 8, and 10 days after when an infested susceptible cultivar infestation served as an indicator of Arrest and dispersal - The settling was surrounded by IC22-CM plants larval movement. response of lepidopterous larvae to (Ampofo 1986). different cultivars can be compared Feeding - Techniques that record subtle with respect to their arrest and changes in insect feeding behavior on dispersal on plants or plant parts. susceptible and resistant plants can be Robinson et al. (1978) placed a sticky useful in identification of resistant trap around maize plants in the germplasm. Such changes in insect 12.5 cm feeding behavior can be determined laboratory and field to measure arrestment or dispersal of O. nubilalis. Thirty first-instar larvae were placed in 30 cm 40 cm either through the measurement of damaged plant parts, or in terms of the whorl of each plant. The number of amount of food ingested. Insect feeding larvae that moved off the plant was in a choice assay (Fig. 3) involves the recorded daily for 4 days, then each determination of insect feeding plant was dissected and the remaining preference among multiple plant larvae were counted. Robinson et al. genotypes. Choice experiments can be (1978) reported that more larvae consistently settled on the susceptible, inbred WF9 than on the highly resistant inbred CI31A. Using similar Figure 2. Arrangement of test plants to measure arrest and dispersal of lepidopterous larvae (Wiseman et al. 1983). useful in the preliminary evaluation of plants. However, no-choice methodology, Kumar et al. (1993) studied larval arrestment of C. partellus on 3-week-old plants of susceptible and resistant maize cultivars. The mean number of larvae recovered from resistant genotypes MP 704 and Poza Rica 7832 was significantly lower than the number recovered from the susceptible control. Ampofo (1986) studied arrestment and dispersal of C. partellus larvae on susceptible and resistant maize plants in field plots. The experiment was Figure 3. Experimental set-up to measure stem borer larval feeding on leaf cuts of susceptible and resistant plants in (a) two-choice and (b) multi-choice tests. A REVIEW OF ENTOMOLOGICAL TECHNIQUES AND METHODS USED TO DETERMINE MECHANISMS AND BASES OF STEM BORER RESISTANCE IN MAIZE 65 experiments are necessary to verify the situation could be influenced by a more sectors on either side. The chamber’s degree of resistance. Insect feeding can attractive plant, the relative non- roof and two vertical end-walls were of be measured either on excised or on preference of a host plant could often glass but open below, the floor being intact plants. be misconstrued for true or genetic formed by the test arena. The front and resistance. To ascertain the presence of rear walls of the central sector were of In a no-choice feeding bioassay, Saxena true resistance in a cultivar, and not the glass and those of the two end sectors (1990) offered a 7 cm long basal relative preference existing only in a were of removable screen. In the field segment of a leaf whorl to 20 neonate choice situation, insect ovipositional the chamber was aligned with its long C. partelluslarvae, or an internode response in a no-choice situation must axis at right angle to the wind direction. segment of a stem to a single 4th instar also be measured. Without it, Test plants were arranged inside one C. partellus larva in a glass vial. After oviposition preference studies are of end compartment in a row along the 72 h, the area of feeding lesions on the very little utility in predicting pest wall. The opposite end compartment leaf was measured using a dotted paper response under field situations. had a similar row of plants of another cultivar in a two-choice test, or sheet or graph paper. The stem segment was removed after 24 h, split open and Saxena (1990) developed and used a contained no plants, but had wax paper the length and width of the cavities three-compartment chamber (Fig. 4) to sheets. Gravid stem borer females were resulting from larval feeding were evaluate the ovipositional response of released in the central compartment measured. In a similar no-choice C. partellus under field conditions. Tests and the eggs laid on the plants and on feeding experiment with excised leaves were conducted in a field with a the wax paper sheets were counted. Kumar et al. (1993) used a photometric constant number of females in the device (leaf area meter) for measuring sector between two equal-sized end feeding. For the bioassay with stems, a maize cultivars can be measured in 210 cm two-choice tests following the method cm pre-weighed, 6 cm long segment of a of Ng et al. (1990), Kumar (1993) and 80 cultivar was offered to a 4th instar C. Ovipositional preference of stem borer adults to susceptible and resistant area of leaves before and after insect Kumar et al.(1993), or in a multiple the uneaten stem was weighed again choice bioassay as described by after the excreta was separated. Stems of each cultivar were also kept alongside the experiment to determine the weight loss from evaporation. The difference between the initial and final weights of stem after adjustment for weight loss from evaporation indicates stem feeding by the larvae. 80 cm partellus larva in a glass vial. After 48 h, Ampofo et al. (1986). Ovipositional ES CS ES Figure 4. A three-compartment chamber for studying ovipositional response of Chilo partellus to sorghum plants in a field. ES, end compartment; CS, central compartment (Saxena 1990). response in a no-choice bioassay can be tested following the methodology of Ampofo (1985). Khan (1994, unpublished) presented cut maize stems(20 cm long) in choice bioassays to ovipositing B. fusca females (Fig. 5). Oviposition - For many phytophagous insects, the selection of an oviposition site is a critical stage in their choice of a host. For most stem borers and other lepidopterous pests, only the adult female has a large and direct influence on host preference/non-preference; therefore, understanding the details of the insect’s oviposition preference is valuable when attempting to identify resistant germplasm in a plant breeding program. However, since an insect’s ovipositional preference under a choice Figure 5. Experimental set-up to measure B. fusca oviposition on stem cuts from susceptible and resistant plants in two-choice test. 66 Z.R. KHAN Antibiosis Even callus tissue from insect- (Zhou et al. 1983; Wilson and Wissink Both chemical and morphological plant susceptible and resistant maize have 1986; Durbey and Sarup 1988; Williams defenses mediate antibiosis, and been used to determine growth of stem et al. 1990; Saxena 1992; Kumar 1993). antibiotic effects of these resistant borer larvae feeding on them. To Fresh, oven-dried or lyophilized leaf plants on the insect pests can range determine the growth of larvae feeding powder, or plant extract is thoroughly from weak to strong. Field and/or on callus initiated from susceptible and blended with a known amount of laboratory experiments have been resistant plants, Petri dishes containing artificial diet. First-instar larvae are fed designed to determine if the mechanism approximately 500 mg to 1 g of callus the amended diets and comparisons of antibiosis is operating within the were infested with 3 to 5 neonate insect growth on diets incorporated resistant plant. The biological criteria larvae. The larvae were weighed after 7 with different susceptible and resistant used most commonly to determine if to 15 days after infestation (Williams et plant materials can be used effectively antibiosis is present or not is growth, al. 1983; Williams and Davis 1985; to assay for antibiosis resistance. By which includes both weight gain and Williams et al. 1987). Williams et al. placing eggs or newly-emerged larvae developmental time of the insect. Other (1983) reported that D. grandiosella on control and treated diets, differences criteria include survival of the various larvae reared for 7 days on calli of in feeding, weight gain, survival, and insect stages, morphological normality resistant maize genotypes were developmental rate can be detected. of growth stages, and fecundity. significantly smaller than larvae reared Techniques for evaluating for antibiosis on calli from susceptible maize Also, some researchers have studied as related to growth utilize intact genotypes. Williams et al. (1987) also the ingestion, digestion, and plants, excised plant tissue, callus reported that D. grandiosella and O. assimilation of plant tissue by the tissue, and artificial diets are discussed nubilalis larvae reared for 7 days on larvae to determine how the resistant as follows. callus initiated from resistant maize plant affects its metabolism. Kumar hybrids weighed significantly less than (1993) and Ng et al. (1993) used the Growth of an insect on susceptible or those reared on callus from susceptible gravimetric method described by resistant plants is commonly hybrids. Figure 6 shows differences in Weldbauer (1968) to calculate determined by measuring the weight growth of fall armyworm larvae after approximate digestibility (AD), gain of the larvae, and the development feeding for 7 days on callus of resistant efficiency of conversion of ingested of larvae into pupae. The latter is and susceptible maize hybrids. food (ECI) and efficiency of conversion of digested food (ECD) of D. quantified as the percentage of larvae transforming into pupae, and the Artificial diets have been widely used grandiosella, C. partellus, and B. fusca. average time period required to do so to detect the presence of stem borer The calculations are done as follows: by those that pupate. Growth rate of larval growth inhibitors in maize plants stem borers on resistant and susceptible varieties of maize has been frequently measured by infesting intact plants of 3 to 4 weeks of age and by removing infested plants after intervals from 7 to 42 days of infestation (Ampofo et al. 1986; Ampofo and Kidiavai 1987; Davis and Williams 1986; Kumar et al. 1993). Each plant was carefully dissected and the number of surviving larvae and their respective growth stages and weights were recorded. Insect growth was also measured on freshly excised leaves and/or stems in laboratory assays of susceptible and resistant plants (Davis et al. 1989; Saxena 1990). Figure 6. Fall armyworm larvae after feeding for 7 days on callus of resistant (left) and susceptible (right) maize hybrids (Williams et al. 1985). A REVIEW OF ENTOMOLOGICAL TECHNIQUES AND METHODS USED TO DETERMINE MECHANISMS AND BASES OF STEM BORER RESISTANCE IN MAIZE AD = (DWF - DWE) / DWF Ba se s of Re sist a nc e 67 the trichomes were left intact. The leaf was then presented to ovipositing ECI = (DWG / DWF) x 100 There is ample evidence to suggest that females, and the number of eggs laid by Where : plant morphological and chemical the females on the hairless side was DWF = Dry weight of food ingested; characters affect normal feeding and compared with that on the intact side. DWE = Dry weight of excreta; and establishment of stem borers on maize The moths laid significantly more eggs DWG = Dry weight gained by insect plants. It is therefore important to on the hairless side than on the side elucidate the causal factors and their with trichomes. ECD = [DWG / (DWF - DWE)] x 100 Tolerance role in insect resistance and Tolerance is unlike non-preference and susceptibility. Chemical bases Artificial diets have been widely used antibiosis in that the plant does not adversely affect the behavior or biology Morphological bases to bioassay the activity of of the insect pest. Tolerance is a Trichomes, also known as hairs or allelochemicals against maize stem response by the plant to compensate for pubescence, are one of the more borers. Water extracts of host plants are damage inflicted by the herbivore. important morphological bases of plant generally added directly to the diet Tolerance can occur in combination resistance to insects. In numerous solution, whereas phytochemicals with the other two mechanisms. species, a negative correlation has been soluble in organic solvents are coated Because of its unique nature in plant established between trichome density onto alphacel, the solvent is then resistance to insects, the quantitative on the plant surface and insect feeding removed from the material under measurement of tolerance is and oviposition. Long and dense vacuum, and the remaining material is accomplished by using entirely trichomes hinder normal feeding and added to the diet as a portion of the different experimental procedures from oviposition. However, the relative alphacel component. those used to study antibiosis or non- contribution of trichome and preference. The study of tolerance nontrichome based resistance to insects Zhou et al. (1984) developed a usually involves comparing yields or may not be well understood unless technique for bioassaying water soluble plant growth characters (e.g. height) trichomes are removed to detect insect maize extracts against O. nubilalis. Diet among genotypes by using infested and resistance. Without the removal of plugs weighing 3 g were cut, frozen at - uninfested plots. trichomes, the effects of plant 10°C for 24 h and lyophilized. The allelochemicals can also be mistakenly shriveled, lyophilized plugs were Chiang and Holdway (1965) studied ascribed to trichome based resistance. dipped in plant extractables and were the relationship between plant height Ampofo (1985) studied the influence of allowed to absorb the extracts for 12 h and yield of field maize as affected by trichomes of certain maize genotypes at 4°C. After each of the diet plugs had O. nubilalis. The resistant cultivar Oh43 on C. partellus oviposition. Trichomes thoroughly absorbed the extract, the x Oh51A suffered less reduction in on the upper and lower surface of odd surplus extract on the outside of each plant height and yield than the numbered leaves were counted and plug was removed and they were susceptible cultivar WF9 x M14 with classified. Generally trichome density infested with larvae of O nubilalis. Zhou the same degree of initial infestation was highest on the resistant genotype et al. (1984) reported that neonate and suggesting that the resistant cultivar ICZ2-CM and lowest on the susceptible second-instar larvae reared for 7 days had tolerance to borer injury in Inbred A. Kumar (1992) reported that on plugs of diets absorbed with extract addition to its well recognized significantly more trichomes on the of resistant maize cultivar weighed less antibiosis which reduces borer survival. upper and lower surfaces of leaves of than the larvae on susceptible plugs. Ajala (1992) estimated tolerance levels resistant maize cultivar ICZ-T were of seven maize cultivars against C. responsible for deterring oviposition by Durbey and Sarup (1988) assessed the partellus using the following formula: C. partellus. Using a thoroughly washed antibiotic effects of resistant maize muslin cloth, Kumar (1992) removed cultivars on C. partellus by the trichomes from one side of the incorporating their water, ethyl alcohol where, YC = Yield of control plants, YI central midrib of the lower surface of or acetone plant extracts into an = Yield of infested plants, and ST = ICZ-T leaf. On the other side of the leaf, artificial diets. The ethyl alcohol fraction Tolerance = 100 x [(YC-YI) / YC] / ST stem tunneling. 68 Z.R. KHAN from a resistant Mex-17 cultivar was Simple bioassays, involving insect the most active in reducing larval and responses on plants with different pupal survival, larval weight, and morphological characters, may not fecundity of females. provide sufficient evidence to prove the real role of plant morphology in insect For O. nubilalis, Czapla and Lang (1990) resistance. Therefore, appropriate used artificial diets to study the effects techniques are needed to prove that a of plant lectins on larval development, true relationship exists between and Houseman et al. (1992) studied the physical factor(s) and resistance rather effects of DIMBOA and MBOA on their than just a simple correlation. growth and digestive processes. Ac k now le dgm e nt s Torto et al. (1991) applied test samples in solvents to both sides of cellulose The author greatly appreciate the acetate disks to study feeding responses assistance of Edna Carraway in of C. partellus. Test disks were dried preparing the manuscript for and then dampened with double publication. distilled water and offered for feeding to third-instar larvae in a no-choice Re fe re nc e s bioassay. Control disks were dipped into solvent only. Test and control disks were weighed before after larval feeding to calculate the amounts of feeding. Sum m a ry As a result of efficient entomological techniques and methods, progress in the development of insect resistant cultivars of maize has recently occurred. A good understanding of the mechanism(s) and bases of resistance is needed for establishing differences among resistant genotypes for these characters and for making intelligent decisions about using resistant germplasm in a breeding program and in integrated pest management. However, it is not unusual to find that combinations of each mechanism contribute to insect resistance, and the absolute contribution of a given resistance mechanism may never be fully elucidated. Similarly, in some cases, it is difficult to demonstrate if a certain morphological character contributes towards insect resistance. Ajala, S.O. 1993. Population cross diallel among maize genotypes with varying levels of resistance to the spotted stem borer Chilo partellus (Swinhoe). Maydica 38: 39-45. Ampofo, J.K.O. 1985. Chilo partellus (Swinhoe) oviposition on susceptible and resistant maize genotypes. Insect Science and its Application, 6: 323-330. Ampofo, J.K.O. 1986. Effect of resistant maize cultivars on larval dispersal and establishment of Chilo partellus (Lepidoptera: Pyralidae). Insect Science and its Application 7: 103-106. Ampofo, J.K.O., and Kidiavai, E.L. 1987. Chilo partellus (Swinhoe) (Lepid., Pyralidae) larval movement and growth on maize plants in relation to plant age and resistance or susceptibility. Zeitschrift fur angewandte Entomologie 5: 483-488. Ampofo, J.K.O., Saxena, K.N., Kibuka, J.G., and Nyangiri, E.O. 1986. Evaluation of some maize cultivars for resistance to the stem borer Chilo partellus (Swinhoe) in Western Kenya. Maydica XXXI: 379-389. Chang, N.T., Wiseman, B.R., Lynch, R.E., and Habeck, D.H. 1985. Fall armyworm (Lepidoptera: Noctuidae) orientation and preference for selected grasses. Florida Entomologist 68: 296-303. Chiang, H.C., and Holdway, F.G. 1965. Relationships between plant height and yield of field corn as affected by the European corn borer. Journal of Economic Entomology 58: 932-938. Czapla, T.H., and Lang, B.A. 1990. Effect of plant lectins on the larval development of European Corn Borer (Lepidoptera: Pyralidae) and southern corn rootworm (Coleoptera: Chrysomelidae). Journal of Economic Entomology 83: 2480-2485. Davis, F.M. 1985. Entomological techniques and methodologies used in research programs on plant resistance to insects. Insect Science and Its Application, 6: 391-400. Davis, F.M., and Williams, W.P. 1986. Survival, growth, and development of southwestern corn borer (Lepidoptera: Pyralidae) on resistant and susceptible maize hybrids. Journal of Economic Entomology 79: 847-851. Davis, F.M., Ng, S.S., and Williams, W.P. 1989. Mechanisms of resistance in corn to leaf feeding by southwestern corn borer and European corn borer (Lepidoptera: Pyralidae). Journal of Economic Entomology 82: 919-922. Durbey, S.L., and Sarup, P. 1988. Effect of different solvent extracts of susceptible maize germplasms on the biological parameters expressing antibiosis in Chilo partellus (Swinhoe) due to their formulation in artificial diet. Journal of Entomological Research 12: 93-97. Houseman, J.G., Campson, F., Thie, N.M.R., Philogene, B.J.R., Akinson, J.A., Morand, P., and Arnason, J.T. 1992. Effect of maize-derived compounds DIMBOA and MBOA on growth and digestive processes of European corn borer (Lepidoptera: Pyralidae). Journal of Economic Entomology 85: 669-674. Huang, X.P., Mark, T.P., and Berger, R.S. 1990. Olfactory responses of lesser cornstalk borer (Lepidoptera: Pyralidae) larvae to peanut plant parts. Environmental Entomology 19: 1289-1295. Kogan, M., and Ortman, E.E. 1978. Antixenosis - a new term proposed to replace Painter’s “non-preference” modality of resistance. Bulletin of Entomological Society of America 24: 175-176. Kumar, H. 1992. Inhibition of ovipositional responses of Chilo partellus (Lepidoptera: Pyralidae) by trichomes on lower leaf surface of a maize cultivar. Journal of Economic Entomology 85: 1936-1739. Kumar, H., Nyangiri, E.M.O., and Asino, G.O. 1993. Colonization responses and damage by Chilo partellus (Lepidoptera: Lepidoptera) to four variably resistant cultivars of maize. Journal of Economic Entomology 86: 739-746. A REVIEW OF ENTOMOLOGICAL TECHNIQUES AND METHODS USED TO DETERMINE MECHANISMS AND BASES OF STEM BORER RESISTANCE IN MAIZE Ng, S.S., Davis, F.M. and Williams, W.P. 1990. Ovipositional response of southwestern corn borer (Lepidoptera: Pyralidae) and fall armyworm (Lepidoptera: Noctuidae) to selected maize hybrids. Journal of Economic Entomology 83: 1575-1577. Ng, S.S., Davis, F.M., and Reese, J.C. 1993. Southwestern corn borer (Lepidoptera: Pyralidae) and fall armyworm (Lepidoptera: Noctuidae): comparative developmental biology and food consumption and utilization. Journal of Economic Entomology 86: 394-400. Painter, R.H. 1951. Insect Resistance in Crop Plants. New York: Macmillan. Robinson, J.F., Klun, J.A., and Brindley, T.A. 1978. European corn borer: a nonpreference mechanism of leaf feeding resistance and its relationship to 1,4-Benzoxazin-3-one concentration in dent corn tissue. Journal of Economic Entomology 71: 461-465. Saxena, K.N. 1985. Behavioral basis of plant resistance or susceptibility to insects. Insect Science and its Application. 6: 303-313. Saxena, K.N. 1990. Mechanisms of resistance/susceptibility of certain sorghum cultivars to the stem borer Chilo partellus: role of behavior and development. Entomologia Experimentalis et Applicata 55: 91-99. Saxena, K.N. 1992. Larval development of Chilo partellus (Swinhoe) (Lepidoptera: Pyralidae) on artificial diet incorporating leaf tissue of sorghum lines in relation to their resistance or susceptibility. Applied Entomology and Zoology 27: 325-330. Torto, B., Hassanali, A., Saxena, K.N., and Nokoe, S. 1991. Feeding responses of Chilo partellus (Swinhoe) (Lepidoptera: Pyralidae) larvae to sorghum plant phenolics and their analogs. Journal of Chemical Ecology, 17: 67-78. Waldbauer, G.P. 1968. The consumption and utilization of food by insects. In W.L. Beament, J.E. Treherne, and V.B. Wigglesworth (eds.) Advances in Insect Physiology, Vol. 5, J., Academic Press. Williams, W.P. , Buckley, P.M., Hedin, P.A., and Davis, F.M. 1990. Laboratory bioassay for resistance in corn to fall armyworm (Lepidoptera: Noctuidae) and southwestern corn borer (Lepidoptera: Pyralidae). Journal of Economic Entomology 83 (4): 1578-1581. Williams, W.P., and Davis, F.M. 1985. Southwestern corn borer larval growth on corn callus and its relationship with leaf feeding resistance. Crop Science 25: 317-319. 69 Williams, W.P., Buckley, P.M., and Davis, F.M. 1985. Larval growth and behavior of the fall armyworm (Lepidoptera: Noctuidae) on callus initiated from susceptible and resistant corn hybrids. J. Econ. Entomol. 78: 951-954. Williams, W.P., Buckley, P.M., and Davis, F.M. 1987. Tissue culture and its use in investigations of insect resistance of maize. Agriculture, Ecosystem and Environment 18: 185-190. Williams, W.P., Buckley, P.M., and Taylor, V.N. 1983. Southwestern corn borer growth on callus initiated from corn genotypes with different levels of resistance to plant damage. Crop Science 23: 1210-1212. Wilson, R.L., and Wissink, K.M. 1986. Laboratory method for screening corn for European corn borer (Lepidoptera: Pyralidae) resistance. Journal of Economic Entomology 79: 274-276. Wiseman, B.R., Davis, F.M., and Williams, W.P. 1983. Fall armyworm larval density and movement as an indication of non-preference in resistant corn, Protection Ecology 5: 135-141. Zhou, D., Guthrie, W.D., and Chen, C. 1984. A bioassay technique for screening inbred lines of maize for resistance to leaf feeding by the European corn borer. Maydica XXIX: 69-75. 70 An Ove rvie w of Re se a rc h on M e c ha nism s of Re sist a nc e in M a ize t o Spot t e d St e m Bore r H. Kumar, Faridabad, Haryana, India Abst ra c t The spotted stem borer Chilo partellus (Swinhoe) (Lepidoptera:Pyralidae) is an important pest of maize in several countries of Asia and Africa. Serious crop losses have been reported, mostly in experiments conducted under artificial infestations at experimental stations. In order to develop economical and environmentally friendly methods of pest management, a large number of maize genotypes with varying level of resistance to C. partellus have been identified. In the identified resistant germplasm, the three components of resistance, namely, non-preference, antibiosis, and tolerance, have been identified. In Asia, various studies have been conducted to elucidate the mechanism of resistance/ susceptibility in the two maize genotypes, Antigua Group 1 (Resistant) and Basi Local (Susceptible), against C. partellus. Several biological parameters including C. partellus larval and pupal survival, larval and pupal weights, larval and pupal period and fecundity were adversely affected due to unknown factors in the resistant source, but not on the susceptible one. An ethanolic extract of Mex 17 has also been reported to inhibit growth and development of C. partellus in comparison to the susceptible genotypes. The studies conducted in Africa show that ovipositional nonpreference by C. partellus on maize genotypes was due to trichomes and surface waxes. A genotype, ICZ-T, with trichomes on both the leaf surfaces was also developed. In some studies, using regression of grain yield reduction on foliar injury due to C. partellus attack on maize genotypes, namely, ICZ1-CM and ICZ2-CM, antibiosis and tolerance were reported to be the components of resistance. In more detailed studies in Africa, non-preference, antibiosis and tolerance types of resistance mechanisms have been reported to be operating within maize genotypes Mp704, Poza Rica 7832 and ER - 29SVR. The resistance mechanisms operating within these sources have also been reported to be expressed in the crosses with agronomically desirable sources. I nt roduc t ion feasible method of crop protection, is • Unawareness among the farmers absent in the commercial maize regarding the existence of plant Maize, Zea mays L., is an important varieties in the developing countries. resistance to insects and the cost/ staple food for millions of people in There are several reasons for the benefits ratios of using plant Africa, Asia and Latin America where it unpopularity of host plant resistance in resistance as a control tactic. serves as a human subsistence crop. these countries: However, the grain yield per hectare is • Intense competition between the Notwithstanding the above problems, low (2.2 t/ha) in comparison to the multinational insecticide exhaustive information has been developed countries (5-6 t/ha). Of the manufacturing companies and the generated on screening of maize various major constraints responsible resource poor national programs of the developing countries. for the low maize production in the developing countries (Table 1), insect • Lack of active collaboration among pests are the most destructive and different members of the unmanageable because the chemical multidisciplinary team needed for control tactics are inaccessible to the the development of resistant farmers. Host plant resistance, which is varieties with good agronomic the cheapest and biologically, background. ecologically, economically and socially Table 1. Major constraints to maize production. 1. Inaccessibility to expensive fertilizers 2. Costs of certified seeds of the improved commercial varieties/hybrids 3. Unreliable and erratic rainfall in the major maize growing areas (>80%) 4. Pests (insects and non insects), diseases, weeds OVERVIEW OF RESEARCH ON MECHANISMS OF RESISTANCE IN MAIZE TO SPOTTED STEM BORER 71 genotypes for resistance to insects as the maize plant suffers damage by two feeding by the larvae in the leaf whorl, well as on the mechanisms of resistance generations of C. partellus. (Fig. 1). The the central shoot dries up and the plant first generation C. partellus attack on can not grow any more. This type of maize commences at the early whorl damage is termed as ‘dead heart’ In Asia and Africa , three major species stage. Neonates hatching from the eggs (Kumar and Asino 1993) (Fig. 3). The of Chilo infest maize (Table 2). Of these, laid by C. partellus on the basal leaves of older larvae leave the leaf whorl and the spotted stem borer Chilo partellus the early whorl stage maize, disperse bore into the stem to cause stem (Swinhoe) is the most important. In the and enter the leaf whorl where they tunneling. The second generation C. literature, the common name of this feed and cause damage to the leaves partellus attack commences at anthesis. stem borer has been too variable. The (Figs. 2 and 3). Because of the extensive Neonates feed inside the leaf sheaths in selected maize genotypes to insects. spotted stem borer should be used irrespective of the crop it infests. It is distributed widely in India, Pakistan, Indonesia, Sri Lanka, Thailand, Oviposition on basal leaves of whorl stage maize (1-2 WAE) Arrival in the central whorl to commence feeding Day 0 Feeding continues until the larvae are in the fourth instar or whorl leaves unfurl fully and tasssel emerges First Generation Chilo partellus (Swinhoe) Temp + Day = 36±1ºC Night = 25±1ºC RH = 60% Arrival in the leaf sheath and stem for feeding Ethiopia, Kenya, Somalia, Tanzania and South Africa (Seshu Reddy 1983; Hamburg 1979) (Table 3). In India, the pest is active during July to September and remains dormant during November to April (Fletcher and Ghose 1920; Rahman 1944) (Table 4). In Africa, Adult eclosion Pupation in stem, leaf Sheath, ear, tassel C. partellus remains active throughout the year (Ampofo 1985). Until harvest, Table 2. Major species of Chilo infesting maize. Chilo partellus (Swinhoe) Asia and Africa Chilo agamemnon (Blezynski) Egypt Chilo orichalcociliellus Strand Coastal Kenya and Madagascar Pupation in stem, leaf sheath, tassel penducle and cob Aestivation Second-generation Chilo partellus Adult eclosion (Day 40) Arrival in the leaf sheath, husks for feeding Oviposition on leaves of plants at flowering or post-flowering stages Enter in the stem and cob for feeding Figure 1. Biological relationships between C. partellus and the maize plant. Table 3. Distribution of C. partellus. 1. India, Pakistan, Indonesia, Sri Lanka, and Thailand (CIA Map 184) 2. Ethiopia, Kenya, Somalia, Sudan, Tanzania and Uganda (Seshu Reddy 1983) 3. South Africa (Hamburg 1979) Table 4. Seasonal occurrence of C. partellus. India (Fletcher and Ghose 1920) Kenya (Ampofo 1985) Peak activity JulySeptember June, August, Dec., January Dormant Period Nov.-April None Figure 2. Foliar damage on maize by stem borers. Figure 3. Dead heart caused by the stem borers to the maize plants. 72 H. KUMAR and ear husks (Kumar 1992b). Older adults to infest maize (Ampofo et al. and exit holes and stalk breakage by C. larvae bore into the stem and ear to 1986), because under natural partellus to distinguish resistant and cause stem tunneling and ear damage. conditions, oviposition by the adults on susceptible genotypes. The ratio of each Reference to the literature shows that the plants is the first step to start the parameter’s value for a test cultivar to maximum mating by C. partellus occurs infestations. However, this method is that for the susceptible check was the first night after the emergence and less practical because large arenas are computed. The relative ratios of all the that maximum oviposition occurs the needed to confine the flying adults on parameters for each genotype were first night after mating (Kumar and the plants. All these methods are useful then averaged to give the overall Saxena 1985b). The mating is confined to elucidate the mechanisms of resistance/susceptibility index (ORSI). to the second half of the night (after resistance in selected maize genotypes The lower the ORSI value of a midnight) while oviposition is to C. partellus. genotype, the greater would be the resistance to C. partellus and vice versa. restricted to the first half of the night (before midnight). Unnithan and Kumar (unpublished data) also However, such a method is not suitable Saxena (!990) monitored C. partellus conducted trials to simulate the natural for rapid screening of maize germplasm populations by using live females in the infestation by planting border rows of in a breeding program. Plus, the traps. The complete chemical nature of the susceptible genotype and infesting secondary damage parameters, like C. partellus sex pheromone has not been with C. partellus. The planting dates of entry holes or stalk breakage, are elucidated yet, although some the test genotypes were then adjusted considered on a par with the primary components have been reported to in such a way that the plants were at 6- damage parameters. Kumar and Asino attract males over short distances (Lux 8 leaf stage when the adults emerged (1993) suggested foliar damage, dead et al. 1994). from the infested border rows and heart and stalk damage on maize by C. started to infest the test genotypes. partellus to clearly distinguish the Grain yield losses due to C. partellus However, this method was not very resistant and susceptible genotypes. attack on whorl stage maize have been successful in the tropical environment, Various workers have used different reported to vary with the cultivars because the survival of the larvae on plant growth stages to screen maize for (Ampofo 1986; Kumar 1988a; Seshu border rows was not sufficient to be resistance to C partellus. Ampofo et al. Reddy and Sum 1992), infestation levels transformed into adults to infest test (1986) used 4 week old plants to infest (Chatterji et al. 1969; Kumar 1988a; genotypes adequately. However, this and screen maize for resistance to C. Sarup et al. 1977; Seshu Reddy and Sum method would be suitable on a small partellus. Kumar and Asino (1993) 1992) and crop phenology at infestation scale in the screen house. Nevertheless, demonstrated that resistant and (Sarup et al. 1977; Seshu Reddy and to distinguish resistant and Sum 1992). Yield losses due to C. susceptible maize genotypes, partellus attack at anthesis have also infestation with larvae has been been reported to vary with cultivars reported as more effective than and infestation levels (Kumar and the egg masses (Kumar, in press). Asino 1994). The hand operated device called “bazooka” can be adapted by the A meridic diet for C. partellus has been entomologist to screen maize developed and used successfully to rear germplasm for resistance to this stem borer (Siddiqui et al. 1977; C. partellus(Fig. 4). Seshu Reddy and Davies 1978; Ochieng et al. 1985). The egg masses at the black A number of parameters have head stage or neonates have been used been used by various workers to to infest maize genotypes to determine assess damage by C. partellus. their resistance or susceptibility to C. Ampofo et al. (1986) used partellus. (Sarup 1983; Kumar 1993a,b, number of egg masses, foliar 1994; Kumar et al. 1993). Some damage, percentage of stem researchers have also used C. partellus length tunneled, number of entry Figure 4. Bazooka for the artificial infestation of maize plants by stem borers OVERVIEW OF RESEARCH ON MECHANISMS OF RESISTANCE IN MAIZE TO SPOTTED STEM BORER • 73 susceptible genotypes were clearly European Corn Borer, Ostrinia nubilalis, distinguished when infested at 2 weeks have been found susceptible to C as non-preference (Painter 1958) and after the germination of the plants. partellus (Ampofo et al. 1986). Several antixenosis (Kogan and Ortman Resistance to C. partellus at the early land races and commercial maize 1978). whorl stage is desirable because hybrids from Kenya have also been economic losses have been reported to found susceptible to C. partellus (Kumar decline with the advance in the age of 1994a). Little information is available the plant (Sarup et al. 1977; Seshu on sources of resistance to second- Reddy and Sum 1992). generation C. partellus. Kumar (1992b) and regeneration of their damaged studied the larval establishment and tissues. • Preference subsequently referred to Antibiosis affecting insects survival, development and egg production on the plants. • Tolerance in plants involving repair Based on the information generated damage by C. partellus on plants at above, many maize genotypes with anthesis. Severe yield losses can occur To establish the above three resistance to C. partellus have been at anthesis because C. partellus attacks components of resistance in plants to identified (Sarup et al. 197; Ampofo et maize directly in the growing ear. insects, certain responses of the insects al; 1986; Kumar 1991; Kumar and Kumar and Asino (1994) and Kumar to the plants can be studied as Saxena 1992; Kumar 1994a,b,c). The (1994c) identified a few sources of explained by Saxena (1969, 1985) and most notable sources of resistance to C resistance to second-generation C. are summarized in Table 6. The partellus are Antigua Group 1, partellus. responses are: Com pone nt s of Re sist a nc e in M a ize t o C. pa rt e llus • • • • • • • Population 590 (Multiple Borer Resistant, MBR) of CIMMYT, Population 390 (Multiple Insect Resistant Tropical, MIRT) of CIMMYT (Table 5 ), and several inbred lines from Painter (1951) proposed three main Mississippi and CIMMYT. Several lines categories of resistance in plants to with a high level of resistance to insects: Table 5. A comparison of infestation and damage (mean ± se) caused by C. partellus among maize cultivars from Kenya and CIMMYT (Mexico). Maize cultivar Inbred A Mp 704 EV SR BC 4/8429 EV SR BC 6/8430 EV SR RSF /8343 EV SR BC 6/8744 EV SR BC 5/8749 Tuxpeño Sequia La Posta Sequia Pool 16 Sequia Hybrid 622 Pwani hybrid Hybrid 511 MIRTb FAM. 1 MIRTb FAM. 2 b MIRT FAM. 18 MIRTb FAM. 99 MIRTb FAM. 136 b MIRT FAM. 170 F (df = 17.17) LSD (P = 0.05) a b Source Kenya Mississippi CIMMYTa a CIMMYT a CIMMYT CIMMYTa a CIMMYT CIMMYTa CIMMYTa a CIMMYT Kenya Kenya Kenya CIMMYTa CIMMYTa a CIMMYT CIMMYTa CIMMYTa a CIMMYT No. of larvae recovered — 4 ± 0.6 12 ± 1.5 8 ± 2.3 7 ± 1.3 6 ± 1.0 4 ± 0.8 7 ± 0.4 9 ± 2.0 5 ± 0.9 11 ± 0.6 5 ± 0.8 8 ± 1.3 4 ± 1.2 4 ± 0.1 8 ± 2.5 6 ± 2.0 4 ± 0.7 7 ± 2.2 4.9 3.2 Foliar damage ratings a 9±0 4 ± 0.5 8 ± 0.3 6 ± 0.4 7 ± 0.4 8 ± 0.6 7 ± 0.1 8 ± 1.1 7 ± 1.3 6 ± 1.0 8 ± 0.8 7 ± 1.2 8 ± 0.2 4 ± 0.2 4 ± 0.2 4 ± 0.6 4 ± 1.0 4 ± 0.7 4 ± 0.9 4.4 1.8 International Maize and Wheat Improvement Center. Multiple Insect Resistant Tropical. Orientation. Feeding. Metabolism of the ingested food. Development of larva. Egg production in the adults. Oviposition. Hatching. Orientation, feeding and oviposition responses by the insects are involved in % plants showing dead hearts % stem length tunneled the non-preference type of mechanisms 74 ± 10 11 ± 11 5 ± 25 10 ± 10 13 ± 13 6±6 0 47 ± 9 0 0 29 ± 11 30 ± 30 18 ± 18 0 0 0 0 0 0 3.06 37.60 — 12 ± 0.9 47 ± 3.5 39 ± 1.5 42 ± 4.5 41 ± 2.1 39 ± 2.5 39 ± 3.5 56 ± 5.5 29 ± 1.0 54 ± 5.5 48 ± 8.5 37 ± 2.5 19 ± 1.0 29 ± 1.5 32 ± 7.0 15 ± 1.5 16 ± 4.5 27 ± 3.0 11.5 11.5 responses. The metabolic responses of of resistance in plants which possess characteristics to inhibit these the insect would involve antibiosis type of mechanisms of resistance in plants which will provide inadequate nutrients or metabolic inhibitors to cause failure of larval development , survival, egg production and hatching of the eggs. Re sponse s of I nse c t s t o Pla nt s Orientation This insect response determines the establishment of the insect on the plant in two ways. Firstly, an insect may be 74 H. KUMAR attracted to a plant or repelled from it Kumar 1994b). Secondly, C. partellus Feeding response because of a certain attractant or larvae emerging from the eggs laid on After the arrival of C. partellus larvae in repellent, respectively. If the insect is the leaves may continue to stay on the the leaf whorls, the establishment of its attracted to a plant, the chances of its plant and reach the feeding sites in the population on the plants would depend establishment on the plant would be leaf whorls, or may depart from the on larval feeding in the leaf whorls. enhanced. On the contrary, if the insect plant during their movements from the Feeding responses of C. partellus on is repelled from the plant, the chances oviposition site (basal leaves) to the plants can be studied in the laboratory, of its establishment on the plant would feeding site (leaf whorl) due to various as well as in the field, as described by be reduced. The attraction/repulsion morphological and biochemical factors. Kumar et al. (1993) and Kumar and could be for feeding in the case of Kumar et al. (1993) compared four Saxena (1992). In the laboratory, the larvae or oviposition in the case of maize genotypes for larval orientation yellow green portions of the unfurled adults. The role of larval orientation in from oviposition to feeding sites (Table whorl leaves of 3 week old plants can determining resistance/susceptibility of 7). The maize genotypes Mp704 and be offered to neonates of C. partellus in maize genotypes has not been studied, Poza Rica 7832 seem to possess glass vials (7.5 cm x 2.5 cm) filled to a but C. partellus adults have been characteristics which suppressed the depth of 2 cm with 2% agar gel. After reported to be attracted equally by the movements of larvae from oviposition 24-48 hours, the area eaten by the larvae resistant and susceptible genotypes for to the feeding sites. on resistant and susceptible maize genotypes can be measured with an oviposition (Kumar and Saxena 1985a; area meter or by a dotted paper sheet Table 6. Mechanisms of resistance as related to the responses of insects or plants. (Letra set International Ltd., UK). Using Categories or mechanisms of resistance (Painter 1951, 1958) demonstrated that C. partellus larvae fed Non-preference [= antixenosis, Kogan and Ortman (1978)] Antibiosis Tolerance Responses of insects or plants involved Orientation of insects Repulsion: avoidance/departure from plants Attraction: arrival and stay on plants Feeding: inhibition stimulation Oviposition: inhibition stimulation Metabolism of food ingested by insects: nutrition metabolic disturbance Development in the larval stage* Survival and egg-production in the adult stage* Repair, regeneration of damaged tissues of plants Differences between resistant (R) and susceptible (S) plants Cultivar Inbred A Mp 704 V-37 Poza Rica 7832 % First instars arrested in 72 h 39.3 ± 9.7a 21.7 ± 3.8b 38.7 ± 2.1a 20.0 ± 4.0b Means ± SE. Means followed by the same letters are not significantly different (P > 0.05; Duncan’s multiple range test). One-way ANOVA of arcsin-transformed data (F = 10.88; df = 3.6; P < 0.01). less on the resistant genotypes (Mp704, V-37 and Poza Rica 7832) in comparison to the susceptible genotype (Inbred A) (Table 8). In the field, the resistant and R>S R<S R>S R<S R>S R<S susceptible genotypes can be grown and 2-3 weeks after plant emergence, the plants are infested with 20 neonates in the leaf whorls. After 24-48 hours, the feeding lesions on the plants are R<S R>S R<S R<S R>S measured as described above. Kumar and Saxena (1992) reported significantly more feeding by C. partellus on the susceptible than the resistant * All failures of insects, survival, development and egg-production (fecundity) need not represent antibiosis; such failures caused by inadequate food-intake would correctly belong to the category non-preference for feeding. Table 7. Chilo partellus larval arrest on different maize cultivars. this technique, Kumar et al. (1993) genotypes. Table 8. Chilo partellus larval feeding responses to different maize cultivars, each offered alone. Cultivar Inbred A V-37 Poza Rica 7832 Mp 704 Fresh wt (mg) of Leaf area (mm2) eaten by 10 first-instars/ 24 h a, c 20.2 ± 4a 2.3 ± 1b 3.3 ± 2b 6.0 ± 3b food ingested by fourth instars/ 48h b,d 1,227 652 766 1,149 ± ± ± ± 254a 107a 86a 158a Means ± SE. Means within a column followed by different letters are significantly different (P < 0.05). a Disk of unfurled whorl leaf of 3-wk-old plant offered to larvae. b A 6-cm-long basal internode of stem of 5-6-wk-old plant offered to larvae for feeding. c One-way ANOVA (F = 31.7; df = 3, 9; P < 0.01). d One-way ANOVA (F = 2.60; df = 3, 27; P > 0.05; LSD = 514.3). OVERVIEW OF RESEARCH ON MECHANISMS OF RESISTANCE IN MAIZE TO SPOTTED STEM BORER 75 Metabolism of the ingested food in each replicate, I, is calculated on a The efficiency with which digested The next step which determines the dry weight basis as: food is converted to body matter (ECD) FI (FC2/FC1) - F2 establishment of C. partellus population is calculated as: on the plant and its successful Where FI = initial fresh weight of food, ECD = (G/I-E) * 100 colonization is the efficient metabolism F2 = dry weight of uneaten food, FC1 = ECI = AD * ECD of the food ingested by the larvae. The fresh weight of control food, and FC2 = experiments are conducted in the dry weight of control food. The only notable report which describes the metabolism of food laboratory with excised leaves of the resistant and susceptible genotypes. The weight gain of the insect, G, is ingested by C partellus on the resistant Only the yellow-green portions of the calculated on a dry weight basis as: and susceptible genotypes is that of W2 - W1 (WC2/WC1) Kumar (1993a). According to this Where W1 = initial weight of the insect report, the dry weight of the food experiments are conducted in plastic before feeding W2 = dry weight after ingested by C partellus larvae on the vials (3 cm high by 4 cm diameter) filled feeding, and WC1 = fresh weight of resistant inbred Mp704 and the single to a depth of about 1 cm with 2% agar control insects. cross hybrid Mp704 x Inbred A was unfurled whorl leaves (minus midrib) of 3 week old plants are used. The lower than the susceptible check Inbred gel. The gel keeps the paper toweling placed beneath the leaf tissue moist and The relative consumption rate (RCR), A (Table 9). The ECI and ECD on the prevents the tissue from wilting. The the amount of feeding relative to time resistant cultivars were also lower than leaf tissue in each vial is presented to 10 and to the mean weight of larvae the susceptible check. Thus the resistant neonates of C. partellus in the form of a during the feeding period, is calculated inbred and the cross involving a disk (2 cm diameter). The initial fresh as: resistant parent had a deleterious RCR = I/(T * W) effects on the ingestion of the food and measured on a Sartorius (R200D) Where I = dry weight of food ingested, its subsequent utilization by the larvae. balance (Sartorius GMBH, Goltingen, T = duration of feeding period in days, The larvae gained less weight on the Germany). After 60 h, the larvae, the and W = mean weight of larva during resistant cultivars in comparison to the uneaten part of the leaf disk, and larval feeding period. susceptible ones. re-weighed. These measurements are The relative growth rate (RGR) is taken on five replicates of 10 larvae calculated as: Survival, growth and development weights of the larvae and leaf disk are frass are collected, dried separately, and each. RGR = G/(T * W) This aspect can be studied in the screen Where G = dry weight gained by larva, house or field. Under field conditions, it To determine the initial dry weight of T = duration of feeding period in days, is difficult to avoid natural infestation food offered to the larvae, the fresh W = mean weight of larva during of the borers and the data gets weights of 10 leaf disks (2 cm diameter) feeding period. confounded. Hence, experiments in the are measured separately for each screen house can help avoid natural cultivar. They are then dried at 60oC for Utilization of food consumed is infestation of the stem borers. The 24 h, and the mean weight per unit calculated by the methods of plants of the resistant and susceptible fresh weight is calculated and is used to Waldbauer (1964) and Okech and genotypes are grown in the screen calculate the initial dry weight of each Saxena (1990) using the data obtained house in a replicated trial. The plants leaf disk offered to the larvae for on food intake described above. are infested at the 6-8 leaf stage with 20 feeding. The fresh weight of 200 larvae per plant. At 15-20 days after neonates in four replicates of 50 each is Approximate digestibility (AD) is infestation, the percentage of larvae measured and dried without feeding. calculated as: recovered from each genotype is (I - E/I) * 100 recorded. On the basis of head capsule Using this information the initial dry Where E = dry weight of frass widths, the recovered larvae are then weight of 10 experimental larvae produced. classified in their respective instars. A offered leaf disks is estimated. The greater percentage of larvae advancing quantity of food ingested by 10 larvae to older instars on the susceptible, 76 H. KUMAR compared to the resistant genotype, The maize lines are grown in a Merck, Germany). Hence, to this would reflect the suitability of former screenhouse. When the plants are 3 sorghum leaf and bean powder and unsuitability of the latter for the weeks old, the leaf whorls are deficient diet, dry maize leaf powders development of the larvae. Using this harvested. After discarding the outer of the maize cultivars are incorporated. technique, Kumar et al. (1993) reported leaf, the whorls are trimmed to 20 cm, The following test diet has been that the percentage of larvae recovered dried in an oven at 60oC for 24 h, and devised: 130 ml distilled water, 1.7 g from the resistant cultivars Mp704, V- ground in an electric blender. brewer’s yeast, 200 mg sorbic acid, 500 mg ascorbic acid, 300 mg methyl-p- 37 and Poza Rica 7832 at 15 days after infestation was significantly lower than The standard artificial diet of C. hydroxybenzoate, 200 mg vitamin E, 83 the susceptible genotype (Fig. 5). Of the partellus (Ochieng et al. 1985) is ml distilled water, 2.12 g agar, and 32 g larvae recovered from the susceptible modified to study the effects of the dry dry maize leaf powder. The artificial genotype, most were in the fourth leaf powders on growth of C. partellus. diet is dispensed into glass vials, (7.5 instar and a few had advanced to the After preliminary experiments, it was by 2.5 cm diameter) fitted with plastic fifth instar. On the resistant cultivars, found that there was no larval survival lids with 40 mesh screen. the percentage of the larvae in fourth if two ingredients of the standard diet, instar was significantly lower than the sorghum leaf powder and bean For each cultivar, eight glass vials filled susceptible genotype (Fig. 5). powder, were removed and with diet to a depth of 6 cm are compensated with an equivalent prepared. On the following morning, amount of cellulose powder (Avicel, E. each of the eight glass vials is infested Similarly, several workers in Asia studied the survival and development of C. partellus in the laboratory (Sharma 100 100 and Sajjan 1987). According to these workers, survival, growth and development of C. partellus on Antigua Group 1 was lower than the susceptible check. % larvae recovered 1980; Durbey and Sarup 1984; Sekhon 80 a 60 40 b b b 2 laboratory by incorporating dry leaf powders of resistant and susceptible maize genotypes into the artificial diet. MP-704 0 V-37 Poza Rica 7832 C. partellus can also be studied in the Inbred-A Survival, growth and development of % larvae in different instars and Chatterji 1971, 1972; Lal and Pant 80 60 40 20 0 2 3 4 5 Inbred-A 2 3 4 5 V-37 2 3 4 5 Poza Rica 7832 2 3 4 5 MP-704 Figure 5. C. partellus larval survival and development on resistant and susceptible maize plants. Table 9. The utilization of leaf tissue from two inbred maize cultivars and their reciprocal crosses by first-instar C. partellus. Cultivar ‘Inbred A’ ‘Mp704’ ‘Mp704’ x ‘Inbred A’ ‘Inbred A’ x ‘Mp704’ Ia 5.2 + 0.3a 2.5 + 0.2b 1.8 + 0.2b 0.6 + 0.2c (RCR)b a (5.0 ) a (4.2 ) (2.5b) b (1.3 ) ADc ECDd ECIe 78.2 + 1.2a 89.4 + 2.9a 64.2 + 6.0b 53.2 + 3.0b 9.2 + 0.2b 1.5 + 0.4c 21.7 + 6.1a 5.7 + 2.0c 7.1 + 0.2b 1.4 + 0.4c 12.5 + 2.4b 3.1 + 0.7c Gf (RGR)g 0.40 + 0.02a 0.03 + 0.006c 0.21 + 0.02b 0.02 + 0.01c Mean + SE (n = 4 replicates of 10 larvae). Means in a column followed by the same letter are not significantly different (P > 0.05) by LSD test. a Dry weight of food ingested. (ANOVA test: F = 62.8; df = 3, 12; P <0.01) LSD = 0.75 b Relative consumption rate. (ANOVA test: F = 17.5; df = 3, 12; P <0.01) LSD = 1.18 c Approximate digestibility. (ANOVA test: F = 13.60; df = 3, 12; P <0.01) LSD = 13.24 d Efficiency of conversion of digested food. (ANOVA test: F = 3.68; df = 3, 12; P <0.05) LSD = 9.48 e Efficiency of conversion of ingested food. (ANOVA test: F = 3.60; df = 3, 12; P <0.05) LSD = 3.92 f Larval growth. (ANOVA test: F = 96.13; df = 3, 12; P <0.01) LSD = 0.043 g Larval growth rate. (ANOVA test: F = 97.92; df = 3, 12; P <0.001) LSD = 0.015 (0.4a) (0.05c) (0.27b) (0.04c) OVERVIEW OF RESEARCH ON MECHANISMS OF RESISTANCE IN MAIZE TO SPOTTED STEM BORER 77 with 15 neonates of C. partellus. The standard diet (Table 10). Among the it dies. Durbey and Sarup (1984) and glass vials are arranged in a completely diets containing dry leaf powders of the Sharma and Chatterji (1971) reported randomized design in a room maize cultivars, the average instar on that fewer eggs were laid by C. partellus maintained at 27-29oC, 40-50% RH and the deficient diet containing ‘Inbred-A’ females which were reared on the a photoperiod of 12:12(L:D)h. Each vial leaf powder was significantly higher resistant Antigua Group 1 in with 15 larvae is considered a replicate. than that on the diets containing leaf comparison to the susceptible Basi After 20 days, the percentage of larvae powders of ‘Mp704’or the F1 hybrids . Local. Sekhon and Sajjan (1987), on the recovered from each vial is recorded. The mean weight of the larvae reared other hand ,did not find any difference The head capsules of the recovered on the diet containing the leaf powder in the fecundity of C. partellus reared on larvae are measured at their greatest of ‘Inbred A’ was significantly greater these two genotypes. widths, with a stereomicroscope fitted than that of those reared on the diet with a calibrated eye piece micrometer. having ‘Mp704’ leaf powder . Larval Ovipositional responses Larvae are measured at a magnification weights on ‘Mp704’ x ‘Inbred A’ and This aspect can be studied in the field of 40X. The head capsule widths can be ‘Inbred A’ x ‘Mp704’ were intermediate by growing the resistant and used to determine the instar of the between those of the two parental lines. susceptible genotypes in the field under larvae collected. Using this This technique can be used only to natural infestation (Ampofo 1985; information, the average instar of C. establish the mechanisms of resistance Kumar 1988b) or by growing and partellus in each treatment is calculated. in maize to C. partellus, but can not exposing the genotypes in the specially Weights of the surviving larvae are also replace the conventional screening constructed cages to the ovipositing measured. Using this technique techniques in the fields. The level of females (Kumar and Saxena 1985) Field (Kumar 1993a) demonstrated that resistance in a genotype established tests by Ampofo (1985) revealed when first instars of C. partellus were with this technique may not conform differences in C. partellus oviposition on reared on the standard artificial diet, with that in the field because of the the resistant and susceptible genotypes. almost 95% of the larvae survived for absence of strong genotype x Durbey and Sarup (1982) reported 20 days after the infestation (Table 10). environment interactions. ovipositional non-preference for certain When dry maize leaf powders of resistant genotypes. In field conditions, Egg production in the adult and their viability the differences observed between the into the sorghum leaf and bean powder-deficient diet, larval survival This aspect can be studied by rearing C. may not necessarily be due to plant was equally high on the diets except for partellus neonates on the susceptible characteristics alone because certain deficient diet + ‘Inbred A’ x ‘Mp704’ and resistant genotypes. Single pairs of non-plant characteristics have also been (Table 10). Of the surviving larvae on adults emerging from the pupae reared reported to influence C. partellus the diets containing leaf powders of the on these genotypes are confined in the orientation and subsequent oviposition maize cultivars, the average instar was oviposition cages to determine the by the females (Kumar 1994b). In more significantly lower than that on the number of eggs laid by the female until detailed studies, Kumar and Saxena different cultivars were incorporated resistant and susceptible genotypes (1985a) compared ovipositional Table 10. Growth and development of C. partellus larvae on artificial diets containing dry maize leaf powders of two inbred cultivars and their reciprocal crosses. Treatment % survival Base diet a SLBPDD + ‘Inbred A’ SLBPDD + ‘Mp 704’ SLBPDD + ‘Mp 704’ x ‘Inbred A’ SLBPDD + ‘Inbred A’ x ‘Mp704’ 95.1 ± 3.5a 91.7 ± 2.7a 86.6 ± 6.7ab 83.4 ± 5.2ab 79.3± 3.9b Instar 4.9 ± 0.03a 4.4 ± 0.08b 4.0 ± 0.06cd 4.1 ± 0.08c 3.8 ± 0.06d Wt (mg) 59.0 ± 2.0a 57.0 ± 1.0ab 34.0 ± 1.0d 54.0 ± 2.0b 49.0 ± 1.0c Mean ± S.E. (n = 8 containers of 15 neonates) measured 20 d after inoculation. Means in a column followed by the same letter are not significantly different. (P > 0.05). ANOVA tests: % survival (F = 5.4; df = 4, 28; P < 0.05; LSD = 5.2), Instar (F = 47.28; df = 4, 28; P < 0.01; LSD = 0.19), Larval weight (F = 50.49; df = 4, 28; P < 0.01; LSD = 4.13). a SLBPDD, sorghum-leaf and bean powder deficient diet. responses of C. partellus to different susceptible and resistant genotypes in the field or screen house in such controlled conditions that the differences in the responses were clearly shown to be caused by the plant characteristics and not influenced by the environment or other stimuli. These workers experimentally demonstrated that variation in the humidity stimuli in the vicinity of the plants was capable of influencing oviposition by C. partellus. 78 H. KUMAR The reduced number of eggs laid by the reports are those of Ampofo (1986) and unit larval weight gain in comparison females on the resistant maize Kumar (1994c). Using regression of to the susceptible Inbred A. The field genotypes was due to contact- grain yield reduction on foliar damage tests revealed that C. partellus perceivable characters (surface waxes, ratings due to C. partellus, Ampofo infestation caused a significant trichomes, etc.) (Table 12) rather than (1986) demonstrated the presence of reduction in the grain yield of the due to distance-perceivable ones tolerance in resistant genotypes ICZ1- susceptible cultivar, but not that of (hygro, visual and olfactory stimuli) CM and ICZ2-CM (Fig. 6). Kumar resistant cultivars (Table 11). (1994c) used regression of functional (Kumar 1994c). plant loss index (FPLI) on leaf feeding T ole ra nc e in M a ize t o C. pa rt e llus Role of Pla nt Cha ra c t e rist ic s in De t e rm ining Re sponse s of I nse c t s damage by C. partellus to elucidate the presence of tolerance of in maize genotypes, ER-29SVR, MBR8637 and This aspect has not been studied Poza Rica 7832 (Fig. 7). There was a adequately well in maize resistance to significant biomass loss by the plant C. partellus although this is the most with unit increase in the larval biomass After determining the components of desirable type of resistance in plants. on the susceptible Inbred A. Besides resistance in plants to insects, the next With tolerance as a mechanism of displaying a moderate degree of step in understanding the mechanisms resistance to insects, the insects are antibiosis against C. partellus, the plants of resistance in plants to insects is to relieved of the strong selection pressure of ER-29SVR, MBR8637 and Poza Rica examine the role of plant characteristics evident in the case of strong antibiosis 7832 lose very little plant biomass per in determining the responses of the in plants to insects. The most notable 100 Percentage of eggs laid (mean ± SE)* Test material A B Inbred A ICZI-CM Inbred A None None ICZI-CM A B 69 ± 4.5 73 ± 8.0 53 ± 4.0NS 31 ± 4.5 27 ± 8.0 47 ± 4.0 Inbred A ICZ 1—CM ICZ 2—CM 80 % Yield reduction Table 11. Ovipositional responses of C. partellus to distance-perceivable characters of a susceptible and a resistant maize genotype. Antibiosis + Tolerance — 60 40 20 Antibiosis + Tolerance + A B Inbred A ICZI-CM Inbred A Glass Glass ICZI-CM Leaf portion* TL TL BU TU BL TL A 81 ± 7 73 ± 5 67 ± 8 72 ± 9 58 ± 11NS 74 ± 7 B 19 ± 7 27 ± 5 33 ± 8 28 ± 9 42 ± 11 26 ± 7 NS = not significantly different from B. * BU and TU = basal and terminal portions of the upper leaf surface respectively. BL and TL = basal and terminal portions of lower leaf surface respectively. 60 Antibiosis + 40 Tolerance — 9 Antibiosis — Tolerance — ŷ = 3.94 + 1.69 x r = 0.332 (n = 120) P _ < 0.01 20 % FPLI Percentage of eggs laid (mean ± SE) 2 3 4 5 6 7 8 Foliar damage at minimum expression Figure 6. Components of resistance in maize to Chilo partellus taking % yield reduction and the foliar damage by the stem borers as the parameters. Table 12. Oviposition responses of C. partellus to contact-perceivable characters of a susceptible and a resistant maize genotype. Test material Antibiosis — Tolerance + 0 1 * Data based on 40-50 females in 4-5 replicates of 10 each. Significantly different from ‘B’ at P = 0.05. NS = not significantly different from ‘B’ at P = 0.05. Antibiosis — Tolerance — 0 -20 -40 Antibiosis + Tolerance + Antibiosis — Tolerance + -20 0 4 8 12 16 Dry weight (mg/larva) 20 Figure 7. Components of resistance in maize to Chilo partellus taking larval weight as an indicator of antibiosis and FPLI as an indicator of tolerance. Empty circles represent Inbred A (susceptible); solid circles represent MBR 8637 (resistant); empty triangles represent ER-29SVR (resistant); and solid triangles represent Poza Rica 7832 (resistant). OVERVIEW OF RESEARCH ON MECHANISMS OF RESISTANCE IN MAIZE TO SPOTTED STEM BORER 79 insects. Excellent reviews are available and these trichomes were equally plants, leaf surface waxes of the on the role of plant characteristics in effective in inhibiting oviposition by the resistant genotype Mp704 were less determining the resistance/ females. effective than those of the susceptible genotype Inbred A (Fig.9) in eliciting susceptibility of the plants to insects (Beck 1965; Thorsteinson 1960; Norris Chemical characters and Kogan 1980; Pathak and Dale Plant chemicals influence the 1983). However, with reference to resistance/susceptibility of the plants in To elucidate the basis of antibiosis in resistance in maize to C. partellus, the several ways: either by determining the maize to C. partellus, Durbey and Sarup information is scattered. orientation, feeding and oviposition (1988) found that alcoholic extracts of behavior of the insects, or by the resistant genotype Mex. 17 Morphological characters determining the metabolism of insects adversely affected growth and Trichomes on the upper leaf surfaces of serving as (a) toxins interfering with the development of C. partellus. Kumar the resistant genotypes have been metabolic processes of insects causing (unpublished data) demonstrated that reported to be related with low failure of the insect survival, C. partellus larval development on the oviposition by C. partellus. oviposition by C. partellus (Durbey and development and egg production on artificial diet containing hexane extracts Sarup 1982; Ampofo 1985). The role of the plant; or (b), nutrients promoting of the resistant genotype Mp 704 was trichomes in inhibiting oviposition by normal metabolic processes resulting in adversely affected (Table 13), but the C. partellus has been experimentally the insect’s normal survival, diet containing methanolic extracts of demonstrated by Kumar and Saxena development and egg production. the resistant genotype did not inhibit (1985a). When the trichome studded Detailed studies conducted by Kumar the growth of C. partellus (Table 14). leaf of the resistant genotype was and Saxena (1985a) and Kumar (1994b) compared with a wax paper, the showed that plant volatiles from the females preferred to lay eggs on the resistant and susceptible maize wax papers. Even when the trichomes genotypes were equally effective in I nhe rit a nc e of Re sist a nc e in M a ize t o C. pa rt e llus on one side of the midrib of a leaf were eliciting oviposition by C. partellus. Both additive and non-additive gene shaved off leaving the other side intact, However, distance-perceivable stimuli effects are important in the inheritance oviposition by the moths on the hairless from the C. partellus infested plants of resistance in maize to C. partellus side was greater than the hairy side of were much more effective than those (Pathak and Othieno 1990). Based on the leaf.(Fig. 8). Kumar (1992a) from the uninfested plants in eliciting the studies of Kumar (1993), developed an inbred line, ICZ-T which oviposition by C. partellus (Kumar 1986; performance of F1 hybrids between had trichomes on both the leaf surfaces Kumar 1994b). After arrival on the susceptible and resistant inbreds was satisfactory. The non-preference and antibiosis types of resistance operating Figure 8. Evidence for the inhibition of C. partellus oviposition by trichomes on the maize leaves. Figure 9. Role of chloroform extracts of a resistant ( Mp704 ) and a susceptible (Inbred A) maize inbreds in determining oviposition by C. partellus. 80 H. KUMAR within the resistant inbred Mp704 was clearly manifested in the single cross hybrids. The accumulation of the desirable additive alleles at loci in a breeding population through S1 or S2 recurrent selection is highly desirable (Pathak and Othieno 1990). Re fe re nc e s Ampofo, J.K.O. 1985. Chilo partellus (Swinhoe) oviposition on susceptible and resistant genotypes. Insect Sci. Applic. 6: 323-330. Ampofo, J.K.O. 1986. Maize Stalk Borer (Lepidoptera: Pyralidae) damage and plant resistance. Environ. Entomol. 15: 1124-1129. Ampofo, J.K.O., K.N. Saxena, J.G. Kibuka, and E.O. Nyangiri. 1986. Evaluation of some maize cultivars for resistance to the stem borer Chilo partellus (Swinhoe) in western Kenya. Maydica 31: 379-389 Beck, S.D. 1965. Resistance of plants to insects. Ann. Rev. Entomol. 10: 207-232. Chattterji, S.M., W.R. Young, G.C. Sharma, I.V. Sayi, B.S. Chahal, B.P. Khare, Y.S. Rathore, V.P.S. Panwar, and K.H. Siddiqui. 1969. Estimation of loss in yield of maize due to insect pests with special reference to borers. Indian J. Entomol.31: 109-115. Durbey, S.L., and P. Sarup. 1982. Ovipositional responses of moths of Chilo partellus (Swinhoe) on different maize germplasms. J. ent. Res. 6: 1-9. Durbey, S.L., and P. Sarup. 1984. Biological parameters related to antibiosis mechanisms of resistance in maize varieties to Chilo partellus (Swinhoe). J. ent. Res. 8: 140-147. Durbey, S.L., and P. Sarup. 1988. Effects of different solvent extracts of resistant and susceptible maize germplasms on the biological parameters expressing antibiosis in Chilo partellus (Swinhoe) due to their formulation in artificial diet. J. ent. Res. 12: 93-97. Fletcher, T.B., and C.C. Ghose. 1920. Borers in sugarcane, rice, etc. Proc. Third Ent. Meeting, Pusa. 1: 354-417. van Hamburg, H. 1979. The grain sorghum stalk borer Chilo partellus (Swinhoe) (Lepidoptera : Pyralidae): seasonal changes in adult populations in sorghum in the Transvaal. J. ent. Soc. southern Africa. 42: 1-9. Table 13. Survival, growth and development of C. partellus larvae on artificial diet containing hexane extracts of four maize genotypes. % larvae surviving Diet + H Diet + EA Diet + EMp Diet + EPR Diet ± EV37 F value (df = 4, 28) LSD at P = 0.05 % larvae in 5th + 6th instar Larval weight (mg/larva) 83.3 ± 6.0a 64.2 ± 5.5b 92.5 ± 2.7a 64.2 ± 4.9b 5.9 ± 6.0b 74.7 ± 4.6a 47.8 ± 9.1b 24.7 ± 3.1c 27.0 ± 7.2bc 46.5 ± 9.1b 53.8 ± 3.9a 58.9 ± 3.1a 53.8 ± 2.2a 56.9 ± 3.5a 57.6 ± 2.2a 8.48** 14.66 7.58** 21.21 0.49 9.54 NS Mean ± SE in a column, means followed by a common letter are not significantly different. 100 g of whorl leaves of 3 week old plant dipped in hexane for 72 hours. Table 14. Survival, growth and development of C. partellus larvae on artificial diet containing methanolic extracts of four maize genotypes. Diet + M Diet + EA Diet + EMp Diet + EPR Diet + EV37 F value (df = 4, 28) LSD at P = 0.05 % larvae surviving % larvae in 5th + 6th instar 74.2 ± 7.8 81.4 ± 4.8 89.0 ± 4.9 80.8 ± 6.1 82.5 ± 3.3 56.8 ± 7.5 45.6 ± 10.1 41.4 ± 2.9 50.4 ± 6.0 38.2 ± 5.5 0.92NS 1.11NS Larval weight (mg/larva) 62.5 ± 3.3b 51.3 ± 1.2c 65.8 ± 1.7ab 69.7 ± 1.3a 63.4 ± 1.3b 13.04** 5.50 Kogan, M., and E.E. Ortman. 1978. Antixenosis - a new term proposed to replace Painter’s “ Non-preference” modality of resistance. ESA Bull. 24. Kumar, H. 1986. Enhancement of oviposition by Chilo partellus (Swinhoe) (Lepidoptera : Pyralidae) on maize plants by larval infestation. Appl. Ent. Zool. 21: 539-545. Kumar, H. 1988a. Effect of stalk damage on growth and yield of certain maize cultivars by the stalk borer Chilo partellus. Entomologia exp. Appl. 46: 149153. Kumar, H. 1988b. Oviposition and larval behaviour of stalk borer Chilo partellus on susceptible and resistant varieties of maize. Indian J. Agric. Sci. 58: 918-921. Kumar, H. 1991. Host plant resistance in maize to first generation Chilo partellus. Annual Plant Resistance to Insects Newsletter 17. Kumar, H. 1992a. Inhibition of ovipositional responses of Chilo partellus (Lepidoptera: Pyralidae) by the trichomes on the lower leaf surfaces of a maize cultivar. J. econ. Entomol. 85: 17361739. Kumar, H. 1992b. Oviposition , larval arrest and establishment of Chilo partellus (Lepidoptera: Pyraliade)on maize genotypes during anthesis. Bull. entomol. Res. 82: 355. Kumar, H. 1993a. Responses of Chilo partellus (Lepidoptera: Pyraliadae) and Busseola fusca (Lepidoptera: Noctuidae) to hybrids of a susceptible and a resistant maize. J. econ. Entomol. 86: 962968. Kumar, H. 1993b. Resistance in maize to Chilo partellus (Lepidoptera: Pyralidae) in relation to crop phenology, larval rearing medium, and larval developmental stages. J. econ. Entomol. 86: 886-890. Kumar, H. 1994a. Field resistance in maize cultivars to stem borer Chilo partellus. Ann. appl. Biol. 124: 333-339. Kumar, H. 1994b. Effects of water stress, nitrogen stress and certain sensory stimuli on infestation and damage by Chilo partellus (Swinhoe) to maize. Ann. appl. Biol. 125: 35-43. Kumar, H. 1994c. Components of resistance in maize Zea mays L. to first and second generation Chilo partellus (Swinhoe). Maydica. 39: 165-170. Kumar, H. 1995. Resistance in maize to Chilo partellus (Swinhoe) (Lepidoptera: Pyralidae) in relation to mode of infestation, larval growth and food utilization. Trop. Agric. (in press). Kumar, H., and K.N. Saxena. 1985a. Ovipositional responses of Chilo partellus (Swinhoe) to certain susceptible and resistant maize genotypes. Insect Sci. Applic. 6: 331-335. OVERVIEW OF RESEARCH ON MECHANISMS OF RESISTANCE IN MAIZE TO SPOTTED STEM BORER Kumar, H., and K.N. Saxena. 1985b. Oviposition by Chilo partellus (Swinhoe) in relation to its mating, diurnal cycle and certain nonplant surfaces. Appl. Entomol. Zool. 20: 218-221. Kumar, H., and K.N. Saxena. 1992. Resistance in certain maize cultivars to first and third instar Chilo partellus larvae. Entomol. exp. Appl. 65: 75-80. Kumar, H., and G.O. Asino. 1993. Resistance of maize to Chilo partellus (Lepidoptera: Pyralidae): Effect of plant phenology. J. econ. Entomol. 86: 969-973. Kumar, H., and G.O. Asino. 1994. Grain yield losses in maize Zea mays L. genotypes in relation to their resistance against Chilo partellus (Swinhoe) infestation at anthesis. Crop Prot. 13: 136-140. Kumar, H., E.O. Nyangiri, and G.O. Asino. 1993. Colonizing responses and damage by a stem borer Chilo partellus (Lepidoptera: Pyralidae) to four variably resistant cultivars of maize. J. econ. Entomol. 86: 739-746 Lal, G., and J.C. Pant. 1980. Laboratory and field testing for resistance in maize and sorghum varieties of Chilo partellus (Swinhoe). Ind. J. Entomol. 42: 606-610. Lux, S., A Hassanali, W. Lwande, and F.N. Njogu. 1994. Proximity of release points of pheromone components as a factor confusing males of the spotted stem borer Chilo partellus, approaching the trap. J. Chem. Ecol. 20: 2065-2075. Norris, D.M., and M . Kogan. 1980. Biochemical and morphological bases of resistance. In F.G. Maxwell, and Jennings P.R. (eds.) Breeding Plants Resistant to Insects, 23-62. New York: J. Wiley. Ochieng, R.S., F.O. Onyango, and M.D.O. Bungu. 1985. Improvement of techniques for mass culture of Chilo partellus (Swinhoe). Insect Sci. Applic. 6: 425-428. Okech, S.H.O., and K. N. Saxena. 1990. Responses of Maruca testulis (Lepidoptera: Pyralidae) larvae on variably resistant cowpea cultivars. Environ. Entomol. 19: 1792-1797. Painter, R.H. 1951. Insect resistance in crop plants. New York: McMillan. Painter, R.H. 1958. Resistance of plants to insects. Ann. Rev. Entomol. 3: 267-290 Pathak, M.D., and A.A. Dale. 1983. The biochemical basis of resistance in host plants to insects. In L.W. Schemilt (Ed.), Chemistry and World Food Supplies: The New Frontiers, 129-142. CHEMRAWN II. Pathak, R.S., and S.M. Othieno. 1990. Inheritance of resistance to the spotted stem borer Chilo partellus (Swinhoe) in maize. Maydica 35: 247-252. Rahman, K.A. 1944. Biology and control of maize and jowar borer Chilo zonellus (Swinhoe). Indian J. Agr. Sciences. 14: 303-307. Sarup, P. 1983. Standardization of techniques for scoring of lines against the stalk borers of maize. In Joginder Singh (ed.), Techniques of scoring for resistance to the major insect pests of maize, 64-72. New Delhi: All India Coordinated Maize Improvement Project. IARI. Sarup, P., V.K. Sharma, V.P.S. Panwar, K.H. Siddiqui, K.K. Marwaha, and K.N. Agarwal. 1977. Economic threshold of Chilo partellus (Swinhoe) infesting maize crop. J. ent. Res. 1: 92-99 Sarup, P., K.K. Marwaha,V.P.S. Panwar, and K.H. Siddiqui. 1978. Identification of sources of resistance to the maize stalk borer Chilo partellus amongst world maize gemplasms comprising international nursery. J. ent. Res. 2: 154159. Saxena, K.N. 1969. Patterns of insect plant relationships determining susceptibility or resistance of different plant to an insect. Entomol. exp. appl. 17: 303-318. Saxena, K.N. 1985 . Behavioral basis of plant resistance or susceptibility to insects. Insect Sci. Applic. 6: 303-313. Seshu Reddy, K.V. 1983. Studies on stem borer complex of sorghum in Kenya. Insect Sci. Applic. 4: 3-10. 81 Seshu Reddy, K.V., and J.C. Davies. 1978. A new medium for mass rearing of the sorghum stem borer Chilo partellus (Swinhoe)(Lepidoptera : Pyralidae) and its use in resistance screening . Indian J. Pl. Prot. 6: 48-55. Seshu Reddy, K.V., and K.O.S. Sum. 1992. Yield infestation relatioship and determination of economic injury level of of the stem borer, Chilo partellus (Swinhoe) in three varieties if maize, Zea mays L. Maydica 37: 371-376. Sharma, V.K., and S.M. Chatterji. 1971. Survival and developmental behavior of Chilo partellus (Swinhoe) on some selected germplasms of maize under laboratory conditions. Indian J. Ent. 34: 11-19. Sharma, V.K., and S.M. Chatterji. 1972. Further studies on the nature of antibiosis in maize Zea mays L against the maize stem borer, Chilo zonellus Swinhoe. Indian J. Entomol. 34: 11-19. Siddiqui, K.H., P. Sarup, V.P.S. Panwar, and K.K. Marwaha. 1977. Evolution of base ingredients to formulate artificial diets for the mass rearing of Chilo partellus (Swinhoe). J. ent. Res. 2: 117131. Sekhon, S.S., and S S. Sajjan. 1987. Antibiosis in maize Zea mays L. to maize borer Chilo partellus (Swinhoe) in India. Tropical Pest Management. 33: 5560. Thorsteinson, A.J. 1960. Host selection in phytophagous insects. Ann. Rev. Entomol. 5: 193-218. Unnithan, G.C., and K.N. Saxena. 1990. Population monitoring of Chilo partellus (Swinhoe) (Lepidoptera : Pyralidae) using pheromone traps. App. Ent. Zool. 11: 793-805. Waldbauer, G.P. 1964. The consumption, digestion and utilization of solanaceous and non solanaceous plants by tobacco horn worm, Protoparce sexta (Johan.) (Lepidoptera: Sphingidae). Entomol. Exp. Appl. 7: 253-269. 82 Phyt oc he m ic a l Ba sis for M ult iple Bore r Re sist a nc e in M a ize D.J. Bergvinson, CIMMYT, Mexico, J.T. Arnason, University of Ottawa, Ottawa, Ontario, J.A. Mihm, French Agricultural Research, Inc., Lamberton, MN, USA and D.C. Jewell, CIMMYT, Harare, Zimbabwe. Abst ra c t One of the major research emphasis’s of the CIMMYT maize improvement program has been the development of germplasm with resistance to multiple generations and species of insects as well as resistance to disease pests. During the past decade, CIMMYT entomologists and breeders in collaboration with Cornell University have developed multiple borer resistant (MBR) populations for the major leaf feeding and stalk-boring pests of maize in temperate, subtropical and tropical areas. Identifying the phytochemical mechanisms of resistance employed by MBR genotypes would serve entomologists, breeders and biotechnologists in identifying new sources of resistance and locating major resistant genes within the genome. For MBR genotypes, the resistance mechanism appears to be nutritional in nature. Leaf tissue of MBR genotypes is tough, which may restrict feeding by early instar larvae. MBR genotypes also tend to have reduced nutritional value (lower nitrogen content), and elevated levels of fiber and cell wall phenolics which may account for the elevated leaf toughness. Cell wall phenolics can cross-link the hemicellulose of the cell wall by the action of peroxidase to produce diferulic acid. Approximately 80% of the variation in field leaf ratings for Ostrinia nubilalis could be accounted for by protein, fiber and diferulic acid content in leaf tissue at the mid-whorl stage in plant development. I nt roduc t ion polygenically controlled and involves Fiber and hemicellulose content of the primarily additive variation (Hinderliter whorl tissue was associated with SWCB Lepidopteran stalk boring larvae cause 1983). Recent diallel experiments with resistance in Caribbean germplasm economically significant losses to maize MBR inbreds have determined that which again did not involve a DIMBOA production throughout the world general combining ability is the most based resistance (Hedin et al. 1984). (Dicke and Guthrie 1988). Host plant important source of variation among F1s resistance is an effective and for leaf feeding resistance and yield The other major group of secondary environmentally safe means of control (Thome et al. 1992, 1994). compounds in maize, the hydroxycinnamic acids, have received for these pests. A source population with multiple borer resistance (MBR) Although the mechanism of MBR relatively little attention as plant was developed by recombination and resistance has not been determined, defense chemicals. Biological activity of recurrent selection under infestation other tropical maize resistant to both soluble hydroxycinnamic acids towards with southwestern corn borer (SWCB), generations of ECB were found to have insects has been investigated (Dowd Diatraea grandiosella, sugarcane borer low levels of the conventional resistance 1990); however, cell wall bound (SCB), D. saccharalis, European corn factor 2,4-dihydroxy-7-methoxy-(2H)- hydroxycinnamic acids have only been borer (ECB), Ostrinia nubilalis, and fall 1,4-benzoxazin-3(4H)-one (DIMBOA) studied in relation to storage insect armyworm (FAW), Spodoptera frugiperda (Sullivan et al. 1974). Silica and lignin pests (Classen et al. 1990). Rumen (Mihm 1985; Benson 1986; Smith et al. content appear to be important in digestion of grass leaf tissue has 1989). Tropical maize resistance to antibiosis-type resistance in tropical demonstrated reduced breakdown of lepidopteran pests appears to be maize (Rojanaridpiched et al. 1984). cell wall materials which have elevated PHYTOCHEMICAL BASIS FOR MULTIPLE BORER RESISTANCE IN MAIZE 83 levels of the hydroxycinnamic acids, p- nutrients less accessible and possibly like DIMBOA, synthetic standards can coumaric and ferulic acid (Akin et al. less desirable to herbivorous insects be made to test the antibiotic and 1990; Jung and Casler 1990). In (Scriber and Slansky 1981). antixenosis effects on the pest organism. For structural defense addition, recent research has shown that cell wall bound phenolic acids can Some of the tools that are available for compounds, the only methods to strengthen the cell wall through a studying and identify host plant substantiate their importance is peroxidase mediated dimerization that resistance mechanisms are depicted in through correlations using a broad cross-links adjacent arabinoxylan Fig. 2. Having access to germplasm range in germplasm or by recurrent molecules with diferulic acid (Fig. 1) with a broad range in field resistance is selection for these structural (Bergvinson 1993). Another phenolic- essential for studying HPR. Having components. based cross-linking mechanism identified the tissues, timing and involves UV-mediated dimerization of conditions when insect feeding is most The primary objective of this study was phenolic acids to produce compounds severe, then plant sampling practices to conduct a phytochemical screening known as truxillic and truxinic acids can then be established to obtain of MBR varieties developed at which may also strengthen plant cell ecologically relevant data on HPR. The CIMMYT and commercially available walls (Hartley et al. 1988; Hartley and most prominent analytical tools of HPR checks to elucidate the phytochemical Ford 1989). Fortification of structural work are gas chromatography-mass components that best predict the components would render energy and spectroscopy (GC-MS) and high observed field resistance, insect performance liquid chromatography bioassay studies and leaf toughness of (HPLC). These tools enable the maize. Although not exhaustive, the list identification and quantification of a of parameters measured included broad range of secondary metabolites resistance parameters studied to date in plants from which inferences can be such as DIMBOA, lignin, fiber, and made on the relative importance a protein. Bound phenolic acid- particular secondary metabolite has on carbohydrate complexes and associated HPR. For soluble defense compounds dimers received special attention. The O OH O OH CH3O OH OH p-Coumaric acid Ferulic acid ara-xyl-xyl O ara-xyl-xyl O OCH3 Field screening OH Agronomic traits OH OCH3 OH O Ferulic acid OH O ara-xyl-xyl Dehydrodiferulic acid R2 R1 COO-araxyl-xyl R1 OH Truxillic acids Tissue sampling OCH3 Peroxidase H2O2 xyl-xylara-OOC Diverse germplasm Damage rating O 2X O R2 OH OH COO-araxyl-xyl- xyl-xyl ara-OOC Truxinic acids Figure 1. Structures of the major phenolic acids, ferulic and pcoumaric acids, and their associated dimers through the action of peroxidase (diferulic acid) or by the absorption of ultraviolet light (truxillic/truxinic acids). Phytochemistry Characterization of Chemical changes NMR (13C and 3H) GC-MS HPLC GC FT-IR NIR Microscopy Localization of Chemical and physical changes white light flourescence microspectrophotometry FT-IR microscopy SEM TEM Synthesize secondary Metabolites Bioassay Active Bioassay Tissue preference (location and maturity) (leaf root, stalk) (kernel, silk, tassel) Acount for field ratings Develop rapid screening technique Incorporate into breeding program -conventional -molecular techniques Figure 2. Schematic of the tools and protocol used for identifying host plant resistance mechanisms. Abbreviations: NMR, nuclear magnetic resonance spectroscopy; GC-MS, gas chromatography-mass spectroscopy; FT-IR, Fourier transform-infrared spectroscopy; NIR, near infrared spectroscopy; SEM, scanning electron microscopy; TEM, transmission electron microscopy. 84 D.J. BERGVINSON, J.T. ARNASON, J.A. MIHM AND D.C. JEWELL second objective was to develop a whorl stage the 13th leaf was harvested standard Instron (model TM-M, Instron simple phytochemical model that from uninfested plants by pulling the Corp., Canton, Mass) was equipped would account for the field resistance to 10th leaf and above whorl out of the with a 2 Kg load cell (Lebow load cell, the ECB. plant and unwrapping the leaves to model 3108, Eaton Corp., Troy Mich.) expose the 13th leaf which was green and a 9 mm chuck to hold the probe. along the exposed half and yellow The probe was lowered at a rate of 1 M a t e ria ls a nd M e t hods along the basal half of the leaf length. cm/s until the probe had punctured the Germplasm and screening The 13th leaf was used for insect leaf (Fig. 3). The leaf was orientated MBR varieties included Across bioassays in the laboratory. Leaf tissue with the undersurface facing up and 86590(IR), Mbita 86590 (Chilo), Poza for phytochemical analysis consisted of held firmly in place using a stainless Rica 86590 (SCB), Across 86590-2 (ECB), the green portion of leaves 10, 11, and steel platform with threaded bolts to Tlaltizapán 85590 (SWCB), CML-135 x 12. The midribs of these leaves were secure the leaf between the platform CML-139, and Ki-3 x Tx601. MBR removed and the leaves were cut into and a Plexiglas plate (Fig. 3). A typical adapted progeny included 6796-13, -49, paper bags. Immature tissue within the force profile is shown in Figure 4, with and -48. Four commercial checks whorl was also cut, placed in paper the force required to puncture the included Fontanelle 6230, Pioneer 3184, bags, frozen on dry ice, and held at - lower epidermis being recorded. Leaf Dekalb 435 and Pickseed 4533. MBR 20°C. Three plants per row were pooled toughness is significantly correlated varieties were developed at CIMMYT as one phytochemical sample. Frozen with field damage ratings for the MBR and provided by J.A. Mihm, northern tissue was thawed for 1 h to hydrolyze adapted inbreds were developed at hydroxamic glucosides and then Agriculture Canada, Ottawa and refrozen for lyophilization. Samples provided by R.I. Hamilton. Planting were milled on a UD cyclone mill (UD occurred in mid-May of 1990 at the Corp., Bolder, CO) with a 1 mm screen. Plant Research Centre, Agriculture Milled samples were stored at -20° C Canada, Ottawa, Ontario, Canada. The until analyzed. rows contained 30 plants spaced over 4.5 m with 0.9 m spacing between rows. Bioassays The soil type was a sandy loam. Four Two leaf sections (3x7 cm) were taken replicates were planted in a complete from the middle of the green and randomized block design. Plants were yellow portions of the 13th leaf. Tissue infested using the larval infestation was stored in water to prevent method developed by Mihm (1983) desiccation and incorporated into insect with ca. 80 larvae per plant. Three bioassays within 6 h of harvest. A weeks after infestation plants were bioassay apparatus was used to rated according to Guthrie’s et al. (1960) measure the area consumed in mm2 9 point scale (1=resistant, 9=very from a 1.2 cm diam. disk of the leaf susceptible). Plants were dissected from tissue exposed to two third-instar late September through early October larvae (for details see Bergvinson et al., to count the number of larvae, number these Proceedings). Mean area and length of tunnels, position of consumed was determined for 40 leaf tunneling and estimated cross-section disks for each genotype and tissue type. of pith excavated by larval feeding. Leaf toughness Sample collection Using the method reported in Since ECB females tend to oviposit on Bergvinson et al. (1994), force the undersurface of the upper whorl measurements were taken from the leaves, these tissues were collected for abaxial leaf surface between veins using phytochemical analysis. At the mid- a 1 mm diam., rounded probe. A Figure 3. Instron apparatus for determining leaf toughness. Stainless steel stage has a 2 cm dia. hole through the plate’s center. Leaf is placed on the stage and covered with a Plexiglas plate with a 2 cm dia. hole through its center. Leaf is pulled taut and Plexiglas plate is tighly secured against stage by wing-nuts. Drill chuck is attached to a 2 Kg load cell. PHYTOCHEMICAL BASIS FOR MULTIPLE BORER RESISTANCE IN MAIZE 85 hybrids (r = -0.82, P < 0.001). For this extracted with ethyl acetate (4 x 50 mL) rotary evaporator and stored at -20°C study, 20 ear leaves from each (BDH, Omni-Solv grade). Ethyl acetate until HPLC analysis. The pellet that genotype were harvested at flowering fractions were pooled and dried by remained after extraction was dried for toughness measurements. rotary evaporator and stored at -20oC and weighed to provide an estimate of until HPLC analysis. fiber content. Protein content was estimated by an After extraction, the pellet that HPLC analysis automatic micro-Kjeldahl nitrogen remained was washed in a Büchner All analyses were performed with a analyzer (Tecator model 1030, funnel with 30 mL each of water, Perkin-Elmer system consisting of an Höganäs, Sweden) on 0.3 g samples methanol and ethyl acetate to remove LC 480 diode scan array detector and a using the conversion factor 6.25 to chlorophyll and provide a crude cell Perkin-Elmer LC250 binary pump fitted estimate protein from nitrogen wall preparation. Cell wall samples with 10 µL injection loop. Separations (McKenzie and Wallace, 1954). One were dried in a desiccator for four days. were achieved using a C18 ODS reverse measurement from each of 3 replicates This preparation was weighed and the phase column (250 x 4.6 mm, 5 µm were taken for both mature and weight loss was used as the gravimetric particle size, Beckman, Fullerton, CA). immature tissue for each genotype. measure of soluble metabolites. Cell Protein determinations wall preparations were shaken in 20 mL Soluble extracts were suspended in 4 Phytochemical analysis of 2N NaOH for 4 h under N2 and mL of 50% methanol and centrifuged at Soluble phenolic conjugates and wrapped in foil to hydrolyze phenolic 500 g for 5 min. The supernatant was hydroxamic acids were extracted from ester linked to hemicellulose. Nitrogen filtered and injected onto the column. a 0.5 g sample of dry leaf tissue. was required to prevent oxidation of The solvent system was comprised of Samples were extracted for 20 s in 70% phenolics and a foil wrapping was methanol (A) and 10 mM H2PO4, pH methanol (4 x 20 mL) and mixed with a required to minimize 2.4 (B) at a flow rate of 1.5 mL/min and polytron mixer (Brinkmann model TC- photoisomerization of phenolic acids. a gradient as follows: 25 to 55% A in 15 1200, Westbury, NY). After Samples were neutralized with 6N HCl min, 55 to 80% A in 5 min, 80 to 100% A centrifugation at 500 g for 10 min. the and the pH lowered to 2.0. After in 2 min, 100% A for 8 min, 100 to 25% supernatants were pooled, methanol centrifugation the supernatant was A in 2 min and 25% A for 3 min. was removed by rotary evaporator extracted with ethyl acetate (3 x 50 mL). DIMBOA (Rt = 10.5 min) and 6- (35ºC), and the pH lowered to 2.0 using The pellet was resuspended in water methoxybenzoxazolinone (MBOA) (Rt 1N HCl. The pH must be lowered to and centrifuged twice with both = 13.6 min) standards were enable phenolic and hydroxamic acids fractions pooled and extracted with synthetically prepared according to to move from a water phase into ethyl ethyl acetate (3 x 50 mL). Ethyl acetate Atkinson et al. (1991). Peak identity was acetate. The water fraction was fractions were pooled and dried by confirmed by on-line UV spectra and spiking of extracts with authentic standards. 0.8 Lower Epidermis Force (N) 0.6 Cell wall bound phenolic acids were suspended in 1 mL of methanol, diluted Upper Epidermis 0.4 10 fold in methanol, filtered and injected onto the column. The solvent system was the same as above except 0.2 the starting mixture was 15% methanol. Standards of E-p-coumaric (Rt = 15.2 min) and E-ferulic acid (Rt = 15.6 min) 0.0 were purchased from Sigma. A typical chromatogram from a cell wall 0 10 20 30 Time (sec) Figure 4. Computer plot of the force profile for a mature maize leaf. Force recorded in newtons (N). extraction of maize leaf tissue is illustrated in Figure 5 as well as the 86 D.J. BERGVINSON, J.T. ARNASON, J.A. MIHM AND D.C. JEWELL characteristic absorption spectra for the Statistical analysis variability for a given genotype was phenolic dimers that cross-link the cell All statistical analyses were performed excessive. Immature leaf tissue was wall carbohydrates. on SAS V. 6.03 (SAS 1988). Leaf rating almost entirely consumed within the 48 data, bioassay consumption, and leaf h bioassay and was not used for further Lignin determinations toughness were transformed by ln analysis. Although the spread in leaf A modified acetyl bromide procedure (x+1) to satisfy the assumptions of the toughness was not large (0.59 to 0.89 outlined by Iiyama and Wallis (1990) general linear model. Forward N), the standard error of the mean was was used. After base hydrolysis, the regressions were done using the low (LSD0.05= 0.083). Leaf feeding fiber pellet that remained was dried in forward option in PROC REG. damage and bioassay consumption of green tissue by ECB larvae were both a dessiccator and 50 mg was used for Re sult s a nd Disc ussion lignin analysis. Tissue was digested negatively correlated (r=-0.58* and 0.81**, respectively) with leaf with 25% acetyl bromide and 4% perchloric acid in acetic acid at 70°C for The mean range of leaf parameters toughness. Likewise, the number of 30 min. After digestion the samples associated with resistance for the MBR larvae, number of stalk tunnels, length were cooled on ice and transferred to a genotypes tested are shown in Table 1. of tunneling and cross-section of pith volumetric flask containing 10 mL of 2 Leaf feeding damage after artificial excavated were negatively correlated M sodium hydroxide and 12 mL of infestation ranged from a low of 2 to a with leaf toughness (data not shown). acetic acid and the volume brought to high of 6, with all plants showing signs These correlations indicate a possible 50 mL using distilled water. The of feeding. Leaf bioassay consumption reduction in the capacity of larvae to absorption at 280 nm was taken and the ranged from 14-78 mm2 but was too establish on genotypes that have value of 20.0 g/L/cm was used for variable despite the large number of tougher leaf tissue. Neonate mortality lignin calculations. replicates. Attempts were made to use often exceeds 80% for the first two days neonate larvae for the bioassay but post-eclosion (Ross and Ostlie 1990). because the ECB is not voracious, the One mortality factor is desiccation (Lee 1.0 0.8 E-pCA 0.04 0.02 Absorbance 0.08 0.06 430 DFA Amax: 230 nm desiccate. Although later instars 0.08 have little difficulty penetrating mature leaf tissue as observed 0.06 in leaf bioassays, neonates may not have the mandibular TA 0.04 DFA strength to penetrate tougher leaves. This incapacity may E-FA 0.02 0.04 account for their migration into 0.02 the whorl of the plant. This 0.00 190 250 310 370 Wavelength (nm) 0.4 shortly after eclosion may 0.06 0.00 190 250 310 370 Wavelength (nm) 0.6 cannot penetrate leaf tissue TA Amax: 230 nm Absorbance at 227 nm Absorbance 0.08 Absorbance at 280 nm 1988), whereby larvae that 0.10 0.00 430 10 20 Time (min.) explain the feeding behavior of the SWCB (Hedin et al. 1984). Z-FA 0.2 reasoning has been used to Plant nitrogen is a major Z-pCA determinant of insect growth and development, with low 0.0 nitrogen possibly serving as a 0 10 20 Time (min.) 30 Figure 5. High performance liquid chromatography (HPLC) run of a cell wall extraction from mature maize leaf tissue. Abreviations: p-CA, p-coumaric acid; FA, ferulic acid; DFA, diferulic acid; TX, truxillic and truxinic acids. plant resistance strategy (Scriber and Slansky 1981). Leaf protein content correlated positively with number of larvae (r=0.55*), length of PHYTOCHEMICAL BASIS FOR MULTIPLE BORER RESISTANCE IN MAIZE 87 tunneling (r=0.56* ) and with cross- differential size and biomass of SWCB acids. With the exception of the light sectional consumption of pith (r=0.53*) larvae grown on resistant (MBR) and activated truxillic acids, cell wall (data not shown). These observations susceptible plants (Davis et al. 1988). phenolics occurred at higher concentrations in immature whorl suggest that more resistant genotypes with lower leaf-protein content may not Mean concentration of major tissue. Weight of soluble components provide sufficient accessible protein to phytochemicals in maize leaf tissue is was greatest for the immature leaf facilitate larval development beyond also reported in Table 1. Cell wall tissue, as were the levels of soluble early instars. This hypothesis is constituents included estimated fiber secondary metabolites such as supported by field observations of content and, hemicellulose bound p- DIMBOA and the glycosides of coumaric, ferulic, diferulic and truxillic phenolic acids (Table 1). Table 1. Means for biochemical and physical resistance factors in mature and immature leaf tissue of maize harvested at the midwhorl stage, 1990. Leaf Genotype Mature Leaf Across 86590(IR) Mbita 86590 (Chilo) Poza Rica 86590 (SCB) Across 86590-2 (ECB) Tlaltizapán 85590 (SWCB) CML135x CML139 Ki3xTx601 Pioneer 3184 Fontanelle 6230 6796-48 6796-49 6796-13 Dekalb 435 Pickseed 4533 Immature Leaf Across 86590(IR) Mbita 86590 (Chilo) Poza Rica 86590 (SCB) Across 86590-2 (ECB) Tlaltizapán 85590 (SWCB) CML135x CML139 Ki3xTx601 Pioneer 3184 Fontanelle 6230 6796-48 6796-49 6796-13 Dekalb 435 Pickseed 4533 † Bio- Rating assay 2.46 20 2.56 Leaf † Force † µg/g dry wt. mg/g dry wt.† Protein (%) PCA FA Tx DFA Soluble Fiber Hx sPCA sFA 0.83 15.59 2.65 2.8 0.85 0.42 300 320 0.89 36 67 22 - 16.98 1.81 2.46 0.97 0.35 240 360 1.13 25 17 2.14 29 0.82 15.89 2.40 2.52 0.73 0.58 340 300 1.45 36 47 2.12 19 0.83 16.07 2.00 2.81 1.04 0.60 300 280 1.17 41 52 2.22 27 0.79 15.65 2.14 2.68 1.13 0.58 300 300 0.95 73 80 2.22 3.61 3.9 5.71 5.11 5.36 2.41 3.47 4.11 14 20 27 40 70 78 44 41 62 0.89 0.68 0.77 0.72 0.64 0.65 0.74 0.67 0.59 16.11 16.47 15.39 18.64 18.37 19.17 15.61 16.28 17.60 2.53 2.07 2.57 1.31 1.76 1.73 1.62 1.78 1.63 3.16 2.14 2.62 1.25 2.48 2.41 1.90 1.65 2.45 1.37 0.73 1.01 0.51 1.52 0.89 0.54 1.20 1.26 0.40 0.41 0.40 0.20 0.18 0.23 0.25 0.28 0.30 320 340 280 320 280 300 380 320 300 300 260 300 280 300 300 260 300 300 0.39 1.2 1.48 .23 0.65 1.06 0.73 1.29 2.12 30 40 115 136 80 31 25 190 140 37 15 64 100 92 122 32 139 173 2.46 20 18.54 0.83 4.07 4.44 0 0.49 440 200 1.06 53 61 2.56 21 18.79 - 3.37 4.17 0.01 0.64 500 160 1.82 70 57 2.14 29 18.28 0.82 2.86 3.24 0.01 0.42 500 200 2.36 109 28 2.12 19 12.38 0.83 4.73 6.93 0.05 0.49 340 240 1.54 35 68 2.22 27 12.84 0.79 5.76 6.97 0.08 0.57 340 200 1.66 60 35 2.22 3.61 3.90 5.71 5.11 5.36 2.41 3.47 4.11 13 20 27 39 70 78 44 41 62 12.68 14.01 11.14 15.27 19.42 21.72 16.27 15.46 17.02 0.89 0.68 0.77 0.72 0.64 0.65 0.74 0.67 0.59 5.59 3.43 5.95 3.82 2.48 2.42 2.49 3.10 3.68 6.45 4.27 7.49 4.24 3.7 3.58 2.98 3.76 5.49 0.01 0 0.08 0 0.02 0 0 0.13 0.02 .039 0.28 0.58 0.51 0.08 0.20 0.21 0.35 0.26 380 560 380 400 500 480 500 460 460 220 160 220 220 160 180 180 180 180 0.87 0.88 0.94 1.65 1.62 1.75 1.83 4.90 3.07 56 53 453 653 30 31 32 79 345 53 20 50 623 40 46 41 117 153 Leaf rating is Guthrie’s (1960) 1-9 scale, bioassay is mm2 tissue consumed, laf toughness of mature ear leaf at tasseling, PCA=p-coumaric acid, FA=ferulic acid, Tx=total cyclobutane dimers, DFA=dehydrodiferulic acid, soluble is gravimetric determination of soluble metabolites on a dry weight basis, fiber is estimated dtergent fiber, Hx=DIMBOA equivalents, sPCA and SFA are soluble conjugates of PCA and FA. 88 D.J. BERGVINSON, J.T. ARNASON, J.A. MIHM AND D.C. JEWELL Soluble phytochemicals such as Lam et al. 1990). Such lignin linkages this photoactivated dimer (Table 2). On DIMBOA, ferulic and p-coumaric acid likely contribute to cell wall the other hand, diferulic acid is conjugates and flavonoids were fortification and tissue toughness. produced by a cell-wall-bound peroxidase which is under genetic positively correlated with leaf feeding and negatively correlated with leaf The most consistent relationship was control and could be manipulated in toughness, with the stronger observed between DFA and variables of the future to increase the production of correlations being observed for mature insect resistance with |r| > 0.66 for phenolic dimers and cell wall leaf tissue (Table 2). One possible mature tissue and |r| > 0.42 for toughness. explanation for this is the possibility immature tissue (Table 2). Diferulic that soluble secondary metabolites are acid, like the cyclobutane dimers, can Forward regressions between acting as host recognition factors and as cross-link cell wall carbohydrates and biochemical parameters as independent such are phagostimulants. Semipurified increase the mechanical strength of the variables and plant resistance extracts of ferulic and p-coumaric acid cell wall (Ishii 1991; Fry 1986; parameters as dependent variables are glycosides from Mbita 86590 (Chilo) Markwalder and Neukom 1976). This is shown in Table 3. All regression models acted as phagostimulants at evident in the positive correlation exceeded an R2 value of 0.7 with only ecologically relevant dosages (10 µg/ between DFA content and leaf three independent variables in the cm2) toughness (r=0.68**, Table 2). Perhaps models. The most common antifeedants at 100 µg/cm2 (Bergvinson because cyclobutane dimer (Tx) independent variables within these 1993). In addition to the phenolic production is largely under models include protein content (PRO), glycosides, DIMBOA can increase environmental control a poor fiber content (CW) and DFA content. consumption by inhibiting digestive correlation was observed for both leaf Incorporating protein, fiber and DFA proteases in the insect, thus requiring toughness and leaf damage rating for content into a fixed regression model and became phytotoxic and greater consumption of leaf tissue to assimilate sufficient nitrogen for larval development (Houseman et al. 1992). All these factors probably contribute to the higher consumption observed for genotypes with higher levels of soluble components. Table 2. Correlations of biochemical parameters with plant damage parameters for 13 maize genotypes, 1990. Tissue Type Independent Variable Field Leaf Ratings† Mature Protein Wt. Solubles DIMBOA pCA (sol.) FA (sol.) Flavonoids Fiber pCA (CW) FA (CW) Tx DFA Lignin Protein Wt. Solubles DIMBOA pCA (sol.) FA (sol.) Flavonoids Fiber pCA (CW) FA (CW) Tx DFA 0.82 -0.13 0.24 0.39 0.63 0.16 -0.14 -0.55 -0.52 0 -0.76 0.06 0.32 0.22 0.11 0.54 0.51 0.31 -0.36 -0.34 -0.25 -0.14 -0.42 Cell wall weight and cell-wall-bound phenolic acids in mature leaf tissue were negatively correlated with field leaf damage ratings and bioassay feeding and positively correlated with leaf toughness (Table 2). Maize-grain resistance to storage pests has been previously correlated with cell wall ferulic acid levels and kernel toughness (Classen et al. 1990). Cell-wall-bound pcoumaric acid (PCA) showed stronger correlations with the dependent variables than ferulic acid (FA). This may be attributed to PCA being more prevalent in secondary cell wall tissue and its prominent role in lignin linkage to polysaccharides (Goto et al. 1991; Immature *** * * * *** * Bioassay Feeding† 0.67 ** 0.08 0.27 0.37 0.69 0.31 -0.13 -0.65 -0.44 0.08 -0.66 -0.07 - Leaf Toughness† -0.65 0.06 -0.45 -0.51 -0.64 -0.45 0.12 0.69 0.51 -0.12 0.68 0.16 -0.46 -0.54 -0.47 -0.19 -0.14 -0.22 0.83 0.59 0.42 0.05 0.66 ** * ** ** *** ** ** *,**,*** P < 0.05, P < 0.01, P < 0.001. † Field rating, bioassay feeding and leaf toughness were transformed by ln(x +1) prior to statistical analysis. PHYTOCHEMICAL BASIS FOR MULTIPLE BORER RESISTANCE IN MAIZE for each tissue type accounts for a (Quisenberry and Wilson 1985) and date, MBR resistance has not broken reasonable amount of the variation, may explain the reduced growth rate of down and some MBR germplasm is with the more consistent and more SWCB larvae feeding on MBR cultivars effective against several borers significant models being observed for compared to those feeding on belonging to different genera. mature leaf tissue (Table 4). Although susceptible cultivars (Davis et al. 1988). 89 Ac k now le dgm e nt s not providing direct evidence for the mechanism of host plant resistance Inheritance of multiple borer resistance employed by MBR varieties, it is appears to be polygenically controlled, We thank Agriculture Canada, Central apparent that much of the variability of and involves primarily additive Experimental Farm for access to field field leaf ratings and leaf toughness can variation (Smith et al. 1989). This plots and micro-Kjeldahl. This work be accounted for by these three proposed inheritance is consistent with was supported by an NSERC strategic parameters. These regressions support our proposed mechanism of resistance grant (Arnason) and subsequently by the hypothesis that MBR varieties which involves three polygenic the NSERC(CRD) program as well as employ a nutritional resistance components, namely protein, fiber and an NSERC Graduate Scholarship to mechanism whereby lower protein cell wall phenolic acid content. A D.J.B. content acts in concert with increased fourth component of this resistance cell wall mechanical strength, model is the peroxidase-mediated manifested through higher fiber content production of DFA which likely and higher levels of cell wall phenolics, involves only a single gene product. to reduce nutrient availability to early The main advantages of polygenic instar larvae. This model is consistent resistance are the reduced likelihood of with earlier reports of FAW showing resistant pest populations developing reduced digestion of bermudagrass and an effective resistance over a with high cell wall content broader spectrum of pest organisms. To Re fe re nc e s Table 3. Forward multiple regressions of biochemical parameters with resistance parameters for resistance to European corn borer in 13 maize genotypes, 1990. Tissue Dependent Type Variable Green Yellow Leaf Rating Bioassay Leaf Toughness Leaf Rating Leaf Toughness Regression Equation † r2 LLR = -0.621 + 0.129(PRO) - 0.541(DFA) + 0.0015(pCA sol.) LBA = 0.605 + 0.223(PRO) - 0.458(FA) + 0.0043(FA sol.) LLT = 0.408 + 0.124(DFA) + 0.061(pCA) - 0.0004(FA sol.) LLR = 2.236 - 0.0033(CW) - 0.850(DFA) + 0.0011(pCA sol.) LLT = -0.167 + 0.0029(CW) + 0.0419(SOL) - 0.0001(pCA sol.) 0.78** 0.79** 0.78** 0.71** 0.89** *,**,*** P < 0.05, P < 0.01, P < 0.001. † Field rating, bioassay feeding and leaf toughness were transformed by ln(x +1) prior to statistical analysis. Table 4. Multiple regressions of protein, fiber and dehydroxydiferulic acid levels with plant resistance parameters for 13 maize genotypes, 1990. Tissue Type Dependent Variable Regression Equation† r2 Green Leaf Rating Bioassay Leaf Toughness Leaf Rating Leaf Toughness LLR = 0.108 +0.136(PRO) - 0.0023(CW) - 0.683(DFA) LBA = 1.794 + 0.188(PRO) - 0.0032(CW) - 1.30(DFA) LLT = 0.664 - 0.015 + 0.0003(CW) + 0.146(DFA) LLR = 1.869 + 0.0075(PRO) - 0.0018(CW) - 0.512(DFA) LLT = 0.221 - 0.00038(PRO) + 0.00179(CW) - 0.016(DFA) 0.78** 0.58* 0.54 0.22 0.69** Yellow *,**,*** P < 0.05, P < 0.01, P < 0.001. † Field rating, bioassay feeding and leaf toughness were transformed by ln(x +1) prior to statistical analysis. Akin, D.E., R.D. Hartley, W.H. 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Efficient mass rearing and infestation techniques to screen for host plant resistance to maize stem borers, Diatrea sp. International Maize and Wheat Improvement Center (CIMMYT), Mexico, D.F., Mexico. Technical Bulletin. Mihm, J.A. 1985. Breeding for host plant resistance to maize stemborers. Insect Sci. Appl. 6: 369-377. Quisenberry, S.S., and H.K. Wilson. 1985. Consumption and utilization of bermuda grass by fall armyworm (Lepidoptera: Noctuidae) larvae. J. Econ. Entomol. 78: 820-824. Rojanaridpiched, C., V.E. Gracen, H.L. Everett, J.G. Coors, B.F. Pugh, and P. Bouthyette. 1984. Multiple factor resistance in maize to European corn borer. Maydica 14: 305-315. Ross, S.E., and K.R. Ostlie. 1990. Dispersal and survival of early instars of European corn borer (Lepidoptera: Pyralidae) in field corn. J. Econ. Entomol. 83: 831-836. SAS Institute Inc. 1988. SAS/STAT User’s Guide. Version 6.03. SAS Institute Inc., Cary, NC. Scriber, J.M., and F. Slansky Jr. 1981. The nutritional ecology of immature arthropods. Annu. Rev. Entomol. 26: 183-211. Smith, M.E., J.A. Mihm, and D.C. Jewell. 1989. Breeding for multiple resistance to temperate, subtropical, and tropical maize insect pests at CIMMYT. In Toward Insect Resistant Maize for the Third World: Proc. Int. Symp. Methodologies for Developing Host Plant Resistance to Maize Insects, 222-234. Mexico D.F.: CIMMYT. Sullivan, S.L., V.E. Gracen, and A. Ortega. 1974. Resistance of exotic maize varieties to the European corn borer, Ostrinia nubilalis (Hübner). Envir. Entomol. 3: 718-720. Thome, C.R., M.E. Smith, and J.A. Mihm. 1992. Leaf feeding resistance to multiple insect species in a maize diallel. Crop Sci. 32: 1460-1463. Thome, C.R., M.E. Smith, and J.A. Mihm. 1994. Yield reduction in a maize diallel under infestation with southwestern corn borer. Crop Sci. 34: 1431-1435. 91 M e c ha nism s of Re sist a nc e in M a ize Gra in t o t he M a ize We e vil a nd t he La rge r Gra in Bore r J.T. Arnason, University of Ottawa, Ottawa, Canada B. Conilh de Beyssac, University of Ottawa, Ottawa, Canada B.J.R. Philogene, University of Ottawa, Ottawa, Canada D. Bergvinson, CIMMYT, Mexico. J.A. Serratos, INIFAP, Mexico. and J. A. Mihm, French Agricultural Research Inc., Lamberton, MN, USA Abst ra c t The mechanism of resistance in maize to the stored product insects such as the maize weevil (MW), Sitophilus zeamais Motsch and the larger grain borer (LGB), Prostephanus truncatus Horn has been investigated in relation to secondary chemistry and other biochemical and physical characteristics of maize genotypes. Performance parameters of weevils (number of eggs laid, number of progeny, Dobie index, grain consumption) were negatively and significantly correlated (r = -0.8, P = 0.05) to the most abundant phenolic of grain, E-ferulic acid. With P. truncatus, the weight loss of grain also showed a negative correlation with E-ferulic acid while percent damage of kernels by insects was negatively correlated to p-coumaric acid. These phenolic acids were found in highest concentration in the pericarp and cell walls of the endosperm by fluorescence microscopy. Phenolic acid content was also found to correlate strongly with hardness of the grain, which may be related to the mechanical contributions of phenolic dimers to cereal cell wall strength. In the aleurone layer phenolic acid amines have been detected that have toxic effects on insects. M e c ha nism s of Re sist a nc e t o Sit ophilus ze a m a is which are fluorescent and highly (number of eggs laid, number of concentrated in the pericarp, as factors progeny, Dobie index, grain in resistance, in addition to previously consumption) in standardized tests A decade ago, while working with described nutritional and mechanical were negatively and significantly Maya farmers of Belize, we noted the factors (Fig. 1). Subsequently, a study correlated (r > -0.8, P = 0.05) to the E substantial resistance of traditionally of 15 CIMMYT pools showed that ferulic acid content of grain varieties used landraces of maize as well as some developmental parameters of weevils (Classen et al. 1990). maize varieties released by the International Maize and Wheat Improvement Center (CIMMYT) to the maize weevil, Sitophilus zeamais as HO OH O O—CH2 the kernel pericarp as observed by 1987). This suggested the role of hydroxycinnamic acids (phenolics) HO O OH O O CH3O HO CH = CH — COOH ferulic acid HO CH = CH — COOH p - coumaric acid OH maize was the intense fluorescence of fluorescence microscopy (Serratos et al. OH O hybrids (Fortier et al. 1982). A remarkable feature of the resistant O O compared to introduced commercial O OH O H3CO OH CH3O HO Figure 1. Hydroxycinnamic acids of maize in their bound form to cereal cell wall arabinoxylans CH3O (upper) and in hydrolysed form (lower). CH = CH — COOH sinapic acid 92 J.T. ARNASON, B. CONILH DE BEYSSAC, B.J.R. PHILOGENE, D. BERGVINSON, J.A. SERRATOS AND J.A. MIHM Protein content and kernel hardness of Our work on the role of phenolics in free acids but are esterified to the cereal varieties were also negatively correlated resistance has recently been reviewed cell wall hemicelluloses in compounds with susceptibility in this (Table 1) and (Arnason et al. 1992). In particular it has such as feruloyl and p-coumaroyl other studies by our group. These have been demonstrated that phenolic arabinoxylans (Fig. 2). These been reported as resistance factors by fluorescence (Sen et al. 1991) can be compounds can be cross-linked by other authors. In a subsequent study used as a rapid indicator for resistance extracellular peroxidases forming a (Arnason et al. 1993) of 31 quality and may be useful for breeders who mechanical cross-link in the cereal cell protein maize genotypes from CIMMYT wish to make a biochemical pre- wall. Resistant genotypes have higher with approximately twice the lysine screening of material for resistance. In concentrations of these diferulic acids and tryptophan content of normal addition, progress has been made on than susceptible materials (Arnason et maize, we found no indication that defining the inheritance of resistance al. 1994). A second bound form of these genotypes were any more parameters and phenolics in a phenolic acids has recently been susceptible on average than generation means analysis (Serratos et localized in the aleurone layer of cell backcrossed material expressing normal al. 1993). walls. Our preliminary results suggest that these phenolic amides, such as protein in the endosperm (Table 2). We also examined a group of 30 Mexican Recent work has shown that the diferuloyl and dicoumaroyl putrescine landraces in an attempt to define phenolics are probably important in may be antibiosis factors to S. zeamais. sources of resistance for future studies. resistance in two ways: through Together these phenolic acid conjugates The ancient indigenous landraces were mechanical resistance and antibiosis. can be detected by new fluorescence the group showing least susceptibility The major hydroxycinnamic acids of imaging techniques which clearly show to weevils (Arnason et al. 1994). maize kernels are ferulic and p- the phenolic barrier to insects in the coumaric acid, which are not found as outer tissues (Fig.3) Table 1. Pearson correlation coefficients (P=0.05) of grain parameters with maize weevil development parameters for 10 genotypes of maize. Grain characteristic E-ferulic acid content protein instron hardness (N) shape index Oviposition (egg plugs/ 100seeds) Weight Loss of Grain (g/100g) -0.71 -0.81 -0.63 -0.75 -0.62 0.67 -0.71 0.76 A. Polysaccharide O Polysaccharide O O H2O2 OH OH OCH3 H3CO 2H2O OH O O Polysaccharide Table 2. Susceptiblity of quality protein maize (QPM) and backcross to normal (QPM x NOR) genotypes to maize weevil. Dobie Index of Suceptibility (S.D) QPM QPM x NOR 8.03 (4.6) 9.13 (1.3) Note: A Dobie Index of 14 indicates very susceptible grain and an Index of 0 is totally resistant. Figure 3. Quantitative imaging map of phenolic acid conjugates detected by microspectophotometry in a cross section of a maize kernel. OCH3 H3CO Figure 2. Formation of diferulic acid crosslinks in maize cell walls. Note: lignin, sugar and lipid content were not significant. Cultivar type O OH O O Polysaccharide MECHANISMS OF RESISTANCE IN MAIZE GRAIN TO THE MAIZE WEEVIL AND THE LARGER GRAIN BORER Re sist a nc e t o t he La rge r Gra in Bore r (LGB), Prost e pha nus t runc a t us H orn 93 example, humidity and partial water instars as well as in determining the content are positively correlated to total weight loss of the grain which is several indices of LGB damage as an important measure of economic observed in the case of MW. Also damage. several LGB development parameters Despite the widespread destruction of were negatively correlated to hardness The importance of hydroxycinnamic grain in Africa by this pest, little was measurements as was found for MW. acids in resistance is also evident with known about grain characteristics The amount of vitreous endosperm is this insect. Weight loss of grain is correlated to susceptibility of grain to negatively correlated to the amount of negatively correlated with ferulic acid the LGB. Seven cultivars from powder produced by adults. Powder content of grain, which may be CIMMYT’s program were assessed for produced by LGB adults is important associated with its importance in cross- the relative susceptibility of maize for the development of early larval linking and strengthening the varieties to the LGB by studying grain damage parameters in standardized tests (% of grains attacked, loss of grain Table 3. Prostephanus truncatus development parameters on maize varieties. weight, powder produced), insect development parameters (mortality, weight of adults, consumption). Development was assessed on five replicate 100g samples of grain equilibrated at 70% relative humidity and 30°C which were infested with 100 unsexed adults for 2 weeks before assessment. Choice tests were performed by releasing 100 insects into Damaged kernals (%) Name of variety Ilonga 8032 Muneng 8128 Cacahuacintle Poza Rica 8121 Across 7740 Across 8035 Ratray-Arnold 8149 43.70b,c 46.16a,b 54.48a 53.88a 42.42b,c 40.42b,c 36.40c Weight loss (g/100g) Powder Choice produced test Consumption (g/100g) (%/variety) (mg/insect/day) 5.02c 7.19a,b 8.62a 5.62b,c 7.92a 6.99a,b,c 5.47b,c 3.6b,c 3.74b,c 8.38a 3.98b,c 4.78b 3.14c 3.26c 8.76 7.26 14.36 10.56 7.45 5.25 10.08 2.50 1.64 3.23 2.61 1.87 1.67 2.41 Values followed by the same letter are not significantly different. an arena with 100g of grain samples of each variety (Table 3). The grain was analyzed for characteristics which may Table 4. Physical characteristics of maize varieties. Hardness Deformation (peak force) before breakage (N) (mm) be correlated to resistance are such as hardness and deformation as measured with by instron, % vitreous endosperm Variety Vitreous endosperm (%) 0.405 0.560 0.714 0.376 0.336 0.418 0.395 0.317 0.304 0.504 0.307 0.286 0.286 0.323 44.5ab 47.4a 41.1bc 33.9d 36.8cd 22.0e 7.84f (gravimetrically). Methods are described in detail elsewhere (Conilh Values followed by the same letter are not significantly different (P = 0.05). and phenolics (by HPLC), total lipids (gravimetrically) protein (estimated by Kjeldahl) and water content 0.433 a,b 0.476 a 0.346 c,d 0.313 d 0.329 d 0.286 d 0.398 c Kernel weight (g) Ilonga 8032 Muneng 8128 Poza Rica 8121 Ratray-Arnold 8149 Across 8035 Across 7740 Cacahuacintle (by quantitative imaging), total sugars 430.4 (a) 418.5 a 318.7 b 289.0 b 285.5 b,c 232.7 c,d 201.3 d Kernel volume 3 (cm ) de Beyssac 1991, 1992). The results were analyzed by ANOVA and Tukey’s or the Kruskal-Wallace tests for the comparison of the means (Tables 3-5). The Pearsons’s correlation coefficients between grain characteristics and insect performance parameters are shown in Table 6. Some of the susceptibility parameters for LGB show the same pattern of correlation as MW. For Table 5: Biochemical characteristics of maize varieties. Name of variety Partial moisture (%) Ilonga 8032 Muneng 8128 Cacahuacintle Poza Rica 8121 Across 7740 Across 8035 Ratray-Arnold 8149 10.45 10.55 10.84 10.42 11.01 10.76 10.79 Total Estimated Lipid moisture Protein Content (%) content (%) (%) 14.4b 14.3c 14.8a 14.3c 14.3c 14.2d 13.9e 11.14c 11.78b 8.99g 10.82d 12.14a 9.85f 10.43c 3.91ab 3.31ab 4.98a 3.82ab 3.64ab 2.56b 4.24ab Total sugar (mg/g) Total phenolics (mg/g) 2.36g 4.36f 5.81d 5.05e 7.53c 8.24b 10.20a 2.10a 1.64bcd 1.53cd 1.93ab 1.35d 1.77a 2.03a Values followed by the same letter are not significantly different (P = 0.05). 94 J.T. ARNASON, B. CONILH DE BEYSSAC, B.J.R. PHILOGENE, D. BERGVINSON, J.A. SERRATOS AND J.A. MIHM hemicelluloses of the outer pericarp of Clear differences with the MW (Classen et al. 1990). Total lipids were the kernel. P-coumaric acid content was situation also are evident. Protein positively correlated to insect choice correlated to % damage of kernels, as content was never significant for and consumption parameters of LGB, well as to the physical parameters of resistance correlations for LGB, suggesting they are attractants or hardness and vitreous endosperm although it is negatively correlated with phagostimulants which is the reverse content in this data set. The importance MW performance (Arnason et al. 1994). of MW (Serratos et al. 1987). Some of of p-coumaric acid may be involved Sugar content was positively correlated the statistically significant relationships with its association with lignin in both to LGB mortality but was not for LGB resistance correlation’s are pericarp and endosperm cell walls. significant for MW in our trials presented in Figures 4-7. Table 6. Pearson correlation coefficients (and P values) of LGB susceptibility parameters to physical and biochemical characteristics of seven genotypes. The significance of these results is that they confirm the importance of the newly discovered phenolic factors in Damaged Powder Weight Choice Weight kernals produced loss test Consumption adults Mortality (%) (g/100g) (g/100g) (#/variety) (mg/day) (mg) (%) Kernels/100g Vitreous endosperm (%) E-ferulic acid (mg/g) p-coumaric acid (mg/g) Total phenolics Total lipids (%) Total sugars (%) Dobie Index Sitophilus zeamais -0.90 (.006) 0.87 (.01) -0.87 (.01) -0.79 (.03) -0.94 (.001) -0.72 (.066) -0.88 (.01) y = 5.571x + 12.745 8 7 6 5 0.8 -.093 (.002) 0.94 (.001) 0.89 (.007) 0.87 (.01) 450 Hardness (n) Hardness (n) 400 350 300 6 300 250 10 20 30 40 Vitreous endosperm (%) Figure 4. Relation of grain weight loss due to LGB and vitreous endosperm content of grain. 50 200 0 1.4 y = 1610.613x + 157.793 350 7 1.3 450 y = 5.570x + 124.058 400 8 0.9 1 1.1 1.2 E-ferulic acid (mg/g) Figure 6. Relation of grain weight loss due to LGB and ferulic acid content of grain. 0.90 (.006) y = 0.071x + 9.059 5 0 9 -0.70 (.006) 9 Weigth loss (g/100g) importance of moisture, hardness, -0.84 (.02) -0.77 (.04) 0.93 (.002) 0.75 (.05) grains as well as defining the Weight loss (g/100g) Hardnessinstron (J) Plasticity of grain (mm) Partial water content (%) Humidity (%) -0.88 (.009) resistance to a second insect pest of 250 10 20 30 40 Vitreous endosperm (%) 50 Figure 5. Relation of grain hardness and vitreous endosperm content of grain. 200 0 10 20 30 40 p-coumaric acid (mg/g) 50 Figure 7. Relation of grain hardness and p-coumaric acid content of grain. MECHANISMS OF RESISTANCE IN MAIZE GRAIN TO THE MAIZE WEEVIL AND THE LARGER GRAIN BORER vitreous endosperm and nutritional factors such as lipids in LGB development or behavior. Ac k now le dgm e nt These investigations were supported with grants from the International Development Research Centre (Canada) and the Natural Science and Engineering research Council (Canada) targeted research programs. Re fe re nc e s Arnason, J.T, J.Gale, B. Conilh de Beyssac, A. Sen, S.S. Miller, B.J.R. Philogene, J.D.H. Lambert, R.G. Fulcher, A. Serratos, and J.Mihm, 1992. Role of phenolics in resistance of maize grain to the stored products insects, Sitophilus zeamais and Prostephanus truncatus. J. Stored Prod. Res. 28: 229-126. Arnason, J.T., J.D.H. Lambert, J. Gale, J.A. Mihm, M. Bjarnason D. Jewell, A. Serratos, J. Fregeau-Reid, and L. Pietrzak, 1993. Is quality protein maize more susceptible than normal maize to the maize weevil? Postharvest Biology and Technology. 2: 349-358. Arnason, J.T., B. Baum, J. Gale, J.D.H. Lambert, D.J. Bergvinson, B.J.R. Philogene, J.A. Serratos, J.A. Mihm, and D.C. Jewell, 1994. Variation in resistance of Mexican landraces of maize to maize weevil in relation to taxonomic and biochemical parameters. Euphytica, 74: 227-236. Classen, D., J.T. Arnason, J.A. Serratos, J.D.H. Lambert, C. Nozzolillo, and B.J.R. Philogène. 1990. Correlation’s of phenolic acid content of maize grain to resistance to Sitophilus zeamais in CIMMYT’S collections. J. Chem. Ecol. 16, 301-315. Conilh de Beyssac, 1991. Analyse de la susceptiblite du grain de mais aux attaques du grand Capucin, M.Sc. thesis, University of Ottawa, Ottawa, Canada. Conilh de Beyssac, B., J.T. Arnason, and B.J.R. Philogene, 1992. Etude de la susceptiblite dugrain de mais aux attaques du grand Capucin, dans, La Poste Recolte en Afrique, K. Foua-Bi et B.J.R. Philogene (Eds), AUPELF 95 Fortier, G., J.T. Arnason, J. Lambert, C. Nozzolillo, and B.J.R. Philogène. l982. Local and improved corn varieties in small farm agriculture in Belize C.A. Phytoprotection 63: 68-78. Sen, A., S.S. Miller, J.T.Arnason, and R.G. Fulcher, 1991. Quantitative determination by HPLC microspectrofluorimetry of phenolic acids in maize, Phytochemical Analysis, 2:225-9. Sen, A., D. Bergvinson, S.S. Miller, J. Atkinson, G. Fulcher, and J. T. Arnason 1994. Distribution, microchemical detection of phenolic acids, flavonoids and phenolic acid amides in maize kernels. J. Agr. Food Chem. 42: 18791883. Serratos, J.A., J.T. Arnason, C. Nozzolillo, J.D.H. Lambert, B.J.R. Philogène, K. Davidson, L. Peacock, J. Atkinson, and P. Morand. 1987. Contribution of phenolic antifeedants to resistance of maize populations to the maize weevil, Sitophilus zeamais. J. Chem. Ecol. 13: 751756. Serratos, J.A., A. Blanco-Labra, J.A. Mihm, L. Pietrzak, and J.T. Arnason, 1993. Generation means analysis for phenolic compounds In maize grain an susceptibility to maize weevil infestation. Can. J. Bot.71: 1176-1181. 96 M e c ha nism s of Re sist a nc e in M a ize t o We st e rn Corn Root w orm J.T. Arnason, J. Larsen, R. Assabgui, Y. Xie, J. Atkinson, B.J.R. Philogene, University of Ottawa, Ottawa, Canada and R.I. Hamilton, Agriculture Canada, Ottawa, Canada Abst ra c t The western corn rootworm (WCR), Diabrotica virgifera virgifera LeConte, is considered a primary pest threatening maize cultivation in North America. Branson et al. (1983) indicated the existence of an unidentified antibiosis factor in resistant germplasm from South Dakota, in addition to the well known tolerance. Our laboratory has identified the hydroxamic acids (Hx): DIMBOA, DIM2BOA, HMBOA and MBOA as antibiosis factors in maize roots. These substances induce larval mortality and delay development of the insect. Behavioral data suggest that Hx also reduce acceptability of maize roots as hosts. Using high pressure liquid chromatography (HPLC), these biochemicals have been located in maize tissue at the rootworm feeding sites. A greenhouse study demonstrated that maize varieties with high Hx content were less damaged than varieties with low Hx content when artificially infested with WCR larvae. This result has been confirmed in the field with 7 inbreds of varying Hx content which were artificially infested with WCR eggs. Pre-screening methods for selection of genotypes based on Hx content are currently being evaluated. Chromosome mapping of resistance and phytochemistry is also being undertaken. antixenosis (behavior modifying) et al. 1991) that allowed an evaluation resistance components have received of their role in maize roots. Using these In many areas of the US and Canada, little attention. A study by Branson et synthetic materials, Y. Xie investigated the Western corn rootworm (WCR) al. (1983) reported antibiosis in several the major hydroxamate compounds Diabrotica vigifera virgifera LeConte has experimental maize hybrids. Our found in maize roots and their possible become the most important insect studies since 1988 have firmly role in resistance. Although they are threat to maize cultivation. Chemical established the role of maize secondary stored as glycosides in vivo, they are control is currently the major strategy metabolites in antibiosis and released as the free aglycones by b- to suppress the insect and the amount antixenosis. glucosidases after damage of tissues, I nt roduc t ion such as maceration or insect feeding. of insecticide used is greater than that for any other pest (Metcalf 1986). Government policy in both countries Phyt oc he m ist ry of M a ize Root s The protocol used for extraction and release of free hydroxamates is described in Figure 1. A gradient HPLC calls for the reduction of pesticide use. Alternative strategies of rootworm We hypothesized that phytochemicals method was developed that management including host plant in maize roots may be contributing to conveniently resolved three resistance are required for widespread the reported antibiosis in resistant hydroxamic acids from the root extracts use. Although there has been interest in varieties. The characteristic secondary (Fig. 2). The main compounds found this area, most previous work has chemicals of maize roots are are 2,4-dihydroxy-7-methoxy-(2H)-1,4- focused on field evaluation of hydroxamic acids. In preparation for benzoxazin-3(4H)-one (DIMBOA), and tolerance, the ability of damaged roots these studies, J. Atkinson of our group its degradation product 6-methoxy- to re-grow after pruning by rootworm developed a synthesis of the major benzoxazoline (MBOA), while the larvae. The antibiosis (toxic) and hydroxamic acids of cereals (Atkinson lactam of DIMBOA, 2-hydroxy-7- MECHANISMS OF RESISTANCE IN MAIZE TO WESTERN CORN ROOTWORM 97 methoxy-1,4(2H)-benzoxazin-3-one the localization showed that they are maize inbreds developed at Agriculture (HMBOA) and 2,4-dihydroxy-7,8- generally found in higher Canada from CIMMYT latitudinal dimethoxy-1,4-benzoxazin-3(4H)-one concentrations in the cortex, which is pools (Table 1). (DIM2BOA) are also present in the site of CRW feeding, than the steele significant quantities (Xie et al. 1991b). (Xie et al. 1991a). (UG/G) 120 DIMBOA 100 A study (Xie et al. 1990) of the phenology of these hydroxamates indicated that HMBOA and DIM2BOA were maximal at about 2 weeks after I n V it ro T ox ic it y a nd Ant ix e nosis of H ydrox a m a t e s t o CRW La rva e HMBOA 80 60 40 germination, while DIMBOA reached its peak at 4 weeks (Fig. 3). The The major hydroxamic acid of maize 20 concentration of these materials is then roots, DIMBOA was found to be toxic 0 diluted by growth of the maize to WCR larvae with an LC50 of 153 (108- seedling. The time course of maximal 209) mg/ml and LD50 of 917 (560-2297) production coincides with the early mg/ml (n = 450). These concentrations development of CRW larvae. Studies of are relevant to natural levels found in DIM2BOA 0 1 2 3 Root age (weeks) 4 Figure 3. Phenology of hydroxamic acid concentration in young maize seedlings. H MeO One gram fresh root 0.02 Homogenized in 3 x 5 mL dH20 2 MeO O Incubated at 25ºC, overnight Filtered through cheesecloth (Discard residue) Absorbance MeO DIM2BOA OH O N OH 1 O OH N O H HMBOA MeO 3 Adjust filtrate to pH 2 with 2N HC1 O MBOA N O H 0.01 MeO O OH N O 4 H H DIMBOA Heat to 65ºC, 1 min; cooled in ice 10 min. 0.00 Filter through filter paper (Whatman No. 42) Extract with 2 x 10 mL ethyl acetate (Discard aqueous phase) Pool organic phase Evaporate under vacuum at 40ºC Dry under stream of nitrogen Resuspend in 1 mL ethyl acetate for storage Completely dry under nitrogen Resuspend in 1 mL methanol; filtered Analysis by HPLC Figure 1. Extraction procedure for hydroxamic acids from maize roots. 0 16 18 20 22 Retention time (min) 24 26 Figure 2. HPLC separation of hydroxamic acids from maize root extracts. Table 1. Concentrations of hydroxamate compounds in roots of maize germplasm of various geographic origins (µg/g fresh wt.) Maize line ITR 3872 NTR-1 3983 ITR 3865 NTR-1 3946 NTR-1 3962 NTR-2 4071 ARGEN 2032 STR 3794 STR 3815 STR 3805 ITR 3862 MEXICO 5 NTR-2 4021 Total 1140.5 444.3 392.8 359.0 296.0 281.0 218.5 191.2 184.3 163.2 143.0 135.6 56.8 A B BC BCD CDE CDEF EFGH EFGHI EFGHI EFGHI FGHI GHI I DIMBOA equiv. 921.1 327.1 296.4 248.7 186.5 215.0 177.9 120.0 115.2 103.2 99.8 100.5 44.9 A B BC BCD DEF CDE DEFG EFGH EFGH EFGH EFGH EFGH H HMBOA 86.9 68.5 30.1 30.1 32.6 22.3 21.4 39.7 41.5 31.4 28.7 19.1 6.1 A B EF EF DE EFGH GH CD C E EFG HI KL DIM2BOA 120.9 35.1 61.7 70.1 69.5 38.8 11.7 26.5 22.5 22.9 10.2 12.3 4.0 A CDE B B B CD FGHI DEFG DEFGH DEFGH GHI FGHI I Means followed by the same letter are not significantly different in Duncan’s multiple range test (P= 0.05). 5 98 J.T. ARNASON, J. LARSEN, R. ASSABGUI, Y. XIE, J. ATKINSON, B.J.R. PHILOGENE AND R.I. HAMILTON A behavioral study was also number of turns decreased after Under artificial infestation with WCR undertaken to determine the effect of treatments of the roots with HMBOA, eggs at four different levels, the high naturally occurring and synthetic Hx DIMBOA, DIM2OA or MBOA. DIMBOA line ITR 3872 showed significantly less damage as indicated on the characteristic search pattern of WCR larvae as they locate maize roots (Fig. 5). Strand and Dunn (1990) I n V ivo Effe c t s of H ydrox a m a t e s by plant growth parameters such as plant height, stem thickness, plant fresh weight, and root fresh weight, than the demonstrated that a decreased search area and locomotory rate and increased A greenhouse study of the role of Hx in low DIMBOA line NTR-2 Germany number of turns occurred after WCR CRW resistance was undertaken with 4042 (Fig. 6). Significantly fewer adult larvae contacted host roots as two elite maize lines with widely WCR emerged from the high DIMBOA compared with non hosts (Fig. 5). A varying Hx content (Xie et al. 1990). line and they had lower mean weights and head capsule widths (Fig. 7). comparison of behavior of larvae towards Hx treated roots and controls (Table 2) indicated that these compounds reduced the host suitability Table 2. Behavioral parameters of rootworm larvae during a 5 min host searching period after removal from treated and control roots. of the roots. In particular, locomotory rate and search area increased while Control HMBOA DIMBOA DIM2BOA MBOA 95 Area searched (mm2) Locomotor rate (mm/min) 58.5 a 36.2 bc 30.4 bc 26.9 cd 25.4 cd 117 ef 158 cde 166 bcd 157 cd 204 ab 20.1 b 27.5 a 31.9 a 28.1 a 22.5 a Means followed by the same letter are not significantly different in Duncan’s multiple range test (P= 0.05) 80 Figure 4. Probit plot of rootworm mortality as a function of DIMBOA concentration (from Xie et al. 1990). Support (3 mm high) A Acetate sheet Filter paper Larva Larva path B Figure 5. Apparatus for larval host seeking experiments (upper) and larval paths (lower) of insects near (A) unsuitable host or (B) suitable host. 4 3 2 1 0 70 50 40 30 20 10 22 15 12 9 6 3 0 0 15 2.4 10 5 0 2 cm Stem thicknes (cm) ITR 3872 NTR-2 Ger. 4042 Root fresh wt. (g) 50 100 200 400 800 Concentration (ppm) 120 100 80 60 40 20 0 Root dry wt. (g) 20 Plant fresh wt. (g) 40 Plant height (cm) 60 Plant dry wt. (g) Mortality (%) 90 Number of turns 1.6 0.8 0 0 400 800 Infestation rate (eggs/pot) 0 400 800 Infestation rate (eggs/pot) Figure 6. Mean plant ‘performances’ of maize lines with different DIMBOA contents infested at different rootworm egg concentrations. Significant difference (P = 0.05) between corn line performance is indicated by (*). (Data from Xie et al. 1990). MECHANISMS OF RESISTANCE IN MAIZE TO WESTERN CORN ROOTWORM A subsequent greenhouse study of seven maize lines with varying Fie ld V e rific a t ion of Ant ibiosis Re sult s 99 A prediction of resistance performance can be made on the basis of Hx content. Using biochemical screening, we DIMBOA content artificially infested with WCR larvae showed significant While the laboratory and greenhouse assessed 18 Ontario check hybrids for negative correlation’s between larval trials had given us some confidence the levels of Hx in roots (Assabgui et al. ‘performance’ and root DIMBOA that hydroxamic acids of maize are an 1993). The results suggested that only content (Table 3). Usually insect antibiosis and antixenosis factor to two cultivars would be predicted to performance is a balance of nutritional WCR, these results could not be have significant antibiosis, nine to be factors such as protein or simple considered useful in an agronomic moderately susceptible and seven carbohydrates against anti-nutritional context until verified in the field. Two susceptible. Two of the extremes were factors such as DIMBOA. We were years of field trials were conducted at tested in field trials and performed as surprised to find no positive correlation the Central Experimental Farm in expected. While the method is between nitrogen and insect Ottawa in 1992-3 (Assabgui et al. 1994). promising, the results highlight the performance, but the results are Seven maize inbreds with varying root rootworm resistance problem that most possibly confounded by the nitrogen levels of DIMBOA were selected and germplasm is not resistant. This may be content of DIMBOA. The negative grown in a randomized block design a result of limited selection for correlation with sugar content suggests with 4 replicates. They were infested rootworm resistance in the past. complex interactions with other factors. with 0, 500, 1000 or 1500 WCR eggs per However, we are now using 30.5 cm of row and damage was biochemical pre-screening on a larger assessed at 8 and 16 weeks after number of crosses of temperate inbreds infestation, by digging roots and to tropical and subtropical germplasm, assessing rootworm damage on the 9 in order to increase the probability of class rating scale of Welch (1977). The introducing and selecting relationship between root damage phytochemically based resistance in rating and the total Hx content of the elite cultivars. 10 ITR 3872 NTR2 Ger. 4042 8 6 * roots was significant and negative (Fig. 4 8). The results demonstrate antibiosis due to Hx in a field context. I nhe rit a nc e of Root w orm Re sist a nc e Fa c t ors Sc re e ning for Ant ibiosis During the 1993 growing season at the 2 0 Weight Head (mg) Capsule (mm) Figure 7. Mean ‘performance’ parameters for rootworms emerged from corn lines in Figure 6. Significant difference (P = 0.05) between insect ‘performance’ on corn lines is indicated by (*). Central Experimental Farm in Ottawa, a The correlation results in greenhouse study was conducted by J. Larsen on and field studies suggested an the inheritance of WCR resistance and important application of biochemical pre-screening to WCR resistance assessment. Rootworm field trials are very labor intensive because of the effort of digging and washing the roots. Table 3. Correlation of larval rootworm performance parameters with nutritional and anti-nutritional factors in seven inbreds. Rootworm performance Mean number of surviving larvae Mean weight of larvae Mean head capsule width Note: n = 7 DIMBOA content of roots Sugar content of roots Nitrogen content of roots r = -0.81 P = 0.02 r = -0.95 P = 0.0013 r = -0.94 P = 0.0016 n.s P > 0.05 -0.895 P = 0.05 n.s. P > 0.05 n.s P > 0.05 n.s P > 0.05 n.s P > 0.05 Root damage ratings (1-9 scale) Number larvae/plant 6 5 r = -0.868 P = 0.0018 n=7 4 3 2 1 1.5 2.0 2.5 3.0 Log [total hydroxamates in root] (UG/G fresh weight) 3.5 Figure 8. Relation between mean root damage rating and total root Hx content performed under field conditions for seven inbreds. 100 J.T. ARNASON, J. LARSEN, R. ASSABGUI, Y. XIE, J. ATKINSON, B.J.R. PHILOGENE AND R.I. HAMILTON Hx. A diallel analysis was conducted Ac k now le dgm e nt s involving seven inbred maize lines, varying in both Hx content and WCR This research was supported by grants resistance. The genotypes used were from the Natural Sciences and SD10, CM7, CO272, ITR3872, ITR3865, Engineering Research Council NTR3983 and NTR4034. Root damage (Canada), the Ontario Government and was assessed according to the nine Pioneer Hi-Bred Inc. point rating scale of Welch (1977) and Hx levels were determined by HPLC Re fe re nc e s according to Xie (1991b). The study found that for root resistance, the general combining ability (GCA) was highly significant and specific combining ability (SCA) was nonsignificant, and for root Hx content GCA and SCA were both significant. Plots of combining abilities against their respective traits showed that those varieties that combine well are also the varieties that perform well for the trait in question. The data for hydroxamates is shown (Fig. 9). The diallel analysis was a preliminary study of the inheritance of WCR resistance and root Hx content and has led to an ongoing study intended to undertake the mapping of quantitative trait loci (QTLs) that significantly affect Total hidroxamic acid content of 3 week-old maize seedling roots (ug/g fr.wt.) resistance to WCR. 500 450 Assabgui, R., J.T. Arnason, and R.I. Hamilton. 1993. Hydroxamic acid content of maize roots of 18 Ontario hybrids and prediction of antibiosis to western corn rootworm. Can. J. Pl. Sci.73: 359-363. Assabgui, R., J.T. Arnason, and R.I. Hamilton. 1994. Hydroxamic acid content of maize roots and field rating resistance to western corn rootworm. Econ. Entomol., In Press. Atkinson, J., P.Morand, J.Arnason, H.M. Niemeyer, and H.R. Bravo. 1991. Analogues of the cyclic hydroxamate DIMBOA: Decomposition to benzoxazolines and reaction with mercaptoethanol. J. Org. Chem., 56: 1788-1800. Branson, T.F., V.A. Welch, G.R. Sutter, and J.R. Fisher. 1983. Resistance to larvae of Diabrotica v. virgifera in three experimental maize varieties. Environ. Entomol. 12: 1509-1512. Metcalf, R.L. 1986. In J.L. Krysan, and T.A. Miller (eds) Methods for the study of pest Diabrotica, 7-15. New York: Springer Verlag. r = -0.81 P = 0.0001 n = 28 400 350 300 250 200 150 100 -200 -150 -100 -50 0 50 100 150 200 General combining ability for total hydroxamic acids Figure 9. Correlation between total Hx of 7 inbreds and general combining ability for Hx content. Strand, S.P., and P.E. Dunn. 1990. Host search behavior of western corn rootworm larve. J. Insect Physiol. 36: 201-205. Welch, V.A. 1977. Breeding for corn rootworm resistance or tolerance. In Proc. 32nd Annual Corn Sorghum Research Conf., 131-142. Washington D.C.: American seed assoc. Xie Y.S., J.T. Arnason, B.J.R. Philogene, J.D.H. Lambert, J.Atkinson, and P. Morand. 1990. Role of DIMBOA in the resistance of corn to western rootworm. Can. Ent. 122: 1177-1186. Xie, Y.S., J.T. Arnason, B.J.R. Philogene, J. Atkinson, and P. Morand. 1991a. Distribution and variation of 1,4 Benzoxazin-3-ones and related compounds in maize root systems. Can. J. Bot. 69: 677-681. Xie, Y.S., J. Atkinson, J.T. Arnason, P. Morand, and B.J.R. Philogene. 1991b. Separation and quantification of 1,4 Bezoxazin-3-ones and benzoxolin-2ones in maize root extracts by HPLC. J. Chromatography. 543: 389-395. 101 M e c ha nism s a nd Ba se s of Re sist a nc e in M a ize t o M it e s T.L. Archer, Texas Agricultural Experiment Station, Lubbock, Texas. F.B. Peairs, Department of Entomology, Colorado State University, Fort Collins. and J.A. Mihm, French Agricultural Research, Inc., Lamberton, MN, USA. Abst ra c t Maize resistance to mites was isolated using recurrent selection, in a population of tropically adapted exotic germplasm accessions, that were crossed with temperately adapted NB 611. Since mite damage is greatest to maize during and following pollination, resistance research must be conducted in the field using plants in the reproductive growth stages. Methods for infesting maize with mites, rating damage, and making selections for resistance are discussed. Also, procedures for determining the mechanisms of resistance in maize to mites are described. Nine sources of resistance to mites have been identified. Preliminary research indicates that mite resistance in maize identified to date is primarily tolerance with some antibiosis involved. from maize anthesis to maturity as determined for mites. It would be very mites exploit changes in plant difficult to grow and screen large Mite pests on maize in the United physiology associated with seed numbers of plants in a greenhouse to States include the Banks grass mite, production and leaf senescence (Perring grain filling stage. Therefore, screening Oligonychus pratensis (Banks), two- et al. 1983; Archer et al. 1986, 1988). for resistance should be done in the M it e Biology field. It is best to create uniform mite spotted spider mite, Tetranychus urticae Koch, and carmine mite, Tetranychus Se le c t ion for Re sist a nc e infestations in the field, because natural mite distribution is too clumped for cinnabarinus (Boisduval), (Ehler 1973). On maize, mite development from egg There are several biological reliable evaluation of plants for to adult is completed in <2 weeks when considerations when designing maize resistance. We have found that the best resistance to mite research: way to obtain large numbers of mites temperatures are 23 to 25oC and <1 week when temperatures are over 30oC Maize is most susceptible to mite for infestation is to collect leaves from a (Perring et al. 1984). Mites usually are damage and yield loss from commercial maize field that is heavily found on the underside of leaves and pollination until dent. infested with mites. We obtain infested • Mites have limited dispersal ability leaves as early in the season as possible outer mesophyll cells by sucking out (walking or being blown by the to avoid collecting predators of mites dissolved nutrients (Jeppson et al. wind), which results in uneven and to get plants, in breeding blocks, 1975). Mite feeding produces chlorotic distribution in a field. infested by mid to late vegetative feed in the epidermal and sometimes • Premature senescence of leaves from growth stages. Only heavily infested portions of leaves and reduce yield abiotic stress (e.g. low fertilizer or leaves are collected to assure rapid (Archer and Bynum 1990, 1993). water stress) may mask mite increase in mite densities on plants Infestations begin on the lowest leaves damage. used for research. Infested leaves are spots on leaves, and may kill all or • Very early or late maturing maize placed in paper sacks and immediately plant as mite abundance increases. Rate cultivars may escape mite damage transported to the research field. These of increase and damage by mites are without being resistant. leaves are laid across leaves in the of plants and spread upwards on the • lower third of plants to be infested. A greatest when weather is hot and plants are water stressed (Perring et al. 1986). Mansour et al. (1993) reported that single infested leaf will usually extend Mite densities are generally greatest plants should be at least pollinating across two to four plants in a row. We before resistance in maize can be 102 T.L. ARCHER, F.B. PEAIRS AND J.A. MIHM prefer to infest every row in the Mites and damage spread up the plant maize kernels dent, we usually make nursery, although one to two rows can over time. Under very heavy mite ratings shortly after denting unless mite be left between infested rows and mites infestations, areas of a leaf or whole damage is slow in developing and will spread across rows after their leaves may die from mite feeding. ratings have to be delayed. The 10 to densities become high on the originally Death usually begins at leaf margins on 20% of the plants receiving the lowest infested plants. Usually 4 to 6 weeks are the distal portions of leaves and damage ratings are advanced to the required to produce an infestation large spreads across and down the leaf. One next cycle. enough to provide enough damage for must be careful not to rate leaves dead resistance selection when infesting from senescence as killed by mites. every row. If predators of mites are Therefore, plants should be rated before found on plants, plots are sprayed with a significant number of leaves We have identified inbreds from nine chlorpyrifos at 0.28 kg ai/ha or senescence naturally as plants approach sources of maize resistant to mites permethrin at 0.22 kg ai/ha to kill the physiological maturity. Since mite (Table 2). These inbreds have been predators. Water and fertilizer stress feeding does not cause yield losses after advanced to the F7 generation. Test M it e Re sist a nt M a ize should be avoided in mite resistance blocks because the symptoms of these stresses can confound accurate ratings Table 1. Mite damage rating scale used to estimate leaf damage from mite feeding on maize. and may affect plant resistance or rate of mite increase. Early maturing maize should be infested early in the season Rating % leaf area damaged/plant 1 1 - 10 2 11 - 20 3 21 - 30 4 31 - 40 mite increase. 5 41 - 50 When we were selecting lines for 6 51 - 60 7 61 - 70 8 71 - 80 9 81 - 90 10 91 -100 or with very heavy numbers of mites to allow time for mite increase and damage to develop before a significant amount of leaf senescence occurs. Late maturing maize should be planted early so that the susceptible growth stages occur while weather is best for resistance, we used single row plots that were 5 m long and replicated three times. During breeding, we do not replicate plots, but select individual plants in a row. Because mite ratings cannot be made on plants prior to pollination, we self plants before we Description of damage A few small mite colonies and associated damage (chlorotic spots) along the midrib of the lowest leaves. Mite colonies and damage spread along the midribs on the lowest leaves on a plant. Mite colonies and damage spreading out from the midrib on the lowest leaves and small colonies may occur on leaves up to the ear. Mites and damage cover most of the leaf area on the 1-2 lowest leaves and mite colonies and damage extend along the midrib to the ear leaf. Mites have killed one leaf, bottom 2-3 green leaves heavily infested and damaged, and mite colonies on 1-2 leaves above the ear. Mites have killed or nearly killed the bottom two leaves and colonies and damage extend beyond the midribs on two leaves above the ear. Mites have killed or nearly killed the bottom three leaves, all leaves up to the ear significantly damaged, and mite colonies and damage found on most to all leaves on the plant. Mites have killed or nearly killed all leaves up to the ear and mites and damage occur on most to all leaves on the plant. Most leaves on the plant killed by mite feeding and only leaves in upper third of plant alive. Very little green area left on plant or plant dead. select for resistance. We infest every plant in a row and self 5 to 10 plants. During the dent growth stage, every selfed plant in a row is rated for mite damage using the 1-10 scale (Table 1). Chlorotic spots on leaves are symptomatic of mite damage. These small spots are caused by mite feeding which drains all nutrients and chlorophyll from individual epidermal cells. Mite infestation and damage will begin on lower leaves on the plant. Table 2. Pedigrees of maize resistant to mites. Source Pedigree 1 2 3 4 5 6 7 8 9 (NB 611 X Valle 411) X (NB 611 X VEN 733) (NB 611 X LOR 9) X (NB 611 X VEN 604) (NB 611 X Arizona 8601) X (NB 611 X VEN 414) (NB 611 X VEN 426) X (NB 611 X Valle 411) (NB 611 X Sin 2) X (NB 611 X Valle 411) (NB 611 X KS 2301) X (NB 611 X Arizona 8601) Bahia Gpo 3 Chiapas 26 Ecuador 569 Key: VEN = Venezuela; Sin = Sinaloa Races (Comun.) (Guaribero) (Piricinco) (Canilla) (?) (Tuxpeno) (Negrito) (Comun.) (Chapalote) (?) (?) (?) Tuson 9 Tepeci 19 Tusilla MECHANISMS AND BASES OF RESISTANCE IN MAIZE TO MITES 103 crosses have been made between Mansour et al. (1993) indicate that this are developed and compared for mites inbreds for most of the sources and research has to be done on older maize on resistant and susceptible lines to MO17 and B73. These crosses were plants beginning at pollination. This determine antibiosis. screened in experiments replicated makes mechanism research difficult three times. Mite damage ratings were because plants have to be grown for Development - Young female mites are about 10 to 20% higher in crosses than over two months to reach pollination collected from the mite culture which is in the inbreds (Table 3). In most cases, before research can be started. When grown on a mite susceptible maize yield and 100 seed weight were as good we began this research, plants were hybrid. Maize is used as the culture in crosses between mite resistant grown in 4 liter pots until 4 to 6 leaves medium because if another plant inbreds and B73 or MO17 and the were free from the whorl and then species is used, the change of host susceptible checks, B73 X MO17 or transplanted into 19 liter pots. We could affect mite development in Deltapine 4673B. The cross between found that plants grew faster and were experiments on maize. Young females source 6 and either MO17 or B73 did more robust if grown from seed in 19 can be separated from the rest of the not yield well. The Deltapine 4673B was liter pots and not transplanted. Plants mites in a colony by placing uninfested damaged by mites which may have were grown in the greenhouse and maize plants among culture plants. In reduced yield. B73 X MO17 was not research was conducted in the 24 hours, mostly young females and a infested by mites. laboratory at 27oC under florescent few nymphs will migrate from heavily infested plants to the previously lights. uninfested plants. These females can be M e c ha nism s of Re sist a nc e Antibiosis transferred individually from the new Research was begun recently to Antibiosis is the measure of the effect plants to development cages on test determine the mechanisms of resistance of the plant on herbivore development, plants with the aid of a small artist’s in maize to the Banks grass mite. survival, and reproduction. Life tables brush. We used a cage similar to the one described by Perring (1983) to Table 3. Mite damage ratings for inbreds and crosses with MO17 and B73 and yields for crosses. Mite damage rating Resistant (R) Susceptible (S) R or S source entry inbred 1 2 3 4 5 6 7 8 9 — 1 2 3 MO17 B73 MO17 B73 MO17 B73 MO17 B73 MO17 B73 MO17 B73 MO17 B73 MO17 B73 MO17 B73 MO17 3 MO17 X B73 Deltapine 4673B3 2.1 — 1.5 — 2.0 — 2.0 — 3.0 — 2.0 — 3.0 — 3.0 — 3.0 — 7.0 4.9±0.4 c-f2 4.4±0.3 c-g 3.7±0.3 e-h 3.7±0.8 e-h 4.0±0.0 d-h 3.0±0.6 gh 6.0±0.0 bc — 5.8±0.4 bcd 5.5±0.2 b-c 3.8±0.2 e-h 2.5±0.2 h 6.0±0.0 bc 3.6±0.8 fgh — — 5.6±0.5 b-e 4.9±0.3 c-f — 7.0±0.0 ab 8.2±0.9 a survival research (Fig. 1). This cage consists of three pieces of 0.3 cm thick 1 RXS cross confine mites during development and Plexiglas. One piece, 18 cm x 5 cm fits Yield (gm) per ear 100 seed weight (gm) on the top of the leaf. Eight 1 cm 144±14 ab2 137± 7 a-d 110±12 a-d 123±16 a-d 132±10 a-d 167± 3 a 135± 7 a-d — 131± 8 a-d 133±16 a-d 85±11 cd 79± 2 d 146±11 ab 143±18 ab 122±37 a-d 138± 4 abc 117±10 a-d 123± 8 a-d — 101± 3 bcd 113± 3 a-d 24.9±1.3 abc2 26.5±1.2 abc 21.4±1.8 c 24.3±2.1 bc 26.8±1.6 abc 26.7±1.5 abc 30.7±0.5 a —25.2±0.7 abc 26.2±0.5 abc 15.8±0.9 d 12.8±1.4 d 27.5±1.0 abc 24.7±2.5 abc 27.7±0.7 abc 26.6±0.4 abc 25.5±1.1 abc 24.8±1.3 abc —29.9±0.8 ab 25.5±0.8 abc Plexiglas. A piece of photograph Damage rated using the 1 - 10 scale listed in Table 1. Means in a column followed by the same letter are not significantly different according to Student-Newman-Kuels multiple range test (P=0.05, SAS PROC GLM). MO17 X B73 and Deltapine 4673B are a cross and a susceptible commercial hybrid, respectively. diameter holes are drilled into the mounting tape with adhesive on both surfaces is attached to the bottom side of the Plexiglas and a 1 cm diameter cork borer is used to drill the eight holes through the tape. The cage is attached to the top surface of the leaf with the mounting tape adhesive. Two 18 cm x 2.5 cm Plexiglas strips are ; @ À ; @ À À ; @ À@;À@;À@;À@;À@;À@;À@;À@;À@;À@;À@;À@; À@;À@;À@;À@;À@;À@;À@;À@;À@;À@; ; @ À ; @ À ; @ À À@; À@;À@; placed on the bottom side of the leaf on either side of the midrib. The sections of Plexiglas Figure 1. Cage used to contain mites to study development for determining antibiosis. 104 T.L. ARCHER, F.B. PEAIRS AND J.A. MIHM sandwich the leaf and are held together Oviposition - Each freshly molted cage, and the cage is attached to a leaf with three strips of masking tape (one female (<24 hours old) is removed from so that mites have access to either the on each end of the cage and one in the the cells and placed in an oviposition top or bottom leaf surface. A middle). arena consisting of a leaf section (3 x 3 susceptible line has to be included in cm) in a Petri dish. Females are each experiment to compare mite Three to four adult female mites are transferred to leaf sections of the same densities and damage between placed into each cell and the cell inbred that they were reared on in the susceptible and resistant lines. We use opening is covered with a single layer development experiment. Two females MO17 as our susceptible check. Weekly of dialysis membrane held in place with are placed on each section of leaf which the leaf area in the susceptible check contact cement. Dialysis membrane is rests on a cotton ball in a pool of water cages is observed for mite damage used because air and moisture will to keep the leaf fresh. The oviposition without removing the cages. When the move through it but mites cannot arena consisted of the bottom or lid of a average damage in susceptible check escape through it. We found that mites 100 x 15 mm Petri dish. We attempt to cages is > 80% of the leaf area, all caged died in the dialysis membrane covered set up at least 25 Petri dishes per run. leaf sections are removed. All leaf 32oC). Every 3 days until female death, sections in cages are removed at the Therefore, fine mesh cloth covers are females are transferred to a new leaf same time. The percentage of each leaf used when temperature is high, but section. At transfer, the number of section damaged by mites is estimated mites can escape through cloth. After 24 females on each leaf section is recorded and mite densities determined by stage. hours, female mites are removed from as live, dead, or missing. Also, the It is essential to relate the percentage of each cell and the number of eggs are number of eggs oviposited on a leaf the leaf area damaged to the number of standardized to 15 per cell. Eggs are section is recorded. The leaf section is mites within the cage when examined with the aid of a held in the Petri dish with water until determining tolerance. If a plant has stereomicroscope. Sixteen cells are egg hatch. The number of larvae or some antibiosis, the number of mites infested for each run of an experiment. nymphs (indication of hatch), or eggs present might be low and damage We determined that the earliest hatch that do not hatch are recorded. The would then be correspondingly low. occurred 4 days after females were experiment is terminated when all This would provide a false indication removed. Therefore, we begin daily females die. of tolerance. female removal. Eggs are observed for Tolerance Antixenosis hatch or collapse. Eggs that do not Tolerance is the most difficult These experiments measure the hatch or have collapsed are considered mechanism to determine because it is a comparative acceptability of resistant dead. The number of larvae produced subjective measurement. For an and susceptible leaf sections. One must are counted and immature mites are accurate measure of be sure to include all resistant sources observed every other day until the first tolerance, it is important that male is detected. Males complete leaf damage percentages be development about a day before related to pest density and leaf females. Then mites are observed daily area available to mites. Five for the last nymphal molt. The number young female mites are placed in of immatures living to adult, number of each of 20 clip-on leaf cages per days to reach adult, and sex of each inbred. Clip-on cages (clear adult are recorded. The number of dead plastic pill boxes) provide a or missing mites is recorded at each means to restrict mite feeding to observation. Female mites produced in a limited area (2.5 x 2.5 cm) on the development study are used for the each line (Fig. 2). Mites are oviposition study. If males are not seen collected from the culture as in a cell as nymphs reach the last molt, described in the antibiosis some are added to mate with females. experiment, placed into each clip-on cells at high temperatures (over observations for egg hatch 3 days after ÀÀÀÀÀÀÀÀÀ ;;;;;;;;; @@@@@@@@@ ;;;;;;;;; @@@@@@@@@ ÀÀÀÀÀÀÀÀÀ ;;;;;;;;; @@@@@@@@@ ÀÀÀÀÀÀÀÀÀ ;;;;;;;;; @@@@@@@@@ ÀÀÀÀÀÀÀÀÀ ;;;;;;;;; @@@@@@@@@ ÀÀÀÀÀÀÀÀÀ ;;;;;;;;; @@@@@@@@@ ÀÀÀÀÀÀÀÀÀ ;;;;;;;;; @@@@@@@@@ ÀÀÀÀÀÀÀÀÀ ÀÀÀÀÀÀÀÀÀÀÀ ;;;;;;;;;;; @@@@@@@@@@@ ;;;;;;;;;;; @@@@@@@@@@@ ÀÀÀÀÀÀÀÀÀÀÀ ;;;;;;;;;;; @@@@@@@@@@@ ÀÀÀÀÀÀÀÀÀÀÀ ;;;;;;;;;;; @@@@@@@@@@@ ÀÀÀÀÀÀÀÀÀÀÀ ;;;;;;;;;;; @@@@@@@@@@@ ÀÀÀÀÀÀÀÀÀÀÀ ;;;;;;;;;;; @@@@@@@@@@@ ÀÀÀÀÀÀÀÀÀÀÀ ;;;;;;;;;;; @@@@@@@@@@@ ÀÀÀÀÀÀÀÀÀÀÀ ;;;;;;;;;;; @@@@@@@@@@@ ÀÀÀÀÀÀÀÀÀÀÀ ÀÀÀÀÀÀÀÀÀÀ ;;;;;;;;;; @@@@@@@@@@ ;;;;;;;;;; @@@@@@@@@@ ÀÀÀÀÀÀÀÀÀÀ ;;;;;;;;;; @@@@@@@@@@ ÀÀÀÀÀÀÀÀÀÀ ;;;;;;;;;; @@@@@@@@@@ ÀÀÀÀÀÀÀÀÀÀ ;;;;;;;;;; @@@@@@@@@@ ÀÀÀÀÀÀÀÀÀÀ ;;;;;;;;;; @@@@@@@@@@ ÀÀÀÀÀÀÀÀÀÀ ;;;;;;;;;; @@@@@@@@@@ ÀÀÀÀÀÀÀÀÀÀ Figure 2. Clip-on leaf cage used for determining tolerance attached to a maize leaf (top and side views). MECHANISMS AND BASES OF RESISTANCE IN MAIZE TO MITES and a susceptible check in the choice Fut ure Dire c t ions test. The order of arrangement of leaf sections must be randomized each time Inbreds of the nine maize sources an experiment is set up. We conduct resistant to mites have been selected. this research in 100 x 15 cm Petri dishes Test crosses of the inbreds and MO17 with a layer of agar about 1 cm thick. or B73 have been made. Yield equal to Each leaf section used in our choice or better than the susceptible cross, B73 experiments is ca. 0.5 cm wide x 3 cm X MO17, was maintained by eight of long. Both ends of the leaf section are the sources. There is little reduction in inserted into the agar to keep the leaf mite resistance in these crosses viable for 48 hours. The leaf sections compared to the corresponding are bowed so that they do not touch the inbreds. We have begun random agar surface, because mites on the mating with resistant inbreds to under side of leaves can get onto the combine genes for greater resistance. wet agar and die. The agar and leaf Random mating will be conducted for sections remain viable longer when a three generations and then we will thin layer of water is kept on the agar begin selfing to extract further resistant surface. A wax paper disc is placed in inbreds. Since mite pest problems are the center of the Petri dish so that its usually most severe when maize is edge touches each leaf section. Ten water stressed, research has been young adult female mites from the started to combine mite resistance and culture are placed on the wax paper drought tolerance. disc and allowed to disperse to leaf sections. Ten Petri dishes are used in Re fe re nc e s each run of the antixenosis experiment. Three runs provide enough individuals to determine antixenosis. We allow mites 48 hours to choose a leaf based on data by Foster et al. (1977). Archer, T.L., and E.D. Bynum Jr. 1990. Economic injury level for the Banks grass mite Acari: Tetranychidae on corn. J. Econ. Entomol. 83: 1069-1073. Archer, T.L., and E.D. Bynum. 1993. Yield loss to corn from feeding by the Banks grass mite and two-spotted spider mite (Acari: Tetranychidae). Exp. Appl. Acarol. 17: 895-903. Archer, T.L., E.D. Bynum Jr., and A.B. Onken. 1988. Abundance of Banks grass mites Acari: Tetranychidae on corn and sorghum fertilized with different rates of nitrogen and phosphorus. J. Econ. Entomol. 81: 300-303. 105 Archer, T.L., E.D. Bynum Jr., and G.C. Peterson. 1986. Influence of sorghum maturity on abundance and damage potential of Banks grass mite, Oligonychus pratensis (Banks). Exp. Appl. Acarol. 2: 217-222. Ehler, L.E. 1973. Spider mites associated with grain sorghum and corn in Texas. J. Econ. Entomol. 66: 1220. Foster, D.G., G.L. Teetes, J.W. Johnson, and C.R. Ward. 1977. Resistance in sorghums to the Banks grass mite. J. Econ. Entomol. 70: 259-262. Jeppson, L.R., H.H. Hartford, and E.W. Baker. 1975. Mites injurious to economic plants. University of California Press, Berkeley, CA. Mansour, F., A. Bar-Zur, and F. Abo-Moch. 1993. Resistance of maize inbred lines to the carmine spider mite Tetranychus cinnabarinus (Acari: Tetranychidae): evaluation of antibiosis of selected lines at different growth stages. Maydica. 38: 309-311. Perring, T.M. 1983. Influences of temperature, humidity, and corn canopy microenvironment on populations dynamics of the Banks grass mite, Oligonychus pratensis (Banks). Ph.D. dissertation, University of Nebraska, Lincoln, NB. Perring, T.M., T.L. Archer, D.L. Krieg, and J.W. Johnson. 1983. Relationships between the Banks grass mite (Acariformes: Tetranychidae) and physiological changes of maturing grain sorghum. Environ. Entomol. 12: 1094-1098. Perring, T.M., T.O. Holtzer, J.L. Toole, J.M. Norman, and G.L. Myers. 1984. Influences of termperature and humidity on pre-adult development of the Banks grass mite (Acari: Tetranychidae). Environ. Entomol. 13: 338-343. Perring, T.M., T.O. Holtzer, J.L. Toole, and J.M. Norman. 1986. Relationships between corn-canopy microenvironments and Banks grass mite (Acari: Tetranychidae) abundance. Environ. Entomol. 15: 79-83. 106 M e c ha nism s a nd Ba se s of Re sist a nc e in M a ize t o Spot t e d St e m Bore r S.S. Sekhon and U. Kanta, Punjab Agricultural University, Ludhiana, India. Abst ra c t Spotted stem borer, Chilo partellus (Swinhoe) is a serious pest of maize, Zea mays L. The mechanisms (antibiosis, antixenosis and tolerance) and bases of resistance to this pest have been investigated in India. Many materials were evaluated for antibiosis and about 20 were reported to manifest this mechanism of resistance. Among these, seven maize materials, namely, Antigua Gr. 1, A1 x Antigua Gr. 1, Antigua Compuesto, Ganga 5, J 22, J 605 and Mex. 17 manifested a higher level of antibiosis. The use of plant materials from this germplasm, as food for rearing C. partellus, adversely affected some vital parameters of the insect’s biology. It reduced larval survival, larval and pupal weight, fecundity and egg viability, prolonged the larval and pupal period, and ultimately reduced the progeny of the pest. A cumulative effect of antibiosis was also observed. Among different plant parts, minimum antibiosis was recorded in ears and maximum in the tassel. Antibiosis was observed to develop and become operative when the plants were 10-15 days old and it increased with plant age. Antixenosis for oviposition occurred in Antigua Gr. 1, A1 x Antigua Gr. 1, Ageti 76, Caribbean Flint Composite and Cuba 11J. The 4-week old plants were less preferred than 2-week old plants. Antigua Gr. 1 and A1 x Antigua Gr. 1 exhibited both antibiosis and antixenosis. Among nine maize varieties tested for tolerance, Vijay ZFS3 appeared to possess this mechanism. Some chemical constituents of maize plants were evaluated in relation to the level of resistance. The germplasm having higher resistance, compared to those possessing lower resistance, had higher contents of silica and iron but lower nitrogen, phosphorous, potash and sugar. Furthermore, the studies showed that the resistance may be due to some toxins. The implications of the results obtained on mechanisms and bases of resistance are discussed. having insect resistance. The research Antibiosis in maize germplasm - work carried out in India on the Antibiosis has been evaluated on the mechanisms and bases of resistance in basis of larval survival by Pant et al. (Swinhoe) is a serious pest of maize, maize to C. partellus is reviewed in this (1961), Kalode and Pant (1966), Mathur Zea mays L., in India. It is one of the paper. and Jain (1972) and Lal and Pant (1980), I nt roduc t ion Spotted stem borer, Chilo partellus and development period by Panwar limiting factors in the successful cultivation of this crop. It is reported to M e c ha nism s of Re sist a nc e and Sarup (1980). The promising germplasm that exhibited antibiosis are cause 24 to 83% loss in maize yield (Chatterjee et al.; 1969; Sarup 1973; Antibiosis presented in Table 1. These include Mathur and Rawat 1981). The This is the most evident, desirable and indigenous collections, indigenously development and use of resistant long lasting mechanism of resistance. It developed hybrids and composites and varieties is the most useful approach to includes all the adverse effects of a introductions from the Caribbean and manage pests. Plant resistance could be temporary or permanent nature on the the USA. explained through three fundamental insect biology resulting from the mechanisms of resistance; antibiosis, ingestion of a plant by an insect. Studies Sharma and Chatterji (1971b), Sekhon antixenosis and tolerance in plants to have been conducted on antibiosis in and Sajjan (1987) and Durbey and insects (Painter 1951). The knowledge different maize germplasm, its Sarup (1984) evaluated different of the mechanisms and bases of expression in relation to plant age and populations and hybrids. In addition to resistance is useful in breeding cultivars in different plant parts and its larval survival they studied the cumulative effects on C. partellus. MECHANISMS AND BASES OF RESISTANCE IN MAIZE TO SPOTTED STEM BORER 107 antibiotic effect of these germplasm on varieties than on Basi local. Further, the generations. The results with respect to other biological parameters, namely production of males out numbered the two resistant (Antigua Gr. 1 and Ganga larval and pupal weight, larval and females on resistant varieties and the 5) and one susceptible line (Basi Local) pupal period, pupal survival fecundity, reverse was the case of Basi Local. The are presented in (Table 3). egg viability, sex ratio and results obtained by Sekhon and Sajjan multiplication rate. They reported that (1987) are presented in Table 2. Durbey The pest multiplied slowly on resistant the resistant varieties, namely Antigua and Sarup (1985, 1988) observed a materials in comparison to the Gr. 1, A1 x Antigua Gr. 1, Antigua similar antibiotic effect on C. partellus susceptible one during the first Compuesto, Ganga 5, J 22, J 605 and when the pest was reared on a diet that generation. The cumulative effect of Mex. 17 reduced larval survival, larval contained powdered dry material and antibiosis for two generations caused a weight and pupal weight, prolonged the ether extract of resistant drastic reduction in the multiplication larval and pupal period as compared to populations, Antigua Gr. 1 and Mex. rate of the pest, which was only 0.4 the susceptible variety Basi Local. The 17. It may be added that Antigua Gr. 1, times on Antigua Gr. 1, 1.8 times on pest multiplied at a slower rate when was used in all six studies on antibiosis. Ganga 5 and about 10.0 times on Basi Local. Apparently, resistant or reared for one generation on resistant Table 1. Maize germplasm showing antibiosis to C. partellus. Cumulative effect of antibiosis - susceptible materials can markedly Information on the cumulative or influence the build-up of the additive effect of antibiosis in maize population in the field. germplasm on C. partellus reared Germplasm Reference AES 805, Ill 1656, K41, nc 27, yellow no. 2 Ganga 101, Arbhavi Local, Jalandhar Local, Rudrapur Local Antigua Gr. 1, A1 x Antigua Gr. 1 Antigua Compuesto Vijay, J 12 Antigua Gr. 1 Jawahar, Ganga 5 Antigua Gr. 1, Mex. 17 J. 605, J 22 Pant et al. (1961) Kalode and Pant (1966) Sharma and Chatterji (1971) Mathur and Jain (1972) Lal and Pant (1980) Panwar and Sarup (1980) Durbey and Sarup (1984, 1985, 1988) Sekhon and Sajjan (1987) continuously on a particular variety for Antibiosis in different plant parts - The more than one generation is of practical antibiotic effect of four plant parts; utility. This will help in identification of stem, whorl, ear and tassel on the the germplasm which may suppress the biological parameters of C. partellus has population build-up of the pest in its been investigated by Sharma and active season. In the Punjab, C. partellus Chatterji (1971b) (Table 4). The multiplies on the spring sown maize percentage survival of the larvae, larval fodder crop for 2-3 generations before it weight, pupal weight, sex ratio shifts to the main rainy season crop. (female/male), fecundity and egg viability were found to be relatively Sajjan and Sekhon (1992a) studied the higher in the case of the larvae reared rate of multiplication of C. partellus on on ears than those on other parts of six maize materials over two plant. Also, larval and pupal period and incubation period were relatively less when reared on ears. The results Table 2. Antibiotic effect of maize germplasm on different biological parameters of C. partellus. suggested that tassel and ear had the maximum and minimum antibiotic Germplasm Larval survival (%) Larval weight (mg) Larval period (d) Antigua Gr. 1 Ganga 5 J 22 J 605 Basi Local 19.5 15.2 17.3 13.1 27.7 43.6 59.9 48.4 66.7 29.8 25.9 26.7 23.5 Germplasm Pupal weight (mg) Pupal period (d) Multiplication ratio Antigua Gr. 1 Ganga 5 J 22 J 605 Basi Local 45.4 53.3 44.3 42.6 52.6 7.0 7.5 6.8 7.3 5.9 1:0.6 1:1.3 1:0.6 1:2.7 Source: Sekhon and Sajjan (1987) effect, respectively. Table 3. Cumulative effect of antibiosis in maize germplasm on C. partellus. Multiplication rate (times) Sex ratio Male Female 1 1 1 1 1 1.1 1.0 0.7 0.8 1.8 Germplasm Antigua Gr. 1 Ganga 5 Basi Local One Two generation generations 1.19 1.38 2.92 Source: Sekhon et al. (1992a) 0.37 1.81 9.96 108 S.S. SEKHON AND UMA KANTA Antibiosis in relation to plant age - old ones. The lack of expression of Chatterji (1971a), Lal and Pant (1980) Plant age has been reported to antibiosis in resistant germplasm and Sekhon and Sajjan (1980) and influence antibiosis (Kalode and Pant during their early growth period has Sekhon and Sajjan (1985). Sharma and 1967). Sekhon and Sajjan (1990) also been observed by Mathur and Jain Chatterjee (1971a) found Caribbean evaluated larval survival of C. partellus (1972), and Singh and Sandhu (1979). Flint Comp. and A1 x Antigua Gr. 1 to be relatively less preferred for on the plants of different ages (5, 10, 15, 20 and 25-days old) in Antigua Gr. 1, Antixenosis oviposition than Basi Local and Ganga 5 and Basi Local (Table 5). There Antixenosis, or non-preference, denotes Antigua Gr. 1. However, Lal and Pant were very small differences among the the plant characteristics and insect (1980a) and Sekhon and Sajjan (1985) lines for larval survival on 5 and 10-day responses that lead to avoidance of a reported Antigua Gr. 1 also to be less old plants. The borer survival, particular plant or variety, for preferred than Basi Local. It may be however, sharply declined on 15-day oviposition, food or shelter or a added that Antigua Gr. 1 and A1 x old plants of resistant populations combination of the three. Kogan and Antigua Gr. 1 have been reported to (Antigua Gr. 1 and Ganga 5) and the Ortman (1978) have proposed this new exhibit antibiosis also. Thus, these may decline continued up to 25 days, but at and appropriate term to replace be usefully exploited in the breeding a lower rate. Thus, the most critical Painter’s term non-preference. program. Antigua Gr. 1 is a parent of double top-cross hybrid cultivar, Ganga time for the development of antibiosis may be when the plants are 10 to 15 Antixenosis in maize for oviposition - 5. In the study of Sekhon and Sajjan days old. Sharma and Chatterjee Differential preference for oviposition (1985), Ganga 5 manifested a fairly high (1971a) also reported more antibiosis in by C. partellus in maize has been amount of antibiosis but showed a little 27-day old plants than that in 15-day reported by Singh (1967), Sharma and antixenosis for oviposition (Table 6), in contrast Ageti 76 expressed a little Table 4. Antibiotic effect of different plant parts of maize germplasm on different biological parameters of C. partellus. Plant part Larval survival (%) Larval weight (mg) Larval period (d) Pupation (%) Pupal weight (mg) 40.9 39.1 47.4 34.7 49.6 45.3 75.0 31.7 18.7 21.1 15.6 22.0 34.5 31.7 40.2 28.6 54.3 55.1 66.7 44.4 Stem Whorl Ear Tassel Plant part Pupal period (d) Moth emergence (%) Sex ratio (f/m) Fecundity (no.) 6.0 6.4 5.4 7.9 76.0 71.9 83.0 80.6 1.13 1.02 1.23 0.88 200.6 188.6 260.1 163.0 Stem Whorl Ear Tassel antibiosis but relatively more antixenosis. Antixenosis in relation to plant age According to Sekhon and Sajjan (1985) 5 day old plants were not preferred at all, but 15 day old plants were the most preferred for oviposition by C. partellus (Table 7). As the plant age increased from 15 days onward, the number of eggs laid by C. partellus went on decreasing so much that it was reduced to one-fourth. Source: Sharma and Chatterji (1971b) Table 5. Antibiotic effect of different maize germplasm in relation to plant age on C. partellus. Table 6. Antixenosis in maize germplasm against oviposition by C. partellus. Larval survival (%) Plant age (d) Antigua Gr. 1 Ganga 5 Basi Local 5 10 15 20 25 7.3 65.0 40.0 31.3 28.8 77.5 76.3 52.5 41.3 38.8 78.5 80.0 75.6 76.3 74.4 Source: Sekhon and Sajjan (1990) Germplasm Antigua Gr. 1 Ganga 5 J 22 Vijay Ageti 76 Basi Local Eggs/ Egg plant (No.) masses (No.) 11.9 39.0 25.2 25.2 20.6 22.7 0.9 2.9 1.8 1.6 0.9 1.8 Table 7. Antixenosis in maize in relation to plant age against oviposition by C. partellus. Plant age (d) Eggs/plant (No.) 5 10 15 20 25 30 0.0 34.8 82.8 36.4 21.6 18.4 Source: Sekhon and Sajjan (1985) Source: Sekhon and Sajjan (1985) MECHANISMS AND BASES OF RESISTANCE IN MAIZE TO SPOTTED STEM BORER 109 Sharma and Chatterjee (1971a) recorded Vijay ZFS3, however, appeared to show an insect, its physiology or may act as relatively more oviposition on 15 day tolerance to this pest in one of the an inhibitor or toxin. In the studies of old plants than on 27 day old plants. experiments, due to the high larval Kalode and Pant (1967), Sharma and Singh and Sandhu (1978) reported survival nearly equal to that on the Chatterji (1971b) and Uma Kanta and maximum oviposition on 16 to 18 day susceptible Basi Local, and the Sajjan (1989), resistance was associated old plants and no oviposition on plants relatively low coefficient of with lower nitrogen content. of age less than 10 days. According to harmfulness. In the case of internal Furthermore, resistant germplasm, in Durbey and Sarup (1982) the maximum feeders like the spotted stem borer it comparison to the susceptible, had egg laying occurred on plants of 7 to 15 may be difficult to maintain a relatively lower sugar (Kalode and Pant 1967a; day old plants, with maximum egg uniform population on the test Sharma and Chatterji 1971c), laying on 7 day old plants. From these varieties, whilst minimizing the phosphorous and potash content, but studies, it seemed that generally the antibiotic effects of the less susceptible higher silica and iron content (Sharma most preferred plant age for oviposition varieties. Maxwell and Jennings (1980) and Chatterji, 1971b). Uma Kanta and was 7-15 days. Therefore, the also remarked that out of the three Sajjan (1989) reported that the nitrogen application of insecticides for the mechanisms of resistance, tolerance is content in the plant decreased with control of this pest must be made on a perhaps the most difficult to quantify. plant age, even in the susceptible 10 to 15-day old crop. Plants of about 15 But this mechanism of resistance needs material (Table 9). days age should be used for studying further investigation. Nutritional deficiency in maize antixenosis in maize germplasm for Sharma and Chatterji (1972) carried out oviposition. Ba se s of re sist a nc e Tolerance Chemical constituents of maize maize in relation to C. partellus This is the ability of the plant to repair Chemical constituents of the plant resistance under both field and injury or grow to produce an adequate affect the survival and developmental laboratory conditions. In field studies, yield, despite supporting an insect behavior of the insect in many ways. they applied solutions of diet population at a level capable of These may affect the normal feeding of ingredients and diets lacking some studies on the nutritional deficiencies in damaging a more susceptible crop. The cultivars exhibiting a moderate level of antibiosis and higher level of tolerance are considered ideal, as they allow the Table 8. Tolerance mechanism of resistance in maize germplasm to C. partellus. survival of an adequate pest Experiment 1 population, large enough to maintain the parasites and predators, but prevent the build up of new biotypes (Horber, 1972). However, little work has been done on this mechanism of resistance. Sekhon and Sajjan (1992) evaluated genotypes having variable resistance to C. partellus (Table 8). The studies revealed that only Antigua Gr. 1 and Ganga 5 resisted the attack of the borer Germplasm Antigua Gr. 1 Ganga 5 Vijay ZFSC3 Basi Local LSD (0.05) Damage Number Loss Damage Number Loss score* of larvae coefficient (%) score of larvae coefficient (%) 4.7 5.2 7.5 8.6 0.6 2.6 3.3 2.8 5.0 1.2 due to antibiosis as revealed by significantly less larval survival on these varieties than that of the others. 4.7 4.5 5.4 7.0 1.2 3.3 2.9 5.2 5.7 0.9 -6.9 14.8 24.5 84.3 Table 9. Nitrogen concentration of plants of maize populations. Nitrogen (%) showed low values for damage grade the resistance in these materials is more 26.2 19.0 100.0 100.0 * 1-9 scale, where 1 = healthy, 9 = dead heart. consistently. Both these materials and the coefficient of harmfulness, but Experiment 2 Whole plant Treatment Antigua Gr. 1 Basi Local LSD (0.05) Stem Leaf 12 DAG 24 DAG 36 DAG 24 DAG 36 DAG 2.27 3.18 0.21 1.96 2.31 0.27 1.56 0.79 0.14 2.13 2.45 0.20 1.55 0.73 0.14 DAG = days after germination Source: Uma Kanta and Sajjan (1989) 110 S.S. SEKHON AND UMA KANTA nutrients to the whorl of resistant plants to resistant plant whorls did not The data given in Table 13 show that plants. In the laboratory, they evaluated improve the survival of borer larvae in the effect of the toxin was suppressed supplemented and deficient diets. field studies. This indicated that the by some dietary components. Dextrose Under field conditions, the addition of differential resistance is probably not and ascorbic acid were the most potent dextrose, ascorbic acid or salt mixture due to the lack of feeding stimulants. suppressers of the toxin. These findings No.2 increased larval survival whereas On the other hand, addition of juice or suggested that the resistance in maize the absence of these nutrients had an ether soluble extracts to the whorl of to C. partellus is probably the result of adverse effect. However, opposite susceptible material from the resistant the amount of toxin present and the effects were obtained in laboratory one decreased larval survival (Table suppression effect of the nutrients in a experiments. 10). This suggested that the resistance is particular germplasm. probably due to the presence of some Nature of antibiosis in maize toxin in the resistant plants. Sharma and Chatterji (1972a) reported Furthermore, laboratory studies that the addition of juice or an ether revealed that larval establishment and soluble extract from the susceptible survival were less in the diet with juice Sharma and Chatterji (1971, 1971a) and ether soluble fraction from the evaluated the relationship of some resistant germplasm (Table 11 and 12). plant characters with resistance (Table The ether soluble fraction was however 14). The germplasms having vigorous more potent. From this it was inferred plants, compact whorl, soft stem and that the toxins probably have a feeding long internode were more susceptible. deterrent or even repellent action on Further, the C. partellus moths preferred the first instar larvae. to lay eggs either on leaves having a Table 10. Effect of susceptible maize populations treated with juice and ether extract of Antigua Gr. 1 on C. partellus. Larvae (No.) Susceptible population Basi Local K T-4 Antigua Gr. 1 Juice Extract 1.05 1.02 Dist. Water 0.97 0.87 Re la t ionship Be t w e e n Pla nt T ra it s a nd Re sist a nc e glabrous surface, trichome density of 5.12 3.52 Table 13. Effect of diets containing ether extract of Antigua Gr. 1 and variable concentration of nutrient on the larvae number of C. partellus. Source : Sharma and Chatterji (1972) Table 11. Effect of diets containing the juice and ether extracts of maize populations on the larval establishment of C. partellus. Larval establishment (No.) Germplasm Juice Extract Antigua Gr. 1 A1 x Antigua Gr. 1 Antigua Comp. Basi Local 0.5 0.8 4.4 35.3 0.0 0.3 3.5 19.4 Larvae (No.) Concentration Nutrients Ascorbic acid Dextrose Casein Salt mixt. # 2 Absent Normal Triple Diet only 0.0 0.0 1.5 2.0 1.5 1.8 1.5 1.6 7.87 10.62 1.87 2.75 14.25 18.00 14.75 15.75 Source: Sharma and Chatterji (1972) Source: Sharma and Chatterji (1972) Table 14. Plant traits in maize populations. Table 12. Effect of diets containing the juice and ether extracts of maize populations on the larval survival of C. partellus. Population Internode length (cm) Antigua Gr. 1 A1 x Antigua Gr. 1 Basi Local Stem hardness (kg/cubic cm) 11.5 11.1 15.5 2.6 2.3 0.79 Larval survival (No.) Germplasm Juice Extract Antigua Gr. 1 A1 x Antigua Gr. 1 Antigua Comp. Basi Local 4.0 6.4 4.3 38.4 0.1 1.0 5.3 29.6 Source: Sharma and Chatterji (1972a) Population Plant vigor Whorl index Leaf width 54.4 60.3 57.8 71.1 0.5 0.7 0.7 1.3 3.8 3.6 3.8 3.3 Antigua Gr. 1 A1 x Ant. Gr. 1 Antigua Comp. Basi Local Source: Sharma and Chatterji (1971) MECHANISMS AND BASES OF RESISTANCE IN MAIZE TO SPOTTED STEM BORER 1.7 mm2, or short, erect trichomes (Durbey and Sarup, 1982b). Durbey and Sarup (1982a) observed that the abaxial leaf surface of the tip portion was the most preferred ovipositional site by the moths. Re fe re nc e s Chatterji, S.M., W.R. Young, G.C. Sharma, I.V. Sayi, B.S. Chahal, B.P. Khare, Y.S. Rathore, V.P.S. Panwar, and K.H. Siddiqui. 1969. Estimation of loss in yield of maize due to insect pests with special reference to borers. Indian J. Ent. 31: 109-115. Durbey, S.L., and P. Sarup. 1982. Ovipositional response of moths of Chilo partellus (Swinhoe) on different maize germplasms. J. Ent. Res., 6: 1-9. Durbey, S.L., and Sarup P. 1982a. Preferential plant sites for oviposition of Chilo partellus (Swinhoe) moths in different maize germplasms. J. Ent. Res. 6: 111-116. Durbey, S.L., and P. Sarup. 1982b. Morphological characters-development and density of trichomes on varied maize germplasms in relation to preferential oviposition by the stalk borer, Chilo partellus (Swinhoe). J. Ent. Res. 6: 187-196. Durbey, S.L., and P. Sarup. 1984. Biological parameters related to antibiosis mechanism of resistance in maize varieties to Chilo partellus (Swinhoe). J. Ent. Res., 8: 140-147. Durbey, S.L., and P. Sarup. 1985. Antibiosis due to powdered dry plant material of maize varieties incorporated in artificial diet for rearing of the stalk borer, Chilo partellus (Swinhoe). J. Ent. Res., 9: 201206. Durbey, S.L., and P. Sarup. 1988. Effect of different solvent extracts of resistant and susceptible maize germplasms on the biological parameters expressing antibiosis in Chilo partellus (Swinhoe) due to their formulation in artificial diet. J. Ent. Res. 12: 93-97. Horber, E. 1972. Plant resistance to insects. Agri. Sci. Rev. 10: 1-10, 18. Kalode, M.B., and N.C. Pant. 1966. Studies on the susceptibility of different varieties of sorghum, maize and bajra to Chilo zonellus (Swinhoe) under field and cage conditions, and the methods of determining it. Indian J. Ent. 28: 448-464. Kalode, M.B., and N.C. Pant. 1967. Effect of host plants on the survival, development and behaviour of C. zonellus (Swinhoe) under laboratory conditions. Indian J. Ent. 29: 48-49. Kalode, M.B., and N.C. Pant. 1967a. Studies on the aminoacids, sugars, nitrogen and moisture content of maize and sorghum varieties and their relationship to Chilo zonellus (Swin.) resistance. Indian J. Ent. 29: 139-144. Kogan, M. 1975. Host plant resistance in pest management. In R.L. Metealf, and W.H. Luckman (Eds.), Introduction to Pest Management, 103-46. New York: John Wiley and Sons. Lal, G., and J.C. Pant. 1980. Laboratory and field testing for resistance in maize and sorghum varieties to Chilo partellus (Swinhoe). Indian J. Ent., 42: 606-610. Lal, G., and Pant, J.C. 1980a. Ovipositional behaviour of Chilo partellus (Swinhoe) on different resistant and susceptible varieties of maize and sorghum. Indian J. Ent. 42 (4): 772-74. Mathur, L.M.L., and H.S. Rawat. 1981. Studies on maize pests with certain observations on the survival of the hibernating larvae of Chilo partellus (Swinhoe) and its incidence in relation to sowing date. Rajasthan J. Pesticides 8: 17-31. Mathur, L.M.L., and P.C. Jain. 1972. Effect of maize germplasm on the survival and development of Chilo zonellus S. under laboratory conditions. Madras Agric. J. 59: 54-56. Maxwell, F.G., and P.R. Jennings. 1980. Breeding Plants Resistant to Insects. John Wiley and Sons, Inc. Painter, R.H. 1951. Insect Resistance in Crop Plants. Mac Millan Co. New York. Pant, N.C., M.D. Pathak,, and J.C. Pant. 1961. Resistance to Chilo zonellus (Swin.) in different host plants. I. Development of the larvae on different hosts. Indian J. Ent. 23: 128-136. Panwar, V.P.S., and P. Sarup. 1980. Differential development of Chilo partellus (Swinhoe) in various maize varieties. J. Ent. Res. 4: 28-33. Sajjan S.S., and S.S. Sekhon. 1992. Occurrence of tolerance mechanism of resistance in maize to Chilo partellus (Swinhoe) (Pyralidae:Lepidoptera) J. Ent. Res. 16: 201-205. Sajjan S.S., and S.S. Sekhon. 1992a. Cumulative effect of antibiosis in maize on Chilo partellus (Swinhoe) (Pyralidae:Lepidoptera). J. Ent. Res. 16: 262-266. Sarup, P. 1973. Strategy for the control of insect pests of maize during the fifth plan. Paper presented at the All India Coordinated Maize Improvement Workshop. Pantnagar, Uttar, Pradesh, India. 111 Sekhon, S.S., and S.S. Sajjan. 1985. Antixenosis (non-preference) mechanism of resistance in maize against oviposition by maize borer, Chilo partellus (Swinhoe). Indian J. Ent. 47: 427-432. Sekhon, S.S., and S.S. Sajjan. 1987. Antibiosis in maize Zea mays L. to maize borer, Chilo partellus (Swinhoe) (Pyralidae:Lepidoptera). Trop. Pest Mgt. 33: 55-60. Sekhon, S.S., and S.S. Sajjan. 1990. Antibiosis in maize to maize borer, Chilo partellus (Swinhoe) in relation to plant age. Indian J. Ent. 52: 579-582. Sharma, V.K., and S.M. Chatterji. 1971. Screening of some maize germplasms Chilo zonellus (Swinhoe) and some varietal plant characters in relation to their differential susceptibility. Indian J. Ent. 33: 299-311. Sharma, V.K., and S.M. Chatterji. 1971a. Preferential oviposition and antibiosis in different maize germplasms against Chilo zonellus (Swinhoe) under cage conditions. Indian J. Ent. 33: 299-311. Sharma, V.K., and S.M. Chatterji. 1971b. Survival and developmental behaviour of Chilo partellus (Swin.) on some selected germplasms of maize under laboratory conditions. Indian J. Ent. 33: 384-395. Sharma, V.K., and S.M. Chatterji. 1971c. Studies on some chemical constituents in relation to differential susceptibility of some maize germplasms to Chilo zonellus (Swinhoe). Indian J. Ent. 33: 419-424. Sharma, V.K., and S.M. Chatterji. 1972. Studies on nutritional deficiencies in maize in relation to stem borer, Chilo zonellus (Swinhoe) resistance. Indian J. Ent. 34: 5-10. Sharma, V.K., and S.M. Chatterji. 1972a. Further studies on the nature of antibiosis in maize Zea mays Linn. against the maize stem borer, Chilo zonellus (Swin.). Indian J. Ent. 34: 11-19. Singh, Gurdip, and G.S. Sandhu. 1978. Oviposition behaviour of maize borer, Chilo partellus (Swinhoe) in the field. Indian J. Ent. 40: 191-196. Singh, Joginder. 1967. Breeding for resistance to stem borer (Chilo zonellus). Ph.D. Thesis, Post Graduate School, Indian Agricultural Research Institute, New Delhi. Uma Kanta and S.S. Sajjan. 1989. Formulation of improved artificial diet for the mass rearing of Chilo partellus (Swinhoe). J. Insect Sci. 2: 98-102. 112 M a ize Re sist a nc e t o t he Le sse r Cornst a lk Bore r a nd Fa ll Arm yw orm I n Bra zil P.A. Viana and P.E.O. Guimarães, EMBRAPA/CNPMS, Sete Lagoas-MG, Brazil. Abst ra c t Maize, Zea mays, is an important cereal crop in Brazil. It is extensively grown throughout the country for food grain, feed, and fodder purposes. Among many factors, insects pests play a major role in limiting maize yields. The lesser cornstalk borer (LCB) and the fall armyworm (FAW) have been considered the most important field pests, being key pests in many of the areas where the crop is grown. The FAW and the LCB have been reared at EMBRAPA/CNPMS to undertake artificial infestation for large-scale studies, including screening for resistance. Several genetic materials were selected for resistance. Sources of resistance such as CMS 23 and CMS 24 to FAW, CMS 15 and CMS 454 to LCB are being used in breeding for resistance. The resistance mechanisms to FAW were studied on four selected maize genotypes. Larvae reared on CMS 14C required longer to develop to the pupal and adult stages and had reduced larval and pupal weights. The genotype Zapalote Chico had fewer larvae feeding on leaf sections than other genotypes tested. The analysis of a diallel cross indicated that gene action conditioning resistance to the FAW appears to be due to additive and non-additive effects. I nt roduc t ion The LCB larva is a semi-subterranean Da m a ge a nd Ec onom ic I m port a nc e feeder, usually attacking a seedling plant at or just below the soil surface. Maize, Zea mays, is an important cereal crop in Brazil. It is extensively grown The FAW larvae attack maize at all Larvae bore into the stem and during throughout the country for food grain, stages, although the most serious feeding, produce tunnels upward and feed, and fodder purposes. The total damage occurs at the mid-whorl stage downward from the entrance hole. area under cultivation in the country (Cruz 1980). According to Carvalho Feeding usually kills the young plant. during 1992-93 was 11.2 million (1970), depending on the stage of the According to All et al. (1982), when hectares, with a production of 26.8 plant when the damage is done, the plants are killed and desiccated, LCB million tons of grain, an average yield yield reduction ranges from 15 to 34%. larvae move to adjacent plants. Several of 2.4 t/ha (Carrieri et al. 1993). In Brazil, among many factors, insect Table 1. Insects damaging maize in Brazil. pests play a major role in limiting Scientific name Common name Spodoptera frugiperda Elasmopalpus lignosellus Sitophilus sp Helicoverpa zea Diabrotica speciosa Diatraea saccharalis Mocis latipes Agrotis ipsilon Rhapalosiphum madis Deois flavopicta Scaptocoris castanea Sitotroga cerealella Several species Several species Fall armyworm Lesser cornstalk borer Weevils Corn earworm Corn rootworm Sugarcane borer Black cutworm Corn leaf aphid Leaf hoppers Angoumois grain moth Wireworms White grubs maize yields. A list of insects attacking maize in Brazil is shown in Table 1. Among the insects attacking maize, the fall armyworm (FAW), Spodoptera frugiperda and the lesser cornstalk borer (LCB), Elasmopalpus lignosellus have been considered the most important field pests, being key pests in many of the areas where maize is grown. *** Key pest; ** occasional; * secondary Pest status *** *** *** ** ** ** * * * * * * * * MAIZE RESISTANCE TO THE LESSER CORNSTALK BORER AND FALL ARMYWORM IN BRAZIL 113 plants may be killed by one larva in this transferred daily from cups to Lesser cornstalk borer way. Damage caused by this insect is oviposition cages and are fed with A modificiation of Burton’s (1969) pinto reported to be from 20 to 50% of the sugar solution through a cotton wick in bean diet cited by Chalfant (1975) planted area (Sauer 1939; Viana 1991) or a 50 ml plastic jelly cup. Recently, we (Table 3) is used to rear LCB larvae at even the entire crop (Jacobsen 1928). are testing split cell modules placed EMBRAPA/CNPMS. The moths lay into the boxes (29 x 29 x 4 cm), as used eggs singularly on napkins placed on at CIMMYT and described by Mihm the top and bottom of the oviposition (1989a), to rear FAW larvae. cage (cylinder of 20 cm diam. x 20 cm). T e c hnique s for M a ss Re a ring, Art ific ia l I nfe st a t ions a nd Eva lua t ion Proc e dure s Napkins with eggs are placed inside a Artificial infestation with FAW is done small plastic bag and kept at 28º C until at EMBRAPA/CNPMS at the 4 to 5 hatch. Newly hatched larvae are mixed The Maize and Sorghum National fully expanded leaf stage. The with fine (# 4) vermiculite and poured Research Center/EMBRAPA at Sete technique used is similar to that into plastic jelly cups containing diet. Lagoas, MG, Brazil, has mass reared described in detail by Mihm (1989b). Larvae average 3 to 5 per cup using this FAW and LCB since the early 1980s, The larval infestation of every plant to method. Preformed trays holding 32 enabling the Institute to undertake be screened is done with 30-40 hatched cups, are left undisturbed until adult artificial infestation for large-scale larvae mixed with maize cob grits, emergence. The number of adults per studies — including screening for using a “bazooka” to deliver the oviposition cage is 30 pairs. The adult resistance and developing biological, neonate larvae into the plant whorl. food (beer) is supplied through 4 cultural and chemical control tactics for Evaluation for resistance to leaf feeding medicine droppers inserted in the pest management programs. is made 14 days after infestation using middle of the oviposition cage. The a visual leaf feeding damage scale oviposition cage is maintained at 28º C Fall armyworm varying from 0 to 9 as suggested by with a 16 hour photoperiod. The FAW is reared at EMBRAPA/ Davis and Williams (1989). For an CNPMS on a modified black cutworm initial screening of materials we usually Screening trials to evaluate maize diet described by Reese et al. (1972) plant one 10 m row where half of each germplasm for LCB resistance are (Table 2). The moths lay eggs on paper row is protected with insecticide. Two conducted in the greenhouse. Ten napkins, placed into a oviposition cage replications are usually planted. maize seeds are planted in 5 L plastic (62 x 62 cm), which are cut into strips pots. When the seedlings emerge, each and placed in plastic jelly cups to be pot is infested with 50 eggs. Plants attacked, number larvae alive and incubated at 28º C. After incubation, one small larva is transferred to an individual plastic jelly cup, containing the diet, and then sealed with flexiglas lids. The cups are placed into trays that hold 32 cups and are kept undisturbed until adult emergence. The adults are Table 2. Ingredients for the FAW diet used at EMBRAPA/CNPMS. Ingredients Pinto beans Torula yeast Wheat germ Ascorbic acid Methyl p-hidroxy benzoate Sorbic acid Agar 40% Formalin Water Amount 333.0 g 101.4 g 158.4 g 10.2 g 6.3 g 3.3 g 41.0 g 8.3 ml 2400.0 ml Table 3. Ingredients for the LCB diet used at EMBRAPA/CNPMS. Ingredients Amount Agar Water Pinto bean Water (hot) Yeast Wheat germ Mold inhibitor Ascorbic acid Methyl paraben Sorbic acid 40% formalin 55% linolenic acid Tetracycline 40 g 1280 ml 420 g 1300 ml 128 g 200 g 10 ml 13 g 8g 4g 8 ml 10 ml 1 capsule (250 mg) 5g Vanderzaant’s vitamin mixture after infestation. Ge ne t ic Sourc e s of Re sist a nc e a nd Bre e ding M e t hodologie s In the mid-1980s research was intensified by EMBRAPA/CNPMS, with a large amount of indigenous and exotic germoplasm and elite lines being tested for resistance to FAW and LCB. The screening work identified several sources of resistance to these insect pests (Viana 1992a; 1992b). The Mold inhibitor ingredients Propionic acid Phosphoric acid (conc.) Water (dist.) weight of larvae are recorded 15 days 418 ml 42 ml 540 ml materials selected are presented in 114 PAULO ALFONSO VIANA AND PAULO E. GUIMARÃES Tables 4 and 5. During the last 8 years, A recurrent selection scheme and mass greenhouse and field at EMBRAPA/ many maize genotypes were infested selection have been used to accumulate CNPMS. Four maize genotypes, CMS and the subsequent leaf damage and desirable genes for resistance to the 23, CMS 14C, CMS 24 and Zapalote percentage of plants alive were FAW and LCB, respectively. A Chico were selected for study in the evaluated for resistance to FAW and summary of the procedures of selection laboratory and greenhouse. Larvae LCB, respectively. Some material that for resistance against these pests at reared on CMS 14C required longer to appeared to sustain less damage than EMBRAPA/CNPMS is presented in develop to the pupal and adult stages. others and showed good agronomic Table 6. Also, larvae reared on leaf tissue of traits was selected for breeding for resistance. Sources of resistance such as CMS 23 and CMS 14C to FAW, CMS 15 CMS 14C presented reduced larval and pupal weights. M e c ha nism s a nd I nhe rit a nc e of Re sist a nc e and CMS 454 to LCB are being used in breeding for resistance. Both choice and non-choice tests were The resistance mechanisms to FAW used to determine if resistant genotypes have been studied in the laboratory, were less preferred by the larvae for Table 4. Maize genotypes selected for resistance to FAW at EMBRAPA/CNPMS. Year Genotypes CMS 23 CMS 14C CMS 24 Zapalote Chico 1987/88 CMS 23 CMS 24 Zapalote Chico CMS 456 BA 03 SE 20 CMS 451 SE 14 CMS 467 1988/89 Amarillo Cristalino WP 1 RR 060 MG 05 1989/90 BR 108 Tuxpeño Comp. Tuxpeño Veracruzano Mata Hambre X Guajira 314 Nõdzob Torê Oaxaca 250 Puerto Rico 5 WP 33 Cuba 45 WP 18 Zapalote Chico 1990/91 077 R2 Guatemala 786 Nõdzob Prê Puerto Rico 13 Composto Arco Iris Guatemala 73 139 R2 1991/92/93 PB 11 WP 16 Rep.Dominicana 248 Zapalote Chico BA 22 PA 008 Damage range 1986/87 4.0 to 7.5 4.1 to 7.2 1.1 to 3.7 4.8 to 7.0 2.2 to 5.5 4.4 to 7.0 Mean ratings 4.0 5.4 5.5 5.5 4.9 4.9 4.1 5.0 5.2 5.3 5.4 5.5 5.5 1.1 1.1 1.4 1.5 5.5 5.4 5.5 4.8 5.5 5.0 5.5 5.5 5.4 5.3 2.2 2.5 2.5 2.5 2.5 2.5 2.5 4.4 4.8 5.2 5.3 5.5 5.5 Table 5. Maize genotypes selected for resistance to LCB at EMBRAPA/CNPMS. Damage range Year Genotypes 1986/87 CMS 454 CMS 15 Baier Zapalote Chico RN 01 BA III Tucson BA 60 Guadeloupe 16 SE 10 CMS 472 Jalisco 274 Cateto Colômbia VII Cohauila 56 CMS 15 PB 13 Zapalote Chico PAG VI - Moroti EW 3151 V.S.C. AC 84 Centralmex J-VIII Composto Jaíba IV Cateto Prolífico IX Composto Cerrado I PB 11 1987/88 1988/89 1989/90 1990/91 1991/92 1992/93 Plants attacked (%) 42 42 50 50 50 50 50 50 50 30 50 40 50 50 40 42 45 54 45 45 45 50 50 50 42 to 100 50 to 100 40 to 100 30 to 100 40 to 100 40 to 100 45 to 100 Table 6. Schemes of selection for resistance used to FAW and LCB at EMBRAPA/CNPMS. Breeding Population Pest methods CMS 14C CMS 23 MIRT CMS 15/ CMS 454 Number of Cycles of progenies selection Year screened selected (1994) FAW FS-S1 87/88 FAW Inbreeding 88/89 Synthetics FAW FS-S1 91/92 200 200 20 20 4 1 180 35 2 LCB 1000 128 3 Mass Sel. 90/91 MAIZE RESISTANCE TO THE LESSER CORNSTALK BORER AND FALL ARMYWORM IN BRAZIL feeding than susceptible genotypes. The Results obtained with 180 S1 progenies results demonstrated that the genotype of the MIRT population tested for Zapalote Chico had fewer larvae resistance to the FAW showed a genetic preferring to feed on leaf sections than heritability of 53% (superior limit) and other genotypes tested. An additional 42% (low limit) (Viana and Guimaraes test was conducted to determine adult 1994), indicating a good range of oviposition preference using the same genetic variability present in these genotypes. The genotype CMS 14C was materials which can be useful to a less preferred for oviposition compared breeding program for resistance to this with the remaining genotypes. pest. A tolerance study was conducted in Conc lusion yield trials where performance under both infested and protected split plots In summary, the plant resistance was evaluated. The results presented in program to maize pests with emphasis Table 7 show a few materials indicating on FAW and LCB at EMBRAPA/ some tolerance to FAW leaf feeding CNPMS has been focussed on the damage. following aspects: • Locating new and better sources of resistance. We have conducted only limited investigations into the inheritance of • leaf-feeding resistance to the FAW. The analysis of a diallel cross of 10 genotypes. • populations (Table 8) grown under artificial infestation indicated that both Properly maintaining the resistant Determining the mechanisms and inheritance of resistance. • Developing suitable breeding general and specific combining ability methodologies for incorporating were significant sources of variation genetic resistance in agronomically (Guimarães and Viana 1994). Gene suitable cultivars. action conditioning resistance to FAW appears to be due to additive and non- Re fe re nc e s additive effects. The mean ratings of FAW damage on the 0 to 9 scale were 2.5 for crosses of resistant populations (Zapalote Chico x CMS 14C) and 4.35 for crosses between susceptible populations (CMS 01 x CMS 02). Table 7. Maize genotypes showing tolerance to FAW at EMBRAPA/ CNPMS. Grain Mean weight (g) Genotypes rating Infested Protected Amarelo Sertão CMS 21 Palha Roxa Mantena CMS 04 6.9 6.6 6.2 6.1 2487 2313 2961 3474 2125 1962 2534 3174 All. J.H., W.A. Gardner, E.F. Suber, and B. Rogers. 1982. Lesser cornstalk borer as a pest of corn and sorghum. In A Review of Information on the Lesser Cornstalk Borer Elasmopalpus lignosellus, 33-42. The University of Georgia. Spec. Publ. Nº 17. Carrieri. A. de P. et al. 1993. Prognóstico agrícola, 1993/94 algodão, amendoim, arroz, feijão, mandioca, milho, soja. Informações econômicas. São Paulo. 23(10): 78-85. Carvalho. R.P.L. 1970. Danos. flutuação da população. controle e comportamento de Spodoptera frugiperda (J.C. Smith) e susceptibilidade de diferentes genótipos de milho em condições de campo. Tese de Doutoramento. ESALQ. São Paulo. Brasil. Chalfant. R.B. 1975. A simplified technique of rearing the Lesser cornstalk borer (Lepidoptera:Phycitidae). Journal Georgia Entomological Society. 10: 33-37. 115 Table 8. Diallel cross of 10 population tested for resistance to FAW at EMBRAPA/CNPMS. 1990/91/92. Genetic Material Zapalote Chico Z. Chico x CMS 01 Z. Chico x CMS 02 Z. Chico x CMS 05 Z. Chico x CMS 06 Z. Chico x CMS 11 Z. Chico x CMS 14C Z. Chico x CMS 15 Z. Chico x CMS 23 Z. Chico x CMS 28 CMS 01 CMS 01 x CMS 02 CMS 01 x CMS 05 CMS 01 x CMS 06 CMS 01 x CMS 11 CMS 01 x CMS 14C CMS 01 x CMS 15 CMS 01 x CMS 23 CMS 01 x CMS 28 CMS 02 CMS 02 x CMS 05 CMS 02 x CMS 06 CMS 02 x CMS 11 CMS 02 x CMS 14C CMS 02 x CMS 15 CMS 02 x CMS 23 CMS 02 x CMS 28 CMS 05 CMS 05 x CMS 06 CMS 05 x CMS 11 CMS 05 x CMS 14C CMS 05 x CMS 15 CMS 05 x CMS 23 CMS 05 x CMS 28 CMS 06 CMS 06 x CMS 11 CMS 06 x CMS 14C CMS 06 x CMS 15 CMS 06 x CMS 23 CMS 06 x CMS 28 CMS 11 CMS 11 x CMS 14C CMS 11 x CMS 15 CMS 11 x CMS 23 CMS 11 x CMS 28 CMS 14C CMS 14 x CMS 15 CMS 14 x CMS 23 CMS 14 x CMS 28 CMS 15 CMS 15 x CMS 23 CMS 15 x CMS 28 CMS 23 CMS 23 x CMS 28 CMS 28 Avg. LSD (0.050) Dp (Gi - Gi) Dp (Sij - Skl) 1 2 Mean rating 3.2 3.1 3.3 2.4 3.2 2.7 2.1 2.1 3.2 3.4 4.2 4.3 3.4 3.5 3.9 3.7 3.6 3.1 3.9 3.5 3.7 3.4 3.7 3.4 3.7 3.2 3.6 3.4 3.4 4.0 3.1 3.9 3.7 4.1 3.7 3.1 3.5 3.7 4.0 3.8 3.9 3.6 4.0 3.6 3.1 4.0 3.6 3.7 4.6 3.6 3.3 3.4 3.0 3.5 SCA 1 GCA 2 -0.56 -0.06 0.27 -0.53 0.19 -0.30 -0.90 -0.82 0.37 0.22 0.19 0.57 -0.33 -0.21 0.15 -0.10 -0.11 -0.52 0.02 0.57 0.16 -0.23 0.03 -0.21 0.12 -0.24 -0.19 0.01 -0.13 0.38 -0.46 0.42 0.26 0.41 0.04 -0.50 -0.10 0.14 0.53 0.02 0.08 -0.04 0.40 0.14 -0.67 0.08 0.00 0.24 0.84 -0.01 0.13 -0.28 -0.01 -0.09 3.8 0.21 3.5 0.9 0.13 0.43 SCA Specific combining ability. GCA General combining ability. 116 PAULO ALFONSO VIANA AND PAULO E. GUIMARÃES Cruz. I. 1980. Impact of fall armyworm, Spodoptera frugiperda (Smith and Abbot. 1797). on grain yield in field corn. M.Sc. Thesis. Purdue University. W. Lafayette. IN. Davis. F.W., and W.P. Williams. 1989. Methods used to screen corn for and to determine mechanisms of resistance to the Southwestern corn borer and fall armyworm. In Toward Insect Resistant Corn for the Third World: Proceedings of the International Symposium on Methodologies for Developing Host Plant Resistance to Corn Insects, 101-108. Mexico D.F.: CIMMYT. Guimarães. P.E.O., and P.A. Viana. 1994. Estudo da herança da resistência de genótipos de milho ao ataque da lagarta-do-cartucho. Spodoptera frugiperda. Rel. Tec. Anual EMBRAPA/ CNPMS. Sete Lagoas. MG. Brasil. 201202. Jacobsen. W.C. 1928. Report for 1927 of the Bureau of Plant Quarantine and Pest Control. Mon. Bull. California Dept. Agric. 16: 633-677. Mihm. J.A. 1989a. Mass rearing stem borers, fall armyworms, and corn earworms at CIMMYT. In Toward Insect Resistant Corn for the Third World: Proceedings of the International Symposium on Methodologies for Developing Host Plant Resistance to Corn Insects, 5-21. Mexico D.F.: CIMMYT. Mihm. J.A. 1989b. Evaluating corn for resistance to tropical stem borer, armyworms, and earworms. In Toward Insect Resistant Corn for the Third World: Proceedings of the International Symposium on Methodologies for Developing Host Plant Resistance to Corn Insects, 109-121. Mexico D.F.: CIMMYT. Reese. J.C. et al. 1972. A method for rearing black cutworms. Journal of Economic Entomology 65: 11047-1050. Sauer. H.F.G. 1939. Notas sobre Elasmopalpus lignosellus Zeller (Lep.: Pyr). seria praga dos cereais no Estado de São Paulo. Arq. Inst. Biol. 10: 199-206. Viana. P.A. 1991. Estimativa de perdas causadas pela lagarta-elasmo. Elasmopalpus lignosellus. em milho. Rel. Tec. Anual EMBRAPA/CNPMS. 19851987. Sete Lagoas. MG. Brasil. 85. Viana. P.A. 1992a. Identificação de fontes de resistência de milho ao ataque da lagarta-do-cartucho. Spodoptera frugiperda. Rel. Tec. Anual EMBRAPA/ CNPMS. 1988-1991. Sete Lagoas. MG. Brasil. 93-94 Viana. P.A. 1992b. Identificação de fontes de resistência de milho ao ataque da lagarta elasmo. Elasmopalpus lignosellus. Rel. Tec. Anual EMBRAPA/CNPMS 1988-1991. Sete Lagoas. MG. Brasil. 93. Viana. P.A,. and P.E.O. Guimarães. 1994. Melhoramento da população - MIRT de milho para resistência a lagarta do cartucho Spodoptera frugiperda. In Congresso Nacional de Milho e Sorgo. 20, 139. Goiânia. GO. Brasil. Resumos. 117 Window s of M a ize Re sist a nc e D.J. Bergvinson, CIMMYT, Mexico Abst ra c t Breeding for maize resistance to insects and disease has been made possible by the broad genetic range in host plant resistance (HPR). However, within a given genotype, resistance can vary considerably over the course of plant development as well as between the different plant tissues for a given stage in development. These temporal and spatial changes in HPR are also reflected in the phytochemical composition of the plant. Using leaf bioassays, the feeding preferences of European corn borer larvae for certain portions of the leaf and stages of maturity were identified. These preferences were then related to phytochemcial composition which included nitrogen, fiber, phenolic acid, and DIMBOA content as well as leaf toughness and epidermal cell wall absorption of ultraviolet light. Disease pests of maize are also influenced by changes in host plant chemistry, in particular the silk and kernel chemistry as it relates to Fusarium sp. Inferences on HPR strategies can be made from these types of studies which in turn can further our understanding of heritable resistance and how to screen germplasm in an efficient manner. This paper also serves to show the importance of sample position and timing when studying phytochemical mechanisms of HPR to insects and disease. 1985). This temporal and spatial This heterogeneity of host plant variation in biochemical resistance resistance in space and time effectively In the extensive cultivation of maize on mechanisms may be heritable and increases defense longevity by two large tracts of land, not all maize plants subject to selection. Interdisciplinary important mechanisms: are of equal quality or suitable for teams consisting of breeders, • insect and disease pests. Variation in entomologists, pathologists, restricted set of preferred tissues, host-plant quality can result from physiologists and phytochemists can thus lowering the contact rate with intrinsic factors, such as genetic or define the different biochemical defensive compounds and reducing ontogeny, or from extrinsic factors such strategies for HPR which are employed selection pressure for the evolution as soil conditions or environmental for different stages in plant of detoxification mechanisms variation. Studies on maize resistance development. With this understanding have documented extensive genetic of HPR, screening and breeding for variation in biochemical defenses resistance can be accelerated. I nt roduc t ion against insect pests (Russell et al. 1975; Feeding activity is concentrated on a (Schultz 1983). • The concentration of herbivores makes non-random searching by biological control agents much easier Reid et al. 1991; Xie et al. 1992; Arnason Additional variation may be introduced et al. 1994), although the inheritance of by environmental factors — such as specific resistance mechanisms is still nutrients, light, water availability and Upon assessing food plant quality not well defined. In addition to genetic temperature — which affect plant through olfaction, gustatory or tactile variability, ontogeny generates both quality and suitability to insect pests responses, herbivorous insects decide if spatial and temporal heterogeneity in (Mattson and Haack 1987). The the plant possesses antixenosis or plant resistance (Kearsley and Whitham proportion of variation in field antibiosis characters, then, if 1989). This is true for maize which resistance explained by environmental undesirable or unpalatable, relocate exhibits biochemical changes between factors in contrast to genetic variation is and sample again (Renwick 1983). If the different tissues and different stages in unknown for most insect-plant systems plant tissue is a poor food source, the development (Reid et al. 1992; Guthrie but is beginning to be understood in insect will seek a more desirable food et al. 1986a; Argandona and Corcuera maize. source in the case of motile herbivores, (Feeny 1976). 118 D.J. BERGIVSON or tolerate the reduced growth influenced by the environment. discussed in this paper. The main associated with poor sites as in the case Greenhouse grown plants have higher objective is to illustrate the dynamic of sessile herbivores (e.g. scales or levels of DIMBOA than field grown changes in biochemical content that aphids) (Schultz 1983). The out come is plants but are more susceptible to leaf occur within the plant for a given tissue the same for both strategies: reduced feeding damage by the ECB (Guthrie et during the course of development (both insect growth. al. 1986b). New mechanisms for maize vegetative and reproductive tissue) and resistance to ECB leaf feeding have the windows of resistance or Since its introduction to North America recently been proposed that are based susceptibility that result from these over 75 years ago, the European corn on phenolic acid - cell wall changes. borer (ECB), Ostrinia nubilalis (Hübner) carbohydrate complexes that act by (Lepidoptera: Pyralidae), has been increasing the mechanical strength of extensively researched and germplasm the leaf (Bergvinson et al. 1994a). One with resistance has been released, e.g. mechanism that has been shown is Germplasm the synthetic BS9 (Russell and Guthrie ultraviolet (UV) light which influenced The maize synthetic BS9(CB)C4 was 1982). Leaf resistance to first generation field resistance by facilitating the used which has the following inbreds in ECB has been attributed largely to 2,4- formation of cyclobutane dimers of its genetic background: B49, B50, B52, dihydroxy-7-methoxy-2H-1,4 phenolic acids esterified to cell wall B54, B55, B57, B68, CI31A, Mo17, and benzoxazin-3 (4H)-one (DIMBOA) hemicellulose (Bergvinson et al. 1994b). SD10 (Russell and Guthrie 1982). which affects ECB feeding (Robinson et A second mechanism involved the al. 1978) as well as growth and action of peroxidase in the presence of development of larvae by peroxide to form dehydrodiferulic acid Plant maturity and leaf profile study noncompetitive inhibition of digestive (Bergvinson et al., these Proceedings). BS9 (C4) was planted in mid-May of proteases (Houseman et al. 1992; M a t e ria ls a nd M e t hods 1992 at the Plant Research Center, Campos et al. 1989). Other resistance The objective of this study was to Agriculture Canada, Ottawa, Ontario, factors that have been studied less demonstrate the importance of spatial Canada. The rows were spaced 0.9 m extensively include silica, lignin and and temporal variation of biochemical and plants spaced 0.15 m apart with 30 fiber content which may act by resistance factors in maize leaf tissue, plants per row. The soil was a sandy reducing the nutritional quality of the using the leaf feeding resistance to ECB loam. Plants were harvested at full leaf leaf or increase tissue toughness and as a model system. Resistance factors extension for the 3, 5, 7, 9, and 10 leaf thereby rendering nutrients less investigated included foliar nitrogen stages of development. Leaf tissue from accessible (Buendgen et al. 1990; content, leaf toughness, fiber content, several plants was bulked to obtain a Rojanaridpiched et al. 1984). soluble and cell-wall-bound minimum of 10 g fresh weight of tissue hydroxycinnamic acids, hydroxamic for each of three replicates. To study the The secondary compounds most acids and cell wall absorbance using a variation in resistance along the length studied in maize have been the microspectropho-tometer to localize of the leaf, the 13th leaf from plants at hydroxamic acids, and a few studies cellular biochemical resistance factors. the 14th leaf stage in development was did investigate their temporal and Biochemical changes over space and harvested according to a previously spatial distribution. The hydroxamic time also extend to many other tissues reported method (Bergvinson et al., acids are known to decline with plant in maize. Results from ear rot studies these Proceedings). For this study the age (Morse et al. 1991; Gutierrez et al. for Fusarium sp. will be used to leaf was aligned with other leaves and 1988; Guthrie et al. 1986a), but these illustrate temporal changes in portioned into eight equal zones, with a changes have not been related to insect biochemical resistance factors of minimum of 10 g wet weight per zone performance on tissue of varying ages. reproductive tissue (silk and kernel) per replicate and three replicates per Plant interaction studies using aphids which may find application to pests zone. The midribs were removed from and lepidopteran larvae have shown such as the corn ear worm (CEW), all samples prior to processing, the induction of hydroxamic acid Helicoverpa zea (Boddie). Although the tissue was placed in perforated paper production during insect feeding environment influences biochemical bags, frozen at -20° C, and lyophilized. (Niemeyer et al. 1989; Gutiérrez et al. mechanisms in maize resistance Samples were milled on a Wiley® mill 1988). Hydroxamic acid levels are also (Bergvinson et al. 1994b) it will not be WINDOWS OF MAIZE RESISTANCE 119 fitted with a 1 mm mesh screen. Milled apparatus. Neonate ECB did not feed 75W) and a series connecting grating samples were stored at -20oC until sufficiently to provide accurate monochromator (bandwidth 10 nm) analyzed. consumption measurements. Third- was used to measure transmittance instar larvae that molt during the spectra at wavelengths between 230 Insect bioassay bioassay are also not desirable due to and 350 nm (5 nm steps). This Leaf tissues described above (time purging of the gut prior to molting microscope can emit a specific study and leaf profile) were which interferes with the bioassay. A wavelength of light that is then incorporated into a bioassay apparatus small plastic covering was placed absorbed by particular groups of illustrated in Figure 1. The percent inside to shade the larvae and the third phytochemicals based on differential consumption of a 1.2 cm2 disk by two, plate was taped over the top to seal the absorbance spectra. Phenolics have a third-instar ECB larvae in a 48 h period larvae into the apparatus. Larvae were strong absorbance at 280 nm in was determined. The bioassay reared and tested under a 16:8 (L:D) addition to specific absorbances of 311 apparatus consisted of three halves of photoperiod at 85% R.H. and a 25°C/ nm for p-coumaric acid and 326 for plastic Petri plates (6 cm dia.) with the 19°C (L:D) temperature. Area ferulic acid (see Bergvinson et al., these bottom plate having a 1.2 cm diam. consumed was measured using 1 mm2 Proceedings for structures). Sections for hole. The top plate is inverted and wet graph paper. Forty bioassays were microspectrophotometry (8 µm thick) cotton is placed inside and covered performed for each leaf stage and were cut from leaf tissue, embedded in with Whatman #1 filter paper. Leaf growth environment. ice and mounted on quartz slides which do not absorb ultraviolet light. The tissue is placed with the top surface down onto the filter paper as the under- Nitrogen determination microscope was fitted with a 100X surface of the leaf is the preferred An automatic micro-Kjeldahl nitrogen ultrafluar Zeiss quartz objective and a substrate in the field. The bottom plate analyzer (Tecator model 1030, 10X ultrafluar Zeiss quartz condenser with the hole is centered onto the leaf to Höganäs, Sweden) was used to lens. The measuring aperture placed expose the feeding surface. Plates were determine percent nitrogen and over the middle of the cell wall was taped together and two early third- estimate protein content of 0.3 g dry 0.32 mm, which provided a measuring instar larvae were placed into the wt. leaf samples using the conversion field diameter of 2 µm. The factor 6.25 for estimating percent microspectrophotometer was adjusted protein from nitrogen content. for 326 nm which gave a high signal to ;;;; ;; 6.0 cm petri dish top Plastic cover noise ratio for taking readings of Leaf toughness epidermal cell walls. More details of the Fifteen leaf samples were taken from microspectrophotometer technique Two larvae each leaf stage of development and have been reported in Bergvinson et al. 6.0 cm petri dish bottom from each leaf section for the leaf (1994c). Leaf without midrib Instron has been described (Bergvinson Phytochemical analysis Filter paper et al., these Proceedings). The phytochemical analyses has been Cotton 6.0 cm petri dish top Assembled assay profile study. The protocol for the previously described (Bergvinson et al., Microspectrophotometer these Proceedings). A computer-controlled Zeiss UMSP-80 microspectrophotometer equipped with a high-pressure xenon lamp (XBO Extraction and quantification of silk waxes Preliminary work using the scanning Figure 1. Bioassay apparatus used to study localized feeding on maize leaf tissue by the European corn borer. Plastic Petri dishes are modified by making a 1.2 cm diam. hole in the Petri dish bottom. Top of Petri dish is inverted and fitted with a wet ball of cotton and filter paper to keep the leaf moist. Leaf tissue is oriented with the top surface facing down. The bottom of the Petri dish is secured with tape to expose only a portion of the leaf. Two third-instar ECB larvae are added and covered by a plastic shelter to enhance feeding. Another top to a Petri dish is secured by tape to seal larvae into the apparatus. electron microscope identified silk waxes as a possible mechanism for resistance to ear rot, Fusarium graminearum. A time study was conducted on one resistant (CO272) and two susceptible (CO265, CO266) 120 D.J. BERGIVSON inbred lines to relate changes in silk used to compare means between that factors other that micro- wax composition, from the time of first different plant development stages. environment are influencing larval silk emergence till 12 days post Regression analyses between larvae preference for feeding within the whorl emergence, to observed field resistance consumption and phytochemical of maize. by artificial fungal inoculation parameters were done using the mean (Hamilton et al., these Proceedings). values for each of the eight leaf sections Other parameters thought to be The husk was peeled and the silk for the profile study. associated with feeding behavior are shown in Figure 3. Protein was lower removed at 2, 4, 6, 8, 10, and 12 days post silk emergence. A second study for the sections around the green- Re sult s a nd Disc ussion yellow interface of the leaf (section 4), was conducted to investigate changes in wax composition along the silk Recently eclosed ECB larvae generally reaching a level as low as 17%. Given length of a resistant inbred (CO272) and move towards the center of the whorl the low protein levels at this location two commercial hybrids (resistant during day light hours (unpublished along the leaf length, one would expect Pride K127, susceptible Dekalb 435). data). Similar observations have been consumption to be higher for this tissue Samples were stored at -20ºC until reported for other stem borers such as so as to fulfill nutritional requirements processed. Silk waxes were extracted Chilo partellus (Swinhoe) (Lepidoptera: for development (Scriber and Slansky from 1.5 g fresh weight samples of silk Pyralidae) in which 95 to 100% of live 1981), but this was not observed during with 2 x 3 ml of chloroform. Each larvae are within the whorl (Ampofo the 48 h bioassay (Fig. 2). Soluble sample was mixed in a vortex mixer for and Kidiavai 1987). A possible metabolites washed from milled leaf 5 s and then decanted into a clean vial. explanation for this behavior is tissue included sugars, soluble proteins, Chloroform was purged with nitrogen avoidance of the hot, dry micro- chlorophyll, phenolic conjugates such and the dry sample was stored at -20ºC environment on the exposed whorl as flavonoids and hydroxycinnamic until analyzed by gas chromatography. leaves, which can desiccate neonate acids, and hydroxamics such as Wax analysis was done on a Varian larvae. This explanation is supported by DIMBOA. The trend for soluble 3400 gas chromatograph with a flame the fact that egg mortality is higher at metabolites is similar to leaf ionization detector (FID) and a Varian lower relative humidities (Lee 1988) consumption (Figs. 2 and 3). Phenolic model 8100 autosampler. A 15 m x 0.53 and larval mortality often exceeds 80% conjugates of maize have been shown mm ID column was packed with 0.1 µm during the first 48 h after eclosion (Ross to be phagostimulants (Bergvinson film of SPB-1 (Supelco, Bellefonte, PA. and Ostlie 1990). 1993) and may account for higher consumption as the level of sugar A 25 min. temperature gradient program starting at 120ºC and A profile of leaf consumption by conjugates of p-coumaric acid are increasing at 5ºC/min. to 220ºC and ECB on BS9(C4) is depicted in Figure 2. higher for immature whorl tissue (Fig. holding at 220ºC for 5 min was used to ECB larvae show the highest separate wax components. The flow consumption rate on immature rate was 24 ml He/min. Eicosene (C20) leaf tissue within the whorl was used as an internal standard. (sections 6 to 8). The leaf Routinely, 45 samples can be extracted section with the lowest and analyzed per day. consumption was at the point All statistical analyses were performed conducting leaf feeding on SAS V. 6.03 (SAS, 1988). Data were bioassays in growth chambers transformed to satisfy the assumptions the effect of relative humidity of the general linear model. Analysis of over the leaf length is variance (ANOVA) was used to controlled and the degree of determine significant differences in feeding on mature tissue is biochemical factors for different stages obviously lower even when in plant development (P < 0.05). The relative humidity is not a Student-Newman-Keuls (SNK) test was restricting factor. This suggests 120 0.4 80 0.2 40 0 Tissue toughness (N) the whorl (section 4). By Leaf consumption (mm2) where the leaf subtends from Statistical analysis 0.6 0.0 2 4 6 8 Green Yellow Leaf Section Figure 2. Line graphs of BS9 leaf toughness profile (dashed line) in relation to leaf consumption (solid line) using the bioassay illustrated in Figure 1. Force is measured in newtons (N). n=4 for each leaf section. WINDOWS OF MAIZE RESISTANCE 121 3). Other soluble secondary metabolites The high feeding preference for tissue avoid desiccation and starvation during such as ferulic acid conjugates or with elevated levels of DIMBOA can be early stages of development. HMBOA fluctuate and showed no rationalized by observing the relative consistent trend (data not shown). absence of physical defense The major cell wall bound phenolic mechanisms in immature whorl tissue. acids are E-ferulic and E-p-coumaric DIMBOA was found to be at the highest Fiber content in immature tissue is very acids which are attached to levels within the yellow whorl tissue low (Fig. 3) and the relative absence of hemicellulose through pentose sugars which was also the most preferred by phenolic fortification in epidermal cell (Kato and Nevins 1985). Both phenolic ECB larvae (Fig. 3). Based on previous walls, as demonstrated by staining and acids reach their highest levels in feeding preference studies, the converse low microspectrophotometer readings sections 5 and 6 (Fig. 4). Cell-wall- would be expected (Robinson et al. (data not shown), render nutrients bound ferulic and p-coumaric acids can 1978). Nutritional studies have shown within the leaf more accessible and form dimers to cross-link cell wall that DIMBOA incorporated into meridic hence make the tissue more desirable carbohydrates either enzymatically diet increased larval consumption while (Scriber and Slansky 1981; Bernays and through peroxidase to form 5, 5'- reducing the efficiency of nutrient Barbehenn 1987). The tissue toughness diferulic acid (Hartley and Jones 1976) assimilation and various fitness profile found in Figure 1 best illustrates or through photochemical reactions to parameters (Houseman et al. 1992). This the absence of mechanical resistance form truxillic and truxinic acids in part may explain the higher factors vis-à-vis fiber and (Hartley et al. 1988; Ford and Hartley consumption rate of immature, yellow hydroxycinnamic acid fortification of 1989). From the profiles in Figure 4 no whorl tissue with elevated DIMBOA cell walls (Fig. 4). individual biochemical component provided a suitable explanation for leaf levels. Elevated levels of DIMBOA did The toughness profile could account for consumption or toughness. However, to larval feeding during a 48 h bioassay, field observations of neonate behavior. when taken together, hydroxycinnamic but may have affected insect Immature whorl tissue would be easier acids provide a biochemical explanation performance through reduced fecundity to consume by neonates than tougher, for feeding performance, with and prolonged development if feeding mature leaf tissue. By migrating to the photodimers cross-linking the was restricted to this tissue throughout inner whorl, larvae would not only be hemicellulose of mature tissue to larval development (Campos et al. in a higher humidity micro- provide structural resistance. For 1989). environment, but would also be able to sections 4 through 6, elevated levels p- easily ingest water and nutrients to coumaric, ferulic and diphenolic acids 25 15 100 4 6 8 DIMBOA (mg/g) 6.0 4.0 2.0 0.0 2 4 6 8 Green Yellow Leaf Section 1.0 0.5 0.0 2 4 6 8 2 4 6 1.0 0.5 0.0 2 4 6 8 Green Yellow Leaf Section Figure 3. Line graphs of BS9 leaf profile for various biochemical factors that are considered important in host plant resistance. n=4 for each leaf section. 1.5 1.0 0.5 0.0 2 8 1.5 E - Ferulic acid (mg/g) 2 Total truxillic acids (mg/gf) 200 20 2.0 E - p-Coumaric acid (mg/g) 300 1.5 Total diphenolic acids (mg/gf) Percent protein 400 E-p-Coumaric acid (mg/g) Fiber content (mg/g) not appear to be a significant deterrent 6.0 4.0 2.0 2 4 6 8 Green Yellow Leaf Section 4 6 8 6.0 4.0 2.0 2 4 6 8 Green Yellow Leaf Section Figure 4. Line graphs of BS9 leaf profile for cell-wall-bound phenolics that are thought to be involved in host-plant resistance by fortifying the cell wall. n=4 for each leaf section. 122 D.J. BERGIVSON sustain leaf toughness. For sections 7 which damage ratings are most severe ECB feeding bioassays it appears that and 8 all cell wall phenolics are at their when infestations are made early in soluble phenolic acids conjugated to lowest levels, corresponding to plant development (Maredia and Mihm sugars act as phagostimulants increases in leaf consumption (Fig. 1). 1991). (Bergvinson 1993). Regression analysis of leaf The hydroxamic acid DIMBOA Protein content dropped significantly consumption against the biochemical dropped to significantly lower levels from the third to the fifth leaf and then parameters identified three parameters after the three leaf stage (Table 1). Since gradually declined with subsequent that could account for over 90% of the the older leaf stages had lower levels of leaves as the plant aged (Table 1). The variation in leaf consumption. These DIMBOA than the younger leaf stages, highest protein content leaves were included epidermal cell wall it is evident that reduced consumption subjected to the most feeding, in absorbance, toughness and fiber of older leaves (Fig. 5) cannot be contrast to low protein content leaves content which are all components or explained by non-preference for which would have been expected to indicators of mechanical resistance. DIMBOA. Similar observations have have elevated feeding to sustain the Fiber not only increases the bulk been reported in other greenhouse insect’s protein/growth requirements. density of the insect’s diet to make studies which have made comparisons Gravimetric determination of soluble nutrient and water requirements less to field-grown maize (Guthrie et al. metabolites provides a crude estimate attainable (Bernays 1986), but would 1986b) or to plants grown under of sugars, soluble secondary also increase the substrate for phenolic elevated artificial light conditions in the metabolites, proteins and chlorophyll cross-linking. Acting in concert, fiber greenhouse (Manuwoto and Scriber (Table 1). The same trend as for protein and hydroxycinnamic acid fortification 1985). From both studies it in epidermal cell wall tissue could appears that in addition to increase leaf toughness of mature leaf DIMBOA, there are other tissue. Neonate larvae would then be biochemical resistance forced to feed on softer, immature mechanisms. Soluble phenolic acids matures, its mandibles may be better conjugated to various sugars able to cope with tougher, mature varied but generally showed a tissue (Bernays, 1986) which has lower reduction with increasing leaf levels of DIMBOA. Based on within- number (Table 1). This trend in leaf variation of feeding preference and soluble phenolic levels has biochemical factors, leaf toughness and been observed for Sorghum the biochemical factors responsible for bicolor (Woodhead 1981). For leaf toughness appear to be the some insects, phenolic acids predominant factors that influence ECB can act as feeding deterrents feeding behavior within maize during (Dowd 1990), but based on the mid-whorl stage of plant development. When considering the biochemical changes over time and their relation to leaf resistance to herbivorous insects, the same trends that were observed in 0.2 c 40 20 3 5 7 9 leaf stage 0.0 10 Figure 5. Bar graph of leaf consumption with the leaf bioassay and leaf toughness using the Instron for BS9 leaves at different stages in development. n=30 for the bioassays and n=15 for toughness measurements of each development stage. Bars topped with different letters within the same development stage are significantly different, SNK (P<0.05). p-Coumaric acid mg/g dry wt.‡ Ferulic acid mg/g dry wt.‡ Percent protein (dry wt.) Soluble metabolites (g/g dry wt.) 2.96 ± 0.04 a 1.43 ± 0.07 d 2.32 ± 0.10 b 1.91 ± 0.09 c 2.03 ± 0.06 c 1.09 ± 0.21 a 0.53 ± 0.02 b 1.06 ± 0.12 a 0.78 ± 0.03 b 0.89 ± 0.02 ab 0.67 ± 0.10 a 0.73 ± 0.03 a 1.48 ± 0.15 b 1.55 ± 0.01 b 0.92 ± 0.04 a 29.2 ± 1.2 a 24.5 ± 0.5 b 23.7 ± 0.1 bc 23.5 ± 0.1 bc 21.5 ± 0.1 cd 0.356 ± 0.013 a 0.319 ± 0.009 a 0.267 ± 0.036 a 0.248 ± 0.043 a 0.283 ± 0.041 a demonstrated in artificially infested ‡ field plots with Diatraea saccharalis in bc 60 DIMBOA Leaf mg/g dry wt.‡ 7th leaf stage (Fig. 5). This preference is larvae prefer plants younger than the 80 0.4 Table 1. Levels of soluble phenolic acid conjugates in BS9(C4) at different stages of development. 3 5 7 9 10 the profile study are evident. ECB 100 c c Consumption Toughness b b a ab a ab Leaf toughness (N) levels of DIMBOA. As the insect Leaf consumption (mm2) whorl tissue which is defended by high ;; ; ;;; ;; ;; ; ;;; ;; ;; ;; ; ;;; ;; ;; ; ;;; ;; ; 120 Means within the same column and treatment followed by the same letter are not significantly different, SNK (P<0.05). WINDOWS OF MAIZE RESISTANCE 123 was observed, suggesting that younger correlated with maize resistance to not increase as insect resistance leaves provide more accessible storage pests (Classen et al. 1990) and to increased which is consistent with these nutrients and water to larvae than do leaf feeding by the ECB (Bergvinson results. older leaves. Fiber content has been 1993). Both phenolic acids reached their hypothesized to increase the bulk highest levels at the 9- and 10-leaf stages The overall impact that cell wall density of the insect’s diet to the point showing levels 2- to 3-fold higher than phenolic acid - carbohydrate complexes that insects cannot ingest sufficient younger leaves (Table 2), which have on leaf toughness is shown in nutrients (Peterson et al. 1988). Fiber correlate with the observed reduction in Figure 5. Leaf stages with the lowest content did not change significantly ECB consumption on mature leaves. levels of cell wall phenolics have the lowest leaf toughness measurements, within the leaf age range tested and would not account for the observed Dimers formed by peroxidase with the 3-leaf stage being the softest of feeding preferences (Table 2). (dehydrodiphenolic acids) or through all stages. It is hypothesized that leaf toughness vis-à-vis phenolic fortification acids) may increase the mechanical of cell wall tissue is the primary defense acids, E-ferulic and E-p-coumaric acids, strength of the cell wall by cross-linking for mature maize tissue against the ECB have been studied as digestibility- hemicellulose (Hartley and Ford 1989). and is likely operating against other reducing factors in ruminants (Hartley Here again a sharp increase in lepidopteran pests of maize given the and Ford 1989) and have been cyclobutane dimers and structural nature of the proposed 2500 2000 ;;;; ; ; ; ;;;;; ; ; ; ; ; ;;;;; Truxillic/truxinic acids c Diferulic acid b b a 1200 a 800 a 1000 500 0 400 0 3 5 7 9 leaf stage 10 Figure 6. Bar graph of phenolic dimers in BS9 leaves at different stages in development. Truxillic and truxinic acids are dimers produced by ultraviolet light and diferulic acid is produced by peroxidase in the presence of peroxide. Both types of dimers are thought to be involved in cell wall fortification as a host plant resistance mechanism in mature leaf tissue. which coincided with Changes in phenolic acids within the reduced insect feeding (Table epidermal cell wall can be estimated 2, Fig. 5). Similar results are using the microspectrophotometer reported for sorghum, with (Akin et al. 1990). Absorbance readings cyclobutane dimer levels showed that the least fortified being highest at later stages epidermal cell walls were found at the in plant development (Goto 3-leaf stage with a dramatic jump in et al. 1991). Lignin content absorbance occurring at the 9-leaf stage did not differ significantly of development (Table 2). Based on (SNK, P > 0.05) between absorbance changes in the epidermal different development stages cell wall this tissue appears to be the (Bergvinson 1993). Buendgen site of major phenolic changes. Leaf et al. (1990) showed that tissue which is most susceptible to ECB between different cycles of S1 feeding tended to have the lowest selection for BS9, lignin epidermal cell wall absorbance and content of whorl tissue did presumably lowest cell wall phenolic content. Table 2. Levels of cell wall bound phenolic acids in BS9(C4) at different stages of development. µg / g dry weight‡ ‡ resistance mechanism. occurs at the 9-leaf stage 1600 b a 1500 b c dehydrodiphenolic dimers Diferulic acid (ug/g) Truxillic/truxinic acids (ug/g) UV absorption (truxillic and truxinic The major cell-wall-bound phenolic Leaf p-Coumaric acid Ferulic acid Epidermal cell wall absorbance at 326 nm 3 5 7 9 10 483 ± 14 a 590 ± 5 a 615 ± 5 a 1163 ± 47 b 1557 ± 66 c 981 ± 20 a 1229 ± 27 b 1490 ± 14 c 2057 ± 82 d 1986 ± 27 d 0.18 ± 0.04 a 0.36 ± 0.08 a 0.41 ± 0.08 b 0.83 ± 0.07 b 0.98 ± 0.11 b Means within the same column and treatment followed by the same letter are not significantly different, SNK (P<0.05). From these spatial and temporal studies of resistance mechanisms in the leaf it appears that leaf toughness is of major importance to ECB larvae in controlled bioassays with environmental variability removed from the HPR equation. Larvae consume immature tissue (whether temporal or spatial) at a higher rate than mature tissue. The differences between different tissues of most significance were leaf toughness 124 D.J. BERGIVSON and epidermal cell wall absorbance. An showed a peak load which As with vegetative tissue, the wax understanding of the variability corresponded to the time silks are most chemistry of the silk changes over its associated with these biochemical susceptible to infection by F. length (Fig. 9). Although no resistance factors of the temporal and graminearum inoculation in the silk commercial hybrid has been found spatial changes in HPR to leaf feeding channel (Fig. 8). Susceptible inbreds which matches the wax levels found in lepidopteran pests will assist in the showed an earlier and much reduced CO272, one commercial hybrid (Pride development of resistant lines. peak in wax load which dropped to K127) does have moderately high basal levels at the time of artificial levels, but only at the point where the Temporal and spatial changes in inoculation. This data illustrates that silk extends outside the husk. If the silk biochemical resistance mechanisms are even structural components such as sample were to be taken at the point of not exclusive to vegetative tissue. HPR wax — which may be considered static attachment to the kernel, the resistant to insect and disease pests of — can change rapidly, and so timing of hybrid would be the most susceptible reproductive structures is also subject phytochemical sampling is as important based on wax content. It is essential for to changes over space and time. One as the time of artificial inoculation. all studies on the biochemical case in point is silk resistance to the ear Other biochemical constituents that mechanisms of HPR to consider the rot pathogen, Fusarium graminearum. By change with silk maturity include time and location of the sample and making morphological comparisons soluble phenolics and flavonoids (Reid that it reflects the interaction which between the resistant inbred (CO272) et al. 1992); these may also have an occurs in the field between the pest and susceptible inbreds it was observed impact on the window of pest organisms and the host. that the silk wax deposits on CO272 susceptibility. For example, the flavone were much greater. Extracting the glycoside maysin has been shown to Although silk wax may not be a waxes in chloroform and analysis by inhibit CEW larval growth (Wiseman et mechanism for CEW resistance, this gas chromatography revealed that the al. 1985). By understanding the study does show the rapid change that composition of silk waxes were less biochemical changes that are occuring occurs in silk biochemistry and which complicated than leaf waxes (Fig. 7) over time, artificial screening should be considered with and that CO272 did have elevated techniques can be developed and used reproductive structures. Kernel levels of wax in comparison with to identify a greater genetic variation in chemistry has also been investigated in susceptible inbreds (Fig. 8). resistant and susceptible genotypes. relation to disease resistance and we have found that major changes in soluble and cell wall chemistry occur Temporal changes in silk wax load was C25 C27 C28 Alkanes C29 C31 √ 9-Nonacosanol 11--Hentriacontanol C29 Alcohols C31 Eicosanal Docosanal C20 C22 Aldehydes Tetracosanal C24 √ hexadecanoic acid C16 Acids 9, 12 Octadecadienoic C18 acid Figure 7. Phytochemical composition of silk wax in maize. Wax composition is largely simple alkanes. Ticks indicate major components in silk wax. CO272 Wax load (mg/g wet wt) √ Pentacosane √ Heptacosane Octacosane √ Nonacosane √ Hentriacontane approximately three weeks post-silking 0.4 (unpublished data). These changes are 0.3 0.2 CO265 0.1 Time of field inoculation CO266 0 2 4 6 8 10 Time (days after silking) 12 Figure 8. Line graph of the temporal changes in silk wax load for one resistant (CO272) and two susceptible (CO265, CO266) inbreds. The greatest differential between resistant and susceptible lines occurs at 6 days when the wax peaks for the resistant inbred line. n=3 for each sampling date. Wax load (mg/g wet wt) of particular interest in that CO272 0.3 CO272 0.2 Pride K127 0.1 DK 435 0 1 2 3 4 Sample position along silk Figure 9. Silk wax profile along the length of the silk channel for one resistant inbred (CO272) and two commercial inbreds (Pride K127 resistant, DK435 susceptible). 5 WINDOWS OF MAIZE RESISTANCE most dramatic in the aleurone layer, a employ qualitative defenses such as factor which may be important when DIMBOA or toxic compounds localized studying HPR to lepidopteran larvae in susceptible tissue. As the plant that feed on the ear. Artificial diet matures and the tissues remain exposed supplemented with sorghum panicles for prolonged periods to pests, more at different stages in development was quantitative resistance mechanisms are shown to reduce FAW performance employed such as the phenolic (time required to complete fortification of cell wall carbohydrates. development, and larval and pupal Consideration of biochemical changes weight) when more mature panicles that are occurring within the plant over were incorporated into the diet time and within a given tissue will (Wiseman 1986). Similar studies in assist in identifying HPR mechanisms maize may indicate the time at which for plant improvement programs in the biochemical resistance mechanisms are future. being expressed in maize kernels against FAW and CEW. Ac k now le dgm e nt s From these studies of temporal and This work was supported by an NSERC spatial changes in biochemical strategic grant (Arnason) and the composition, it is evident that maize Ontario Ministry of Agriculture and resistance is toxin-related during early Food, an NSERC Graduate Scholarship stages of tissue development and and an NSERC PDF to D.J.B. structurally-related in mature tissue. The transition from one resistance Re fe re nc e s strategy to another represents a continuum that is illustrated in Figure 10. The immature or young tissue is rather ephemeral and would likely Immature tissue Mature tissue Early stages in plant development Later stages in plant development Qualitative Resistance Mechanisms Quantitative Resistance Mechanisms Figure 10. Proposed host plant resistance strategy used by maize during different stages of development. Based on Feeny’s (1976) theory of quantitative and qualitative defense, maize tissues appear to employ different resistance strategies depending on tissue maturity and stage of plant development. Changes in defense strategy from qualitative to quantitative defenses represent a continuum with both types of defenses being present but varying with plant/tissue maturity. Akin, D.E., N. Ames-Gottfred, R.D. Hartley, R.G. Fulcher, and L.L. Rigsby. 1990. Microspectrophotometry of phenolic compounds in bermudagrass cell walls in relation to rumen microbial digestion. Crop Sci. 30: 396-401. Ampofo, J.K.O., and E.L. Kidiavai. 1987. 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Biological effects of developing sorghum panicles in a meridic diet on fall armyworm (Lepidoptera: Noctuidae) development. J. Econ. Entomol. 79: 1637-1640. Woodhead, S. 1981. Environmental and biotic factors affecting the phenolic content of different cultivars of Sorghum bicolor. J. Chem. Ecol. 7: 1035-1047. Xie, Y., J.T. Arnason, B.J.R. Philogene, H.T. Olechowski, and R.I. Hamilton. 1992. Variation of hydroxamic acid content in maize roots in relation to geographical origin of maize germplasm and resistance to western corn rootworm (Coleoptera: Chyrsomelidae). J. Economic Entomol. 85: 2478-2485. 127 Genetic Basis of Silk Resistance (Antibiosis) to the Corn Earworm in Six Crosses of M aize Lines: Statistical M ethodology K. Bondari, Coastal Plain Station, University of Georgia, Tifton, GA. and B.R. Wiseman, USDA-ARS, Tifton, GA. Abst r a c t The genetic basis of resistance (antibiosis) in maize silks to larvae of corn earworm (CEW), Helicoverpa zea (Boddie), was studied in six crosses of resistant and susceptible inbred lines of maize, Zea mays (L). For each breeding line, crosses were made between parental (P1 and P2) lines to produce F1 seed. The F1 plants were selfed to produce F2 seed and backcrossed to each parent to produce BC1 (F1 x P1) and BC2 (F1 x P2) seed. No attempt was made to produce reciprocal crosses since no evidence of significant maternal effects for these crosses existed. Silk from plants of all six generations (P1, P2, F1, F2, BC1, and BC2) was evaluated by recording 8-day weight of CEW larvae fed on a silk diet. A three-parameter additive-dominance model and a six-parameter digenic additive-dominance/ epistatic model were used to analyze generation means by the method of variance-weighted least squares. The genetic control of resistance to CEW larvae was determined in terms of additive-dominance gene action as well as contributions due to epistatic effects of genes at different loci. Results of the simple and joint scaling tests indicated genetic control for silk resistance to CEW, but the gene action differed from one type of cross to another. In the cross Zapalote chico x PI340856, the three-parameter additive-dominance model proved adequate and genes controlling resistance in PI340856 are dominant to those of Z. chico. However, in most crosses, non-allelic interactions were present, thus the fit of the additive-dominance model to the data was considered inadequate. Int roduct ion Although the biological effects on CEW parameter model may not accurately of antibiosis in corn silks are well describe the underlying genetic control The biological effects (antibiosis) of documented, the genetic basis of in most cases. The authors further resistant corn silks on CEW may be resistance remains elusive. According concluded that in several crosses the measured in several ways, including: to Widstrom et al. (1992), maize genetic resistance was complex and • Population reduction via mortality breeders will seldom practice selective perhaps controlled by several pairs of of eggs, larvae and pupae (Widstrom breeding in maize with the primary genes at different loci. et al. 1977; Wiseman et al. 1978, 1983; objective of enhancing its genetic Wilson et al. 1984) and decreased resistance to insects, since any progress The primary objective of this study was fecundity over several generations made will most likely be at the expense to determine the genetic basis of (Wiseman and Isenhour 1990). of other agronomic traits. antibiosis resistance via the 8-day • • weights of CEW larvae fed on silk diet Reduced larval weight (8-10 days after hatching) and pupal weight Wiseman and Bondari (1992, 1995) (dry silk mixed in diluted pinto bean (Wiseman and Isenhour 1990, 1991; studied the genetic basis of antibiosis to diet). The study also determined the Wiseman and Bondari 1992, 1995). CEW in maize silks from several relative importance of additive and Increased larval period (Wiseman crosses of resistant and susceptible non-additive genetic effects. The results and Isenhour 1990). inbred lines of maize and concluded should have important implications, that the additive-dominance three- contributing among other things to 128 K. BONDARI AND B.R. WISEMAN reduced CEW damage and a reduced to provide sufficient silk for evaluating are commonly designated as [d] = net need for pesticides to control CEW. individual ear samples in a feeding trial additive deviation and [h] = net using CEW larvae. Two-day-old dominance deviation. The method of unpollinated silks (ear covered with variance-weighted least squares, shoot bag) were excised from the ear tip employing three- and six-parameter of each experimental plant, oven dried models, was used to estimate these seven parent lines: at 41°C for about 10 days, ground parameters from the generation means. • (1 mm) by using a Cyclotec® 1093 The goodness-of-fit of each genetic Zapalote chico (P2, resistant silk). sample mill, and stored at -10°C. model was tested by a weighted chi- Zapalote chico 2451# (PC3) Weights of individual larvae were square (χ2) comparing observed and originates from a collection of Z. determined after 8 days of feeding on expected generation means. The t-test chico from Mexico maintained in silk diets (for further details see was used to test the significance of each Tifton, Georgia, and Ab18 was used Wiseman and Bondari 1992; Wiseman estimated genetic parameter and in a cross reported by Wiseman and and Widstrom 1992; Wiseman et al. contrast (linear relationship among Bondari (1992). 1993; Wiseman and Bondari 1995). generation means). The three contrasts M aterials and M ethods The study involved six crosses among • • • • • Ab18 (P1, susceptible silk) x GT114 (P1, resistant silk) x GT119 among generation means computed (P2, susceptible silk), both inbred Statistical analysis were A = 2BC1 - (P1 + F1) B = 2BC2 - lines recently released (Widstrom et Generation means and standard errors (P2 + F1) C = 4F2 - (2F1 + P1 + P2). al. 1988). (SE) of the means for each cross were Standard errors of these contrasts were Z. chico (P1, resistant silk) x computed from a one-way analysis of computed assuming independence of PI340856 (P2, resistant silk). variance using PROC GLM of SAS (SAS generation means included in each PI340856 (PI) is a popcorn plant Institute Inc. 1989). Within-cross contrast. introduction that has shown comparisons of generation means were dominance in the F1 with over 20 made using the PDIFF option of SAS. We developed a computer program dent maize inbreds for low larval Generation means, SE of means, and using several PROCs from SAS to weight (Wiseman et al. 1992; number of observations associated with perform generation means analyses, Wiseman and Bondari 1995). each mean were used in the variance- employing both three- and six- Z. chico (P1, resistant silk) x GT114 weighted least squares procedure to parameter models, based on the (P2, resistant silk). perform three-parameter and six- method described by Mather and Jinks Z. chico (P1, resistant silk) x CI64 parameter scaling tests (Mather and (1982). Other computer programs (e.g., (P2, resistant silk). CI64 was Jinks 1982). Each generation mean was a BASIC program by Mosjidis et al. developed from a cereal weighted by the reciprocal of the 1989) are also available to perform introduction (Wiseman et al. 1992; variance of the mean for that these analyses. The method of weighted Wiseman and Bondari 1995). generation. Genetic parameters least squares used in the analysis of GT3 (P1, susceptible silk) x PI340856 estimated from this procedure were generation means has been described (P2, resistant silk), GT3 was used in genetic models to determine the by several authors, including Mather developed in Georgia (Wiseman et adequacy of the additive-dominance and Jinks (1982), Rowe and Alexander al. 1992; Wiseman and Bondari model for resistance to CEW larvae. (1980), and Beaver and Mosjidis (1988). The procedure uses weights that are 1995). Following the notation of Fisher et al., equal to the reciprocals of the standard In addition to P1 and P2 (parental (1932) and Mather and Jinks (1982), errors of the generation means. The generations), F1, F2, BC1 (backcross to genotypes of AA, Aa, and aa are analysis method is based on Fisher P1 or F1 x P1), and BC2 (backcross to P2 assigned quantitative phenotypes +d, h, (1941), with the average effect of a gene or F1 x P2) generation seeds from each and -d, respectively and the origin of substitution using expected coefficients cross were produced in the maize measurement is the mid-homozygote of the gene effects proposed by breeding nursery. Bulk plantings of (m) which is the mid-point value from Hayman and Mather (1955) and seed of each generation were made which measurements can be expressed Hayman (1958). For instance, the using a completely randomized design as deviations. These genetic parameters expected coefficients of gene effects for GENETIC BASIS OF SILK RESISTANCE (ANTIBIOSIS) TO THE CORN EARWORM 129 the three-parameter model (m=mean, that variation exists among generations that the three-parameter additive- [d]=additive, and [h]=dominance) are within each cross and that variation dominance model does not provide an presented below: exists among crosses possessing adequate description of genetic control various degrees of CEW resistance. for the 8-day CEW larval weight. Generation means and their standard None of the [h], [i], [j], or [l] parameters errors and the results of the joint were significant for the two crosses scaling test and estimates of gene involving PI as the parental line (Tables effects based on a three-parameter 1 and 3). PI340856 is a highly resistant additive-dominance genetic model for maize line and when crossed with Z. Generation means and appropriate each cross assuming A,a alleles (m = chico, also possessing resistant silk, F1 weights (reciprocals of squared mean, d = additive gene effect, and h = and P2 (PI inbred line) means do not standard error of the means) are added dominance gene effect) are presented in differ (Table 1). When F1 is backcrossed to these coefficients to perform the Table 1. The A, B, and C contrasts to PI340856, the BC2 mean does not Gen Cross m [d] [h] P1 P2 F1 F2 BC1 BC2 P1 x P1 P2 x P2 F1 x P2 F1 x F1 F1 x P1 F1 x P2 1 1 1 1 1 1 1.0 -1.0 0 0 0.5 -0.5 0 0 1.0 0.5 0.5 0.5 χ2 differ from P2 or F1 (Table 1). weighted least-squares analysis. The among generation means and generation means were used as the computed for the test of goodness-of-fit Furthermore, [h] is negative and almost dependent variable and the coefficients of the additive-dominance model are of equal magnitude to [d]. These of the genetic parameters as the presented in Table 2. These results findings indicate that the PI340856 independent variables. PROC MATRIX indicate that: genes controlling silk resistance are dominant to the Z. chico genes and that (SAS version 5) or PROC IML (SAS version 6) was used to obtain solutions At least one of the three contrasts and the three-parameter model seems for the unknown parameters. A test of the χ2 statistic were significant for five adequate to predict generation means goodness of fit described by Cavalli of the six crosses (Table 2), indicating for this cross. (1952) and Mather and Jinks (1982) was performed to verify the adequacy of the three-parameter additivedominance model. For all six crosses, a % 100 three-parameter model was fitted first, 80 but even when a good fit was observed, 60 a six-parameter model was also fitted. In the presence of significant epistatic effects, fitting a six-parameter model alone would not provide meaningful estimates of the main effects. 40 20 0 % 100 80 Results and Discussion 60 40 The distribution of 8-day weights of CEW larvae over six generations 20 0 (parents, P1 and P2; F1; F2; and backcrosses, BC1 and BC2) of each cross is presented in Figure 1. The 8- % 100 day weights of larvae fed the silk-diet 80 are classified into three groups (<100, 60 100-200, and >200 mg). Distributions of these weight classes clearly indicate a genetic control of the 8-day larval weight by the host plant. It is evident 40 20 0 ; ; ; ; ;; ;; ; ;; ; ; ; ; ;; ; ;;; ;; ;;;; Weight (mg) <100 100-200 >200 P1 P2 F1 F2 BC1 BC2 Ab18 (P1, s) x Z. Chico (P2, r) r = resistance s = susceptible P1 P2 F1 F2 BC1 BC2 GT114 (P1, r) x GT119 (P2, s) P1 P2 F1 F2 BC1 BC2 Z. Chico (P1, r) x P1 (P2, s) % 100 ; ; ; ;; ; ; ;;;; ;;; ; ;;;; ;; 80 60 40 20 0 P1 P2 F1 F2 BC1 BC2 Z. Chico (P1, r) x GT114 (P2, r) % 100 80 60 40 20 0 P1 P2 F1 F2 BC1 BC2 Z. Chico (P1, r) x C164 (P2, r) % 100 ;;;; ;; 80 60 40 20 0 P1 P2 F1 F2 BC1 BC2 GT3 (P1, s) x P1 (P2, r) Figure 1. Distribution of eight-day weight of corn earworm larvae. 130 K. BONDARI AND B.R. WISEMAN Results of the second cross involving The remaining four crosses involve a Although resistance to CEW larvae PI340856 (GT3 x PI340856) differs from significant contrast and a significant χ2 varied among inbred lines used in the first one. GT3 possesses susceptible (Table 2) and some types of non-allelic these crosses, the biometric genetic silk and the significant B and C interactions (Table 3), indicating that procedures employed seemed best χ2 genetic control for resistance in these suited for crosses of parents quite indicate that the three-parameter model crosses may involve two or more loci. diverse in degree of resistance. Another is inadequate for this cross. However, The type of non-allelic interaction important consideration in this study the six-parameter digenic model does varied from one cross to another. For was the ability to control not detect any significant non-allelic instance, GT114 x GT119 involved an environmental variations. All interaction effect (Table 3). Neither F1 additive x additive genetic effect; Z. generations of each cross were grown contrasts and statistic (Table 2) nor BC2 means differ from the PI340856 chico x CI64 involved additive x under as similar environmental mean (Table 1), [h] is negative and dominance non-allelic interaction and conditions as possible and the diet trial similar in magnitude to [d] and, thus, more than one interaction effect was was conducted under controlled PI genes for this cross also act dominant significant for Ab18 x Z. chico and Z. environmental conditions. to GT3 genes, as they did in PI340856 x chico. x GT114. For these four crosses, Z. chico. However, the number of loci both additive and non-additive genetic In conclusion, the generation means involved in the genetic resistance is not effects were found to play a significant analysis indicates that resistance to silk- clear for this cross; thus the inheritance role in the inheritance of antibiosis feeding by CEW larvae is under genetic may involve a different genetic resistance in corn silks to CEW larvae. control of the host plant, but gene mechanism. Table 1. Number of observations (n), mean corn earworm larval weight, and standard error of the mean (SE) for parental (P1 and P2), F1, F2, and backcross (BC1=F1 x P1 and BC2=F1 x P2) generations of six maize crosses. Ab18 x ZCy Gen z P1 P2 F1 F2 BC1 BC2 n Mean 15 15 15 78 48 48 388.9 22.5d c 116.9 b 179.8 225.2b d 36.8 a ZC x PIy GT114 x GT119 SE n 22.1 3.4 10.1 14.4 19.1 4.9 15 15 15 80 48 48 Mean ZC x GT114y SE n Mean SE 38.7 3.9 358.9a 12.8 c 143.4 9.3 b 244.7 13.5 113.5c 9.7 b 271.2 17.4 48 36 48 93 100 84 72.6 38.6c c 35.1 b 51.6 65.9a c 36.8 a 5.9 5.3 6.5 5.4 5.6 7.1 d n Mean ZC x CI64y GT3 x PIy SE n Mean SE n 30 82.2 7.0 52 106.7a 10.3 b 49 82.2 7.9 b 97 64.2 5.2 97 105.3a 9.2 b 95 75.9 6.6 54 45 50 103 87 98 56.1 100.7a d 26.8 cd 33.5 38.3cd c 41.7 b 5.5 8.8 2.1 3.0 3.8 5.1 49 36 51 101 101 94 b Mean a 358.0 43.3de cd 60.9 c 93.1 176.8b e 23.2 SE 23.7 4.6 17.0 12.8 15.9 2.7 Genetic Effects from Joint Scaling Test (monogenic 3-parameter model) z P Estimate SE Estimate SE Estimate SE m [d] [h] 207.7** 188.7** -120.2** 9.4 9.3 12.8 207.2** -166.0** -44.2** 6.2 6.2 10.8 57.9** 19.4** -17.5** 3.6 3.6 7.2 Estimate SE 85.4** -2.0 -13.0 5.3 5.3 9.8 Estimate SE 60.6** -11.0* -36.2** 3.5 3.9 4.5 Estimate SE 194.0** 9.5 154.0** 9.1 -183.6** 13.0 y ZC=Zapalote chico 2451 # PC3, and PI=PI340856. Generation means, within a column, bearing different superscript letters differ (P<0.05). *,** Estimated genetic parameter significant at the 0.05 (*) or 0.01 (**) probability level. z Parameters are: m = mean, [d] = additive, and [h] = dominance effects. a,b,c,d,e Table 2. Generation means contrasts (C) from the joint scaling test using the three-parameter additive-dominance model and standard error of the contrasts (SE) for six maize crosses. Ab18 x ZCy GT114 x GT119 ZC x PIy ZC x GT114y ZC x CI64y GT3 x PIy C Estimate SE Estimate SE Estimate SE Estimate SE Estimate SE Estimate SE A B C -55.4 -65.8** 74.0 45.3 14.5 65.0 44.9* 40.3 294.4** 21.9 38.2 58.7 24.1 -0.1 24.8 14.2 16.4 26.4 46.1* 21.2 -37.0* 18.5 -96.3** 28.9 -6.3 9.7 -44.1** 13.6 -76.6** 16.4 -65.3 43.2 -57.9** 18.4 -150.6* 66.0 22.3** 24.4** 10.7* χ2 3 y 26.9** 27.4** 3.2 ZC=Zapalote chico 2451 # PC3, and PI=PI340856. *,** Estimated contrast (A, B, or C) or chi-square (χ2) with 3 df significant at the 0.05 (*) or 0.01 (**) probability level. 131 GENETIC BASIS OF SILK RESISTANCE (ANTIBIOSIS) TO THE CORN EARWORM action controlling resistance may differ from one type of cross to another. Because of the dominance nature of the gene action, genetic resistance associated with the PI340856 inbred line may be easily transmitted to other commercial inbred lines. Using a breeding program that relies on pedigree and backcrossing should result in progress toward breeding maize resistant to CEW larvae. The end result would be reduced CEW damage, enhanced food safety, reduced pesticide use, and more environmentally sound agronomic practices for maize production. Industry has already made progress in transferring the resistant gene from this popcorn line to one of their “elite” dent inbred lines through 3-4 generations of backcrossing. The new inbred has been further crossed with several other inbred lines to produce hybrid combinations without ill effects from genes from the popcorn line. Furthermore, increasing silk resistance to CEW larvae may lead to the enhancement of resistance to some other maize pests or toxins as well. Re fe re nce s Beaver, R.J., and J.A. Mosjidis. 1988. Important considerations in the analysis of generation means. Euphytica 39: 233235. Cavalli, L.L. 1952. An analysis of linkage in quantitative inheritance. In E.C.R. Reeve, and C.H. Waddington (eds.)Quantitative Inheritance, 135-144. London: HMSO. Fisher, R.A., F.R. Immer, and O. Teding. 1932. The genetical interpretation of statistics of the third degree in the study of quantitative inheritance. Genetics 17: 107-124. Fisher, R.A. 1941. Average excess and average effect of a gene substitution. Ann. Eugen. 11: 53-63. Hayman, B.I., and K. Mather. 1955. The description of genetic interaction in continuous variation. Biometrics 11: 6982. Hayman, B.I. 1958. The separation of epistatic from additive and dominance variation in generation means. Heredity 12: 371-390. Mather, K., and J.L. Jinks. 1982. Biometrical Genetics, 3rd Ed. London, New York: Chapman and Hall. Mosjidis, J.A., C.K. Huzar, and D.A. Roland. 1989. A BASIC program to calculate genetic effects with the use of generation means analysis. J. Hered. 80: 67. Rowe, K.E., and W.L. Alexander. 1980. Computations for estimating the genetic parameters in joint-scaling tests. Crop Sci. 20: 109-110. SAS Institute, Inc. 1989. SAS/STAT user’s guide, version 6, 4th edition. Vol. 1 and 2. Cary, N.C. Widstrom, N.W., B.R. Wiseman, and W.W. McMillian. 1977. Response of corn earworm larvae to maize silks. Agron. J. 69: 815-817. Widstrom, N.W., B.R. Wiseman, and W.W. McMillian. 1988. Registration of six corn earworm resistant germplasm lines of maize. Crop Sci. 28: 202. Widstrom, N.W., K. Bondari, and W.W. McMillian. 1992. Hybrid performance among maize populations selected for resistance to insects. Crop Sci. 32: 85-89. Wilson, R.L., B.R. Wiseman, and N.W. Widstrom. 1984. Growth response of corn earworm (Lepidoptera: Noctuidae) larvae on meridic diets containing fresh and lyophilized corn silk. J. Econ. Entomol. 77: 1159-1162. Wiseman, B.R., and K. Bondari. 1992. Genetics of antibiotic resistance in corn silks to the corn earworm (Lepidoptera: Noctuidae). J. Econ. Entomol. 85: 293-298. Wiseman, B.R., and K. Bondari. 1995. Inheritance of resistance in maize silks to the corn earworm. Entomol. Exp. Appl. (In Press). Wiseman, B.R., and D.J. Isenhour. 1990. Effects of resistant corn silks on corn earworm (Lepidoptera: Noctuidae) biology: A laboratory study. J. Econ. Entomol. 83: 614-617. Wiseman, B.R., and D.J. Isenhour. 1991. Microtechnique for antibiosis evaluations against the corn earworm. J. Kansas Entomol. Soc. 64: 146-151. Wiseman, B.R., W.W. McMillian, and N.W. Widstrom. 1978. Potential of resistant corn to reduce corn earworm populations. Florida Entomol. 61: 92. Wiseman, B.R., M.E. Snook, and D.J. Isenhour. 1993. Maysin content and growth of corn earworm larvae (Lepidoptera: Noctuidae) on silks from first and second ears of corn. J. Econ. Entomol. 86: 939-944. Wiseman, B.R., M.E. Snook, D.J. Isenhour, J.A. Mihm, and N.W. Widstrom. 1992. Relationship between growth of corn earworm and fall armyworm larvae (Lepidoptera: Noctuidae) and maysin concentration in corn silks. J. Econ. Entomol. 85: 2473-2477. Wiseman, B.R., and N.W. Widstrom. 1992. Resistance of dent corn inbreds to larvae of the corn earworm (Lepidoptera: Noctuidae). J. Econ. Entomol. 85: 289-292. Wiseman, B.R., N.W. Widstrom, and W.W. McMillian. 1983. Influence of resistant and susceptible corn silks on selected developmental parameters of corn earworm (Lepidoptera: Noctuidae) larvae. J. Econ. Entomol. 76: 1288-1290. Table 3. Estimates of genetic parameters (P) and standard error of the estimates (SE) from a six-parameter digenic model for 8-day weights of corn earworm larvae fed silk diets from six maize crosses. Ab18 x ZCy Px Estimate m [d] [h] [i] [j] [l] 401 183** -600** -195** 10 316** SE 70.7 11.2 162.2 69.8 45.3 102.2 GT114 x GT119 Estimate 409 -160** -390* -210** 6 124 SE 67.4 6.7 162.4 67.1 42.0 98.9 ZC x PIy ZC x GT114y ZC x CI64y GT3 x PIy Estimate SE Estimate SE Estimate SE Estimate 57* 17** 2 -1 24 -23 28.4 4.0 69.4 28.1 19.7 44.7 -11 -12* 208** 105** 83** -115* 31.4 6.2 80.8 30.7 25.8 53.7 52* -22** -50 26 38* 24 18.2 5.2 46.3 17.5 16.5 30.3 173** 157** -208 27 -7 96 SE 61.7 12.1 137.7 60.6 40.4 92.4 *,** Genetic parameter estimate differs from zero at the 0.05 (*) or 0.01(**) probability level (t-test). x Parameters are: m = mean, [d] = additive (Add) and [h] = dominance (dom) effects and [i] = add x add, [j] = add x dom, and [l] = dom x dom epistatic effects. y ZC = Zapalote chico 2451 # PC3, and PI = PI340856. 132 Genetics of M aize Grain Resistance to M aize Weevil J.A. Serratos, INIFAP, Chapingo, Mexico. A. Blanco-Labra, Centro de Investigación y de Estudios Avanzados del IPN., Irapuato, México. J.T. Arnason, University of Ottawa, Ontario, Canada. and J.A. Mihm, French Agricultural Research, Inc., Lamberton, MN, USA. Abst r a c t The genetics of maize grain resistance to the maize weevil, Sitophilus zeamais Motsch., infestation was analyzed by means of additive linear models which considered genetic contributions of maize caryopsis through embryo, endosperm and pericarp. Specific traits associated with these grain tissues were: phenolic acids (pericarp, embryo), proteinase inhibitors (endosperm, embryo) and hardness of grain (pericarp, endosperm, embryo). The susceptibility of the grains to weevil infestation was measured by feeding, consumption and reproductive activities of insect populations. Inbred lines of quality protein maize (QPM), contrasting in resistance to maize weevil infestation, were used for the genetic analysis of resistance. Concentrations of phenolic acids in grain have a highly negative and significant correlation with indices of susceptibility of maize to the maize weevil. However, the correlation between susceptibility of grain and contents of proteinase inhibitors in the endosperm is low, although negative and significant. Resistance of pericarp-testa to compression forces was the only rheological trait of grain inversely correlated with susceptibility of maize to colonization by maize weevils, but neither the correlation coefficient nor the significance was high. The negative relationship of biochemical and biophysical traits of maize grain with feeding and reproductive activities of insects on the grain, suggests detrimental effects of these grain characteristics on the colonization success of insect populations. The estimated genetic parameters for additivity of endosperm and dominance of pericarp associated with the expression of phenolic acid concentration in the grain were highly significant and inversely correlated to estimated susceptibility parameters of genetic action. Estimated parameters of genetic action for proteinase inhibitor concentration in endosperm were nonsignificant, likewise estimated parameters for rheological traits of maize grains had very low significance. Int roduct ion Several models have been proposed, caryopsis because the selection, but most of them have oversimplified colonization, feeding and reproductive Quantitative genetic analysis of any or ignored the intricate genetic activities of the insect take place seed trait is a difficult task because of interaction among maternal, entirely on and within the maize grains. the complexity of the seed structure. In cytoplasmic, endosperm and embryo Adult weevils feed, mate and oviposit maize, as with all cereals, the caryopsis structures (Mather and Jinks 1982; on the maize grain, whereas the larvae contains seed coat, endosperm-aleurone Mosjidis et al. 1989). Although, recent feed, grow and develop inside the and embryo tissues which correspond studies (Huidong 1988; Foolad and grains. Thus, the genetic analysis of to two different generations. Pericarp Jones 1992) have paid more careful resistance of maize grains to S. zeamais belongs to the n generation whereas attention to the genetics of maternal, infestation, implies the analysis of all endosperm and embryo represent the cytoplasmic and endosperm variation. grain components. there are two types of zygosity in grain: Resistance of maize grain to maize The role of plant secondary products on the caryopsis, embryo and seed coat are weevil (S. zeamais Motsch.) infestation plant-insect interactions is well diploid while the endosperm is triploid. is a trait connected to the whole documented (Fraenkel 1959; Dethier n+1 generation within the grain. Also, 133 GENETICS OF MAIZE RESISTANCE TO MAIZE WEEVIL 1980; Guthrie and Russell 1989), and Some types of proteinase inhibitors many instances of well studied cases of have been described and characterized phytochemistry, ecology and in maize grain (Blanco-Labra and Maize material biochemistry of plant secondary Iturbe-Chinas 1981; Baker 1982; Maize generations were derived from compounds and their significance to Richardson et al. 1987), although their controlled crosses between quality herbivorous insects exist (Berenbaum effect upon stored grain insect pests protein inbred maize lines, resistant 1978, 1981; Waiss et al. 1979). have not been well established. and susceptible to maize weevil M aterials and M ethods infestation, as described in Serratos et Molecular biology investigations into The objective of this paper is to attempt al. (1993). These crosses yielded 14 the mechanisms and modes of action of the genetic analysis of maize grain generations as follows: P1, P2, F11, F12, plant defenses, focusing on regulation resistance to maize weevil infestation BC11, BC12, BC21, BC22, RBC11, RBC12, of phenylalanine ammonia lyase, which by estimating genetic variation in RBC21, RBC22, F21 and F22 (Fig. 1). Pools is the key enzyme in the biochemical and biophysical characters of grains shelled from maize ears phenylpropanoid pathway, have been and susceptibility indices of selected harvested at random in entries from 10- undertaken (Lamb et al. 1989; Xiaowu et genotypes of maize kernels, through row plots represented the generations. al. 1989). However, these molecular three types of linear genetic models. Samples of 3 to 5 g from maize ears of genetic studies have concentrated on the plant-plant pathogen microorganism interaction. P1 In maize grain, phenolics are an Seed coat endosperm embryo indicator of resistance to maize weevil infestation (Serratos et al. 1987), and sources of resistance have been traced to “Ancient Indigenous” and “Prehistoric Mestizos” groups of maize landraces containing high concentrations of P1 G(1) RBC11 G(2) RBC11 G(2) to be related to the resistance of maize Proteinase inhibitors have often been referred to as protective substances of plants to pathogens and insect pests (Ryan et al. 1986; Broadway et al. 1986; proteinase inhibitors, and their action against proteinases of insects, represents plant P1 G(1) P2 G(1) P2 G(1) RBC22 G(2) RBC22 G(2) BC22 F11 F11 G(2) BC11 G(3) BC11 G(3) F11 G(2) F12 F12 G(2) BC22 G(3) BC22 G(3) F12 G(2) BC21 F11 G(2) BC21 G(3) BC21 G(3) Seed coat endosperm embryo P2 G(1) F12 G(2) F12 G(2) BC11 Ryan 1990). The theory of plant defense based upon induced synthesis of P2 G(0) 2n P2 G(1) 3n P2 G(1) 2n RBC22 P1 G(1) F11 G(2) F11 G(2) 1994).In addition, phenolic acids seem 1992; Xie et al. 1991). P1 G(0) 2n P1 G(1) 3n P1 (G(1) 2n P2 RBC11 hydroxicinnamic acids (Arnason et al. to other pests and pathogens (Reid et al. Seed BC12 plant plant P2 G(1) P1 G(1) RBC21 F12 G(2) BC12 G(3) BC12 G(3) RBC12 P2 G(1) RBC21 G(2) RBC21 G(2) P1 G(1) RBC12 G(2) RBC12 G(2) a dynamic plant-insect interaction (Ryan et al. 1986; Ryan 1992). The presence of constitutive proteinase inhibitors in dormant tissue (e.g. seed) F21 F11 G(2) F21 G(3) F21 G(3) F22 F12 G (2) F22 G (3) F22 G (3) represents an interesting recent discovery in plant-insect interactions. Figure 1. Generations derived from a cross between two inbreed lines of maize differing in resistance to infestation to maize weevil. Caryopsis is represented by the three compartment block. G(n) indicates the nth generation, while 2n and 3n stands for diploid and triploid zygosity. 134 J.A. SERRATOS, J.T. ARNASON. A. BLANCO-LABRA AND J.A. MIHM each generation were used for incubated for 10 min, the substrate was determined as described previously biochemical determinations. Fifty then added to this enzyme-inhibitor (Dobie, 1974; Classen et al. 1990). grains from each of 5 to 10 ears complex and the mixture incubated at Weight loss of grain was the difference harvested were used in the analysis of 30 oC for 2 h. The reaction was stopped in weight of grain samples before and biophysical-hardness of grain. by adding an alkaline reagent after the infestation of weevils in no- (Sandoval, 1991). choice trials (Serratos 1987). A parameter of resistance (b) was Biochemical analysis of maize grain traits Proteolytic and inhibitory activities calculated as described in Serratos Maize grain phenolics - Phenolics were were measured using routine (1987). This parameter compares the determined gravimetrically by high spectrophotometric methods in a rate of consumption of grain by insect performance liquid chromatography Beckman DU-50 spectrophotometer. populations in a confinement test. (HPLC) and quantitative imaging of Inhibition was directly correlated to phenolics was carried out by inhibitor concentration for the aliquot Genetic analysis - The estimation of microspectrofluorimetric methods tested. Amounts required for 50% additive and dominance genetic using a Carl Zeiss UMSP80 inhibition were determined from the parameters were carried out applying microspectrophotometer, as described linear portion of percent inhibition weighted multiple linear regression to in Sen et al. (1991), Serratos et al. (1993) plots. One unit of enzymatic activity three linear genetic models described in and Arnason et al. (1994). was defined as the amount of enzyme Mather and Jinks (1982), Huidong that catalyzed an increase of 0.01 (1988), Foolad and Jones (1992), and Assay of insect proteinases and maize absorption units under the described modified by Serratos et al. (1993). The proteinase inhibitor - Proteinase assay conditions. One unit of inhibitory matrices of coefficients assigned to the inhibitor was extracted from 1 g of activity was defined as the amount of generations for each one of the linear ground defatted grains. The flour was inhibitor that inhibited one unit of models are described in Table 1. In the sieved with a 1 mm mesh sieve and enzyme activity. present study, the 14 generations extracted with deionized water at 4oC derived (Fig. 1) were pooled into 6 generations (P1, P2, F1, BC1, BC2, and for 12 hours. The crude extract was Rheological methods - Rheological used to determine inhibitory activity of characteristics of grain were F2) for the model of Mather and Jinks insect proteinase inhibitors from grain. determined using an universal (1982). To accomodate the genetic texturometer, INSTRON (Instron Corp., model for expression of endosperm Weevil larvae were collected from Canton Massachussets, USA). A traits as descibed in Huidong (1988), infested grain under controlled compression cell (strainsert 1000 lb) the 14 generations were also pooled infestation schedules to obtain 10 g of together with a force indicator into 9 generations (P1, P2, F1, F1R, BC1, third-instar larvae. Whole larvae were (Daytronic-3278) integrated to a BC1R, BC2, BC2R, and F2). Because of homogenized in a 0.2 M Succinate transducer of mechanical signal missing data for proteinase inhibitor buffer solution at pH 4.5 (1.5 p/v) using (Daytronic 9000) were used. The and maximum force of compression of a Ultra-turrax homogenizer at Texture Program Analysis Software grain, the regression analysis was 4oC. package used to analyze the data was carried out directly on the generation Homogenates were clarified by developed at the Institute of means of Table 2, together with the centrifugation at 15,000 rpm for 25 min Engineering and Food Science, Ottawa coefficients of Table 1. The linear at 4oC. Research Station, Central Experimental genetic models were as follows: maximum speed for 1 min at Farm, Agriculture Canada (Buckley et Proteolytic and inhibitory activities of al. 1984).The 50 kernels from each proteinases, that function in acid maize ear were tested individually. medium, were assayed as indicated in Sandoval (1991). This method requires Indices of susceptibility of maize to hemoglobin as a substrate, with 0.2 M weevils - Grain samples were prepared citrate buffer at pH 2.5. The extracts of as described in Serratos et al. (1993). enzymes from weevil larvae and the The index of susceptibility to weevil inhibitor from maize grain were pre- infestation (I = 100 x (ln F)/D) was (1) pi = m + [ a ] + [ d ] (Mather and Jinks, 1982) (2) pi = m + [a] + [d e1] + [d e2] (Huidong, 1988) (3) pi = m + [a] + [ae] + [apc] + [d ee] + [d p] (Serratos et al., 1993) In these models pi is the expected phenotypic value of a generation, m is GENETICS OF MAIZE RESISTANCE TO MAIZE WEEVIL 135 the midparent of two homozygous summarized in Table 2. The matrix of with different levels of resistance, as parents, a indicates the disomic additive correlations (Table 3) between grain related to different concentrations of phenolic acids and proteinase inhibitor. effect, ae and apc (equation 3) are traits and indices of susceptibility to the additive parameters of the endosperm maize weevil show that there exists an and pericarp-cytoplasm. The disomic excellent negative connection for Combining the values in Table 2 and dominance effect is represented by d, phenolics and proteinase inhibitor the matrix of coefficients for each whereas de1, de2 (equation 2), dee and d p concentration with susceptibility of model, genetic parameters were (equation 3) represent, the first and maize to maize weevil. To explore estimated using weighted multiple second dominance effect in the further the relationship between linear regression. The estimated genetic endosperm, the main effects of phenolic acids, proteinase inhibitor of parameters for each model are shown in dominance attributed to embryo and grain, and the index of susceptibility of Table 4. All three models adequately endosperm and dominance effects of grain to the maize weevil, the estimated describe the observed results since pericarp, respectively. values for these variables, as generated more than 90% of variation in each by each model, were plotted in three model is explained by the regression dimensional graphs as shown in Figure and F ratios are significant. The 3. The values in the graphs were estimated m values for all variables are The generation means of seven traits smoothed by means of an inverse highly significant for all models. With analyzed for the maize generations regression function to represent a Mather and Jinks (1982) (MJ) and specified in each model are response surface of maize generations Huidong’s (1988) (HU) models, Results and Discussion estimated additive parameters are Table 1. Matrix of coefficients used with multiple linear regression to estimate parameters of genetic action for 3 linear genetic models. Parameters of genetic action are specified in the materials and methods section. significant for phenolics, proteinase (Mather and Jinks, 1982) Generation Crosses infestation, whereas rheological traits of P1 P2 F1 BC1 BC2 F2 P1 self P2 self P1 x P2 F1 x P1 F1 x P2 F1 self (Huidong, 1988) Generation Crosses P1 P2 F1 F1R BC1 BC1R BC2 BC2R F2 P1 self P2 self P1 x P2 P2 x P1 F1 x P1 P1 x F1 F1 x P2 P2 x F1 F1 self (Serratos et al., 1993) Generation Crosses P1 P2 F11 F12 BC11 BC12 BC21 BC22 RBC11 RBC12 RBC21 RBC22 F21 F22 P1 self P2 self P1 x P2 P2 x P1 F11 x P1 F12 x P1 F11 x P2 F12 x P2 P1 x F11 P1 x F12 P2 x F11 P2 x F12 F11 self F12 self m a d 1 1 0 inhibitor, and the three indices of susceptibility to maize weevil grain were non-significant. Estimated 1 -1 0 parameter of additivity of endosperm 1 0 1 for phenolics in grain was the only 1 1/2 1/2 1 -1/2 1/2 significant additive parameter in the 1 0 1/2 Serratos et al. (1993) (SE) model. On the m a de1 de2 1 1 1/2 0 0 1 -1 1/2 0 0 contrary, none of the dominance parameters for all variables in either MJ or HU models were significant, whereas dominance of endosperm-pericarp for 1 1/2 1 0 1 -1/2 0 1 1 1/2 0 1/2 1 1 1/2 0 1 -1/2 1/2 0 1 -1 0 1/2 1 0 1/4 1/4 m a ae apc dee dp 1 1 1 2 0 0 phenolics in grain, and dominance of pericarp for phenolics, maximum force of compression and index of susceptibility were highly significant in the SE model (Table 4). 1 -1 -1 -2 0 0 1 0 1/3 2 2 0 1 0 -1/3 -2 2 0 The physiological and metabolic processes occurring during development of seeds have an enormous impact on the presence and 1 1/2 1/3 1 1 1 1 1/2 1/3 -1 1 1 accumulation of metabolites such as 1 -1/2 -1/3 1 1 1 1 -1/2 -1/3 -1 1 1 phenolics and proteinase inhibitors. The 1 1/2 2/3 2 1 0 enzymes producing and accumulating 1 1/2 2/3 2 1 0 1 -1/2 -2/3 -2 1 0 these substances in the different tissues 1 -1/2 -2/3 -2 1 0 of the grain are coded by their specific 1 0 0 1 1 1 1 0 0 -1 1 1 136 J.A. SERRATOS, J.T. ARNASON. A. BLANCO-LABRA AND J.A. MIHM genes. In this sense, enzymes breeding strategies, due to the catalyzing phenolics or proteinases are estimation of genetic parameters useful the same regardless of the grain tissue. for plant breeders, it should be However, endosperm, embryo or emphasized that more detailed pericarp have different metabolic molecular and biochemical knowledge environments which imply different of maize mechanisms of resistance is substrate concentrations and required. differences in activities and inductions for the catalytic activities of these Re fe re nce s enzymes — all of which necessarily affects the additive and dominance behavior of polygenes. In this context, the lack of significance for most of the dominance parameters of biochemical traits with all models should be considered with some caution. In conclusion, although the information generated in this report contributes to a better design and efficiency of plant Arnason J.T., B. Baum, J. Gale, J.D.H. Lambert, D. Bergvinson, B.J.R. Philogène, J.A. Serratos, J. Mihm, and D.C. Jewell. 1994. Variation in resistance of Mexican landraces of maize to maize weevil Sitophilus zeamais, in relation to taxonomic and biochemical parameters. Euphytica 74: 227-236. Baker J.E. 1982. Digestive proteinases of Sitophilus weevils (Coleoptera : Curculionidae) and their response to inhibitors from wheat and corn flour. Can. J. Zool. 60: 3206-3214. Berenbaum M. 1978. Toxicity of a furanocoumarin to armyworms: A case of biosynthetic escape from insect herbivores. Science 201: 532-534. Berenbaum M. 1981. Toxicity of angular furanocoumarins to swallowtail butterflies: Escalation in a coevolutionary arms race? Science 212: 927-929. Blanco-Labra A., and F. A. Iturbe-Chinas. 1981. Purification and characterization of an amylase inhibitor from maize. J. Food Biochem. 5: 1-17. Broadway R.M., S.S. Duffey, G. Pearce, and C.A. Ryan. 1986. Plant proteinase inhibitors: A defense against herbivorous insects? Entomol. Exp. Appl. 41: 33-38. Buckley D.J., G.E. Timbers, M. Kloek, and M.J.L. Lalonde. 1984. Texture profile analysis with curve smoothing using a personal computer system. J. Texture Studies 15: 247-261. Table 2. Mean values of maize kernel traits grouped according to generations used for genetic analysis of three linear models. All the F ratios from the analysis of variance for all variables are significant at P < 0.001. Generations Phenolic acids [µg/g] (Mather and Jinks 1982) 404.50 P1 P2 54.39 146.83 F1 BC1 361.92 184.72 BC2 F2 292.85 (Mo Huidong 1988) 404.50 P1 54.39 P2 F1 201.86 91.81 F1R BC1 353.45 369.97 BC1R BC2 243.05 111.80 BC2R F2 292.85 (Serratos et al. 1993) 404.50 P1 54.39 P2 F11 201.86 91.81 F12 BC11 337.67 373.18 BC12 BC21 247.36 238.73 BC22 RBC11 369.97 99.64 RBC21 RBC22 132.08 280.03 F21 F22 305.68 Proteinase inhibitor [PIU/g] Maximum force of compression [Newtons] Time of resistance to breakage of seed coat [seconds] 187.24 76.67 100.00 197.92 119.63 164.06 92.63 126.08 132.88 180.47 174.02 158.55 .958 .717 .772 .607 .562 .813 9.51 13.28 11.59 11.11 11.94 10.27 1.78 5.59 4.32 4.54 4.93 4.51 0.56 1.20 0.94 0.99 1.08 0.99 187.24 76.67 111.39 65.83 161.83 258.06 123.50 114.79 164.06 92.63 126.08 146.14 119.61 209.59 125.95 185.97 159.08 158.55 .958 .717 .828 .717 .481 .783 .591 .524 .813 9.51 13.28 10.40 12.78 11.01 11.21 11.08 13.02 10.27 1.78 5.59 4.58 4.06 4.46 4.44 4.12 5.94 4.51 0.56 1.20 1.00 0.89 0.97 0.98 0.91 1.29 0.99 92.63 126.08 146.14. 119.61 203.38 217.36 173.05 198.89 125.95 162.90 152.73 162.81 154.3 .958 .717 .828 .717 .478 .484 .695 .488 .783 .454 .642 .953 .673 9.51 13.28 10.40 12.78 11.14 10.86 10.36 11.80 11.21 13.07 12.94 10.32 10.22 1.78 5.59 4.58 4.06 5.03 3.73 4.00 4.23 4.44 6.18 5.52 4.61 4.41 0.56 1.20 1.00 0.89 1.11 0.81 0.88 0.93 0.98 1.34 1.21 1.02 0.97 187.2 76.7 111.4 65.8 177.5 138.3 132.5 110.0 258.1 78.3 151.3 155.4 172.7 Index of Weight susceptibility loss of grain [I] [grams] Parameter of resistance [b] GENETICS OF MAIZE RESISTANCE TO MAIZE WEEVIL Susceptibility index 13.0 12.0 11.0 10.0 85 110 135 Proteinase 160 inhibitor 440 369 298 227 185 210 Susceptibility index 13 85 156 Phenolics 12 11 10 85 110 460 135 Proteinase 160 inhibitor 387 185 168 210 Susceptibility index 13.5 12.5 11.5 95 314 241 Phenolics 137 Classen D., J.T. Arnason, J.A. Serratos, J.D.H. Lambert, C. Nozzolillo, and B.J.R. Philogène. 1990. Correlation of phenolic acid content of maize to resistance to Sitophilus zeamais, the maize weevil in CIMMYT’s collections. J. Chem. Ecol. 16: 301-315. Dethier V.G. 1980. Evolution of receptor sensitivity to secondary plant substances with special reference to deterrents. Am. Nat. 115: 45-65. Dobie, P. 1974. The laboratory assessment of inherent susceptibility of maize varieties to post-harvest infestation by Sitophilus zeamais (Coleoptera:Curculionidae). J. Stored Prod. Res. 10: 183-197. Foolad M.R., and R.A. Jones. 1992. Models to estimate maternally controlled genetic variation in quantitative seed characters. Theor. Appl. Genet. 83: 360366. Fraenkel, G. 1959. The raison d’être of secondary plant substances. Science 129: 1466-1470. Guthrie W.D., and W.A. Russell. 1989. Breeding methodologies and genetic basis of resistance in maize to the european corn borer. In Toward insect resistant maize for the Third World: Proceedings of the International Symposium on methodologies for developing host plant resistance to maize insects, 192-203. Mexico D.F.: CIMMYT. Huidong, M. 1988. Genetic expression for endosperm traits. In B. Weir, E.J. Eisen, M.M. Goodman, and G. Namkoong (Eds.) Proceedings of the 2nd International Conference on Quantitative Genetics, 478487. Sutherland Mass. Sinauer Associates Inc. Lamb C.J., M.A. Lawton, M. Dron, and R.A. Dixon. 1989. Signals and transduction mechanisms for activation of plant defenses against microbial attack. Cell 56: 215-224. Mather K., and J.L. Jinks. 1982. Biometrical Genetics. The study of continuous variation. London, U.K.: Chapman and Hall, 3rd. ed. Mosjidis J.A., J.W. Waines, D.M. Yermanos, and A.A. Rosielle. 1989. Methods for the study of cytoplasmic effects on quantitative traits. Theor. Appl. Genet. 77: 195-199. 10.5 85 105 125 145 Proteinase 165 185 inhibitor 205 Figure 2. Three dimensional graph of the estimated values of phenolic acids, proteinase inhibitor and index of susceptibility, obtained applying three linear genetic models. a) model of Mather and Jinks 425 (1982); b) model of Huidong (1988); c) model of 375 325 Serratos et al. (1993). Axis X represents phenolic 275 acid concentration [µg/g], axis Y is the 225 175 concentration of proteinase inhibitor [PIU/dg], and Phenolics 125 225 75 in axis Z the estimated values of susceptibility index have been plotted [I]. 138 J.A. SERRATOS, J.T. ARNASON. A. BLANCO-LABRA AND J.A. MIHM Reid L.M., D.E. Mather, J.T. Arnason, R.I. Hamilton, and A.T. Bolton. 1992. Changes in phenolic constituents of maize silk infected with Fusarium graminearum. Can. J. Bot. 70: 1697-1702. Richardson M., S. Valdes-Rodriguez, and A. Blanco-Labra. 1987. A possible function for thaumatin and TMVinduced protein suggested by homology to a maize inhibitor. Nature 327: 432-434. Ryan C.A., P.D. Bishop, J.S. Graham, R.M. Broadway, and S.S. Duffey. 1986. Plant and fungal cell wall fragments activate expression of proteinase inhibitor genes for plant defense. J. Chem Ecol. 16 (5): 1025-1036. Ryan C.A. 1990. Protease inhibitors in plants: Genes for improving defenses against insects and pathogens. Annu. Rev. Phytopathol. 28: 425-449. Ryan C.A. 1992. The search for the proteinase inhibitor-inducing factor, PIIF. Plant Mol. Biol. 19: 123-133. Sandoval-Cardoso M. L. 1991. Purificación y caracterización de enzimas larvales de 4 insectos que atacan al maíz durante el almacenamiento. B. Sc. Thesis. CIEIAA, Universidad de Guanajuato, Guanajuato, México. Sen A., S.S. Miller, J.T. Arnason, and G. Fulcher. 1991. Quantitative determination by HPLC and microspectrofluorimetry of phenolic acids in maize kernels. Phytochem. Anal. 2: 225-229. Table 3. Matrix of correlation coefficients for biochemical and biophysical traits of the maize kernel against indices of susceptibility to maize weevils for three genetic models. Correlation coefficients were obtained using data from Table 2. Model Phenolic acids [µg/g] MJ HU SE MJ HU SE MJ HU SE - 0.901** - 0.844** - 0.823** - 0.738* - 0.653* - 0.655* - 0.750* - 0.642* - 0.648* a Index of susceptibility [ I ] Weight loss of grain [ g ] Parameter of resistance [ b ] a Maximum Time of resistance Proteinase Force of to breakage of inhibitor compression seed coat [PIU/g] [Newtons] [seconds] - 0.830** - 0.590* - 0.564* - 0.605* - 0.366 - 0.340 - 0.619* - 0.340 - 0.306 0.230 0.001 - 0.030 0.632* 0.413 0.268 0.603* 0.363 0.194 - 0.620* - 0.524 - 0.467 - 0.752* - 0.602* - 0.462 - 0.747* - 0.577* - 0.421 Serratos J.A. 1987. Resistance of indigenous races of maize to infestation by maize weevil Sitophilus zeamais Motsch. M.Sc. Thesis. University of Ottawa, Faculty of Science. Ottawa, Ontario, Canada. Serratos J.A., J.T. Arnason, C. Nozzolillo, J.D.H. Lambert, B.J.R. Philogène, G. Fulcher, K. Davidson, L. Peacock, J. Atkinson, and P. Morand. 1987. Factors contributing to resistance of exotic maize populations to maize weevil, Sitophilus zeamais. J. Chem Ecol. 13 (4): 751-762. Serratos J.A., A. Blanco-Labra, J.A. Mihm, L. Pietrzak, and J.T. Arnason. 1993. Generation means analysis of phenolic compounds in maize grain and susceptibility to maize weevil Sitophilus zeamais infestation. Can. J. Bot. 71: 11761181. Waiss A.C., B.G. Chan, C.A. Elliger, B.R. Wiseman, W.W. McMillian, N.W. Widstrom, M.S. Zuber and A.J. Keaster. 1979. Maysin, a flavone glycoside from corn silks with antibiotic activity toward corn earworm. J. Econ. Entomol. 72: 256-258. Xiaowu L., M. Dron, C.L. Cramer, R.A. Dixon, and C.J. Lamb. 1989. Differential regulation of phenylalanine ammonialyase genes during plant development and by environmental cues. J. Biol. Chem. 264 (24): 14486-14492. Xie Y.S., J.T. Arnason, B.J.R. Philogène, J. Atkinson, and P. Morand. 1991. Distribution and variation of hydroxamic acids and related compounds in maize (Zea mays) root system. Can. J. Bot. 69: 677-681. Abbreviations are: MJ (Mather and Jinks 1982); HU (Huidong 1988); SE (Serratos et al. 1993) Table 4. Estimated genetic parameters using three linear genetic models. ** indicates significance at P < 0.001; * indicates significance at P < 0.05. The values without asterisk are non significant. Estimated genetic parameters Phenolic acids [µg/g] (Mather and Jinks 1982) m 261.80** a 175.48** d -50.25 (Huidong 1988) m 277.27** a 118.75** -80.68 de1 -84.54 de2 (Serratos et al. 1993) m 251.26** a -33.58 224.67** ae -8.76 apc -41.30** dee dp 87.15** Proteinase inhibitor [PIU/g] Maximum force of compression [Newtons] Time of resistance to breakage Index of of seed coat susceptibility [seconds] [I ] 147.68** 59.88* -16.23 126.97** -12.09 41.13 .787** .105 -.116 162.05** 44.75* -33.04 -53.64 135.31** -8.49 27.15 19.89 160.96** 6.21 42.56 7.40 -21.67 8.45 119.19** -7.71 7.37 -5.22 11.76 54.01** Weight loss of grain [grams] Parameter of resistance [b] 11.26** -1.68** .06 3.92** -1.60** 0.87 .92** -.27** .10 .745** .070 -.002 -.129 11.30** -1.05** -0.37 0.75 4.07** -0.97** 0.99 0.28 .95** -.17* .12 .0 .777** .051 -.111 .080 -.033 -.116 11.60** 1.16 -2.33 -0.18 0.10 -0.91** 4.15** 0.69 -2.45 0.21 0.32 -0.13 -.97** .18 -.52 .05 .03 -.05 139 Improving Two Tropical M aize Populations for Resistance to Stunt Complex R. Urbina A., Regional Maize Program for Central America and the Caribbean, Managua, Nicaragua Abst r a c t Caused by mycoplasmas, spiroplasmas, and maize fine stripe virus, maize stunt complex is endemic throughout the tropical lowlands of Central America and poses a potential danger for maize production in the region. To counteract the damaging effects of the disease in commercial maize plots, the Regional Maize Program for Central America and the Caribbean (Programa Regional de Maíz, PRM/CAC) has undertaken a collaborative stunt resistance breeding project, with the principal objective of developing high yielding, disease resistant cultivars. A tropical late white dent population (Pop. 73) and a tropical intermediate white flint population (Pop. 76), in their fifth and third improvement cycle, respectively, are being improved using an S1-S2 recurrent selection scheme. Research conducted independently in El Salvador and Nicaragua is aimed at developing S1 lines, advancing them to S2, recombining the best segments of each population, and forming experimental synthetic varieties. Lines are evaluated in both countries during normal crop cycles under heavy disease pressure. Lines developed each cycle are tested in countries in the region facing stunt problems. Synthetics developed during the latest breeding cycles (SC3P73 N, SC2P76 N and SC3P73 R) out-yielded resistant cultivar NB-6 by 15.5%, 11.7%, and 17% respectively. A variable percentage (1-20.5%) had fewer stunted plants and ears. In disease free environments, performance of resistant cultivars was statistically similar to that of susceptible high yielding hybrids used as reference checks. Resistant cultivars show outstanding performance under disease pressure in less favored environments, without any loss in yield potential in favored ones. The effects of stunt on commercial national maize programs of Nicaragua maize plots were quantified for the first and El Salvador initiated a One of the most devastating maize time in Nicaragua in 1986. That year, collaborative breeding project aimed at diseases, stunt is a production area lost or partially affected totaled developing stunt resistant cultivars. constraint in tropical and subtropical 27,682 ha; the foregone grain (not Comparing the stunt response of three environments of the American produced on this area) was 29,445 tons, selection cycles with cycle 0 at sites in continent. It is found in areas situated equivalent in economic terms to a loss Nicaragua and El Salvador, average from sea level to mid- and high of US$5,005,700 (DGB-MIDINRA 1986). reductions of 16, 28, and 19% were Int roduct ion altitudes, between 40o N to 30o observed in the number of stunted S latitude (De León, 1981). In Central In regions where stunt is endemic, the plants in populations 73, 76, and 79, America and the Caribbean, the disease risk of loss increases when farmers respectively (De León et al. 1984). can reach critical levels of incidence, delay planting due to a late-starting principally in regions where farmers rainy season. Disease resistant cultivars Collaborative efforts begun in 1975 led sow local varieties with low input must be planted to counteract the to the 1984 release in Nicaragua of levels, and where climatological detrimental effect of the disease on variety NB-6 (Santa Rosa 8073), conditions such as low rainfall, high commercial maize cropping and to released subsequently under the name temperatures, and low relative ensure sustainable production. Lujosa B-101 in Honduras and Santa Rosa in Mexico and Venezuela humidity favor development of the disease vector. Because of this problem, in 1975 the (Córdova et al.1986). Reports from CIMMYT Maize Program and the Nicaragua indicate that NB-6, planted 140 R. URBINA A. on 2,000 ha, yielded 3.5 t/ha, whereas it failed to achieve any significant gains synthetic experimental variety. Efforts stunt susceptible hybrids yielded only in resistance, likely due to a lack of a are made to select unrelated lines to 1.5 t/ha (Urbina 1991). high frequency of resistance genes. A avoid narrowing the population’s group of lines derived from Santa Rosa genetic base. Obvious progress has been achieved in 8576 was substituted for Pop. 22; the breeding for stunt resistence using an genetic background of Santa Rosa 8576 Full-sib families are planted during the S1-S2 recurrent selection scheme. included improved germplasm from period of high disease incidence, and Therefore in the second improvement the Taiwanese Technical Agricultural each family is selfed. Fifteen days after phase, begun in 1985 by the PRM/ Mission and the Nicaraguan Maize flowering is completed, selfed plants CAC, the same methodology with Program. From the time it was showing stunt symptoms are certain variations is being used in the incorporated into the project it was eliminated. The remaining plants are short term to: referred to as Population 76, because it harvested and used for further testing in • Eliminate or reduce the frequency of contained a good percentage of the the next breeding cycle. deleterious recessive genes in TIWF population. breeding populations; Increase the frequency of favorable Breeding methodology Changes in the recurrent selection scheme alleles involved in stunt resistance. Populations are being improved using First change - In cycle 3 of Pop. 73 and Develop high yielding stunt an S1-S2 recurrent selection scheme. cycle 2 of Pop. 76, S1 lines were resistant cultivars. Since this is a collaborative project, recombined through half-sibbing. This breeding is carried out in the was done to break up undesirable participating countries, but linkage groups that in the future might responsibility for managing each obscure the selection of favorable traits The second phase of the collaborative population resides with one country. and, at the same time, to reduce stunt resistance breeding project was Thus El Salvador is handling inbreeding in the population. Full-sibs re-initiated by the PRM/CAC in 1985 Population 73, and Nicaragua handles of half-sib families were formed through in El Salvador and the Dominican Population 76. direct and reciprocal crosses for testing • • M aterials and M ethods in international trials in different Republic, with Nicaragua joining in 1986. At the beginning of the first cycle, 400 countries in two seasons: one normal S1 lines of each population were and the other under disease pressure. Germplasm generated. Four nurseries with lines Once the international testing of full-sib This phase began with two white and from each population were formed for families was completed, a normal cycle two yellow populations, but this paper testing in Nicaragua and El Salvador of recurrent selection was begun. refers only to white populations using two sowing dates (one normal, improved in El Salvador and and the other late with high disease Second change - Starting with cycle 4 of Nicaragua. Both populations were incidence). A simple 20 x 20 lattice Pop. 73 and cycle 3 of Pop. 76, S1 lines formed based on S1 lines derived from design with two replications was used. were advanced to S2. The S1 lines were the following experimental varieties: Plot size was a 5 m row. For each line, sown in 6 m rows during the period of data were recorded on agronomic traits, stunt incidence. A high seeding rate (15 stunt response (number of stunted cm between plants) is used on half the plants and ears, and disease severity row to evaluate the line, and a low score), and grain yield. seeding rate (30 cm between plants) is Pop. 22 (Bulk Tropical White) Pop. 73 (Tropical Late White Dent) Across 8222 Los Baños 8222 Los Baños (1) 8222 Gwibi(1) 8222 Gwibi (2) 8222 Cycle IV (50%) Maracay 8222 Suwan 8222 Suwan (1) 8222 Cuyuta 8073 Porrillo 8073 Santa Rosa 8073 Tlatizapán 8073 Bulk of Pop. 73 used on the other 3 m to allow selfing. Pooled data of all test variables were Undesirable families are eliminated used to select the superior fraction of before and after flowering; at harvest, each population (40 lines), which was only healthy plants are selected for then planted the following cycle in each inclusion in the following cycle’s yield country to recombine S1 lines through and phytosanitary trials. As a result of Population 22 was eliminated from the full-sibbing. Likewise, each cycle the 10 this change, 225 S2 lines are being project after two selection cycles when best lines were selected to form a evaluated in 2.5 m rows with two 141 IMPROVING TWO TROPICAL MAIZE POPULATIONS FOR RESISTANCE TO STUNT COMPLEX replications in two sites and using two incidence. Negative values indicate that Selection improvement of Population planting dates. lines selected for recombination in the 73, shown in Table 3, has increased following cycle have higher levels of grain yield in environments with high Breeding progress in both populations disease resistance than the population disease pressure by an average of 306/ is measured indirectly by testing as a whole. kg/ha/cycle (10.4% per cycle). This increase is associated with a 10% experimental synthetics developed in the latest selection cycles, along with Over the cropping cycles, advances on reduction in the number of stunted composite varieties from each cycle and economically important characteristics plants each cycle. The regression resistant varieties and hybrids indicate that recurrent selection of S1 between yield and number of stunted developed in previous years, using lines is effective for eliminating plants (Table 4.) indicated that for each susceptible high yielding commercial deleterious recessive alleles that limit diseased plant, yield is reduced by hybrids as reference checks. selection progress (Córdova et al. 1986). approximately 75 grams (Aguiluz and Considerable gains were observed in Urbina 1992). Trials including these materials are the selected fraction in terms of grain evaluated in Guatemala, El Salvador, yield and reduced disease damage to After three selection cycles and under Nicaragua, Panama, and the Dominican plants and ears, as a result of moderate stunt incidence, per cycle Republic during normal sowing cycles capitalizing on favorable alleles gains of 11% in disease resistance and and periods of high disease incidence. conferring resistance (Tables 1 and 2). 4.3% in yield were achieved. These results show that selection has been Complete randomized blocks with four replications are used; plot size is four 5 Results from the previous stunt effective for improving varietal m rows. Data are recorded on resistance breeding program (De León performance under disease pressure agronomic traits, stunt response, and et al. 1984) confirmed that a scheme although at the expense of slightly grain yield of each entry. combining recurrent selection, lower yield potential in optimal evaluation, and recombination of S1 environments. Statistical analyses lines is effective for accumulating stable Analysis of variance (site specific and resistance levels. combined), stability analysis, mean comparison using the Tukey test, orthogonal contrasts, simple regression Table 1. Mean yields and stunt response of a selected fraction of Population 73. Combined data analysis from El Salvador and Nicaragua, 1989 analysis and class frequencies, Cycle 1 calculation of selection and stunt indices, were performed on the data. Results and Discussion Selection based on inbred progenies (S1, S2, etc.) is theoretically effective for bringing about changes in the Population mean Selected fraction mean Exp. variety mean Selection differential 1 kg/ha % st.1 kg/ha % st.1 3670 3697 4257 27 27 18 12 -9 3252 3829 3898 577 75 38 28 -37 1924 2717 3027 793 23 12 10 -11 Percent stunted plants. Table 2. Statistical data for 225 full-sib families from Population 76, cycle 2, Nicaragua, 1991. 1991-A and Miranda 1981). Recurrent selection of both populations was effective as stunted plants was negative but Population mean Selected fraction mean Selection differential Maximum Minimum Standard deviation Checks NB-12 B-833 variable due to erratic disease 1 of stunted plants and ears. Tables 1 and 2 show the selection differential for grain yield increasing over the test cycles for Pop.73 and Pop. 76, respectively, the differential for percent Cycle 3 % st.1 frequency of additive genes (Hallauer evidenced by grain yield, and numbers Cycle 2 kg/ha 1991-B kg/ha %st. pts.1 % st. ears1 kg/ha % st. pts.1 % st. ears1 3827 4488 662 5825 2097 695 48 35 -13 93 9 15 22 12 -10 65 0 13 3749 4035 556 5170 1177 734 54 41 -13 91 1 16 11 5 -6 53 4 8 4226 911 55 100 26 94 3627 1389 76 97 27 92 Percent stunted plants and ears (respectively). 142 R. URBINA A. Comparing the best synthetics from produced higher grain yields and had Cultivars improved for stunt resistance both populations developed during the lower percentages of stunted plants and during the last breeding cycles showed last breeding cycle with stunt resistant ears than check varieties NB-6 and H-53 marked performance differentials commercial varieties and high yielding (Table 4). These results objectively compared to hybrids and resistant hybrids clearly shows that synthetics demonstrate the progress achieved in varieties under severe disease SC3P73 N, SC2P76 N, and SC3P73 R that test cultivars performed better than conditions. Synthetics SC3P73 R and the check varieties, which are widely NB-12 had the lowest yield reductions used by farmers. when shifted from an environment Table 3. Grain yield and stunt response of synthetic and composite lines derived from Population 73 evaluated in seven locations of Central America, 1991 Yield (kg/ha)1 Genotype Composite C3 Synthetic C3 Composite C2 Synthetic C2 Synthetic C1 Synthetic C0 NB-6 B-833 1 % over % st. NB-6 plants 4247 a 4175 a 4093 a 3175 a 3634 b 3559 b 3459 b 2708 c 23 21 18 7 5 3 0 -22 37.6 33.2 39.6 42.4 38.2 47.7 35.7 58.8 Yields with the same letter are statistically similar at 5% probability using the Tukey test. with stunt incidence to another with high disease incidence. It is important to note that the synthetics show improved performance under high disease incidence in Given this evidence, there is no doubt unfavored environments, but do not that cultivars now available for farmer lose their yield potential in favorable use are superior to the ones currently ones. Synthetics SC3P73 N, SC2P76 N, being grown. and SC3P73 R yielded the same as hybrids B-833 and HN-879 in diseaseoutperforming them under high disease conditions, sometimes by more than 2.0 t/ha (>100%) (Tables 4 and 5). Table 4. Mean yields and stunt response of maize cultivars evaluated in Nicaragua, Panama, and El Salvador, 1991. Cultivar Yield kg/ha1 % over NB-6 SC3P73 N SC2P76 N SC3P73 R NB-12 H-53 NB-6 B-833 HN-879 Mean yield 4221 a 4219 a 4055 ab 4014 ab 3730 ab 3666 b 2990 c 2795 c 3711 15 15 11 9 2 0 -18 -24 1 2 Regression dev S2di 0.24 ** 0.05 ns 0.12 * 0.04 ns 0.13 * 0.15 * 0.79 ** 0.81 * 0.29 Regression coeff Bi % st. pts.2 % st. ears 0.66 ns 0.51 * 0.36 * 0.55 * 1.19 ns 1.20 ns 1.78 * 1.75 ** 1.00 38.5 42.3 42.8 49.5 56.8 54.0 78.8 82.6 55.7 21.7 17.0 18.3 20.9 50.4 38.8 72.8 76.2 39.5 Yields with the same letter are statistically similar at 5% probability using the Tukey test. Mean of four environments with stunt stress. Table 5. Effect of stunt on grain yield of maize cultivars evaluated at the H. Tapia B. experiment station, Managua, Nicaragua, June and September, 1991. Cultivar SC3P73 N SC2P76 N SC3P73 R NB-12 H-53 NB-6 B-833 HN-879 1 Favorable Unfavorable environment (kg/ha) environment (kg/ha) 4896 4593 4326 4053 4607 4831 3524 2827 Re fe re nce s free environments, while significantly 2873 3026 3298 2902 1379 1747 1234 789 % yield reduction Stunt resistance index1 41.3 34.1 23.8 28.4 70.3 63.8 65.0 72.1 0.59 0.66 0.76 0.72 0.30 0.36 0.35 0.28 Stunt resistance index = 1-(Y1-Y2)/(Y1), where, Y1 = Yield in favorable environments; and Y2 = Yield in unfavorable environments. Aguiluz, A., and R. Urbina. 1992. Evaluación de ciclos de selección para resistencia al achaparramiento en la Población 73. In Síntesis de Resultados Experimentales 1991. Programa Regional de Maíz para Centro América y el Caribe. Guatemala, 3: 59-65. Córdova, H., J. Lothrop, and M. Gutiérrez. 1986. Mejoramiento integral para cobertura y pudrición de mazorca en los complejos germoplásmicos de CIMMYT. XXXII Reunión Anual del PCCMCA, San Salvador, El Salvador. Córdova, H. 1990. Desarrollo y mejoramiento de germoplasma para resistencia a factores adversos bióticos y abióticos y producción de semilla. Estrategias y logros 1986-1991. Programa Regional de Maíz para Centro América y el Caribe. Guatemala. De León, C. 1981. Mejoramiento de poblaciones de maíz para resistencia al achaparramiento y al mildiú. XXVII Reunión anual del PCCMCA, Santo Domingo, República Dominicana. De León, C., L. Pineda, and R. Rodríguez. 1984. Resistencia genética: una alternativa contra el achaparramiento del maíz. XXX Reunión Anual del PCCMCA, Managua, Nicaragua. DGB-MIDINRA. 1986. Incidencia del achaparramiento en el cultivo del maíz y su impacto en el país. Managua, Nicaragua. Hallauer, A.R., and J.B. Miranda. 1981. Quantitative genetics in maize breeding. Ames: Iowa State University Press. Urbina, R. 1991. Incidencia y efectos del achaparramiento en la producción de maíz en Nicaragua. CNIGB-MIDINRA. Separata. 143 Response to Selection for Resistance to Leaf Feeding by Fall Armyworm in PopG, a Guadeloupe M aize Population C. Welcker, J.D. Gilet, D. Clavel, and I. Guinet, INRA, Pointe-a-Pitre, Guadeloupe. Abst r a c t Fall armyworm, (FAW) Spodoptera frugiperda (J.E. Smith) is a serious insect pest on maize, Zea mays L., in the Central American tropical lowlands and the Caribbean. Development of populations of maize with effective levels of resistance to damage by FAW larvae appears essential for sustainable maize farming. In the Guadeloupe Archipelago, recurrent S1 selection for resistance to leaf feeding by FAW larvae was conducted with a local maize composite, PopG. Genetic variability, heritability and predicted genetic gain were estimated from S1 progeny performance tests, and response to selection following three selection cycles was evaluated. Genetic progress was determined from a multilocal, replicated evaluation of populations per se, which were generated by recombinations from each selection cycle. Heritability estimates reached 0.22 for C1 and C2 cycles, whereas S1s and predicted genetic gains were 0.10 and 0.35 respectively. The regression of leaf damage ratings on selection cycles gave a significant b value of -0.16 units per cycle of selection. Advanced cycle PopG should be a good source of resistance with intermediate level to leaf feeding by FAW larvae. source of resistance to insects. Several pressure, were bulked in an original populations and inbreds, derived from population. This population (PopG), In the Guadeloupe archipelago, an Caribbean genetic germplasm with which was well adapted to Caribbean effort to enhance maize germplasm resistance to FAW have been identified conditions, was then subjected to a and to develop adapted varieties to the (Widstrom et al. 1972; Wiseman et al. recurrent breeding scheme for FAW Caribbean was initiated by the French 1979; Scott et al. 1981). The resistance (Welcker 1993). National Institute of Agricultural development and control of artificial Research (INRA) at the end of the infestation has enabled the screening of Our main objectives were to evaluate 1970s. For the last five years, in a large number of original populations actual progress for resistance to FAW collaboration with the Center for (Mihm 1983) Techniques such as: after 3 cycles of recurrent S1 selection in International Cooperation in selfing within populations and crosses this population PopG, and to estimate Agricultural Research for Development among populations; recurrent selection genetic variance, heritability and (CIRAD), France, the research program among S1 and half-sib families within expected gain from S1 progenies of has focused on resistance in maize to broad-based populations; have opened PopG-C2. leaf feeding by fall armyworm (FAW), up selection possibilities (Mihm 1989; Spodoptera frugiperda J.E. Smith, one of Smith et al. 1989; Williams and Davis This approach enabled us to estimate the main pest contraints in the 1989; Widstrom et al. 1992). available genetic variation in PopG, to Int roduct ion assess expected selection effectiveness, Caribbean. Native maize samples were collected in and eventually to redirect selection Caribbean maize has long been the Guadeloupe Archipelago in 1983 scheme parameters. recognized as an important breeding and several samples, showing relatively material for lowland tropics and as a good performance under insect 144 C. WELCKER, J.D. GILET, D. CLAVEL, I. GUINET families of the population, as male experimental station, on black cotton plants. Then, 500 self-pollinations were soil, during two different seasons (dry The plant material chosen was a made in 1992 and tested as S1 progenies and warm season) and, second, at composite formed from local ecotypes in 1993 using 10 lattice linked trials Duclos experimental station, on identified as the most resistant samples with 2 replications under heavy natural ferralitic soil in a wet area, during the to FAW and/or corn earworm, infestation. Forty-six progenies were warm season. This multilocation test Helicoverpa zea Boddie (CEW), in 1993 selected and recombined to form a C2 was designed to obtain the optimal and 1985 in the Guadeloupe population as shown in Figure 1. This screening of the three cycles of selection Archipelago. After three generations of half-sib family structure allowed a and to characterize their behavioral recombined mating, and one of random maternal link to be maintained, so variabilities in different locations. mating, the population was labeled inbreeding development could be PopG. controlled. Three hundred self pollinations of The third cycle was initiated in 1994. with 10 replications at each location (2 PopG were made in 1989 (plant Plant and family selections were made rows of 5 m per plot). This design takes selection based on resistance and vigor) in C2 based on a performance rating into account genotype x location and evaluated as S1 progenies in 1990 in scale of 0 to 9 (Williams and Davis interaction effect, commonly observed a randomized experiment under 1989) of plants growing under artificial in host-plant resistance experiments natural infestation. Plants were rated infestation. 300 S1 progenies were sown (Mihm 1989; Widstrom et al. 1992). 20, 30 and 40 days after sowing on a in June 1994 and the best ones were scale of 1 (no damage) to 5 (heavy selfed for evaluation at the S2 level. M aterial and M ethods The experimental design was a randomized complete block experiment The S1 progenies of PopG-C2 were evaluated at Godet in the warm season, damage). The 50 best performing progenies were recombined to form a The three cycles of selection C0, C1 and on six connected 7 x 8 lattices with two C1 population in 1991. Crosses were C2, are considered as varieties- replications (10 plants per plot, resistant realized using a bulk of the different populations formed by mass and susceptible checks randomly multiplication of a natural population included) This structure was chosen to and well adapted to their selection control potential location heterogeneity. environment. Artificial infestations were applied to 1985 Irish method 1989 Ecotypes composite Intercrosses (x 3) PopG-C0 300 S1 16% selection intensity 1992 PopG-C1 500 S1 10% selection intensity these trials (5 leaves, 25 larvae). The Plant selections were based not only on stage of 5-7 leaves appears to be the resistance parameters (under heavy most susceptible one to FAW (Davis et natural infestation during the first al. 1989). Larval damage was rated for cycles C0 and C1, and under artificial each plant on a scale of 0 (no damage) to infestation for the cycle C2), but also on 9 (heavy damage), as reported by agronomic characters such as vigor, Williams and Davis (1989). Plant and plant height, ear productivity. A family selections were based on 7 and significant improvment was obtained 14 Days After Infestation damage for resistance evaluation in the last ratings (DAI). cycle with the development of artificial infestation and individual plant to Response to selection was evaluated plant observations. from standard regression procedures of damage rating on selection cycles from 1994 PopG-C2 46 HS families The initial population C0, and the C0 through to C2 (Widstrom et al. populations issued from the two cycles 1992). This regression procedure 300 S1 15% selection intensity of selection, C1 and C2, were evaluated permits estimation of the effective 250 S2 15% selection intensity in a multilocal test which included genetic gain obtained from the three different environments in beginning of recurrent S1 selection. Figure 1. PopG recurrent selection scheme. Guadeloupe: first, at Godet RESPONSE TO SELECTION FOR RESISTANCE TO LEAF FEEDING BY FALL ARMYWORM 145 Standard analyses of variance, used to comparable with FAWCC progress effects, observed in multilocal analysis, analyse leaf damage ratings at each (0.18 units reduction on the two first might be contributing to the observed location, were combined based on selection cycles) obtained by Widstrom differences. For this reason, the S1 homogeneity of error variances. Both et al. (1992). progenies test could provide useful populations and locations were assumed to be random variables. information. (Table 1). These results confirm that three cycles of S1 recurrent selection seem sufficient Heritability, based on genetic estimates Components which estimated genetic to obtain a good level of resistance, as reached 0.22. This value seems to be variance and phenotypic variance were mentioned by Hallauer (1992). similar to results obtained by obtained from the software package SELECT (developed by INRA). We Widstrom et al. (1992) on FAWCC. This Genetic parameters estimated from S1 progenies test result appears to be low, but it should calculated genetic parameters from a statistical model of the genetic value Although the analysis of our data environment interactions are based on maternal plant effects within (based on individual observations and considered, no appropriate PopG-C2 i.e. genetic variance- taking into account plant-to-plant experimental design could significantly covariance components, heritabilities, variation) show significant genetic reduce the estimate of h2. and, Best Linear Unbiaised Predictor of variation within the populations of genetic value (BLUP) of each of the 300 PopG for resistance to larval feeding by The low genetic variances of PopG-C2 S1 of popG-C2. Heritability was FAW, it is possible that the high were not encouraging, even though we genotype by environment interaction later determined a larger mean estimated according to the formula: h2= G /s2 P. Additionally, genetic gains were estimated according to the formula GS = k s2 P h2, in which k=1.76 for 10% selection intensity. Results and Discussion 5 Damage rating s2 be borne in mind that when b = -0.26 ± 0.22 R2 = 0.85 Damage rating (index) 5 4.5 4 4.5 2.5 C0 Genetic gain after three cycles of selection 6.5 performances of the three cycles of selection per se and on the most discriminant environment, indicated significant progress for resistance to larvae feeding by FAW, at 7, 14 DAI, Damage rating Regression results, based on the three populations per se test environments, indicated a reduction of 0.16 units in damage per cycle (Fig. 3) This reduction indicates significant additive genetic variation and is 2.5 b = -0.16 ± 0.09 R2 = 0.60 C0 C1 Cycle C2 Figure 3. Selection response for reduced leaf-feeding damage by FAW to two cycles of recurrent selection in PopG at different locations. 6 Damage rating multilocal regression b value, based on b = -0.22 ± 0.06 R2 = 0.98 5.5 C0 conditions, the response of 0.23 units After location adjustment, the 4 6 and 14 DAI ratings) (Fig. 2). In these effectiveness of the selection process. C2 5 and with our selection index (mean of 7 reduction in damage per cycle attests C1 Cycle C1 Cycle C2 b = -0.23 ± 0.09 2 R = 0.95 5.5 Table 1. Heritability, genetic variance component estimates and predicted responses to S1 selection for resistance to leaf feeding by FAW in PopG-C2. H ∃ Ô 2G Ĥ 2G Predicted responses 0.15 0.18 0.24 0.22 0.33 0.35 2G 5 4.5 C0 C1 Cycle C2 Figure 2. Selection response for reduced leaf-feeding damage by FAW to two cycles of recurrent selection in PopG within the most discriminant environment. 7 DAI 14 DAI Index 7 + 14 DAI 0.63 rG = 0.58 ** ** 7DAI rating - 14DAI rating genetic correlation estimated from the S1 progenies of PopG-C2. 146 C. WELCKER, J.D. GILET, D. CLAVEL, I. GUINET selection response by using a genetic estimations of their BLUP, the expected Variation within families appeared to gain test. However, the main fact was progress reaches 0.32 units, confirming be 10 times higher than the variation the increasing of genetic variance for the first SELECT evaluation based on between families. High variation PopG-C2 from the initial pool. This the 300 S1s. Hence, highlighting the between S1 plants was observed. variance seems to be sufficient to value of BLUP estimations in the Therefore, self-pollinations were made suggest that recombination generated evaluation of the last S1 selection, and advancing the selected families to the S2 additional genetic variance. the use of these estimates in the level. The objective of this was also to potential organization of further get a more precise evaluation of their Expected genetic gain, estimated from selection schemes (Fig. 4). These results resistance levels, taking into account the genetic variance and heritability for demonstrate the effectiveness of the high environmental variance. This resistance evaluated 7 DAI and 14 DAI selection process for reduced leaf environmental variation, estimated appears to be promising. Its high level feeding, but also its slowness. This is from the residual value of inbred (0.35) and variability within PopG allow probably a consequence of: checks, underlines the importance of us to conclude that sufficient genetic A compromise between variability the check choice and the necessity to variation remained in PopG to justify preservation and selection intensity on increase the number of test sites or additional selection. This result affirms the main character and, replications, to improve the accuracy of the benefits of artificial infestation and Lower quality of the estimation of this genetic parameters. experimental design in cycle C2, when character during the first steps of the used to aid the selection process in an S1 selection scheme. Our results tend to show that faster progress could be obtained, if more testing procedure. This result confirms also the interest of individual-family Analysis of variance of S1 progenies importance is given to the 14 DAI rating combined selection and the maintaince indicated the presence of significant in the index estimation. However, this of a maternal link. Results obtained variation for resistance to larval feeding could increase the risk of lost using the SELECT software seem to by FAW within PopG-C2. Widstrom et information on resistance mechanisms, confirm that great progress was al. (1992) indicated similar values of potentially characterized by the 7 DAI obtained, from the initial pool and the genetic variance for FAWCC Cycle 3 rating. This was confirmed by a 7 DAI first selection steps. and Cycle 4, which allowed significant rating-14 DAI rating genetic correlation progress in this population in the of 0.58 estimated from the S1 progenies further cycles. (Fig.4). We selected the 10% best S1 based on our selection index. From the It does appear that continued progress should be possible in PopG. These 1 results underline the interest of this Genetic effects of S1 - 14DAI original Caribbean population as a new source of resistance to insects, with high 0.5 adaptability to the Caribbean. Therefore, PopG appears to be a 0 promising source of inbreds with an intermediate to high level of resistance to FAW. -0.5 Re fe re nce s -1 -1.5 -1.5 -1 *0.5 0 Genetic effects of S1 - 7DAI 0.5 1 Figure 4. Genetic variation for leaf-feeding damage by FAW between S1 of PopG-C2 - INRA - Godet 1994. Davis, F.M., Ng S.S., and Williams W.P. (1992) Visual rating scale for screening whorl-stage corn for resistance to fall armyworm. Mississippi Agricultural and Forestry Experiment Station technical bulletin 186. RESPONSE TO SELECTION FOR RESISTANCE TO LEAF FEEDING BY FALL ARMYWORM Hallauer A.R. (1992) Use of genetic variation for breeding populations in cross-pollinated species. In H.T. Stalker, and J.P. Murphy (Eds.) Plant Breeding in the 1990s, 37-117. London: CAB international. Mihm, J.A. (1983) Efficient mass rearing and infestation techniques to screen for resistance to fall armyworm, Spodoptera frugiperda. Maize program report. CIMMYT, Mexico, 12-23 Mihm, J.A. (1989) Evaluating maize for resistance to tropical stem borers, armyworms, and earworms. In Toward Insect Resistant Maize for the Third World: Proceedings of the International Symposium on Methodologies for Developing Resistance to Maize Insects, 109-121. Mexico D.F.: CIMMYT. Scott, G.E., and Davis F.M. (1981b) Registration of MpSWCB-4 population of maize. Crop Sci. 21: 148. Smith, M.E., Mihm J.A., and Jewell D.C. (1989) Breeding for multiple resistance to temperate, subtropical, and tropical maize insect pests at CIMMYT. In Toward Insect Resistant Maize for the Third World: Proceedings of the International Symposium on Methodologies for Developing Resistance to Maize Insects, 222-234. Mexico D.F.: CIMMYT. Welcker, C. (1993) Breeding for resistance in maize to fall armyworm in Caribbean region. Plant Resistance to Insects News Letter 20: 19-20. Widstrom, N.W., Wiseman B.R., and McMillian W.W. (1972) Resistance among some maize inbreds and single crosses to fall armyworm injury. Crop Sci. 12: 290-292. 147 Widstrom, N.W., Williams W.P., Wiseman B.R., and Davis F.M. (1992) Recurrent selection for resistance to leaf feeding by fall armyworm on maize. Crop Sci. 32: 1171-1174. Williams, W.P., and Davis F.M. (1989) Breeding for resistance in maize to southwestern corn borer and fall armyworm. In Toward Insect Resistant Maize for the Third World: Proceedings of the International Symposium on Methodologies for Developing Resistance to Maize Insects, 207-210. Mexico D.F.: CIMMYT. Wiseman B.R., and Davis F.M. (1979) Plant resistance to the fall armyworm. Florida Entomologist 62: 123-130 148 Location and Effect of Quantitative Trait Loci for Southwestern Corn Borer and Sugarcane Borer Resistance In Tropical M aize M. Khairallah, D. Hoisington, D. González-de-León, CIMMYT Int., Mexico M. Bohn, A. Melchinger, University of Hohenheim, Stuttgart, Germany D.C. Jewell, CIMMYT Int., Mount Pleasant, Zimbabwe J.A. Deutsch, ICI Seeds, Marshall, MO, USA and J. Mihm, French Agricultural Research, Inc., Lamberton, MN, USA Abst r a c t Development of multiple insect resistance in tropical and subtropical maize represents a major effort of the maize breeding program at CIMMYT. Resistance to the southwestern corn borer (SWCB), an aggressive feeder, appears to be polygenically controlled and has been widely considered to involve primarily additive gene action. Some of the components of resistance to SWCB seem to confer resistance to other important Lepidopteran maize pests, including the sugar cane borer (SCB). Our objective was to map, using restriction fragment length polymorphism (RFLP) markers, the quantitative trait loci (QTL) involved in the resistance to SWCB and SCB as a first step towards the use of marker-assisted selection in the breeding for such complex traits. Two distinct F2 populations were developed, each from a cross between a susceptible (S) and a resistant (R) line: the population derived from the Ki3 (S) and CML139 (R) cross was comprised of 476 F2 individuals and was evaluated for SWCB. The population derived from the CML131 (S) and CML67 (R) cross consisted of 215 individuals and was rated for SWCB and SCB. F2 individuals were genotyped using close to 100 genomic and cDNA maize probes. F3 families were rated for leaf-feeding damage (1-10 scale) after artificial infestation for two or three consecutive years at one or two locations. The QTL analyses were conducted using single-factor ANOVAs and a maximum likelihood approach (MAPMAKER/QTL). Several chromosomal regions were found to be involved in the resistance to SWCB and SCB. Not all regions were shared by the two populations for SWCB and some QTL were common in the resistance to both insects. Most of the QTL showed additive and dominance effects. or all of these insects would provide an feeders, appears to be polygenically effective way of increasing maize controlled and is thought to involve About 30 out of 55 million hectares production in affected areas, while primarily additive variation (Scott and planted with maize in developing keeping down the cost to the farmer Davis 1978; Williams et al. 1989; Thome countries are seriously affected by and reducing the impact of chemicals et al. 1992). Moreover, some of the insect problems. Lepidopteran insects on the environment. components of resistance to SWCB Int roduct ion seem to confer resistance to other insect are among the most important pests species, including the sugarcane borer affecting this crop worldwide. For Development of multiple insect instance, typical annual losses resistance in tropical and subtropical (SCB), Diatraea saccharalis F., and to estimated at over 4 million tons in maize represents a major effort of the other Lepidopteran species against Brazil and 1 million tons in Mexico maize breeding program at CIMMYT. which Caribbean materials were tested result in an overall cash loss of more Resistance to the southwestern corn (Smith et al. 1989). Breeding for than US$ 600 million (CIMMYT, 1988). borer (SWCB), Diatraea grandiosella resistance to SWCB and SCB has been Improved germplasm resistant to some Dyar, one of the most aggressive laborious and time consuming because LOCATION AND EFFECT OF QUANTITATIVE TRAIT LOCI FOR SOUTHWESTERN CORN BORER AND SUGARCANE BORER RESISTANCE IN TROPICAL MAIZE 149 it has required recurrent selection with protocols described in Hoisington et al. consisted of 619 entries: 476 F3 families, at least four to five cycles of infestation (1994). DNA was extracted from 36 of parent A and 35 of parent B used in order to recover and verify a lyophilized ground leaf tissue then as parental checks. In addition, 72 desirable level of resistance. This has digested with one of two restriction entries of an S1 bulk of a white seeded also implied the need for insect mass- endonucleases, EcoRI and HindIII. DNA hybrid (CML61xCML62) were used as a rearing facilities. In order to assist in fragments were separated by gel physical check to control planting the breeding efforts for borer resistance, electrophoresis in 0.7% agarose gels errors in the field and/or loading and our goal was to map, using restriction then transferred onto non-charged handling errors in the lab. The trials fragment length polymorphism (RFLP) nylon membranes by Southern blotting. were grown in a RCBD with two markers, the quantitative trait loci Genomic and cDNA maize clones from replications in the summer of 1990 (QTL) involved in the resistance to the University of Missouri, Columbia (Tl90B), and the winters of 1991 (Tl91A) SWCB and SCB as a first step towards (UMC), Brookhaven National and 1992 (Tl92A). The CxD trials the use of marker-assisted selection Laboratory (BNL) and the Native Plants consisted of 240 entries: 215 F3 families, (MAS) in the breeding for such complex Inc. (NPI) collections were used as 12 of parent C and 13 of parent D, traits. probes to detect RFLPs. These clones which were grown in a 24x10 a-lattice were amplified by PCR and labeled design with two replications during the with 2.5% digoxigenin-dUTP. After winter seasons of 1992 (Tl92A) and 1993 overnight hybridizations, RFLPs were (Tl93A). M aterials and M ethods Populations detected with the antidigoxigenin- Four maize lines, two susceptible to alkaline phosphatase-AMPPD Entries were grown in 2.5 or 5 m single- SWCB and SCB and two resistant ones, chemiluminescence system. The same row plots, 0.75 m apart. Plants were were used to form the two populations blots were hybridized to several thinned to a distance of 25 cm and were used in this study (Table 1). Crosses consecutive probes by first infested at the mid-whorl stage with 30- were made between the susceptible and stripwashing the last probe off the blot. 40 neonate SWCB or SCB larvae. These the resistant lines: Ki3xCML139 (AxB), RFLP data were captured and verified were applied as a larvae-grit mixture and CML131xCML67 (CxD) and two F1 using HyperMapdata, software with a mechanical dispenser (Mihm, ears from each cross were selfed to developed at CIMMYT. 1983). Leaf feeding damage by the insects was assessed 15-24 days after produce the F2 populations. For the Insect damage rating of the F3 families infestation using the 1 (no visible leaf harvested from single F2 plants which were then selfed to produce F3 seeds. F3 SWCB and SCB infestation trials were (1-9 as in Davis and Williams 1989). plants of each family were sib-mated conducted at CIMMYT’s Tlaltizapán and seeds pooled for planting in station in the State of Morelos, Mexico Data analyses replicated trials for the evaluation of (18.41oN, 940 masl, 830 mm average Insect damage ratings from the insect leaf feeding damage. rainfall). In addition, one SCB trial was individual plants were averaged to give planted at the Poza Rica station in the a mean value per F3 family. Lattice RFLP analysis, leaf tissue was damage) to 10 (dead plant) rating scale tropical part of the State of Puebla, analyses of variance were performed RFLP genotyping was done on 475 and Mexico (20.34oN, 60 masl, 1200 mm for the CxD field trials on the data from 190 F2 individuals for the cross AxB average rainfall) during the winter cycle each experiment. Adjusted entry mean and CxD, respectively, using the of 1993 (PR93A). The AxB trials squares and effective errors were then Genotyping the F2 individuals Table 1. Some characteristics of the maize lines used to generate the populations for the mapping of SWCB and SCB resistance (DR=Dominican Republic) Designation Line Reaction to SWCB, SCB Origin Adaptation Maturity Grain type A B Ki3 CML139 Susceptible Resistant Suwan1 DR Grp. 1/ Antigua Grp.2 Tropical Subtropical Late Intermediate Yellow, flint Yellow, semi-flint C D CML131 CML67 Very susceptible Very resistant Pop. 42 Antigua Grp.2 Subtropical Tropical Intermediate Late White, dent Red/yellow semi-dent 150 M. KHAIRALLAH, D. HOISINGTON, D. GONZÁLEZ-DE-LEÓN, M. BOHN, A. MELCHINGER, D.C. JEWELL, J.A. DEUTSCH, AND J. MIHM used to compute the combined analyses Mapping of QTL and estimation of due to insufficient insects at the time of of variance and covariance across their genetic effects were performed the artificial infestation and to poor environments for SWCB and SCB according to interval mapping using growing conditions in the 1990B trial. experiments. For the AxB 1990 and 1991 the package MAPMAKER/QTL Therefore, neither variance components trials, SWCB leaf feeding damage was (Lander and Botstein 1989). The nor heritabilitites were computed for evaluated in only one replication, presence of a putative QTL in a given these two trials (Table 2). Although the therefore, only a combined analysis of genomic region was declared when the three trials were artificially infested, the variance was performed on the data LOD score exceeded a threshold of 2.5. damage was most severe in 1991, less from the three experiments. Gene action was determined based on severe in 1990 and a very light damage Heritabilities were computed according the ratio of dominant to additive resulted in 1992. These differences are to Hallauer and Miranda (p. 90, 1981): genetic effects and the criteria used by expressed by a significant GxE Stuber et al. (1987). The AxB data was interaction and consequently a medium also analyzed by one-way ANOVA low heritability, h2=0.39. It is important using the SAS PROC GLM (SAS to note that the 1990 and 1991 AxB Institute, 1988). trials were sown in poor soil in the σ̂2g h2 = σ̂2g + σ̂2ge e + σ̂2 , re station, and plants were seen to be where r = no. of reps, e = no. of Results and Discussion environments, σ̂ = error variance, σ̂2g = genotypic variance, and σ̂2ge = genotype x environment variance. affected by iron deficiency particularly in the rainy season (Tl90B trial). Mean ratings of insect leaf feeding damage on the two F3 populations In contrast, the CxD SWCB trials were exhibited near normal distributions grown on better soils using a more An RFLP linkage map was constructed with apparent transgressive efficient experimental design and for each population using the software segregation in the case of the therefore the results were more similar package MAPMAKER (Lander et al. population derived from AxB. The across seasons, although the 1993 trial 1987). For declaration of linkage, a LOD mean parental values and the range showed slightly more severe damage. (log10 of the likelihood ratio) threshold and mean for the F3’s in the separate However, albeit the GxE interactions of 3.00 and a maximum recombination trials are shown in Table 2 for SWCB were significant, h2 was moderately frequency of 0.40 were used. Genetic and in Table 3 for SCB. high at 0.64 (Table 2). The SCB trials were also similar in terms of distances between markers were estimated with the Haldane mapping For the AxB population, unfortunately distribution and resulted in a h2 function. A combined map was also there is data from only one replication estimate of 0.64 across the three trials constructed by pooling the genotypic for the 1990 and 1991 trials. This was (Table 3). data from the two populations. Table 2. Means and standard errors for SWCB ratings of the four parents and the 476 F3 families in the AxB population and the 215 F3 families in the CxD population in the individual trials. Variance components and heritabilities were computed for the individual trials and across trials. AxB Parameter Means ± SE P1 P2 F3 lines Range, F3’s Tl91A1 Tl92A Combined Tl92A Tl93A Combined 8.5 ± 0.13 6.1 ± 0.11 6.8 ± 0.06 4.0 - 10.0 8.9 ± 0.03 7.0 ± 0.10 8.0 ± 0.03 5.9 - 9.5 6.2 ± 0.06 4.2 ± 0.04 4.8 ± 0.02 3.5 - 6.3 — — — — 9.1 ± 0.07 3.6 ± 0.06 6.2 ± 0.07 4.0 - 8.4 8.6 ± 0.14 5.5 ± 0.14 7.5 ± 0.05 4.8 - 8.8 8.9 ± 0.18 4.6 ± 0.16 6.9 ± 0.05 — 0.11** — 0.19 0.54 0.12** 0.42** 0.19 0.39 1.17** — 0.50 0.70 0.54** — 0.20** 0.73 0.33** 0.20** 0.35 0.64 Variance components and heritabities (F3 lines) — — s 2g 2 s ge — — — — s2 — — h2 1 CxD Tl90B1 Data from only one replication ** Significant at the 0.01 probability level. LOCATION AND EFFECT OF QUANTITATIVE TRAIT LOCI FOR SOUTHWESTERN CORN BORER AND SUGARCANE BORER RESISTANCE IN TROPICAL MAIZE 151 Phenotypic correlations between SWCB resistance are presented in Table 4. In portion of the genetic variance, were and SCB mean leaf ratings on the F3 the three AxB trials, several putative detected. These were located on families of the CxD cross was 0.5 QTL were detected, most explaining a chromosomes 1, 5, 7 and 9. One of the (significant at the 0.01 probability level) small portion of the total variance for QTL on chromosome 1 was detected in for both the Tl92A and Tl93A trials. As SWCB leaf feeding damage. These were both trials as well as in the AxB cross shown by earlier work (Thome et al. spread throughout the genome and (Tl90B). Both additive and dominance 1992), this relatively high correlation only three regions on chromosomes 3 effects were present and all additive between the damage caused by SWCB, and 8 were common to two or three effects contributing to the increased a very aggressive feeder, and SCB may trials. The QTL exhibited both additive resistance came from the resistant allow some progress to be made in and dominance effects. With the parent. Surprisingly, dominance effects breeding for multiple borer resistance exception of the QTL on chromosome 4 were almost as important as the by selecting only under infestation with detected in the 1990 trial, all additive additive ones and contributed to an SWCB. The selections could then be effects contributing to increased increase in the rating scale or a decrease verified for multiple resistance by resistance came from the resistant in resistance (Table 4). subsequent testing with other insects. parent. Most dominance effects contributed to increased resistance. A The QTL for SCB resistance are A total of 128 and 97 RFLP loci were few of the effects were from Ki3, the summarized in Table 5. Putative QTL placed on the AxB and CxD linkage susceptible parent in the AxB cross, and were located on chromosomes 1, 2, 5, 9, maps respectively. The two maps were this was reflected in the transgressive and 10. Again, the variance at each of consistent in locus order with each segregation observed for leaf feeding these QTL included both additive and other and also with other published ratings in this population. Results from dominant effects and most alleles for maize maps (e.g., Maize Genetics the one-way ANOVA were very increased resistance to SCB were Cooperation Newsletter no.68, 1994). consistent with those from the interval contributed by CML67. In this case, The combined map included 166 loci mapping analysis in determining dominance effects also were exhibited (60 loci in common between both regions of the genome containing as an increased resistance. populations) and spanned a distance of putative QTL with the exception of the 2041 cM resulting in an average marker QTL on chromosome 2 (Tl91A) and the Most of the gene action at the putative distance of 12.4cM (Fig. 1). The one on chromosome 4 (Tl90B) where QTL detected in both populations for individual maps provided a relatively the F-test did not show any locus to be both insects ranged from partial to dense framework for mapping QTL, as significantly correlated with the SWCB overdominance with the exception of discussed below. damage rating. the QTL on chromosome 3 in the AxB SWCB Tl92A trial and the QTL on Results of the interval mapping For the CxD cross, a smaller number of chromosome 9 in the CxD SCB Tl92A analyses for QTL responsible for SWCB putative QTL, each explaining a larger trial. These results do not fully agree with results from the combining ability Table 3. Means and standard errors for SCB ratings of the two parents and the 215 F3 families in the CxD population in the individual trials. Variance components and heritabilities were computed for the individual trials and across trials. Parameter Tl92A Tl93A PR93A combined Means ± SE P1 P2 F3 lines Range, F3’s 8.3 ± 0.09 4.3 ± 0.17 6.2 ± 0.05 4.2 - 8.1 8.6 ± 0.19 4.3 ± 0.18 6.3 ± 0.06 4.0 - 8.5 8.1 ± 0.20 5.2 ± 0.25 6.6 ± 0.05 4.4 - 9.3 8.3 ± 0.20 4.6 ± 0.10 6.4 ± 0.00 — 0.47** — 0.37 0.56 0.24** 0.22** 0.36 0.64 Variance components and heritabilities (F3 families) 0.59** 0.95** s 2g — — s2ge 2 s 0.37 0.36 0.62 0.73 h2 ** Significant at the 0.01 probability level. studies for SWCB and SCB resistance where additive gene action was found to be more important (Scott and Davis 1978; Williams et al. 1989; Thome et al. 1992). Up to 53% of the genetic variance of any one trait in any one trial could be explained in terms of the set of regions detected for resistance to SWCB or to SCB. The estimated heritabilities do not appear to provide a valid criterion to predict how many QTL will be detected in particular environments and which percentage of the phenotypic variance 152 Figure 1. Combined RFLP linkage map of the genome of tropical maize Ki3 x CML139 and CML67 x CML131 (loci names on the right and distances in cM on the left of each linkage group). C2 C3 umc53a umc94a umc32a umc6 2.7 6.8 5.9 11.0 npi286 umc29b umc371 umc131 umc8b 3.8 4.4 9.0 0.4 7.4 umc303 umc336a 11.0 umc5a umc167 umc104c umc67 umc177° umc338#1° umc357 umc59° umc58 9.0 7.8 umc22a umc328a 4.8 19.2 umc16a umc150b 21.8 8.0 umc23a umc33a umc128 10.3 umc308a 17.1 umc392 umc126a 8.8 umc51a 11.6 8.6 5.7 umc96 umc336b umc308b 1.4 umc321c 13.3 umc 360a npi253a 28.8 umc32b 15.5 18.1 umc65a 9.1 5.1 2.1 7.5 umc338#3° bnl8.23° 6.8 umc 95 umc323 umc48a 6.4 7.4 umc 18b bnl5.62 umc 130 umc 155 bnl7.49b umc188 umc 64 5.0 3.3 5.9 5.7 11.9 umc38b 11.1 umc146 umc44a 7.9 20.8 umc 378a umc30a 23.0 13.5 13.9 umc 340 umc150a bnl16.06 1.3 9.3 umc 114 umc153 umc 380 umc 338#2° 12.3 33.3 umc313b bnl5.47a umc38a 21.4 umc81 6.4 1.0 2.2 17.0 umc12a bnl15.27b umc328b umc125b bnl14.07 10.6 9.0 3.0 5.4 15.6 12.4 bnl6.06b umc 364 umc 386 9.1 bnl15.21 2.9 7.0 19.7 11.7 umc21 umc369a umc 105a 19.6 umc116a 7.7 npi285 25.2 umc103a 28.2 bnl3.04 13.1 29.0 umc 358a bnl5.09 bnl14.28 5.5 4.9 bnl10.24b 14.0 bnl7.49a 13.2 14.8 umc35 30.5 umc132a umc334 33.8 24.7 5.3 36.6 umc389 umc68 umc39c umc 337b 16.1 14.8 19.8 umc366a bnl13.05a C1 0 umc 113a umc310 bnl8.45b umc 133a C9 bnl15.40 15.0 22.6 umc15a 6.7 umc83a 8.1 1.4 10.8 17.7 umc19 4.7 umc327#3° umc312 20.5 9.9 28.5 24.4 bnl5.71a umc318b umc353 23.1 12.6 umc49a umc85 umc44c umc379 npi361° umc66c umc318a 22.1 umc63 8.8 umc83b C8 8.2 umc347 22.1 17.0 umc389#1° bnl6.16 umc3b 1.2 12.3 5.5 bnl10.24a umc137a 25.4 5.9 45.4 umc47 9.5 umc140#2° 13.3 umc27a 13.2 13.2 7.8 umc18a umc322 umc384b umc374 umc90 umc72a umc365 C7 umc152#1° bnl5.37 5.9 3.0 umc372c umc382 bnl6.25 10.6 8.7 6.1 umc321b bnl13.05b umc10 15.6 umc55a 36.3 umc31a 13.5 11.0 2.5 11.1 6.5 0.8 1.9 13.1 47.0 umc50 34.0 C6 umc132b umc350 umc313a 17.7 55.5 umc11 umc378c umc8a 2.2 2.4 13.7 13.3 36.6 4.3 9.1 0.9 0.2 5.0 3.5 4.4 12.8 umc123 umc121 npi97a 4.1 C5 9.6 14.2 7.5 C4 M. KHAIRALLAH, D. HOISINGTON, D. GONZÁLEZ-DE-LEÓN, M. BOHN, A. MELCHINGER, D.C. JEWELL, J.A. DEUTSCH, AND J. MIHM C1 umc3a umc127b umc317a 46.1 29.8 umc140a umc388 11.3 umc104b umc106a they explain. For example, in the case of the CxD cross, in which heritabilities were relatively high and similar, only one putative QTL could be detected across environments, the rest being specific to particular environments (Tables 2 and 4). When comparing QTL for SWCB resistance detected across the two populations, only those on º indicates an unassigned locus designator chromosomes 1 and 5 were in the same bnl6.32 475 and 240 F2 individuals Haldane 166 loci 2041 cM 12.4 cM regions of the genome. Whether this 18.6 Population: Mapping Function: # Loci: Total Length: Average Density: means that CML139 and CML67 have bnl8.29a different attributes for resistance to umc161a 11.6 SWCB or is merely a reflection of the 35.1 QTL x Environment interactions is not umc327a LOCATION AND EFFECT OF QUANTITATIVE TRAIT LOCI FOR SOUTHWESTERN CORN BORER AND SUGARCANE BORER RESISTANCE IN TROPICAL MAIZE 153 clear. When looking across insects, QTL are used as cofactors in order to reduce For a given population and trait, there on chromosomes 1, 5 and 9 were the noise produced and better define was wide variation in the detection of detected for both SWCB and SCB the location of the QTL. some regions from one trial to another; resistance in the CxD population and this may indicate a highly plastic Prospects for marker-assisted selection genotype-environment interaction with population. This indicates that at least some of the factors controlling These data, as many other in the under certain conditions. In a MAS resistance to one borer also control literature (Schön et al. 1993), confirm scheme, it will be critical to ascertain resistance to the other, and is in the complexities of analyzing QTL which are the most important regions agreement with results reported by inheritance and expression patterns, enhancing the trait of interest under a Thome et al. (1992). and raise many questions as to the given environment. These may be practical approaches needed for the enough to provide an economically We are now in the process of analyzing successful application of marker- sufficient level of resistance, while these data for QTL detection using assisted selection (MAS). other, minor regions, which may in on chromosomes 1 and 5 with the AxB some regions only becoming “active” alternative methods such as composite some cases be false positives, may be interval mapping where some markers ignored for practical purposes. Table 4. Putative QTL for SWCB resistance and their genetic effects in the separate trials of the AxB (476 F3 families) and the CxD (215 F3 families) populations. Genetic effects are expressed as the change in the leaf feeding damage scoring due to the contribution of an allele from the resistant parent (a=additive, d=dominant, p=partial, od=overdominant). Genetic effects Chromosome Flanking markers Position in interval (cM) Max. LOD Phenotypic variance score explained % AxB Tl90B 1 3 3 4 5 5 7 8 umc23a - umc83a bnl10.24a - umc389 umc16a - umc63 umc123 - umc31a umc382 - bnl6.25 umc318a - umc68 bnl6.06b - umc328b umc103a - umc32b 10 8 16 18 0 12 8 12 2.52 2.80 2.80 3.69 2.54 4.09 2.57 4.24 Total AxB Tl91A 1 2 3 3 5 8 9 umc388 - umc161a umc6 - umc371 bnl10.24a - umc389 umc16a - umc63 bnl6.25 - umc90 bnl13.05a - umc321c bnl5.09 - umc337b 18 0 6 12 0 2 12 AxB Tl92A 1 3 5 6 8 9 bnl8.29a - bnl6.32 umc16a - umc63 umc392 - umc126a umc65a - umc21 umc103a - umc32b umc95 - umc378a CxD Tl92A 1 1 CxD Tl93A 1 5 7 9 Additive Dominant Gene action 3.8 3.8 4.0 14.4 2.7 5.6 3.1 5.8 43.2 -0.33 -0.37 -0.37 0.29 -0.29 -0.44 -0.22 -0.39 -2.12 -0.38 0.08 -0.18 -1.64 -0.28 -0.36 -0.66 -0.58 -4.00 d pd pd od d d od od 3.20 2.93 4.32 3.87 2.84 2.59 2.60 Total 5.7 3.0 5.9 5.3 2.8 3.2 3.8 29.7 -0.22 -0.10 -0.23 -0.21 -0.14 -0.11 -0.17 -1.18 -0.14 -0.36 0.10 -0.16 -0.20 0.36 -0.24 -0.64 pd od pd pd od od od 12 20 10 12 10 8 2.53 8.93 4.61 5.02 3.96 11.40 Total 4.1 10.5 7.1 6.5 5.7 18.4 52.4 -0.06 -0.21 -0.12 -0.14 -0.13 -0.25 -0.91 -0.34 -0.02 -0.34 -0.20 -0.24 -0.32 -1.46 od a od od od od umc67-umc357 umc58-umc33a 0 18 5.01 5.86 Total 13.9 19.3 33.2 -0.22 -0.37 -0.59 0.27 0.36 0.63 od d umc33a-umc128 umc318a-umc68 bnl14.07-bnl16.06 umc380-umc340 0 4 6 0 3.66 3.12 2.94 2.53 Total 9.9 9.7 10.2 6.7 36.5 -0.21 -0.24 -0.28 -0.23 -0.96 -0.08 0.30 0.14 0.50 0.86 pd od pd od 154 M. KHAIRALLAH, D. HOISINGTON, D. GONZÁLEZ-DE-LEÓN, M. BOHN, A. MELCHINGER, D.C. JEWELL, J.A. DEUTSCH, AND J. MIHM We have now embarked on a pilot experiment, reported elsewhere in these proceedings (Willcox et al.), in which we are examining the relative efficiency of MAS in transferring insect resistance from CML67 (parent D) into African elite germplasm. We believe that some of the intricacies of the expression of regions detected in the study reported here may well be clarified as we backcross them in specific combinations into susceptible backgrounds. Thus, in a very pragmatic fashion, we shall determine the feasibility and value of MAS for such complex traits as insect resistance. These traits have required many years of intensive, laborious and costly breeding to advance to the current levels of resistance and MAS may well prove to increase the speed and effectiveness of transfers to a wider germplasm pool. Re fe re nce s CIMMYT. 1988. Maize production regions in developing countries. Maize Program Internal Document. CIMMYT, Mexico. Davis, F.M. and W.P. Williams. 1989. Methods used to screen maize for and to determine mechanisms of resistance to the southwestern corn borer and fall armyworm. In Toward Insect Resistant Maize for the Third World: Proceedings of the International Symposium on Methodologies for Developing Host Plant Resistance to Maize Insects, 101-108. Mexico, D.F.: CIMMYT. Hallauer, A.R., and J.B. Miranda Fo. 1988. Quantitative Genetics in Maize Breeding. 2nd ed. Iowa State University Press, Ames, IA. Hoisington, D., M. Khairallah, and D. González-de-León. 1994. Laboratory protocols: CIMMYT Applied Molecular Genetics Laboratory. Second Edition, CIMMYT, Mexico, D.F. Lander, E.S., and D. Botstein. 1989. Mapping mendelian factors underlying quantitative traits using RFLP linkage maps. Genetics 121: 185-199. Lander, E.S., P. Green, J. Abrahamson, A. Barlow, M.J. Daley, S.E. Lincoln, and L. Newburg, 1987. MAPMAKER: an interactive computer package for constructing primary genetic linkage maps of experimental and natural populations. Genomics 1: 174-181. Mihm, J.A., 1983 Efficient mass rearing and infestation techniques to screen for host plant resistance to maize stem borers, Diatraea spp. CIMMYT, El Batán, Mexico. SAS Institute Inc. 1988. SAS/STAT™ User’s Guide, Release 6.03 Edition. Cary, NC: SAS Institute Inc. Schön, C.C., M. Lee, A.E. Melchinger, W.D. Guthrie, and W.L. Woodman, 1993. Mapping and characterization of quantitative trait loci affecting resistance against second-generation European corn borer in maize with the aid of RFLPs. Heredity 70: 648-659. Scott, G.E., and F.M. Davis, 1978 Secondbrood damage by southwestern corn borer in a corn diallel cross. Crop Sci. 18: 335-336. Smith, M.E., J.A. Mihm, and D.C. Jewell. 1989. Breeding for multiple resistance to temperate, subtropical, and tropical maize insect pests at CIMMYT. In Toward Insect Resistant Maize for the Third World: Proceedings of the International Symposium on Methodologies for Developing Host Plant Resistance to Maize Insects, 222-234. Mexico, D.F.: CIMMYT. Stuber, C.W., M.D. Edwards, and J.F. Wendel. 1987. Molecular markerfacilitated investigations of quantitative trait loci in maize. II. Factors influencing yield and its component traits. Crop Sci. 27: 639-648. Thome, C.R., M.E. Smith, and J.A. Mihm, 1992. Leaf feeding resistance to multiple insect species in a maize diallel. Crop Sci. 32: 1460-1463. Williams, W.P., P.M. Buckley, and F.M. Davis, 1989 Combining ability for resistance in corn to fall armyworm and southwestern corn borer. Crop Sci. 29: 913-915. Table 5. Putative QTL for SCB resistance and their genetic effects in the separate trials of the CxD population (215 F3 families). Genetic effects are expressed as the change in the leaf feeding damage scoring due to the contribution of an allele from the resistant parent (a=additive, d=dominant, p=partial, od=overdominant). Chromosome Flanking markers Position in interval (cM) Max. LOD score Phenotypic variance explained % Genetic effects Additive Dominant Gene action Tl92A 5 9 umc126a-umc318a umc105a-umc153 6 16 3.61 8.32 Total 11.5 26.3 37.8 -0.30 -0.51 -0.81 -0.19 -0.01 -0.20 pd a Tl93A 2 9 10 umc53a-umc6 umc340-umc358a umc44a-bnl7.49a 0 6 24 4.09 7.66 2.75 Total 10.8 21.7 9.1 41.6 -0.18 -0.45 -0.30 -0.93 -0.60 -0.69 -0.13 -1.42 od od pd PR 93A 1 1 2 9 umc167-umc67 umc58-umc33a umc131-umc22a umc113a-umc105a 2 12 10 24 2.63 3.61 5.32 3.52 Total 7.4 13.2 20.6 11.3 52.5 -0.11 -0.25 -0.31 -0.28 -0.95 0.13 -0.33 -0.65 -0.24 -1.09 d od od d Developing Insect Resistant Germplasm Using RFLP Aided Breeding Techniques D.L. Benson, ICI Seeds, Thomasville, Georgia Abst r a c t The molecular markers known as restriction fragment length polymorphisms (RFLPs) can be utilized to identify the chromosomal locations of genes controlling traits of agronomic importance. Among the traits that ICI Seeds has mapped are those responsible for resistance to European corn borer, Ostrinia nubilalis Hübner, stalk tunneling, (ECB2). This information can be used to develop elite resistant germplasm. Families derived from a resistant x susceptible cross were utilized to map the chromosomal locations of the genes for resistance. The families of plants were screened against the specific insect damage, ECB2. Leaf tissue was taken from the families for DNA extraction and RFLP characterization. Insect damage ratings were regressed against RFLP data to map gene locations and identify gene action. Using this molecular marker data concurrently with insect screening, the technique was successfully used to introduce ECB2 resistance into elite ICI Seeds inbreds. The development of insect resistance in breeders the ability to fine map germplasm. Identify how the maize, Zea mays L., for the US corn belt quantitative trait loci (QTLs), and use germplasm responds. Evaluate has been an ongoing process. Some of this information to integrate traits into germplasm over time, define the the first studies that tried to map the elite germplasm with minimal linkage rate of progress over time for each genes controlling resistance to drag from donor sources. To have a entry. Is the resistance tolerance, European corn borer, Ostrinia nubilalis, viable marker assisted selection antibiosis or non preference? (ECB) used reciprocal translocation program for insects, the following steps studies to locate genes controlling should be taken for each pest for which When resistant germplasm is resistance. The studies identified six resistance is to be developed. The basic identified, there is additional chromosome arms associated with first components any breeding project needs information to obtain before efficient generation resistance (ECB1) (Scott et for developing new insect resistant product development can take place: al. 1966) and seven chromosomal arms germplasm can be summarized as associated with second generation follows (Mihm 1983): resistance. What is the dominance resistance (ECB2), (Onukogu et al. 1978). This information confirmed the • Establish reliable production of complexity of the trait but use of mass reared insects for infestation information, in particular reciprocal that mimics the vigor and variability translocations, in commercial breeding of the naturally occurring programs to develop ECB2 resistance population. was limited. The objective of • Identify the gene action of • Develop screening techniques for and/or additive nature of resistance? • How does the resistance act in inbreds vs. hybrids? • What is the inheritance of resistance? commercial hybrid development large scale germplasm evaluation programs is to develop elite high and become familiar with the rating Once these questions have been yielding stable products. Rapid scales used to rate resistance and answered one can select appropriate classify germplasm. breeding methodologies for developing conversion of elite inbreds to either ECB1 or ECB2 resistance would be • Screen germplasm: Identify resistant the desired end product. At this point, highly beneficial. Molecular markers, and susceptible germplasm. breeding new resistant elite germplasm such as restriction fragment length Determine whether resistance exists can commence. The development of polymorphisms (RFLPs), have given in adapted and/or exotic new germplasm requires use of the 156 D.L. BENSON • Identify the chromosomal regions The third is that pre-anthesis selection coupled with infestation, rating and responsible for resistance using the of plants allows continuous selection. For hybrid development RFLP linkage analysis. Fine map backcrossing. This minimizes the programs, infestation and rating of those regions identified with meiotic events and therefore reduces testcross hybrids is essential. resistance to precisely locate the the chances of introgressing donor line genes controlling resistance. DNA into the developing line. Once the appropriate breeding techniques • Identify gene number, gene action desired genetic arrangement has been developed, one must identify the and the contribution of each loci to achieved, one generation of selfing is reasons why and evaluate the the trait. required to fix the trait in a If new resistant germplasm cannot be homozygous state. (Greaves et. al. feasibility and costs of alternative 1993) approaches. It is at this point that the Once the mapping and gene action use of molecular markers, RFLPs, studies have been completed, the should be considered. Remember that appropriate breeding strategy can be The approach for introgressing the use of markers is only possible selected to transfer the resistance genes multiple genes into an elite background when reliable infestation and rating of into elite germplasm, although each is similar. However it requires more germplasm can be obtained. trait introgression program carries its knowledge of gene action and the effect own specific challenges. each gene has on the trait. Resistance to stalk boring by the ECB2 has been Marker assisted selection using RFLPs requires mapping the chromosomal ICI Seeds has successfully used marker shown to be dominant or partially so location of the gene(s) controlling the assisted selection (MAS) to develop (Guthrie et al. 1971). In other cases trait of interest (Greaves et. al. 1993). lines and hybrids with resistance to additive factors play a significant role This requires: insects, diseases and herbicides. Two and heterosis for resistance was also • cases will be discussed: First, for a shown (Jennings et al. 1974). Seven A segregating mapping population single dominant gene, second, for a chromosomal arms were shown to of plant families derived from a multigenic trait. contain genes for resistance to ECB2 (Onukogu et al. 1978). Relying on this resistant x susceptible cross. The • • • parents need to be fixed for the trait Backcrossing a desired trait into an elite published data and internally and have a maximum number of line can be accomplished rapidly. This generated information, ICI Seeds polymorphic RFLP loci. was the case with ICI Seeds decided to initiate studies on using The evaluation requires introgression of the IT (ALS2) gene, a MAS for introgressing ECB2 resistance approximately 200 or more single dominant gene, into an elite into elite germplasm. This program segregating families. This number of inbred. This example of the impact of was initiated in 1987. families gives enough replication of biotechnology on plant breeding used the genetic classes for good data an interdisciplinary approach which The initial F1 cross of a resistant (R) quality. Plant and collect leaf tissue involved molecular markers, combined source inbred to an elite susceptible (S) for DNA extraction from each with plant breeding and physiology. inbred was selfed to generate an F2 family. For insects, infest and rate The project developed elite IT inbreds population. Leaf tissue was taken from each family. in as few as four generations, including the F2 plants for RFLP analysis. The F3 With insects it is wise to use families the F1. This was possible because of families derived from the sampled F2 planted ear to row where the row is three factors. First the trait can be plants were infested with ECB2. The infested and the genetic structure of screened for in the seedling stage, linkage map was generated by the family is evaluated against the greatly reducing the number of families regressing F3 family data on the F2 mean rating of the family. that need to be mapped. Second, the plant RFLP marker data. The linkage Selection of RFLP probes to molecular marker data can be obtained data indicated that there were more generally cover the genome; spacing on individual plants pre-anthesis. This than five major and many minor loci of 20-30 centimorgans is sufficient. allows the selection of plants with associated with resistance. opportunistic crossovers near the IT Additionally, some of the loci for gene and the recipient parent resistance had close linkage with background on all other chromosomes. DEVELOPING INSECT RESISTANT GERMPLASM USING RFLP AIDED BREEDING TECHNIQUES 157 unfavorable alleles from the resistant new hybrid was equal to the When an initial linkage map is parent. Families were selected for susceptible hybrid. Table 2 indicates developed for any multigenic trait, advancement based on the presence of that for the new inbred with a different there may be a desire to introduce the resistance loci, a favorable elite tester the ECB2 resistance was equal to trait into other elite backgrounds. This background from the RFLP data, the the resistant source. Yield was could be accomplished by crossing resistance data from field infestations intermediate between the resistant plants, selected with RFLPs for and hybrid testcross data. Repeating source and the susceptible inbred. The resistance loci and a high level of the process of selection with RFLP agronomics for the new line were favorable background from the markers and field infestations, an improved over the susceptible line mapping populations, to selected lines. inbred with a favorable elite though not significantly so. Data for the The new F1s can be backcrossed to the background and resistance to ECB2 inbreds per se, Table 3, shows that the selected elite lines and/or selfed. The was developed in four generations of new inbred has resistance to ECB2 that subsequent families could then be selfing. The inbred contained some but is equal to the resistant source and analyzed with RFLPs and screened for by no means all of the mapped significantly different from the the trait. Selection with RFLPs should resistance loci. susceptible line selected for conversion. be used to retain favorable crossovers. Trait screening data can be used to pull In 1992, testing of the new inbred in In 1993, EXP 1 had higher stalk lodging through the alleles for the desired trait. hybrid combination and per se was and lower yield than comparable Further development can be initiated. The data are presented as checks (data not shown). This was due accomplished without using RFLPs by follows: Table 1 and Table 2 present to anthracnose stalk rot, Colletotrichum selecting with trait screening and yield testcross data using different testers. graminicola, introduced into the stalk at trials. Table 3 is the inbred data per se. the point of initial ECB2 feeding. Other researchers have also reported this From the development of the ECB2 Table 1 indicates that the new inbred interaction between insects and disease resistant hybrids and the IT hybrids the has the ECB2 resistance of the resistant as well (Keller et. al. 1986; Carruthers following conclusions can be drawn: source. Additionally, the yield of the et. al. 1986). • the field at every generation Table 1. 1992 yield trial data. EXP 1 is the newly developed resistant hybrid. Entry EXP 1 Check 1 Check 2 Susceptible testcross Resistant testcross LSD ECB2 Yield 2.3 4.0 3.9 4.4 2.4 1.3 11.196 11.762 11.447 11.133 11.447 0.881 % moisture 21.0 20.4 21.5 20.9 23.3 1.1 % SL % RL 4.9 3.5 1.9 4.2 3.4 3.0 1.4 1.7 0.8 1.2 1.1 2.4 possible. This eliminates the 0.0 0.0 0.0 0.0 0.0 0.1 between the selection markers and the gene controlling the trait. • The interaction of the trait with other factors, such as yield or usefulness of the newly developed germplasm. ECB2 Yield % moisture % SL % RL % DE 2.3 2.9 3.8 4.0 2.4 1.2 11.951 11.951 12.328 13.334 10.504 1.258 22.9 19.9 20.5 20.5 22.7 1.3 5.5 5.4 1.9 8.1 2.3 7.7 1.2 3.7 0.7 7.2 8.9 7.5 0.0 0.0 0.0 0.0 0.3 0.3 Check 3 and Check 4 are commercial hybrids and share a common tester with EXP 1. lines that have crossovers occurring disease, can significantly limit the Table 2. 1992 yield trial data. EXP 2 is the newly developed resistant hybrid. EXP 2 Check 3 Check 4 Susceptible testcross Resistant testcross LSD possibility of selecting developing % DE ECB2 rating = cm of tunneling per internode for the four internodes above and four internodes below the ear. Yield = t/ha. Moisture, stalk lodging (SL), root lodging (RL), and dropped ears (DE) are in percent. Check 1 and Check 2 are commercial hybrids and share a common tester with EXP 1. Entry It is essential to screen for the trait in Table 3 Resistance rating for ECB2 damage of the newly developed resistant inbred, the elite susceptible and the resistant source. Inbreds NEW Elite susceptible Resistant source LSD ECB2 2.7 5.8 2.5 1.3 158 • D.L. BENSON With a trait controlled by multiple Ac know le dgm e nt s alleles it may not be necessary to • have all the alleles present in the Work reported in this paper which finished line. An economically involves ICI Seeds European corn borer significant level of resistance can be second generation resistance achieved with only a portion of introgression effort was jointly favorable alleles with a large effect performed by David Foster, G. Keith present. Rufener II and L. Von Kaster. The Selection of plants with RFLPs for author gratefully acknowledges their opportunistic crossovers and elite efforts. background early in the development and using field screens Re fe re nce s and testcrosses to fix the trait can greatly increase the probability of developing useful germplasm with a multigenic trait. Carruthers, R.I., G.C. Bergstrom, and P.A. Haynes. 1986. Accelerated development of the European corn borer induced by interactions with Colletotrichum graminicola, the causal fungus of maize anthracnose. Annals of the Entom. Soc. of America. 79: 385-389. Greaves, J.A., G.K. RufenerII, M.T. Chang, and P.H. Koehler. 1993. Development of resistance to Pursuit herbicide in corn— the IT gene. Proc. Ann. Corn Sorghum Res. Conf., 48th; 104-118. Guthrie, W.D., W.A. Russell, and C.W. Jennings. 1971. Resistance of maize to second-brood European corn borer. Proc. Ann. Corn Sorghum Res. Conf., 26th; 165-179. Jennings, C.W., W.A. Russell, and W.D. Guthrie. 1974. Genetics of resistance in maize to first- and second-brood European corn borer. Crop Sci. 14: 394398. Keller, N.P., G.C. Bergstrom, and R.J. Carruthers. 1986. Potential yield reductions in maize associated with an Anthracnose/European corn borer pest complex in New York. Phytopathology 76: 586-589 Mihm, J.A., 1983. Efficient mass rearing and infestation techniques for host plant resistance to maize stem borers, Diatraea sp. Centro Internacional de Mejoramiento de Maiz y Trigo. El Batan, Mexico. Technical Bulletin. Onukogu, R.A., W.D. Guthrie, W.A. Russell, G.L. Reed, and J.C. Robbins. 1978. Location of genes that condition resistance in maize to sheath-collar feeding by second generation European Corn Borers. J. Econ. Entomol. 71: 1-4. Scott, G.E., F.F. Dicke, and G.R. Pesho. 1966. Location of genes conditioning resistance to leaf feeding of European Corn Borers. Crop Sci. 6: 444-446. Construction of a Bioinsecticidal Strain of Pseudomonas fluorescens Active Against Sugarcane Borer G. Herrera, AECI, Modderfontein, South Africa S.J. Snyman, SA Sugar Association Experiment Station, Mount Edgecombe 4300, South Africa J.A. Thomson, University of Cape Town, South Africa Abst r a c t A cryIA(c) gene was cloned from a native Bacillus thuringiensis strain which showed activity against the sugarcane borer Eldana saccharina. The sequence of the cloned gene was very similar to that of the B. thuringiensis subsp. kurstaki HD-73 cryIA(c) gene. The gene was introduced into an isolate of Pseudomonas fluorescens capable of colonizing sugarcane, on two broad host range plasmids, pDER405 and pKT240, having copy numbers of 13 and 28 respectively. The cry gene was introduced into the chromosome of P. fluorescens isolate 14 using an artificial transposoncarrying vector, Omegon-Km. Bioassays on Eldana larvae showed that the strain carrying the gene integrated into the chromosome was as toxic as the one carrying it on pKT240. Glasshouse trials indicated that sugarcane treated with P. fluorescens 14::Omegon-Km-cry were more resistant to Eldana damage than untreated sugarcane. Int roduct ion Many strains of Bacillus thuringiensis insects which may explain the medium (polymixin pyruvate egg yolk specificity of the toxins (Höfte and mannitol bromothymol blue agar Whiteley 1989; Van Rie et al. 1990). [Holbrook and Anderson 1980]). Pseudomonas strains were isolated from produce crystalline inclusions during sporulation which contain proteins Eldana saccharina Walker sugarcane by growth on King’s exhibiting highly specific insecticidal (Lepidoptera:Pyralidae) is an endemic Medium B (King et al. 1954) and activity (Höfte and Whiteley 1989). The species in Africa, the larvae of which confirmed by API tests using the API inclusions dissolve in the larval bore into the stalks of sugarcane and 2ONE identification strips. midgut, releasing one or more can cause considerable crop loss. It was Spontaneous nalidixic acid (Nal) and insecticidal proteins called δ- decided to screen local isolates of B. rifampicin (Rif) resistant mutants were endotoxins. Most are protoxins which thuringiensis for activity against E. isolated. are proteolytically converted into saccharina larvae and develop a smaller toxic polypeptides. The biological control agent. Laboratory toxicity bioassays M aterials and M ethods fed on an artificial insect diet in which activated toxins appear to generate pores in the midgut epithelium cells of Two-week-old E. saccharina larvae were susceptible insects, thus disturbing the osmotic balance. The cells swell and different concentrations of freeze-dried bacteria were incorporated (Black and lyse, resulting in larval death. In some Bacterial strains and growth conditions instances, specific high-affinity binding Strains of B. thuringiensis were isolated in plastic 32-cell trays for five days at sites have been shown to exist in the from soil samples around insect- 30°C after which mortality was midgut epithelial cells of susceptible infested sugarcane and from dead E. recorded. saccharina larvae by growth on PEMBA Snyman 1991). Larvae were incubated 160 G. HERRERA, S.J. SNYMAN, AND J.A. THOMSON Purification of the δ-endotoxin plant mass. This corresponded to a Results and Discussion decrease from 1 x 107 to 8 x 104 cfu/g δ-Endotoxin crystals from B. from cultures grown on nutrient agar Cloning the δ-endotoxin gene of B. thuringiensis isolate 234 thuringiensis isolate 234 were isolated fresh mass. None of the other isolates tested showed more efficient for 48 to 72 h at 30°C using gradient More than 50 local isolates of B. colonization. The cry gene from pGH37 centrifugation through Urografin 60% thuringiensis were subjected to was cloned into pKT240 (Rawlings et (Schering) following the method of screening assays on E. saccharina larvae al. 1986) and introduced into isolate 14 Gonzalez et al. (1982). and isolate 234 was identified as a by tri-parental conjugation. The potential candidate for the isolation of resultant strain was found to express Isolation of DNA from B. thuringiensis a cry gene. Crystals isolated from B. the cry gene (Herrera et al. 1994). isolate 234, construction and screening thuringiensis isolate 234 were of a genomic library, immunological bipyramidal and the d-endotoxin had As horizontal spread of the cry gene detection of δ-endotoxin production, an apparent Mr of 135 kDa (results not could occur when it is carried on a and molecular techniques, These were shown). A gene library was screened mobilizable plasmid, we decided to as described by Herrera et al. (1994). by colony hybridization using a 32P- integrate it into the chromosome of labelled 2.1-kb PvuII fragment from isolate 14 using the artificially Colonization assays pES1 as a probe, as B. thuringiensis generated interposon Omegon-Km Three-month old sugarcane plants subsp. kurstaki HD-1, from which pES1 (Fellay et al. 1989). The Omegon were dipped in stationary phase was derived (Schnepf et al. 1987), also module consists of the W interposon, cultures of P. fluorescens strains showed some toxicity towards Eldana flanked with synthetic inverted 28-bp containing one drop of Tween 80 per 50 larvae (results not shown). Plasmid ends of IS1, which can transpose if IS1 ml culture. Plants were harvested at pGH37 was chosen for further analysis. gene products are supplied. Omegon- various time intervals by cutting off at Comparisons between the DNA and Km is carried on the plasmid pJFF350 ground level, weighing, cutting into deduced amino acid sequence of its cry which has an origin of transfer pieces and shaking vigorously on a gene and other d-endotoxin genes allowing mobilization into Gram- wrist-action shaker in sterile flasks showed that the 234 cry was almost negative bacteria. The ‘disabled’ IS1 containing glassbeads and sterile water identical to that found in B. element on pJFF350 cannot itself for 5 min. Bacteria were enumerated by thuringiensis subsp. kurstaki HD-73, transpose, but enables transposition of plating on King’s Medium B containing cryIA(c) (Adang et al. 1985). There were the Omegon-Km module. Thus P. Nal (100 mg/ml) and Rif (50 mg/ml). only 4 different nucleotides at positions fluorescens carrying the cry gene in the 978 (A to C), 981 (G to T), 1102 (T to G) chromosome is stable cry+. DNA and 1020 (T to C), but these did not sequence analysis of the cry gene lead to any amino acid changes. The cry showed that it was carried on a 3.7-kb Effect on E. saccharina of sugarcane inoculated with P. fluorescens 14::Omegon-Km cry gene, an allele of cryIA(c), will shortly NdeI fragment. This fragment was be given a number by the Cry Gene cloned into the NdeI site of the Six-month-old sugarcane plants grown Nomenclature Committee. integration vector, pJFF350. pJFF350-cry in pots in the glasshouse were sprayed with 100 ml of a suspension of either P. was conjugally mobilized into isolate 14, selecting for KmR exconjugants. As After two weeks each plant was Isolation of sugarcanecolonizing Pseudomonas fluorescens and construction of P. fluorescens cry+ strains inoculated with 300 E. saccharina eggs Colonization studies showed that a placed by hand behind a leaf sheath at number of isolates of P. fluorescens were the base of the stalk. Stalks were able to survive on sugarcane. Isolate 14 Southern blot analysis of isolate 14 sampled four weeks after egg was selected as one of the strains carrying the cry gene integrated into placement, and larval numbers and the which, after 60 days, showed only a fluorescens 14 or P. flourescens 14::Omegon-Km-cry at 2 x 109 cfu/ml. 107 105 number of internodes that had been decrease in titer from 8 x bored were recorded. cfu per plant despite a 42% increase in to 9 x the plasmid cannot replicate in this host, Km selects for integration of the Omegon-Km-cry cassette into the chromosome. the chromosome showed that the gene could be integrated at single sites (Herrera et al. 1994). It was of interest 161 CONSTRUCTION OF A BIOINSECTICIDAL STRAIN OF PSEUDOMONAS FLUORESCENS ACTIVE AGAINST SUGARCANE BORER to note that a strain carrying the Quantification of δ-endotoxin promoter (Ge at al. 1990) and the integrated gene was as toxic to E. production in triplicate cultures using construct introduced into the saccharina as a strain carrying the gene ELISA indicated that it represented chromosome, is underway. In addition on pKT240, despite the fact that the 3.5% (SD 0.185%) and 3.7% (SD 0.153%) the potential of an obligate sugarcane copy number of pKT240 in isolate 14 is of the total dissolved protein in isolate endophyte, Acetobacter diazotrophicus 28. It is possible that the increased 14 carrying pKT240-cry, and Omegon- (Cavalcante and Dobereiner 1988), as a expression of the cry gene integrated Km-cry respectively. recipient for the cry gene is being investigated. into the chromosome was due to the deletion of 1.4 kb of DNA 5' to the gene Acknow le dgm e nt s the 3.7-kb NdeI cry fragment into The effect of P. fluorescens 14::Omegon-Km-cry-inoculated plants on E. saccharina pJFF350. Two AT-rich regions of dyad As the toxicity of isolate 14::Omegon- The authors wish to thank Di James for symmetry occur upstream of the NdeI Km-cry was similar to that of the strain DNA sequencing and analysis, and site of the 234 cry gene and were carrying pKT240-cry, it was used in Kevin G Black for technical assistance removed during the subcloning into glasshouse trials. Apart from the cry in the bioassays. We thank the South pJFF350. Support for our hypothesis gene being a stable integration into the African Sugar Association for partial comes from a previous experiment in chromosome in this strain, it is more funding of the project. which we cloned the entire 6.7 kb acceptable from a bio-safety which occurred during the cloning of BamHI fragment carrying the cry gene consideration as the cry gene is not on a and the upstream region into pJFF350 mobilizable plasmid. A comparison of and integrated it into the chromosome the number of Eldana larvae recovered of isolate 14. No detectable toxin was and the damage to stalks between found on Western blot analysis (data plants sprayed with isolate 14 and not shown). 14::Omegon-Km cry is shown in Figure 2. These glasshouse trials showed that Western blot (immunoblot) analysis there was a decrease in the presence of confirmed the expression of the cry larvae and consequent damage of gene in the exconjugants (Herrera et al. approximately 60% after 4 weeks 1994). P. fluorescens isolate 14 carrying compared with the control strain. These pKT240-cry and Omegon-Km-cry were results are promising. A further toxic to E. saccharina larvae (Fig. 1). improvement to the biocontrol strain, in which the cry gene will be cloned yy ;; ;; yy downstream of the efficient tac Re fe re nce s Adang, M.J., M.J. Staver, T.A. Rocheleau, J. Leighton, R.F. Barker, and D.V. Thompson. 1985. Characterized fulllength and truncated plasmid clones of the crystal protein of Bacillus thuringiensis subsp. kurstaki HD-73 and their toxity to Manduca sexta. Gene 36: 289-300. Black, K.G., and S.J. Snyman. 1991. Biomass yield and insecticidal activity of a local Bacillus thuringiensis isolate in six fermentation media. Proc. S. Afr. Sug. Technol. Ass. 65: 77-79. Cavalcante, V.A., and J. Dobereiner. 1988. A new acid-tolerant nitrogen-fixing bacterium associated with sugarcane. Plant & Soil 108: 23-31. 70 yy ;; ;; yy ;; yy ;; yy ;; yy 40 30 20 10 0 yy ;; ;; yy ;; yy ;; yy ;; yy ;; yy MG. Freeze Cultures/ dried ml yy ;; ;; yy ;; yy ;; yy ;; yy ;; yy ;; yy Insect diet Figure 1. Toxicity of P. fluorescens 14 (pKT240-cry) and P. fluorescens 14::Omegon-Km-cry against E. saccharina larvae. Results are the means of three replicates. Bars above the histograms represent standard deviations. 4 yy ;; ;; yy ;; yy ;; yy ;; yy ;; yy ;; yy yy ;; ;; yy yy ;; ;; yy yy ;; ;; yy yy ;; ;; yy yy ;; ;; yy yy ;; no bacteria 3 2 1 0 14::OmegonKm-cry No. Eldana per pot No. Eldana per stalk yy ;; ;; yy ;; yy ;; yy ;; yy ;; yy ;; yy ;; yy ;; yy % Damaged internodes per pot 40 yy ;; ;; yy ;; yy ;; yy ;; yy ;; yy ;; yy ;; yy 30 20 10 % internodes damaged/stalk 50 14 (pKT240-cry) 14::Omegon-Km-cry No. Eldana larvae/pot % Mortality 60 0 % Damaged internodes per stalk Figure 2. Eldana damage to sugarcane pretreated with no bacteria or with P. fluorescens 14::Omegon-Km-cry. 162 G. HERRERA, S.J. SNYMAN, AND J.A. THOMSON Fellay, R., H.M. Krisch, P. Prentki, and J. Frey. 1989. Omegon-Km: a transposable element designed for in vivo insertional mutagenesis and cloning of genes in Gram-negative bacteria. Gene 76: 215226. Ge, A.Z., R.M. Pfister, and D.H. Dean. 1990. Hyperexpression of a Bacillus thuringiensis delta-endotoxin-encoding gene in Escherichia coli: properties of the product. Gene 93: 49-54. Gonzalez, J.M. Jr., B.J. Brown, and B.C. Carlton. 1982. Transfer of Bacillus thuringiensis plasmids coding for dendotoxin among strains of B. thuringiensis and B. cereus. Proc. Natl. Acad. Sci. USA. 79: 6951-6955. Herrera, G., S.J. Snyman, and J.A. Thomson. 1994. Construction of a Bioinsecticidal strain of Pseudomonas fluorescens Active against the Sugarcane Borer, Eldana saccharina. Appl. Environ. Microbiol. 60: 682-690. Höfte, H., and H.R. Whiteley. 1989. Insecticidal crystal proteins of Bacillus thuringiensis. Microbiol. Rev. 53: 242-255. Holbrook, R., and J.M. Anderson. 1980. An improved selective and diagnostic medium for the isolation and enumeration of Bacillus cereus in foods. Can. J. Microbiol. 36: 753-759. King, E.O., M.K. Ward, and D.E. Raney. 1954. Two simple media for the demonstration of pyocyanin and fluorescein. J. Lab. Clin. Med. 44: 301307. Rawlings, D.E., I.-M. Pretorius, and D.R. Woods. 1986. Expression of Thiobacillus ferrooxidans plasmid functions and the development of genetic systems for the Thiobacilli. In H.L. Ehrlich, and D.S. Holmes (eds.), Workshop on biotechnology for mining, metal-refining and fossil fuel processing industries. Biotechnology and Bioengineering Symposium 16, 281-287. New York: John Wiley and Sons. Schnepf, H.E., H.C. Wong, and H.R. Whiteley. 1987. Expression of a cloned Bacillus thuringiensis crystal protein gene in Escherichia coli. J. Bacteriol. 169: 4110-4118. Van Rie, J., S. Jansens, H. Höfte, D. Degheele, and H. van Mellaert. 1990. Receptors on the brush border membrane of the insect midgut as determinants of the specificity of Bacillus thuringiensis d-endotoxins. Appl. Env. Microbiol. 56: 1378-1385. Developing M aize w ith Resistance to European Corn Borer J. Sagers, M. Edwards, B. Bolan, A. Wang, I. Mettler, L. Barrett, C. Garrett, Northrup King Company Research Center, Stanton, MN, USA D. Mies, Northrup King Company, St. Joseph, IL, USA. Abst r a c t The European corn borer (ECB), Ostrinia nubilalis Hübner, causes hundreds of millions of US dollars in crop losses in the United States and Europe. With these large losses in mind, Northrup King began a multifaceted approach to develop commercial hybrids with resistance to ECB damage. A combination of conventional breeding tactics, molecular marker assisted breeding and transgenic technology have been employed to develop long lasting, effective resistance to this pest. Successes have been made using conventional pedigree breeding with an emphasis on ECB resistance, high yield, and good agronomic health. However, conventional breeding relies on artificial infesting with ECB, and it is resource intensive. Thus, we have actively pursued molecular-marker assisted breeding for stalk tunneling resistance to ECB. Molecular marker assisted selection allows 1) advances in selection in years with low ECB damage in the field; 2) more than one selection cycle in a year; 3) use of effective backcross breeding tactics for complexly inherited traits; and 4) reduced field evaluation. Transgenic technology has allowed the production of hybrid corn containing an insecticidal gene from Bacillus thuringiensis Kurstaki. During three years of field testing, corn plants containing this gene have provided excellent full-season control of ECB larvae. The combination of conventional breeding, molecular marker assisted breeding, and transgenic technology will result in stable, highly insect resistant hybrids. These should help us manage ECB and perhaps other lepidopteran pests into the future. Int roduct ion • The European corn borer (ECB), Ostrinia nubilalis Hübner, reportedly • causes hundreds of millions of US • Physiological yield loss due to leaf, used a multifaceted approach that sheath, stalk, ear shank and kernel includes conventional breeding, feeding damage. molecular marker-assisted selection, Harvest losses due to dropped ears and transformation technology. or lodged plants. Through combined research efforts, it Costs associated with application of is our primary goal to develop stable, each year in Europe and the US. During chemical insecticides to prevent high yielding, durable ECB resistant the 1991 growing season, losses of $196 damage. hybrids. dollars loss in maize (corn), Zea mays L., million were estimated in Minnesota (MN) alone (K. Ostlie, Personal In addition, stalk rot pathogens are Communication, 1992). MN growers often associated with damage by the planted slightly less than 10 percent of corn borer. These pathogens further the total US corn acreage in 1991 (1991 compromise the yield and standability Various conventional breeding USDA Annual Crop Summary, January of maize (Showers et al. 1989). techniques have facilitated significant Conventional Breeding for Insect Resistant M aize improvements in resistance to 1992). Therefore, in years with high ECB populations such as 1991, loss due With this huge potential loss in mind, European corn borer. Often the to ECB damage could surpass one Northrup King Company has breeding method of choice is a form of billion US dollars throughout the world aggressively pursued the development recurrent selection. Using recurrent corn growing regions. Losses to ECB of ECB resistant hybrid corn. We have selection, the selected resistant are extensive including: 164 J. SAGERS, M. EDWARDS, B. BOLAN, A. WANG, I. METTLER, L. BARRETT, C. GARRETT, AND D. MIES • Progeny may be screened against progenies are intercrossed to increase Figure 1 shows a general pedigree the frequency of favorable resistance breeding procedure for developing alleles. Barry et al. (1983, 1984, 1985) lines with improved levels of ECB and Klenke et al. (1986a) reported resistance. We often use this or a successful use of recurrent selection to similar method when crossing an elite produce testcross hybrid seed and produce improved sources of resistance (adapted) insect-resistant inbred line to sometimes to advance generations to ECB. Various modifications of an elite susceptible inbred line. The pedigree breeding systems also have goal is to develop inbred lines with been used to develop ECB resistant improved resistance levels relative to resistance under artificial ECB lines and hybrids. Russell and Guthrie the susceptible elite line. Ultimately, pressure. (1979) reported success using pedigree through insect efficacy testing and breeding to develop inbred line B86. yield testing procedures, a useful yield performance across multiple Also, Hawk (1985) developed ECB hybrid product may result. Figure 1 is locations, throughout the testing resistance source DE811 using a self-explanatory for the most part, but procedure. pedigree breeding approach. There are some details that are not evident many effective conventional breeding include: Principal selection criteria include: methods that may be used to improve • • Artificial infesting with 300-600 ECB resistance to insect pests. However, the larvae begins at the F3 (S1) source of resistance utilized and the generation. both leaf (first generation) and stalk (second generation) ECB damage. • without ECB selection pressure. • • Testcross hybrids are evaluated for Testcross hybrids are evaluated for Improved insect resistance as a “line” per se. • The ability to convey resistance to hybrid progeny produced using the exact goal of the breeding program “line”. must be considered. (Year 1) Cross susceptible inbred by resistent inbred Self F1 • High general combining ability and ultimately specific combining ability with one or more other inbreds. Self F2 Improved resistant line available (Year7-8) Winter nurseries may be used to (Year 2) Screen 150+F3 progenies self poll. & select top 10-15% • • Good agronomic appearance. Agronomic appearance includes features such as late season intactness, strong root systems, late Test cross F4 (Winter nursery) and high grain quality. Numerous Foundation seed (year7) (Year 3) 1. Screen F4 progeny 2. Yield test F4 hybrids 3. Screen F4 TC hybrids 4. Self & select top 10-20% Breeder seed variations of this pedigree breeding protocol may be implemented according to personal preference and the goals of the breeding project. (Winter nursery) (Winter nursery) season staygreen, disease resistance, (Year 4) 1. Screen F6 progeny 2. Yield test F5 TC hybrids 3. Screen F5 TC hybrids 4. Self & select top 10-20% Expand yield testing with more testers Results of Conventional Breeding Efforts Conventional breeding technology has (Year 5) (F8) Repeat contributed greatly to reducing loss to the European corn borer. Barry et al. (1991) tested 400 commercial corn (Winter nursery) hybrids over a four year period. They (Year 6)(F10) Repeat Expand yield testing found that 90% of the hybrids had intermediate or better resistance to leaf Hybrid development Year 7-9 Expand yield/performance testing Year 7-10—Commercial hybrid available with improved resistance. Figure 1. Conventional pedigree breeding for ECB resistant lines. feeding damage by ECB. Of the tested hybrids, 75% had intermediate or better resistance to sheath and stalk tunneling damage. DEVELOPING MAIZE WITH RESISTANCE TO EUROPEAN CORN BORER Figure 2 displays an inbred line with displays variation seen in resistance to ECB leaf feeding damage. segregating F3 progeny rows. These This inbred displays a leaf feeding F3 progenies were the result of a rating of 3, using Guthrie’s 1-9 scale, cross between a susceptible inbred where 1 represents no damage or a few line and the resistant inbred line pinholes and 9 represents severe leaf shown in Figure 2. Both of the damage on several leaves (Guthrie et al. displayed plants were infested with 1960). This line also has strong over 250 ECB larvae around resistance to ECB stalk tunneling anthesis. The resistant F3 progeny damage. Damage remains consistently row (top of photo) displayed an below 5 centimeters per plant on average of only 2.5 cm of tunneling average. Compare this to a susceptible damage per plant. In contrast, the inbred line with a leaf damage rating of susceptible F3 progeny (bottom of 9 shown in Figure 3. The resistant photo) displayed an average of 36.6 inbred line was developed using a cm of damage. The variability that conventional pedigree breeding exists in early generations of a cross technique with selection under ECB between a resistant and susceptible feeding pressure. Both natural ECB parent allows useful selection for pressure and artificial ECB pressure more resistant genotypes. 165 aided selection as this line was developed. Maintaining stalk damage resistance in agronomically Figure 4 displays an example of ECB acceptable genotypes throughout stalk tunneling resistance. This figure the inbreeding process is labor Figure 2. Inbred line with natural European corn borer leaf feeding resistance. Rates a “3” on 1-9 scale, where 1 = no damage or a few pinholes. Figure 4. F3 segregants produced by crossing susceptible inbred line by resistant line shown in Figure 2. Segregants show variation in levels of resistance to stalk and ear shank tunneling damage by European corn borer. Left plant is resistant to both types of damage. Right plant is susceptible to both types of damage. Each plant artificially infested with over 250 neonate ECB larvae at anthesis. Figure 3. Inbred line showing high susceptibility to European corn borer leaf feeding damage. Rates a “9” on 1-9 scale, where 1 = no damage and 9 = several leaves shredded by ECB. 166 J. SAGERS, M. EDWARDS, B. BOLAN, A. WANG, I. METTLER, L. BARRETT, C. GARRETT, AND D. MIES intensive and difficult. Often resistance using them. Therefore, substantial gains quantitatively inherited trait which is alleles are lost during inbreeding and have been made using conventional conditioned by at least five alleles selection processes. Sometimes breeding methodology. The future (Schön et al. 1993; Onukogu et al. 1978; improved resistance to ECB is holds additional improvements Northrup King Company research, negatively correlated with grain yield, through conventional breeding to 1987-present). Therefore, backcross especially if yield is not a selection develop improved resistance sources. breeding would not normally be criterion during development (Klenke After repeated cycles of inbreeding, considered a practical approach for et al. 1986a). Often crosses are made selection, yield testing, and advance, developing plants with improved between a resistant inbred and a both inbred lines and commercial resistance. However, with the susceptible inbred to produce F1 hybrids with improved resistance can assistance of molecular probes to track commercial hybrid seed. Heterosis be developed. movement of both favorable resistance alleles and recurrent parent alleles, masks some susceptibility to ECB damage, but if hybrid progenies are not screened for ECB resistance specifically, the F1 hybrid will often be more M olecular M arker-Assisted Breeding for ECB Re sist a nc e feasibility of backcross breeding for a complexly inherited trait improves. Figure 5 shows a typical backcross breeding procedure which may be used susceptible than desired. When crossing resistant by susceptible lines, it Scientists have demonstrated that in conjunction with molecular marker- is preferable that resistance genes act resistance to second generation ECB assisted selection. with at least partial dominance to stalk and sheath damage is a convey useful resistance to the F1 hybrid progeny of the cross (Guthrie et al. 1985, 1989). Finally, labor demands associated with developing ECB stalk damage (Resistant line) R x s F2 x S BC1 x S Self Analyze F2 resistance conflict with other essential operations in plant breeding programs. Artificial infesting for stalk tunneling BC2 the same time hand pollinating activities typically occur in a breeding (seasonal assistants) have returned to Self x S MM assisted selection BC5 x S repeat damage to approximately one-third inbred lines B86 and DE811 conveyed S Self successfully reduce ECB stalk tunneling (1985, 1989) demonstrated that resistant x MM assisted selection BC4 breeders and entomologists can Communication, 1994). Guthrie et al. BC3 Self harvest, and occurs after students susceptible parent (D. Mies, Personal S MM assisted selection (or) splitting) also conflicts with hand that sustained by the original x Self nursery. Damage evaluation (stalk In spite of these difficulties, plant MM probes used to identify most favorable BC1 progeny to cross with “S”. MM assisted selection damage evaluations occurs at anthesis, school. (Susceptible adapted line) repeat New line with most resistance alleles of “R”. Similar to “S” but not identical. repeat Line with resistance alleles of “R” and identical to “S”. improved leaf feeding and stalk damage resistance to hybrids produced Figure 5. Molecular marker assisted backcross breeding procedure to select for ECB resistant lines. DEVELOPING MAIZE WITH RESISTANCE TO EUROPEAN CORN BORER 167 Benefits of M olecular M arker-Assisted Selection for ECB Resistance follow resistance alleles in progeny of a larvae. This is the process of developing Although molecular marker technology Using a combination of conventional a quantitative trait loci (QTL) model. To is not likely to replace conventional breeding tactics, artificial infestation, date, we have developed several QTL techniques and field testing altogether, and molecular markers, plant breeders models for various sources of ECB it may enhance these efforts and entomologists have the tools to resistance. These QTL models are significantly. Molecular marker assisted successfully reduce damage caused by currently being used to help develop selection may: ECB and other lepidopteran pests of lines and hybrids with improved • Allow advance in resistance maize. These improved sources of development even in years with low natural resistance combined with natural ECB pressure (or low transgenic technology should provide a pressure from artificially infested formidable source of ECB resistance. Using artificially infested field trials, molecular markers (probes) are identified that are associated with cross between a resistant parent and the susceptible parent you wish to improve. resistance to stalk damage by ECB natural resistance to ECB damage. Identification of molecular probes is ECB). typically performed as follows: • 200 (or more) F2 (or later generation) • • • Transform at ion Te chnology per year, since field evaluation is not resistant parent and a susceptible essential each cycle. Northrup King Company’s corn plant • Allow the use of more efficient transformation research began in 1987, tunneling resistance. backcross breeding strategy for when we obtained the first genes from DNA samples from the same quantitatively inherited multigenic Bacillus thuringiensis Kurstaki. Between progeny are cut into fragments using traits. 1987 and 1990, Northrup King and • Reduce workload associated with other private organizations invested Fragments are analyzed using a artificial infestation (which coincides significantly in the development of broad set of molecular marker probes with breeding nursery hand- insect resistant transgenic plants. developed by Northrup King and pollinating) and fall damage During that period several obstacles assorted public and private evaluation (stalk splitting, which had to be overcome. They included: institutions. coincides with harvest). • • restriction enzymes. • Allow two or more selection cycles progeny of a cross between a parent are analyzed for ECB stalk • • Polymorphic probes that distinguish Cloning the Bt gene. Construction of functional expression vectors. between the two parental genotypes Scott et al. (1967), Jennings et al. (1974), are identified. and Sadehdel-Moghaddam et al. (1983) Regions that are significantly demonstrated that resistance is associated with resistance to ECB conditioned predominantly by additive feeding damage are identified using gene effects. However, the exact least squares analysis (e.g. regression number and location of resistance analysis) and computer programs factors (loci) vary according to the such as Mapmaker QTL (a software source of resistance utilized. Therefore, program designed to link molecular for each different resistance source markers to phenotypic traits). utilized, molecular marker probes must Between 1990 and 1992 ballistic and Lander and Botstein (1989) describe be identified that are associated protoplast transformation methods details of Mapmaker QTL software specifically with that source’s ECB became available which allowed use for these types of analyses. Lee et resistance alleles. These probes need to successful recovery of fertile al. (1989) and Schön et al. (1993) be polymorphic so they differentiate transformed maize plants. Figure 6 describe specific details of between the alleles of the resistant and schematically displays two common methodology surrounding restriction susceptible genotypes in chromosome methods of transformation. In the fragment length polymorphism regions linked to resistance genes. ballistic method, microscopic tungsten (RFLP) analysis. Provided these conditions are met, particles coated with foreign DNA are molecular marker probes can be used to forcefully propelled through the cell • Improving protein expression in transformed plant tissues. • Modification of the gene itself (changing the nucleotide sequences to make them more plant like. • Developing successful maize transformation techniques. 168 J. SAGERS, M. EDWARDS, B. BOLAN, A. WANG, I. METTLER, L. BARRETT, C. GARRETT, AND D. MIES Ballistic Metod Microscopic Bullets Wall Membrane ;; ;; ; ; ; ;;;;; ;;;; ;;; ;; ;; ;;;; ; ;; chambers, magenta boxes are opened so plantlets are exposed to air. This helps Ballistic the leaves adapt to the less protected Method environment they will be exposed to in DNA DNA the greenhouse. Approximately 4.5 months post-transformation, seedlings are transplanted to soil and moved to greenhouses to grow to maturity. As CELL Enzyme CELL Electric Current or PEG CELL either self-pollinated or crossed to other elite non-transformed lines. Depending on which transformation technique is DNA Electric or PEG Method soon as anthesis begins, plants are Protoplast Figure 6. Two methods of plant transformation. Ballistic method shown on top. Protoplast method shown on bottom. used this entire process, from transformation of cells to seed production, requires approximately 4-7 months. wall into the cytoplasm and nuclei of on selective culture medium, calli are Following initial transformation and cells. In the protoplast method, the cell transferred to regeneration medium. production of fertile plants, Bt genes walls are first removed. Then the cell Approximately 4 months following were backcrossed into elite parental membrane becomes readily permeable transformation, small seedlings are lines to develop commercial hybrids to foreign DNA. Movement of foreign transferred to magenta boxes, which expressing resistance from Bt genes. DNA through the cell membrane is allow upright growth and normal Throughout backcrossing and breeding facilitated either by applying an development of roots, shoots, and procedures, selective herbicides acted electrical current (electroporation) or leaves. During the last days in growth as highly effective tools for selecting adding Polyethylene Glycol (PEG). Foreign DNA in solution surrounding the cells passes through the cell membrane, with some of it being incorporated into the nuclei of cells. Following either method of transformation, approximately 120-210 days pass prior to harvesting seed from the primary transformed plants. Photographs that demonstrate an approximate timeline of critical steps following protoplast transformation are shown in Figure 7. First, the transformed cells are placed on nurse cell cultures. These nurse cell cultures supply nutrition and provide a suitable osmotic environment for the fragile transformed cells. Often, a selective agent is included in the cell culture medium to kill non-transformed cells. After approximately 3 months growth Figure 7. Approximate timeline for recovery of seed following polyethylene glycol mediated protoplast transformation. 0 months, transformed cells placed on nurse cell culture containing selectable agent; 2 months, transformed calli multiply; 3 months, healthy calli transferred to regeneration medium; 4 months, plantlets regenerate and are transferred to magenta boxes; 4.5 months, upright plantlets are transferred to soil in greenhouse; 7 months, transformed plants produce seed. DEVELOPING MAIZE WITH RESISTANCE TO EUROPEAN CORN BORER 169 transformed plants. Plants grown in the zone of plants. Multiple applications average tunneling score > 5.1 cm, 10 greenhouse or field were sprayed with were spread over a 2 week period. plants were dissected from each plot at appropriate selective herbicides to that site. Only six locations met this eliminate those that were not Leaf feeding damage was evaluated minimal damage threshold throughout transformed. using a 1-9 whorl leaf damage rating, the Midwest testing region. However, where 1 represents no damage and 9 these trials provided useful stalk After several years of developmental represents several leaves with severe damage data that were analyzed across research by Northrup King and leaf shredding (Guthrie et al. 1960). all trials of similar maturity. These trials contributions by several other private Stalk tunneling damage was evaluated were divided into two groups; northern companies, we conducted our first field by dissecting stalks from US Corn Belt adapted hybrids or trials in 1992. Transgenic corn plants approximately 4 nodes above the southern US Corn Belt adapted hybrids. were field tested against ECB, the primary (top) ear down to the ground. primary lepidopteran pest of U.S. Total ECB tunnel length was estimated maize. in inches and converted to centimeters. M aterials and M ethods for Field Evaluation of Transformed Corn Plants Natural pressure ECB efficacy trials Field testing of transformed corn To gain information on the effects of Bt- conducted during the past three years. Bt-Transgenic Field Trial Results versus ECB against ECB, the target pest, has been maize on natural populations of ECB, Excellent full season ECB control has Artificially infested ECB efficacy evaluation an observation range was planted at all been the result each year. Leaf feeding 1994 field test sites. At each site, we damage has been limited to a few During the past three years, planted approximately eight Bt hybrids pinholes on one or two leaves. Figure 8 transformed maize has been screened and eight representative non-Bt control compares a non-transgenic plant (left) against ECB using similar protocols hybrids. At each site, natural ECB to a Bt-transgenic plant (right). Neonate each year. Seeds were planted to result feeding pressure was monitored by ECB larvae only took a few bites of the in a final plant stand of 30 plants per 5.7 dissecting 10 plants each, of 2 different transgenic tissue before they stopped meter row (0.77 m row width). Two- non-Bt hybrids (20 plants in total). If feeding. Within 24 hours the neonate row plots were planted to leave an either control hybrid displayed an insects were dead. In artificially infested uninfested buffer row between infested rows. Typically, 2 or 3 replicates of each entry were planted in randomized complete block design experiments. Replicate trials were planted at multiple locations. To evaluate leaf feeding damage, approximately 250 neonate larvae were applied to each plant in the first row of the 2-row plot. Infestation began as the plants reached the fifth leaf of development. A modified “bazooka” (Davis and Oswalt 1979; Mihm 1983) was used to infest approximately 50 larvae per plant per application. Larvae were applied every 3 days over a 2 week period. Plants were infested again at anthesis to simulate infestation for stalk damage. Approximately 250 larvae were applied directly to the leaf axils around the ear Figure 8. Bt corn (right) shows no leaf feeding damage by ECB. Non-Bt control plant (left) shows first symptoms of severe leaf feeding damage. (Photo taken 10 days following infestation with approximately 200 neonate ECB larvae). 170 J. SAGERS, M. EDWARDS, B. BOLAN, A. WANG, I. METTLER, L. BARRETT, C. GARRETT, AND D. MIES trials over the past two years, average Bt control hybrids. Bt hybrids have leaf feeding ratings on transgenic displayed tremendous reductions in hybrids have been 1.06. In contrast, stalk tunneling damage and non-transgenic control hybrids have improvements in late season plant displayed an average leaf damage intactness, relative to non-Bt hybrids. rating of 3.71 (LSD = 0.58, a=0.05)(Fig. Figures 11 and 12 display these 9). improvements, respectively. Larvae infested at anthesis to simulate Non-Bt quite rapidly. Very few live larvae were 8 years. Average tunneling damage was only 0.15 cm per Bt-hybrid versus 4.53 cm per non-transgenic hybrid control (LSD 2.16 cm, a=0.05) (Fig. 9). Damage found in the stalks of thousands of Bt hybrids dissected over the past three ;; ;; ;; ;; 10 ECB stalk tunneling damage also died Bt 6 4 2 0 In naturally infested ECB observation trials, results were equally dramatic (Fig. 10). Bt hybrids adapted to northern U.S. Corn Belt growing regions displayed only 0.10 cm of stalk tunneling damage on average. The nonBt control hybrids displayed 5.20 cm of damage on average. Southern U.S. Corn Belt Bt hybrids displayed 0.3 cm of tunneling damage on average, compared to 9.7 cm of damage in non- 5 Northern ; ; ; ; ; Southern Figure 10. Stalk tunneling damage on plants under natural ECB pressure. Combined multi-location data. Hybrids were divided into two groups: those adapted to the northern U.S. corn belt and those adapted to the southern U.S. corn belt. Stalk damage expressed as average centimeters tunneled per plant. LSD (a=0.05): northern hybrids = 1.8 cm; southern hybrids = 5.6 cm. Figure 11. Bt hybrid (right) displays no stalk damage by ECB. Non-Bt hybrid (left) with live larva and associated stalk tunneling damage. Non-Bt Bt Damage 4 3 2 1 0 Stalk Leaf Figure 9. 1993 and 1994 combined ECB trial results across multiple locations and hybrids. Hybrid plants artificially infested during whorl stage of growth and at anthesis. Stalk damage expressed as average centimeters tunneled per plant. Leaf damage expressed on 1-9 scale where 1 = no damage or a few pinholes. Figure 12. Bt hybrid (left) shows substantial improvement in late season plant intactness relative to non-Bt hybrid (right). Natural ECB feeding pressure at SW Iowa trial site, 1993. DEVELOPING MAIZE WITH RESISTANCE TO EUROPEAN CORN BORER Discussion Tremendous gains have been made in developing natural sources of ECB resistance. Additional gains remain to be made using conventional breeding techniques. Also, the future holds a range of new tools to aid selection for resistance (e.g. molecular marker technology) and complementary, novel sources of resistance incorporated through transformation technology. Plant breeders and entomologists should be able to develop durable sources of plant resistance using a combination of: • Conventional resistance sources and breeding procedures. • New resistance sources from other species or novel proteins. • Molecular marker technology to track resistance genes. These new tools and sources of resistance will enhance the efficiency with which we can breed for resistance to insect pests. In turn this should help us manage insect pests of maize into the future. Re fe re nce s Barry, D., M.S. Zuber, A.Q. Antonio, and L.L Darrah. 1983. Selection for resistance to the second generation of the European corn borer (Lepidoptera: Pyralidae) in maize. Journal of Economic Entomology 76(2): 392-394. Barry, D., and M.S. Zuber. 1984. Registration of MoECB2(S1)C5 maize germplasm. Crop Science. 24: 213. Barry, D., M.S. Zuber, and L.L. Darrah. 1985. Registration of Mo-2ECB-2 maize germplasm. Crop Science. 25: 715-716. Barry, D., and L.L. Darrah. 1991. Effect of research on commercial hybrid maize resistance to European corn borer (Lepidoptera: Pyralidae). Journal of Economic Entomology. 84(3): 1053-1059. Davis, F.M., and T.G. Oswalt. 1979. Hand inoculator for dispensing lepidopterous larvae. United States Department of Agriculture, Science and Education Administration. Advances in Agricultural Technology. AAT-S-9. Guthrie, W.D., F.F. Dicke, and C.R. Neiswander. 1960. Leaf and sheath feeding resistance to the European corn borer in eight inbred lines of dent corn. Ohio Agricultural Experiment Station Bulletin 860. Wooster, Ohio. Guthrie, W.D., W.A. Russell, J.L. Jarvis, and J.C. Robbins. 1985. Performance of maize inbred line B86 in hybrid combinations: Resistance to first- and second-generation European corn borers (Lepidoptera: Pyralidae). Journal of Economic Entomology. (78)1: 93-95. Guthrie, W.D., J.A. Hawk, and J.L. Jarvis. 1989. Performance of maize inbred line DE811 in hybrid combinations: Resistance to first- and secondgeneration European corn borers (Lepidoptera: Pyralidae). Journal of Economic Entomology. (82)6: 1804-1806. Hawk, J.A. 1985. Registration of DE811 germplasm line of maize. Crop Science 25: 716. Jennings, C.W., W.A. Russell, W.D. Guthrie, and R.L. Grindeland. 1974. Genetics of resistance in maize to second-brood European corn borer. Iowa State Journal of Research. 48(3): 267280. Klenke, J.R., W.A. Russell, and W.D. Guthrie. 1986a. Recurrent selection for resistance to European corn borer in a corn synthetic and correlated effects on agronomic traits. Crop Science. 26: 864868. 171 Klenke, J.R., W.A. Russell, and W.D. Guthrie. 1986b. Grain yield reduction caused by second generation European corn borer in BS9 corn synthetic. Crop Science. 26: 859-863. Lander, E.S., and D. Botstein. 1989. Mapping Mendelian factors underlying quantitative traits using RFLP linkage maps. Genetics. 121: 185-199. Lee, M., E.B. Godshalk, K.R. Lamkey, and W.W. Woodman. 1989. Associations of restriction fragment length polymorphisms among maize inbreds with agronomic performance of their crosses. Crop Science. 29: 1067-1071. Mihm, J.A. 1983. Efficient mass rearing and infestation techniques to screen for host plant resistance to maize stem borers, Diatraea sp. Technical Bulletin. CIMMYT, Mexico. Onukogu, F.A., W.D. Guthrie, W.A. Russell, G.L. Reed, and J.C. Robbins. 1978. Location of genes that condition resistance in maize to sheath-collar feeding by second-generation European corn borers. Journal of Economic Entomology. 71(1): 1-4. Russell, W.A., and W.D. Guthrie. 1979. Registration of B85 and B86 germplasm lines of maize. Crop Science. 19: 565. Sadehdel-Moghaddam, M., P.J. Loesch, Jr., A.R. Hallauer, and W.D. Guthrie. 1983. Inheritance of resistance to first and second broods of the European corn borer in corn. Proceedings of the Iowa Academy of Science. 90(1): 35-38. Schön, C.C., M. Lee, A.E. Melchinger, W.D. Guthrie, and W.L. Woodman. 1993. Mapping and characterization of quantitative trait loci affecting resistance against second-generation European corn borer in maize with the aid of RFLP’s. Heredity. 70: 648-659. Scott, G.E., W.D. Guthrie, and G.R. Pesho. 1967. Effect of second-brood European corn borer infestation on 45 single-cross corn hybrids. Crop Science. 7: 229-230. Showers, W.B., J.F. Witkowski, C.E. Mason, D.D. Calvin, R.A. Higgins, and G.P Dively. 1989. European corn borer development and management. North Central Regional Extension Publication No. 327. May 1989. Iowa State University, Ames, IA, USA. The Expression of a Synthetic CryIA(b) Gene in Transgenic M aize Confers Resistance to European Corn Borer J.J. Estruch, N.B. Carozzi, N. Desai, G.W. Warren, N.B. Duck, and M.G. Koziel, CIBA Agricultural Biotechnology, NC, USA. Abst r a c t Pest control constitutes a major area of interest for the biotechnology industry. Genes encoding insecticidal proteins have been cloned and they are being introduced in crop plants. At CIBA Agricultural Biotechnology, we have introduced a truncated form of the cryIA(b) gene obtained from Bacillus thuringiensis into an elite line of maize. A synthetic version of the gene was made to increase CryIA(b) protein levels in transgenic maize. The expression of the cryIA(b) gene was targeted to the pollen, pith, and green tissues by using appropriate tissue specific promoters. The resulting transgenic maize plants were evaluated for resistance to European corn borer (ECB), Ostrinia nubilalis, under field conditions. Plants with high levels of the CryIA(b) protein exhibited excellent resistance to repeated heavy infestations of the pest. and, considering their record in efficacy genes whose sequences have been and safety, they are now prime targets optimized for plants under the control Propagation of plant varieties for the for plant biotechnology. δ−Endotoxins of new promoters including tissue- purpose of improving certain traits has are the product of single genes and they specific promoters. Advances in been the main goal of plant breeding. constitute the seminal tool to engineer transformation techniques has allowed Successful breeding programs consist plants resistant to insects. to expand insect-control programs to Int roduct ion monocots, in particular maize. of multi-step processes where plants are crossed and crossed again until the The first generation of transgenic Bt desired character(s) is obtained. plants is represented by transgenic Recently, genetic engineering has tobacco (Vaeck et al. 1987; Barton et al. provided the means to obtain genetic 1987; Adang et al. 1987) or tomato Transformation vectors information about those favorable (Fischhoff et al. 1987) where the Vectors used to transform maize are all traits. One of the most important expression of native cryIA(b) genes was derivatives of pUC18 or pUC19. They applications of genetic engineering to driven by constitutive promoters. The contain a truncated-synthetic version of crops has been the production of insect- resulting transgenic plants conferred the cryIA(b) gene from Bacillus resistant plants in one step. good protection towards tobacco thuringiensis var. kurstaki placed under hornworm, Manduca sexta. However, it the control of either the CaMV 35S Bacillus thuringiensis (Bt) is a Gram- was clear that higher levels of cryIA(b) promoter or tissue-specific promoters positive, spore-forming bacterium, expression would be needed to achieve (Fig. 1). which produces parasporal crystals control of other agronomical important during sporulation. These crystals are pest such as tomato fruitworm, formed by proteins (known as δ− Heliocoverpa zea, and tomato pinworm, Transformation and embryo rescue endotoxins) which posses insecticidal Keiferia lycopersicella. Immature embryos (maize inbred activities when ingested by certain M aterial and M ethods CG00526) were excised 2 weeks after insects. Indeed, Bt strains have been The second generation of transgenic Bt pollination and plated scutellum up on used since 1938 as insecticidal sprays plants involves expressing δ-endotoxin 2DG4 + 5 mg/l chloramben. Plasmid THE EXPRESSION OF A SYNTHETIC CRYIA(B) GENE IN TRANSGENIC MAIZE CONFERS RESISTANCE TO EUROPEAN CORN BORER 173 DNA was deposited onto reached anthesis, a second round of sheath and collar tissue. Larvae begin microprojectiles as described in the infestations took place. 300 neonates per to tunnel into the stalk after three to six DuPont Biolistic manual. Generally, 6 plant each week for four weeks were weeks, often in the ear region and this mg of DNA are used per 50 ml of applied to emulate a second generation is where the feeding causes severe microcarrier. Delivery of the infestation. One hundred were yield losses from stalk breakage and/or microprojectiles is performed using the deposited into the leaf axil of the ear dropping. PDS-1000He Biolistic Gun with rupture primary ear, one node above and below disks of 1550 psi. After bombardment, the primary ear. The extent of the embryos are kept for one day in the internal ECB tunneling damage was Optimizing δ−endotoxin expression in transgenic maize dark at 25ºC, and then transferred to a assessed in 90 cm sections of the stalk. Increasing the GC content of B. callus initiation medium containing 3 thuringiensis insecticidal protein genes mg/l of phosphinothricin (PPT). CryIA(b) protein determinations leads to better expression in plants Resultant embryogenic callus was Quantitative determinations of the (Perlak et al. 1991). The Insect Control transferred to 2DG4 supplemented with levels of CryIA(b) protein were Group at CIBA decided to make a 0.5 mg/l of 2,4-dichlorophenoxyacetic performed by ELISA (Clark et al. 1986). synthetic version of the cryIA(b) gene acid. About twelve weeks later, tissue Immunoaffinity-purified polyclonal increasing the GC content from 38% in was transferred to MS medium rabbit and goat antibodies specific for the native gene to 65% in the synthetic containing 3% sucrose and hormones the CryIA(b) protein were used. The version. The gene encodes the first 648 (Koziel et al. 1993). Transformed plants sensitivity of the double sandwich- amino acids (aa) of the 1155 aa protoxin were identified by PCR for sequences in ELISA is 1-5 ng CryIA(b) per mg of and it produces the same active the promoters and the synthetic cryIA(b) soluble protein from crude plant insecticidal toxin as the full-length gene. Positive plants were moved to the extracts. Extracts were prepared as protoxin, once it is processed in the greenhouse for additional tests and described by Carozzi et al. (1992). insect gut. The expression of the synthetic cryIA(b) gene is driven either crosses with various inbreeds. Sixteen days after pollination, the ear tips were by a constitutive promoter (35S), or by Re sult s tissue-specific promoters (see Fig. 1): a removed, the embryos excised and plated on B5 medium containing 2% Biology of the target maize phosphoenolpyruvate sucrose. The principal target pest for CIBA has carboxylase (PEPC) promoter, which been European Corn Borer (ECB), expresses in green tissues, a maize Insect infestations Ostrinia nubilalis. ECB is a major pest in pollen specific promoter, which Lab-grown ECB larvae were used to Europe and North America causing expresses in pollen (Estruch et al. 1994), infest plant material. Infestations yield losses ranging from 3 to 20%. ECB and/or a pith-preferred promoter. started when maize plants were about has two generations annually, but three 40 cm high. For four weeks, 300 or even four generations can occur Chimeric cryIA(b) genes were neonates each week were mixed with depending on the area of distribution. introduced into proprietary inbred corn cob grits and introduced into the ECB larvae migrate into the whorl and lines by microprojectile bombardment whorl of each plant. When plants feed on leaf material. First-instar larvae of immature embryos (Koziel et al. CaMV 35S synthetic crylA(b) [648 aa] Maize PEPC synthetic crylA(b) [648 aa] Maize pollen synthetic crylA(b) [648 aa] Maize pith synthetic crylA(b) [648 aa] Event 171 Event 176 Figure 1. Versions of the maize optimized cryIA(b) gene under different promoters. The synthetic gene encodes the amino terminal 648 amino acids of CryIA(b) protein from Bacillus thuringiensis var. kurstaki HD-1. The promoters driving cryIA(b) gene expression are a green tissue-specific, a pollenspecific, and a pith-preferred promoter. tunnel into the stalk 1993). The bar gene, used to confer where they will feed resistance to PPT, was used as and pupate. Adult selectable marker. The material moths emerge over the obtained was then analyzed summer period and thoroughly for PPT resistance, deposit their egg masses CryIA(b) levels, and ECB resistance. on the abaxial side of the leaves close to the ear node. Neonates Evaluation of transgenic maize plants in the field generally move to the Germination of immature embryos was leaf axils and feed on used to produce the F1 hybrid plantlets accumulated pollen, for planting in the field. When plants 174 J.J. ESTRUCH, N.B. CAROZZI, N. DESAI, G.W. WARREN, N.B. DUCK, AND M.G. KOZIEL reached about 40 cm in height, they microprojectile bombardment of introduced into commercial maize lines. were infested with neonate ECB. A total immature embryos. The possibility of Transgenic plants will therefore of 2,400 larvae per plant were applied transforming inbred lines represents a represent an invaluable tool to use in during the eight week treatment (300 significant advantage over the regular integrated pest management strategies. per week). This represents 10 to 100 breeding programs. fold the economic threshold of second Re fe re nce s generation ECB. As indicated by the The Bt gene encoding the δ−endotoxin severe foliar and stalk damage CryIA(b) has been optimized for produced in the control plants, the ECB expression in maize plants. Maize pressure employed was strong enough plants transgenic for the cryIA(b) to evaluate the performance of the synthetic gene are protected from heavy transgenic maize for cryIA(b). Of the infestations of European Corn Borer. different transgenic maize lines, the This protection is observed in plants offspring coming from the cross hemizygous as well as homozygous for CG00554 x 176 provided the best the cryIA(b) gene, so hybrid maize resistance, where no leaf damage could obtained from a transgenic parent will be observed (see also Koziel et al. 1993). inherit the protection trait. Concerning the second generation of ECB, whose principal target is the stalk, Tissue-specific expression of the transgenic maize and in particular cryIA(b) gene is achieved by using CG00554 x 176, offered an excellent green, pollen and pith-preferred tissue resistance against ECB. For example, specific promoters. The use of these while a control plant had 59 cm of promoters allows expression of the tunneling damage on average, the insecticidal protein in parts of the plant transgenic line had less than 2 cm. where ECB feeds while minimizing expression of the insecticidal gene in The best performers among the seeds. The presence of the CryIA(b) transgenic maize plants were protein in pollen is particularly thoroughly analyzed for cryIA(b) gene important because it constitutes the expression and CryIA(b) protein levels. main diet during the first and second Transgenic maize for the cryIA(b) gene instar of the ECB (Showers et al. 1989). under the PEPC and pollen specific The effectiveness of the transgenic promoter produced over 1000 ng maize plants against ECB infestation CryIA(b) protein per mg of total protein has also been tested under field (they could contain up to 4 times more) conditions. Several transgenic maize in leaves and up to 400 ng/mg in lines have been produced, in particular pollen. While the expression of the pith- line 176, that are very resistant to ECB preferred promoter led to lower levels even under infestation pressures several of CryIA(b) protein (around 35 ng/mg orders of magnitude higher than those in pith), it was sufficient to control ECB. occurring naturally. In addition, the CryIA(b) protein could not be detected in kernels in these Our group at CIBA Agricultural plants expressing the gene under Biotechnology has created the tissue-specific promoters. framework to introduce traits into maize. Transgenic maize plants Discussion resistant to ECB are now a reality, and as improved insecticidal genes become Chimeric genes were introduced into elite inbreds of maize via available, they can be rapidly Adang, M.J., E. Firoozabady, J. Klein, D. Deboer, V. Sekar, J.D. Kemp, E. Murray, T.A. Rocheleau, K. Rashka, G. Staffield, C. Stock, D. Sutton, and D.J. Merl. 1987. Application of a Bacillus thuringiensis crystal protein for insect control. In C.J. Arntzen, and C, Ryan (eds.), Molecular Strategies for Crop Protection, 345-353. New York: Alan R. Liss. Barton, K.A., H.R. Whiteley, and N. Yang. 1987. Bacillus thuringiensis δ− endotoxin expressed in transgenic Nicotiana tabacum provides resistance to lepidopteran insects. Plant Physiology. 85: 1103-1109. Carozzi, N.B., G.W. Warren, N. Desai, S. M. Jayne, R. Lotstein, D.A. Rice, S. Evola, and M.G. Koziel. 1992. Expression of a chimeric CaMV 35S Bacillus thuringiensis insecticidal protein in transgenic tobacco. Plant Molecular Biology. 20: 539548. Clark, M.F., R.M. Lister, and M. Bar-Joseph. 1986. ELISA Techniques. Methods in Enzymology. 118: 742-766. Estruch, J.J., S. Kadwell, E. Merlin, and L. Crossland. 1994. Cloning and characterization of a maize pollen specific calcium-dependent calmodulinindependent protein kinase. Proceedings of the National Academy of Sciences, USA. 91: 8837-8841. Fischhoff, D.A., K.S. Bowdish, F.J. Perlak, P.G. Marrone, S.M. McCormick, J.G. Niedermeyer, D.A. Dean, K. KusanoKretzmer, E.J. Mayer, D.E. Rochester, S.G. Rogers, and R.T. Fraley. 1987. Insect tolerant transgenic tomato plants. Bio/Technology. 5: 807-813. Koziel, M.G., G.L. Beland, C. Bowman, N.B. Carozzi, R. Crenshaw, L. Crossland, J. Dawson, N. Desai, M. Hill, S. Kadwell, K. Launis, K. Lewis, D. Maddox, K. McPherson, M.R. Meghji, E. Merlin, R. Rho- des, G.W. Warren, M. Wright, and S. Evola. 1993. Field performance of elite transgenic maize plants expressing an insecticidal protein derived from Bacillus thuringiensis. Bio/ Technology. 11: 194-200. Vaeck, M., A. Reynaerts, H. Hofte, S. Jansens, M. De Beuckeleer, C. Dean, M. Zabeau, M. Van Montagu, and J. Leemans. 1987. Transgenic plants protected from insect attack. Nature. 328: 33-37. Sustaining Host Plant Resistance Derived Through Conventional and Biotechnological M eans K.M. Maredia, Michigan State University, East Lansing, MI, U.S.A. Abst r a c t Globally, during the last four decades, large investments and long-term research efforts have been put into plant breeding to develop pest resistant varieties and hybrids of crop plants to substitute for the use of toxic chemical pesticides. More recently, new tools of biotechnology have been added to the plant breeding programs to speed up this process. Many pest resistant varieties and hybrids have been released, and in a few years, genetically engineered transgenic varieties and hybrids are expected to be commercialized and released worldwide. Pests can adapt to any management tactic depending on the selection pressure exerted on them, so deployment strategies must be designed and implemented to delay or prevent the breakdown of resistance. Some of these strategies may include use of multiple genes, combining the host plant resistance (HPR) derived through conventional and biotechnological means to pyramid or stack resistance genes, rotation or alteration of genes, use of different gene promoters, and manipulation in the levels of expression (spatial and temporal) of genes. In addition, these HPR deployment strategies must be integrated into an overall integrated pest management (IPM) program that incorporates multiple tactics (cultural, biological, mechanical, chemical, etc.) to diversify pest mortality sources and reduce subsequent selection pressure on the pests. Pest resistance management must be viewed within the context of IPM. If IPM is successfully adopted and implemented at a community or landscape level, the objective of resistance management will be automatically achieved. Hence, IPM should become a part of national agricultural policy. Int roduct ion plant resistance (HPR), cultural control pests. The development of transgenic and mechanical control have been plants has given a new dimension to Food losses due to insect pests investigated to substitute for chemical HPR. represent a major threat to global food pesticides. Globally, during the last four security. Sustaining global food decades, large investments and long- In a few years, genetically engineered security will be of even greater concern term research efforts have been put into transgenic varieties and hybrids are in the future as the world’s population plant breeding to develop pest resistant expected to be commercialized and continues to grow. With the advent of varieties and hybrids in both the public released worldwide. However, pests the insecticide, DDT, in the late forties, and private sector. can adapt to a host plant resistance mechanism if sufficient selection toxic chemical pesticides have been extensively used in agricultural Many pest resistant varieties and pressure is exerted on them. In this landscapes to manage pests and help hybrids developed through paper we discuss different strategies for reduce food losses. However, due to a conventional plant breeding have been delaying or preventing the breakdown negative impact of pesticides on the released; and undoubtedly this will of resistance. environment and human health, the continue in the future. More recently, global community has been actively the new tools of genetic engineering looking for alternatives to toxic have been added to plant breeding chemical pesticides. programs to speed up this process. Host Plant Resistance (HPR) as a Tool of Pest M anage m e nt These new tools of biotechnology allow Several different approaches such as us to incorporate alien genes into crop Plant resistant to insects is composed of biological control, breeding for host plants to impart resistance to insect genetically inherited qualities that result in a plant of one cultivar of a 176 K.M. MAREDIA species being less damaged than a More recently transgenic plants have metabolic mechanisms of resistance to susceptible plant, which lacks this been developed by incorporating alien HPR factors (Kogan 1976; Smith 1989), quality (Smith 1989). Three types of genes such as Bacillus thuringiensis (B.t.) cultural control (Ostlie 1987), biological resistance are recognized: non- from bacteria, and trypsin inhibitor control agents (Maund & Hsiao 1991), preference (for shelter, food and genes into crop plants. B.t. is an and insect controlling pathogens (Dunn oviposition), antibiosis (adverse effects aerobic, gram positive, spore forming 1986). of the plants on the biology of insects), bacterium commonly found in the and tolerance (ability of the plant to environment (McGaughey and Whalon In the case of HPR developed through withstand damage or recover from 1992). The presence of a number of conventional plant breeding, rice damage caused by populations of insect toxins in B.t. has been well brown plant hopper, Nilpervata lugens, insects that would decimate a documented. The most distinctive of have been reported to have overcome susceptible plant). Non-preference these are protein crystals formed the resistance in varieties developed by prevents insects from occurring, during sporulation (Feitelson et al. the International Rice Research antibiosis prevents them from 1992). Gene transformation offers a Institute (IRRI) and many national establishing at high levels, and potential method of delivery for the agricultural research programs in Asia tolerance protects the host from large toxin. Using genetic engineering (Heinrichs 1986; Saxena 1987). In the yield reductions. techniques, the B.t. genes have been case of maize, the only reported case of inserted into many plant species pest overcoming HPR is corn leaf The use of resistant varieties or hybrids including maize, tobacco, tomato, aphids Rhopalosiphum maidis (Smith offers an economic, stable and potato, and cotton. 1989). There is optimism for the future of agriculture due to developments in ecologically sound approach to minimizing losses from insect pests. Problem of Pest Resistance plant biotechnology (e.g. new crop varieties that use B.t. genes to impart This method is particularly appropriate for subsistence farmers in sub-tropical Pest resistance is the adaptation of plant defense mechanisms). But this and tropical regions of developing pests to management tactics. Pests can new technology is already at risk since countries who often have limited adapt to any management tactics resistance to B.t. toxins has developed resources and inadequate knowledge depending on the selection pressure in the field (Tabashnik et al. 1991). of, or access to pesticides. In addition, exerted on them. Although all living HPR, unlike pesticides, is compatible organisms have an ability to respond to In the USA, the DIMBOA mechanism with all other pest management tactics. their environment, arthropods are of HPR to European Corn Borer in among the most successful. The ability maize has remained stable for the last These arguments have justified large of insects to utilize a variety of niches three-four decades and has not broken investments and long-term efforts by also allows them to compete with down. This is mainly due to the fact the global community in developing human beings for food and fiber. In that in the USA, not all varieties pest resistant varieties through response, humans have used a variety planted are resistant and maize is only conventional and biotechnological of tactics to reduce the impact of pest grown once a year which limits the means. Through conventional insects. However, as with any selection number of generations of corn borers to breeding, resistance genes have been pressure placed on a population, the 2-3 per year. Hence, the selection identified from plants within the same insect’s response has been to adapt to pressure has been very low and slow. species and wild relatives. Some of an altered environment. However, the warm and humid climates of tropics and subtropics these sources have been successfully incorporated into elite germplasm and Pest resistance is a consequence of (where most developing countries are varieties. In the case of maize in natural evolutionary processes and is located) are more conducive to pest developing countries, excellent not limited to a particular agricultural development. Pests reproduce rapidly progress has been made in identifying system. Thus, it has become a global and produce multiple generations in a sources of resistance to many phenomenon. Examples of pest given season or a year, exerting higher important pests through conventional resistance abound. Insects have selection pressure. At present, few pest means. developed behavioral, physiological, or resistant maize varieties have been released in developing countries. SUSTAINING HOST PLANT RESISTANCE DERIVED THROUGH CONVENTIONAL AND BIOTECHNOLOGICAL MEANS However, when resistant varieties are • Making pest resistance management Deployment strategies must be more common, the chances of pests a part of the national biosafety designed from the onset of HPR overcoming resistance will be higher policy. programs to delay or prevent the problem of pest resistance. The than in temperate countries, although the effects are likely to be mediated if multiple genes are involved in conferring the resistance. M anagement of the Pest Resistance Problem 177 Strategies to Integrate HPR in Integrated Pest M anage m e nt (IPM ) Program s following HPR strategies may be deployed: • Use of multiple genes. • Combining the HPR derived Preventing pests from overcoming through conventional and HPR will require reduction in selection biotechnological means to pyramid Widespread development of pest pressure on pests. This can be or stack resistance genes. resistance could seriously diminish the accomplished by adopting an • Rotation or alteration of genes. economic value of HPR and force integrated system of pest management. • Use of different gene promoters. continued reliance on chemical IPM is a comprehensive approach to • Manipulation in the levels of pesticides. This is particularly true for pest management that uses multiple polyphagous insect pests, where tactics to avert or reduce the pest breakdown of B.t. genes in one crop problems in agroecosystems. will diminish the value of the same Conventional and biotechnological genes in other crops. The deployment derived HPR must be used along with strategies must therefore be designed other means of pest management strategies into an overall IPM and implemented in HPR programs to (cultural, biological, mechanical, program delay or prevent the breakdown of chemical etc.). For example, in the case resistance. of maize stem borer, coupling HPR expression (spatial and temporal) of genes. • Preservation of susceptible pest genes through refuges. • Integration of HPR deployment IPM : A National Policy with biological, cultural and chemical Resistance management strategies controls can be accomplished to reduce Pest resistance management must be the selection pressure due to the viewed within the context of IPM. In Pest resistance management prevents intensive use of any one tactic. Overall, order for both conventional and or delays the adaptation of pest species HPR sources of mortality should be just biotechnological means of pest to any defense mechanisms. Resistance one component of a stem borer management to last longer, they must management strategies must be based management scheme (Table 1). be integrated and utilized within the on the following five principles: • Reduction of selection pressure from each mortality mechanism to the target pests. • Diversification of mortality sources so that a selection pressure is divided between multiple mortality mechanisms; it is known that single gene traits are quickly overcome. • Maintenance of susceptible pest individuals by providing refuges or promoting immigration of susceptibles. • Development of resistance level estimation and/or prediction through the development of diagnostic tools and monitoring. context of IPM. This will reduce the Table 1. Integrated management program for European Corn Borer in the MidWestern United States. Cultural Control Adjustment of planting date Destruction of stubble’s (use of animals) Design of landscape Biological Control Egg parasites (e.g. Trichogramma, Minute pirate bug) Egg predators (e.g. Spotted lady beetles) Larval parasites (e.g. Eriborus terebrans) Larval predators (e.g. Big eyed bug) Larval pathogens (e.g. Nosema pyrausta, Beauvaria bassiana) Host Plant Resistance DIMBOA mechanism Antigua sources B.t. genes (Transgenic hybrids) Pesticides Biopesticides Chemical pesticides (Reduced rate of less toxic chemicals based on monitoring and economic threshold levels) 178 K.M. MAREDIA selection pressure on the pest and • USAID IPM CRSP: The United • GPRM: The Global Pest Resistance hence help increase the life span of new States Agency for International Management (GPRM) program has innovations. This will not only help in Development (USAID) has been developed at Michigan State the management of resistance to these established a collaborative research University. Using the “train the strategies, but also to other IPM tactics program in IPM. The program trainers” approach, this program by diversifying the pest mortality includes a consortium of several conducts an annual two-week mechanisms. If IPM is successfully public and private institutions, summer institute in pest resistance adapted and implemented at a NGOs and national programs of management and provides training community or landscape level, the selected countries in Asia, Africa to scientists from around the world objective of resistance management and Latin America. The goal of the (Wierenga et. al. 1994). will be automatically achieved. Hence, program is to reduce use of chemical IPM should become part of national pesticides through non-chemical Agricultural Biotechnology for agricultural policy. Also, many national approaches based on ecological Sustainable Productivity (ABSP) programs are revising their national principles. project at Michigan State University • • USAID ABSP: The USAID CGIAR IPM Task Force: This task is assisting developing countries in biotechnology innovations. Pest force consists of CGIAR centers and the use and management of resistance management must also is coordinated from IITA’s agricultural biotechnology’s with become an integral component of any Biological Control Center in Benin. emphasis on insect and disease national biosafety framework. The goal is to design and implement resistance. The ABSP project has IPM programs that will be based on incorporated resistance farming systems rather than specific management strategies in its crops. This program will also foster product oriented research programs biosafety frameworks to incorporate International Initiatives in IPM in potatoes and maize. interactions and information exchange across centers. From the experience with DDT and • • B.t. working group: This U.S. based The International Organization of Pest group consists of members from community needs to be made aware Resistance Management (IOPRM) is a industry with an advisory panel that no single management tactic can Washington, D.C. based non-profit from academia. This group is provide lasting solutions to the pest organization developed to assist the developing deployment strategies problem. A large investment has been global community in pest resistance for B.t. (used both conventionally made in HPR (derived through management. The IOPRM extends and transgenically) to delay or conventional and biotechnological its membership to all institutions, prevent the development of means) and other ecologically sound including public and private sectors resistance to these new and pest management tools (biological and international development control, biopesticides, etc.) to substitute agencies. synthetic pyrithroids, the global • expensive technologies. • The World Bank/Rockefeller Global IPM Service: The Consortium Foundation/UNDP Initiative: In interest of the global community that for International Crop Protection October 1993 the World Bank, these tools of pest management endure. (CICP) and the USDA’s National Rockefeller foundation and United Otherwise, the world’s farmers will be Biological Impact Assessment Nations Development Program forced to continue to rely on toxic Program (NBIAP) has formed a (UNDP) sponsored an international chemical pesticides. In this context, strategic partnership to assemble workshop on biotechnology and during the last few years, many and support global information and IPM in Italy. The purpose was to international initiatives have been communication on IPM research, assist the likelihood of new started to integrate these tools into an teaching, training, and biotechnology’s being usefully overall IPM program. These initiatives implementation of technology and incorporated into pest management are designed to strengthen national policy. This program has initiated an programs. The workshop also program capabilities in IPM and international IPM electronic data discussed the types of new influence policy-makers to integrate base and communication service biotechnology which would be most IPM in national agricultural policies: which can accessed via the internet. useful to facilitate the wider use of for toxic chemical pesticides. It is in the IPM strategies. SUSTAINING HOST PLANT RESISTANCE DERIVED THROUGH CONVENTIONAL AND BIOTECHNOLOGICAL MEANS • National IPM centers: Several national programs have taken The Need for Regional and Global Cooperation initiatives and formed national IPM • centers to foster networking, Since insects do not respect political provide training and facilitate boundaries, implementation of pest information exchange related to resistance management strategies will IPM. As an example, India has require both regional and global formed a national center of approaches and cooperation. In this Integrated Pest Management under context, the need for global networking the Indian Council of Agricultural to foster cooperation, and structural Research, which plays an active role adjustments in institutions to in promoting IPM at the national encourage multi-disciplinary and level and tries to influence policy systems approaches to pest makers in this area. management will become critical. Regional IPM programs: During the last few years, several regional It is hoped that the initiatives at programs in IPM have been national and international levels will initiated. For example, the foster this philosophy and sustain HPR Cooperative Program for the technologies. It is also hoped that HPR Development of Agricultural would play a key role in the pest Technology in the Southern Cone management programs of the 21st (PROCISUR) region of Latin century and contribute to the America has formed a regional enhancement of global food security collaborative program in IPM. FAO and long-term sustainability of has also successfully implemented a agroecosystems. regional IPM program in southeast Asia. Ac k now le dgm e nt This publication was made possible through support provided by the office of USAID/Cairo/AGR/A, under cooperative agreement No: 263-0152-A00-3036-00. 179 Re fe re nce s Dunn, P.E. 1986. Biochemical aspects of insect immunity. Annual review of Entomology. 31: 321-339. Feitelson, J.S., J. Payne, and L. Kim. 1992. Bacillus thuringiensis: Insects and beyond. Biotechnology (N.Y.). 10: 271275. Heinrichs, E.A. 1986. Perspectives and directions for the continued development of insect resistant rice varieties. Agric. Ecosyst. Environ. 18: 936. Kogan, M. 1976. The role of chemical factors in insect/plant relationships. In Proceedings of the 15th International Congress of Entomology, 221-227. Lanham, MD: Entomological Society of America. Maund, C.M., and T.H. Hsiao. 1991. Differential encapsulation of two Bathyplectes parasitoids among alfalfa weevil strain, Hypera postica (Gyllenal). Canadian Entomologist. 123: 197-203. McGaughey, W.H., and M.E. Whalon. 1992. Managing insect resistance to Bacillus thuringiensis toxins. Science. 258: 1451-1455. Ostlie, K.R. 1987. Extended diapause: Northern corn rootworm adapts to corn rotation. Crops and soil Magazine. 39: 2325. Saxena, R.C., and A.A. Barrion. 1987. Limitations of host plant resistance: Insect biotypes. In Proceedings of the 11th International Congress of Plant Protection, Volume 1, 541-545. Manila, Philippines. Smith, C. M. 1989. Insect biotypes that overcome plant resistance. In Plant Resistance to Insects: A Fundamental Approach, 221-239. John Wiley and Sons. Tabashnik, B.E., Cushing, N.L., Finson, N., and M.W. Johnson. 1991. Field development of resistance to Bacillus thuringiensis in diamondback moth (Lepidoptera: Plutellidae). Journal of Economic Entomology. 83: 1671-1676. Wierenga, J.M., M.E. Whalon, and K. Maredia. 1994. Resistance management training for international scientists. Pesticide Outlook. 5: 23-25. Insect Resistant M aize: A New Paradigm for Conducting Research J.E. Foster and S. Ramnath, University of Nebraska-Lincoln, Lincoln, Nebraska. Abst r a c t A paradigm is defined as a “model, pattern, or example.” Our thesis is that the model for conducting maize research is changing. In this presentation we look at past models for conducting maize research, review some of the current models and introduce a suggestion for a future model. We are convinced that the team approach will be the hallmark of the twenty-first century, as it has been for the past several decades. Defining the team will be critical. Biotechnology offers a scientific paradigm shift that we in maize research and agriculture can use to our advantage. Innovation is the way teams gain a Past model competitive edge. As researchers we The past model was “one scientist - one A paradigm has been defined as: “A set have all taken pride in our innovative project”, and most work of this type of rules and regulations that does two accomplishments. Innovation coupled involved one insect and one crop. things. Firstly it establishes or defines with excellence is a powerful Research under this model has boundaries; and secondly it tells you combination. Excellence and provided excellent data and results, as how to behave inside the boundaries so innovation, however, are not enough. evidenced by even a casual review of Int roduct ion the literature. Compelling arguments as to be successful” (Barker 1992). Barker describes three keys to the Anticipation provides teams with the can made against this model: for future of any organization. We believe information that allows them to be in example, it lifted-up the excellence of these principles apply to researchers the right place at the right time with an an individual; obviously, though, one and research organizations, both excellent innovative idea or service. scientist cannot possess all the formal and informal, including maize Anticipation is the final key element of information, knowledge or skills for researchers. The keys to the future are: this triad. This triad allows us to success. Furthermore, this model excellence, innovation and anticipation. predict our future needs, to provide sometimes promoted the dominance or All keys three are necessary, as shown innovative products or services, and to importance of one academic discipline below. produce those products and services in over all others. Such a model was in an excellent manner. These three team our opinion doomed to fade into the Excellence is the basis for research. It attributes are necessary for us to past and it essentially has become part has been in the past and will be even survive in the twenty-first century. of our history. M ode ls Present model more important in the future. Excellence provides the competitive edge for awhile and then it becomes the The second model, the present, has ticket price of entry into research. The We believe there are minimally three been and continues to be very basic components of excellence are models to conduct research: the past, successful. The hallmark of this model continuous improvement, bench the present and the future. The three promotes the “team approach”. It is marking, the continuous pursuit of models will be discussed briefly and uncertain where or when this approach excellence, and the capability of then will be drawn on for comparative started with respect to insect resistance knowing how to do the right thing the purposes to illustrate our point and studies. Historically, teams of two or first time. support our arguments. more people have probably existed INSECT RESISTANT MAIZE: A NEW PARADIGM FOR CONDUCTING RESEARCH 181 since the time of the first co-authored support and collaborative capacity. We propose that most scientists paper. Though Painter may have That role has certainly changed over conducting research on maize know the actually served as the catalyst for the time to one of full research partner; in rules of team work and have shared in promotion of this approach with the many cases serving as the main thread one or more aspects of its success. publishing of the first book on plant to cooperative efforts between states. Further, we would venture that many resistance to insects (Painter 1951). Some researchers have gone so far as to have not contemplated the possibility of However, Painter was quick to give say that the USDA-ARS has been the a shift in the way we conduct research. credit to earlier work, as he pointed to mainstay, holding disparate state Kuhn (1962), stated, “Men whose research on the woolly apple aphid, “A efforts together by providing research is based on shared paradigms case for team research” (Hatton et al. collaborate leadership. Missing in are committed to the same rules and 1937). This model, at least initially, Painters’ depiction of a model team standards for scientific practice”. promoted the basic research team of an was a defined role for private and or Paradigms give us a set of expectations agronomist or horticulturist, an corporate breeding programs. about what probably will occur based entomologist and a plant breeder. The Correspondent to the efforts of state on our shared set of assumptions. Those plant breeder had already successfully and federal research teams being committed to team work on maize worked as a team member with plant formed, private or corporate breeding know how we work and get things pathologists. From this model a programs such as Pioneer Hi-bred Inc., done. Smith (1975) says, “ When we are number of successes in several cereal Dekalb Inc. and others were in the middle of the paradigm, it is hard crops, including maize, have resulted established. Their efforts have grown to imagine any other paradigm”. We in the release of a large number of high with amazing rapidity, adding not only are in the middle of the present model; yielding germplasms, varieties, and an ever increasing number of desirable i.e., we are in the middle of a paradigm hybrids. It has continuously evolved traits to maize but always increase that we know and have become familiar and today many disciplines are yield. The trait package of corporate with. The present model provides us involved. It is also responsible for the breeding programs is a true success with the, “basic way of perceiving, growth of the sub-discipline of story in agricultural research. As a thinking, valuing and doing associated entomology we call plant resistance to result of state, federal and corporate with a particular vision of reality” insects. The importance of cooperation efforts, the collective grain yield (Harmon 1970). The ability of the maize between investigators working on the increases for maize in the USA have researchers to do team research is well plant was central to the model, and it averaged about 2-3 % per year over the documented. We know the boundaries detailed the work to be handled by last 50 years. and how to perform within those boundaries as defined by our paradigm; each investigator, outlined the facilities for the work and even suggested the A parallel success story is the i.e., we know what we can expect from division of labor assigned to each development of the international universities, the USDA-ARS, CIMMYT discipline. We believe it is unfortunate research centers such as CIMMYT. It is and other centers conducting corn that some plant breeders interpreted interesting to note that Painter pointed research. Further, we each know and their role to be more important because out that two crop teams had excelled have developed linkages with corporate of being central. using this team model, namely the corn breeding and improvement entities. insects and wheat insects research “The dominant paradigm is seldom if Worthy of note in Painters’ detailed teams (Painter 1951). We are pleased to ever stated explicitly; it exists as an effort to outline such a program for have served as a member of the wheat unquestioned, tacit understanding that breeding for insect resistance was the insects research efforts, in collaboration is transmitted through culture and to relationship between breeders (being with CIMMYT, and to now be a part of succeeding generations through direct central) and entomologists, plant the effort on maize. experience rather than being taught.”(Harmon 1970). Our dominant pathologists, and the United States Department of Agriculture-Agriculture The scenario we have just gone paradigm or model is one of Research Service. It is also interesting through is relevant in the following cooperative team research. We propose to note that in Painters’ model the way. We offered up the definition of a that maize researchers consider that we USDA was positioned to serve in a paradigm as described by Barker(1992). 182 J.E. FOSTER AND S. RAMNATH are in a very changing research The third and obvious force that will make with all components of the new environment. The name of the game is affect the way we conduct future maize team or teams will determine our changing . “A paradigm shift, then, is a research concerns intellectual property success. change to a new game, a new set of rights. Obviously, the way we share rules”(Barker 1992). and exchange both information and A challenge for future teams will by genetic resources will be affected by necessity be budgetary constraints. this issue. How to finance research will be a Future model serious consideration. The answer may The pertinent question is, what is the new game? Before that can be answered The fourth force, and one of potentially be hidden in the key of excellence. In let us take a quick look at a few selected enormous impact for maize research, is that key we noted that “bench forces that will impact on us as maize the use of fiber optics. Our ability to marking” and “knowing how to do it researchers working in the future. communicate, share information, the right the first time” are basic These forces are not exclusive, but ease of moving data, our ability and components of that key. Bench marking rather are a selected minimal number ease to publish faster, and even is more than recording our successes that will make our research lives more distance learning and conferences will and reporting them. Listing our complex. all be enhanced by this technology. accomplishments is not enough. Firstly, there is a trend toward All of the above, when combined, will discussing impacts and the return on regionalization of world economics and have hitherto unimagined effects on investment. The returns on team reduced funding for research. No the way we conduct research research investment are often matter what state, country or region we programs. substantial (Roberts et al. 1983,1988), Somehow we must do a better job of yet there is a dearth of reports for most are from, we will be affected in research by a relative decline in monetary We indicated that the keys to the future crops. Returns on investment are both resources. Research administrators are excellence, innovation, and direct and indirect. Direct measures are express it in at least two ways. We will anticipation. The first two of these keys difficult, but not impossible to estimate. be doing more with less and we will be are evidenced by the accomplishment For example, Roberts et al. (1988) doing it with fewer people, i.e. of many. The last key will be discussing wheat research, “rightsizing”. determined by how we answer the conservatively estimated the direct question of the new game. Convincing return on research dollars invested to Secondly, biotechnology may be the arguments can be made for one of be $4.6 million per person-year input. most immediate and observable many positions. The structure of new Indirect measures can be determined influence on maize researchers in the team efforts will be as varied as the by number of publications such as future. For example, take a quick look at number of teams and their objectives, refereed scientific journal articles, the Proceedings of the International however all will have to take into bulletins, published abstracts and Symposium on Methodologies for consideration the factors that affect the graduate theses. Also, informal Developing Host Plant Resistance to new model. exchanges of information can be tallied by newsletters, conference records, Maize Insects held here in Mexico in 1987. The term biotechnology did not The future model will be built on the invitational seminars, and numerous appear in a single title listed in the table existing models, with change occurring reports from regional efforts and of contents! Today, it is highly probable in the third key, anticipation. working groups. that transgenic corn will be on the Components of the new team(s) will market in a couple of years. This is a comprise university researchers, the The second hidden message in the key powerful technological accomplishment USDA-ARS, corporate breeding of excellence may be in the phrase, in agriculture. Biotechnology offers us entities, international centers, and now “knowing how to do it right the first the genetic diversity that we as maize a whole new set of players, such as time”. Recently, Nelson (University of researchers have so long sought. biotechnology firms, lawyers and Nebraska Agricultural Research regulatory agencies. The linkages we Division Newsletter) stated,” we are INSECT RESISTANT MAIZE: A NEW PARADIGM FOR CONDUCTING RESEARCH answering questions that nobody is asking”. He was referring to a luxury that we at universities can no longer afford. We must be accountable to our research financiers. Re fe re nce s Barker. J.A. 1992. Future edge: Discovering the new paradigms of success. William Morrow and Company. Harmon, W. 1970. An Incomplete Guide to the Future. W. W. Norton Co. Hatton, R.G., W.S. Rogers, R.M. Greenslade, M.B. Crane, A.M. Massie, H.M. Tydeman, and W.A. Roach. 1937. The problems raised by the woolly aphid of the apple-A case for team research. Ann. App. Biol. 24: 169-210. Kuhn, T.S. 1970. The Structure of Scientific Revolutions. University of Chicago Press. Painter, R.H. 1951. Insect Resistance in Crop Plants. University of Kansas Press, Lawerence, KS. Patterson, F.L., G.E. Shaner. H.W. Ohm, and J.E. Foster. 1990.. A Historical Perspective for the Establishment of 183 Research Goals for Wheat Improvement. J. Prod. Agric. 3: 30-38. Roberts, J.J., F.L. Patterson, J.E. Foster, and W.J. Hinsman. 1983. Increased Productivity of Purdue-USDA Soft Red Winter Wheat Cultivars—A Major Return from Research. Purdue Univ. Agric. Stn. Bull. 424. Roberts, J.J., J.E. Foster, and F.L. Patterson. 1988. The Purdue-USDA Small Grain Improvement Program—A Model of Research Productivity. J. Prod. Agric. 1: 239-241. Smith, A. 1975. Power of the Mind. Ballantine Books. Improved Technologies for Rearing Lepidopterous Pests for Plant Resistance Research F.M. Davis, USDA-ARS, Mississippi State, USA. Abst r a c t Two major advances in rearing lepidopterous insects have recently been made at the Crop Science Research Laboratory (USDA/ARS) located at Mississippi State, Mississippi. First, a multicellular tray made of 15 mil polyvinyl chloride plastic with a perforated polyester heat seal lid has replaced the 30 ml plastic cups with paperboard insert caps for rearing larvae to pupation. The new rearing container with 32 individual rearing cells is cheaper and saves time and space. Second, a solution to the human health hazard created by loose moth scales inherent in lepidopterous rearing programs has been obtained. This second technology involves a separate facility to house the moth colonies, large moth cages designed to allow free exit of scales, an improved air filtration system, and appropriate sanitation procedures to deal with trapped and residual scales. Int roduct ion Procedures used during this era were European corn borer (ECB), Ostrinia described by Davis (1976). nubilalis (Hübner), were tried. Our experience with production of SWCB We have reared lepidopterous insects for plant resistance research for 25 In 1976, significant support was and FAW in large, common containers years at the Crop Science Research obtained for increasing research on was highly variable. Contamination of Laboratory (USDA/ARS, Mississippi plant resistance to SWCB. This required the diet by microbes and larval State, MS). Our goals have been to: a dramatic increase in the number of cannibalism were major problems. • Have the capability and reliability to SWCB for the program. In the early produce the number of insects 1980's we also began artificial rearing of The ‘third era’ (since 1988) features the required. fall armyworm (FAW), Spodoptera development and use of a new rearing Rear an insect which is frugiperda (J.E. Smith), for use in container and the completion of our physiologically and behaviorally providing uniform infestations to system for managing loose moth scales equivalent to its feral counterparts. screen maize for leaf feeding resistance. and body fragments. I report herein on Rear the insects in as efficient and We continued using the cup and cap the origin of the new rearing container, cost effective manner as possible. rearing containers, but developed semi- its use and benefits, and the system that automatic equipment to increase we now use to manage loose moth Our rearing program has evolved rearing efficiency and allow for scales in the building where the adult through three distinct eras. During the increased production. This rearing colonies are housed. first (before 1976), our rearing system system, used during the ‘second era’ was simple and capable of producing (1976 to 1987), was described in detail only small numbers of southwestern at the previous international corn borer (SWCB), Diatraea grandiosella symposium at CIMMYT on insect Dyar. The rearing container was a 30 ml resistance in maize (Davis 1989). • • Origin of New Rearing Cont aine r In the fall of 1985, we were asked by personnel of the Gast Insect Rearing clear plastic cup with a paperboard insert cap coated on one side to prevent In the early 1980's, we anticipated the Facility (Southern Field Crops Insect moisture loss. It was chosen primarily need for less expensive, more efficient Management Laboratory, USDA/ARS) because of its availability and the need rearing containers. Large plastic at Mississippi State, MS to join them on to separate the SWCB larvae because of containers, such as the dishes described a research project to improve an their strong cannibalistic nature. by Guthrie et al. (1971) for rearing the existing multicellular rearing container. IMPROVED TECHNOLOGIES FOR REARING LEPIDOPTEROUS PESTS FOR PLANT RESISTANCE RESEARCH 185 This container had been developed by 49504), a vendor that specialized in Mylar® would be adequate for rearing Sparks and Harrell (1976) for use in various types of lidding material, the above species because we observed their in-line form-fill-seal machine that including Tyvek®, helped us develop a that most of the SWCB larvae exiting was modified for mass rearing of suitable lid. this thickness of Mylar® did so just before pupation and returned to their lepidopterous insects. It consisted of a rearing cells to pupate. tray formed from 20 mil thick, high Many types of lidding materials were polystyrene. The tray contained 32 tested including perforated and non- individual rearing cells. The top, or lid, perforated papers, paper with tin foil Two additional steps were required for the tray was a commercially backing that had been perforated, and after a suitable lidding material was available product (Tyvek®), commonly polyesters of various thickness. selected. First, we needed to determine used for many purposes (e.g., to control Polyesters were emphasized because the spacing between pinholes which moisture in homes). Tyvek®, with an Ignoffo and Boening (1970) reported would provide sufficient air exchange adhesive on one side, was sealed to the some success in rearing an array of so that the diet would dry slowly top of the plastic tray by applying heat insects, including lepidopterans, in during larval development. Secondly, and pressure. Because this rearing compartmentalized disposable plastic we worked with personnel of Oliver container consisted of separate rearing trays (used in the food industry to Products Company in testing for an cells, we considered it potentially useful provide individual servings of jelly and adhesive that would hold its seal to the for rearing SWCB. other foods) that had a lid made of the plastic tray for several weeks and peel polyester, Mylar®. Their lidding back easily from the tray at pupal The problems with their multicellular material was a clear 0.5 mil Mylar® harvest. Different spacing of the rearing container were: film with one side coated with a heat pinholes was tested to determine the • The diet dried out too fast, resulting sensitive adhesive. The lid was sealed best for desired dry down of diet. in poor larval development. to the plastic tray using a Teflon®- Lidding with pinholes arranged 5 mm Many of the larvae exited the rearing coated tacking iron. Since Mylar® film apart was selected based on cells by chewing through both the lid is nonporous, they punctured the film, developmental data for the four and plastic tray under our rearing after sealing, with a specially lepidopterans. Diet moisture • environment (27.6°C and 50-60% RH). constructed board containing a series of requirements specific to a species were The plastic used to form the tray was nails. Ignoffo and Boening (1970) further adjusted by the time the diet opaque, so one could not see clearly encountered problems with was allowed to dry before infesting and what was happening inside the lepidopterans that had a strong sealing the lid. For example, FAW rearing cells. tendency to leave their compartments larvae do not develop satisfactorily if prior to pupation. They solved this the diet is too moist during the latter Technicians of the Gast facility made a problem by placing 0.16 cm mesh wire instars. Extra drying time of diet in new die for forming the plastic tray that screening or 0.3 cm plywood covers unlidded trays, under a clean air hood, was similar to the one used by Sparks between trays. solves this problem. By the end of 1987, • an improved multicellular rearing and Harrell (1976). Their tray is 15.24 cm wide by 27.94 cm long. It consists of We tested Mylar® of 1, 2, 3, and 5 mil container had been created (Fig. 1). 32 individual cells that are 3.0 cm deep thickness as lidding material. The 1 mil Also, data to support its suitability as a by 3.8 cm wide. A search was then Mylar® was not thick enough to rearing container for the SWCB, FAW, made to find a suitable plastic to form prevent larval exit. Larvae of the FAW CEW, and TBW had been generated the trays. I found a polyvinyl chloride and two other test species, the tobacco (Davis et al. 1990). (PVC), clear plastic (15 mil thick) that budworm (TBW), Heliothis virescens (F), formed a tray strong enough to prevent and the corn earworm (CEW), In 1987, Oliver Products Company larval escape. Developing a lid strong Helicoverpa zea (Boddie), were unable to made the polyester lidding (with enough to prevent larval escape but exit their rearing cells when 2 mil perforations and adhesive coating) porous enough to permit the diet to dry Mylar® lidding was used, but SWCB available to the public for use in insect down slowly as the larvae developed larvae were able to exit. None of the rearing. In the same year, James White, was difficult. Oliver Products Company above species could cut through the 3 an entomologist with CIBA-GEIGY (445 Sixth St., N.W., Grand Rapids, MI mil Mylar®. We decided that the 2 mil Seed Division (Bloomington, IL) 186 F.M. DAVIS assisted us in getting Dixon Paper lid onto the plastic tray. Oliver medicated maize-cob-grits using a Company (4402 Locust Avenue, Products Company fabricated a hand- bazooka (Davis et al. 1990). The infested Lubbock, TX 79408) to be a vendor for operated sealer for us (Fig. 2). Over the trays are then placed individually in the the 32-cell tray formed from 15 mil PVC years, we have made adjustments to the sealer’s tray well (Fig. 2, see arrow). Just plastic. Since then, Stephen Gould Corp. sealer to improve its seal. Oliver before initiating the sealing process, a (91480 Deerecho Road, Lutherville, MD Products Company is now marketing a moist sponge is lightly wiped over the 21093) has also become a vendor for the semi-automatic sealer with improved top surface of the tray and the 32-cell PVC plastic tray. sealing capabilities. underside of the lidding film to Use of New Rearing Cont aine r The diet is prepared and dispensed into the maize-cob-grits to be strongly each rearing cell of the tray using the attracted to the lid surface. The lidding equipment described by Davis (1989). material is then placed over the tray The 32-cell tray with its polyester film After dispensing, the diet-filled trays and the top of the lidder containing the lid replaced cups and caps as our are placed under clean air hoods for heating pad is brought down onto the standard rearing container in 1987. The cooling and drying of diet, after which lid and held for about 10 seconds. After only new equipment that we had to the rearing cells are infested with sealing, the lidding material on the tray purchase was a sealer for securing the neonate larvae mixed in autoclaved and is cut from the unsealed film with a eliminate static electricity which causes sharp knife. After the lids have been sealed onto the trays, the containers are stacked in upright, portable racks (Fig. 3). Each rack holds 20 rearing containers. Fabrication of these racks was described by Davis et al. (1990). Pupae are removed from the containers by simply peeling back the lidding material (Fig. 4) and emptying the contents of the tray cells into a large plastic container. The pupae are then hand picked from the residual diet and frass. Figure 1. New rearing container consisting of a 32-cell clear plastic tray with a perforated Mylar® lid. Benefits of the New Rearing Cont aine r Cost savings Multicellular trays and Mylar® lidding material cost significantly less than an equivalent number of the previously used cups and caps. For example, we use approximately 10,000 multicellular containers at a cost of approximately $3,800 (including transportation) to rear FAW and SWCB. The cost of an equivalent number of rearing cells (320,000 30 ml cups and paperboard caps) is approximately $11,600, a saving of $7,800 that can now be used to offset Figure 2. Semiautomatic lidder to seal Mylar® film to the top of the plastic tray. other costs, such as diet and labor. IMPROVED TECHNOLOGIES FOR REARING LEPIDOPTEROUS PESTS FOR PLANT RESISTANCE RESEARCH 187 Time savings Space savings containers have made our rearing The time required to process the new Rearing containers must be stored prior program more efficient and cost container with 32 rearing cells from to use. Storage can require substantial effective. dispensing the diet to harvesting the space, especially when purchasing pupae is significantly less than that large quantities to receive a volume required for cups and caps. This is discount. Multicellular trays and because it is much more efficient to Mylar® lidding require significantly handle a single container with 32 less space than storing cups and caps. Moth scales and other body fragments rearing cells than to handle 32 cups and For example, 320,000 cups and caps are well known allergens and pose a caps, individually. This time saving has require 2.5 times more storage space serious health hazard for sensitive allowed us to significantly reduce our than 10,000 multicellular rearing workers in artificial rearing programs permanent rearing personnel and containers (trays and lids). (Wirtz 1980, 1984; Bellas 1981; Lugo et allowed us to increase our rearing to M oth Scale Colle ct ion Syst e m al. 1994). For years we tried to develop include some cooperative rearing for Space in environmentally controlled a system to manage loose scales the Cotton Host Plant Resistance rooms to hold rearing containers generated by the moths, but only Research Unit, within our Crop Science during larval development is often a recently has the system evolved into Research Laboratory. Presently, we rear factor limiting increased production. one that solves the problem. for them about the same number of TBW and CEW as our own Type of rearing container and type of structure to stack or hold lepidopterous species. containers are important considerations. Again, the One rearing technician maintains the 4 multicellular rearing containers colonies of insects primarily alone require significantly less space during the off-season. During the than a comparable number of 30 spring, when colony size must be ml plastic cups. The multicellular increased to provide eggs for the peak rearing containers are held in rearing period, 1 to 2 additional part- racks that are 30.5 cm wide by 30.5 time workers are needed. During peak cm long by 30.5 cm high (Fig. 3). production, the technician, plus three Each rack holds 20 containers or full-time, temporary employees, trays that contain a total of 640 comprise the rearing work force. The individual rearing cells. In about bottom line is that it is more efficient to the same space, only 210 cups can process the multicellular containers be stacked in Styrofoam cup than cups and caps, and this results in holders (30 cups per holder). savings in personnel requirements and, Savings on cost, labor, and space ultimately, in research dollars. by using the multicellular rearing Figure 4. Removal of Mylar® lidding material from the plastic tray. Figure 3. Portable rack for holding multicellular rearing containers. 188 F.M. DAVIS Our present system involves a separate Status of Rearing Program facility for housing the moth colonies, and Forestry Experiment Station. oviposition cages that facilitate the exit We have at last attained our goals of of scales and other body fragments, an capability and reliability, efficiency and improved air filtration system, and cost effectiveness, and the production sanitation procedures to eliminate of high quality insects. Given the trapped and residual scales. Even excellence of the present rearing during peak moth production (20,000 system, no further substantial research or more individuals), the air in our efforts are envisaged in this area. This ‘moth house’ is lower in suspended does not mean, however, that we do particulate matter than the air just not have to monitor carefully each outside the building. The air filtration rearing phase (i.e., production build-up system (Fig. 5) takes in literally millions plans, infusion of wild genes into the of scales and other debris particles laboratory colony, diet contamination created by the moths, especially during by microbes and diseases) to ensure the scotophase cycle. Our tests show standards are maintained. that the filtration system removes 95 to 100% of particles from 0.5 to 5.0 PS-8637 of the Mississippi Agricultural Ac know le dgm e nt s microns. Details of this highly efficient and relatively inexpensive system are Appreciation is expressed to my described elsewhere (Davis and Jenkins technicians, Thomas Oswalt and Susan 1995), and would be of interest for both Wolf, for their assistance in developing small- and large-scale laboratories. the rearing system. I am also thankful for the opportunity to work with Stan Malone, the late Bill Jordan, and Dan Harsh of the Gast Rearing Facility in creating an improved multicellular rearing container. Also, I appreciate the cooperation of the employees of Oliver Products Company (especially Eloy Cantu and David Haines) for helping us develop a suitable lid for the multicellular tray and making it available to the public. Also, appreciation is extended to my secretary, Edna Carraway, for preparation of this manuscript. This article is a contribution of the Crop Science Research Laboratory, Agricultural Research Service, U.S. Department of Agriculture in cooperation with the Mississippi Agricultural and Forestry Experiment Station. It is published with the approval Figure 5. Air filtration unit used to remove moth scales and other debris from the air. of both agencies as Journal no. Re fe re nce s Bellas, T.E. 1981. Insects as a cause of inhalant allergies: A bibliography. CSIRO Australian Div. Entomol. Rept. No. 25. Davis, F.M. 1976. Production and handling of eggs of the southwestern corn borer for host plant resistance studies. Mississippi Agric. and Forestry Exp. Stn. Tech. Bull. 74. Davis, F.M. 1989. Rearing the southwestern corn borer and fall armyworm at Mississippi State. In Toward Insect Resistant Maize for the Third World: Proceedings of the International Symposium on Methodologies for Developing Host Plant Resistance to Maize Insects, 27-36. Mexico, D.F.: CIMMYT. Davis, F.M., and J.N. Jenkins. 1995. Management of scales and other insect debris, an occupational health hazard in a lepidopterous rearing facility. J. Econ. Entomol. 88(2): 185-191. Davis, F.M., S. Malone, T.G. Oswalt, and W.C. Jordan. 1990. Medium-sized lepidopterous rearing system using multicellular rearing trays. J. Econ. Entomol. 83: 1535-1540. Guthrie, W.D., W.A. Russell, and C.W. Jennings. 1971. Resistance of maize to second-brood European corn borers. Proc. Annu. Corn Sorghum Ind. Res. Conf. 26: 165-179. Ignoffo, C.M., and O.P. Boening. 1970. Compartmented disposable plastic trays for rearing insects. J. Econ. Entomol. 63: 1696-1697. Lugo, C., C. Cipolla, R. Bonfiglioli, C. Sassi, S. Maini, M. Pia Cancellieri, G. Battista Raffi, and E. Pisi. 1994. A new risk of occupational disease: allergic asthma and rhinoconjunctivitis in persons working with beneficial arthropods. Int. Archiv. Occup. Environ. Health 65: 291294. Sparks, A.N., and E.A. Harrell. 1976. Corn earworm rearing mechanization. USDAARS Technical Bulletin 1554. Wirtz, R.A. 1980. Occupational allergies to arthropods - documentation and prevention. Bull. Entomol. Soc. Am. 26: 356-36. Wirtz, R.A. 1984. Health and safety in arthropod rearing. In E.G. King, and N.C. Leppla (eds.), Advances and Challenges in Insect Rearing, 263-268. USDA-ARS. A New Technique for Evaluating Southwestern Corn Borer Damage to Post-Anthesis M aize F.M. Davis and W.P. Williams, USDA–ARS, Mississippi State, USA. Abst r a c t An effective and efficient technique for evaluating plants’ susceptibility to an insect pest is essential to screening for resistance. For many years, the accepted method of evaluating the resistance of maize, Zea mays (L.) at post-anthesis to southwestern corn borer (SWCB), Diatraea grandiosella Dyar, has been to measure the extent of stalk tunneling damage 35-to-40 days after infestation with eggs or neonate larvae. Given the failure thus far to identify and develop germplasm that possesses resistance after anthesis using this method, we developed a new technique. Studies have shown that SWCB larvae up to 14 days old feed primarily on leaf sheath and ear tissue (especially husk–leaves) on post-anthesis-stage plants. The larvae make feeding lesions on these tissues similar to those made on whorl leaves. A visual rating scale was developed utilizing type and number of feeding lesions on the outer three husk–leaves of the top ear and its associated leaf sheath. The leaf sheath and husk–leaf rating scales and their utility as an evaluation technique are discussed. Int roduct ion At the previous international symposium on insect resistant maize at CIMMYT in 1987, we stated that we had made significant progress in identifying and developing maize with resistance to leaf feeding by SWCB. By 1990, we had released nine germplasm lines and one population with leaf–feeding resistance (Williams and Davis 1989; Williams et al. 1990). We also stated at the last symposium that we had not made progress in identifying sources of postanthesis resistance to SWCB in maize. We felt that progress had been hampered by inadequate techniques for identifying resistance and a lack of significant resistance in the germplasm we have screened. In the last seven years, we have devoted much time and effort to improving our screening techniques. We report herein on: • Problems in screening for resistance to SWCB in maize after anthesis. • A new approach to evaluation using visual ratings of leaf sheath and husk–leaf damage. • Progress in identifying reliable silking, or to infest genotypes susceptible check genotypes and separately as each reaches the pre– potentially resistant genotypes. selected stage. Either option has advantages and disadvantages. For Screening Problems example, advantages of infesting all genotypes on the same day include the Screening involves two components: 1) fact that all larvae come from the same infesting plants with the test insect; group of eggs and survive and develop and 2) evaluating the insect/plant on the plants under the same interaction after a selected period of environmental conditions. A time. Evaluation can be done by disadvantage is that there may be a 2– determining either the effect of the to-3-week difference between when the insect on the plant (damage estimate) first and last genotype reach the pre– or the effect of the plant on the insect selected growth stage. An advantage of (survival and/or development). infesting genotypes when each reaches the pre–selected growth stage is that A problem that occurs with infestation the larvae have the opportunity to at post-anthesis (as opposed to the survive and grow on plants of the same whorl stage) is that maturity physiological stage. Disadvantages are differences result in plants of various that the larvae used for infesting genotypes not all being at originate from different groups of eggs approximately the same state of and the larvae must survive and grow development. In dealing with this, the on the plants under different researcher has two options: to infest all environments. In this approach, plants in an experiment after all staggered plantings of susceptible genotypes have reached a pre–selected check genotypes could be used to growth stage, such as 7 days after 50% provide a series of rows in different 190 F.M. DAVIS AND W.P. WILLIAMS physiological stages for comparison once each year. Even then, infestations insect on leaves of whorl-stage plants. with test genotypes. Experiments can be made over a period of only a Also, I observed different types of should be conducted to compare few weeks, thus limiting the number of lesions on the husk–leaves. These damage and/or survival/growth of genotypes that can be screened. If a observations stimulated us to begin larvae on different physiological stages growing season is missed because of evaluating post-anthesis maize by of the plant and thus indicate which inclement weather, then it is necessary visually rating the extent of damage on approach is better. When known, to wait another year unless the the leaf sheaths and husk–leaves, similar genotypes with similar maturities researcher has access to a winter to the technique described by Guthrie et should be screened together. nursery or collaborates with someone al. (1978) for evaluating leaf sheath collar Unfortunately, maturity (days to outside of the temperate zone. damage by European corn borer (ECB), Ostrinia nubilalis, Hübner. anthesis) is not known for many genotypes prior to planting in the screening nursery, and environment A New Evaluation Te chnique The first step in developing this approach was to characterize the feeding significantly influences maturity. Before describing the new evaluation lesions from different-aged SWCB larvae When analyzing our old technique of technique involving leaf sheaths and on leaf sheaths and husk–leaves. Also, evaluating stalk damage (primarily by husk–leaves, we want to discuss its information was needed on: splitting stalks and measuring extent of origin briefly. For many years we had • tunneling 35-to-45 days after known that SWCB larvae feed on leaf plant after releasing neonates in the infestation), we realized that the sheath and ear (primarily husk–leaves) axil of the top ear leaf. behavior of non-diapausing and tissues prior to entering the stalks of diapausing larvae influences the degree maize plants at post-anthesis (Davis et of stalk damage. SWCB larvae in the al. 1972). During this study we failed, early instars feed primarily on leaf however, to describe the larval feeding different ages, to determine how long sheath and ear tissues, regardless of lesions made on these tissues. the insect/plant interaction should diapause status. The behavioral • Larval establishment sites on the Whether damage varied among the different husk–leaves of the top ear. • The degree of damage from larvae of last before evaluating larval feeding. difference is that some non-diapausing One day in the late 1980s, I (Davis) was larvae will continue feeding on ear walking through some maize plots in We observed that SWCB larvae make tissue and pupate there without which plants had been infested for different feeding lesions depending on entering the stalk to tunnel, thus their about two weeks with SWCB larvae their age. Lesion types were the same on damage is not reflected in stalk released as neonates in the axil of the both husk–leaves and leaf sheaths. tunneling measurements. On the other top ear leaf. Some hand, almost all later-stage diapausing interesting-looking, larvae enter the stalk to tunnel and large, elongated lesions prepare an overwintering site at the on the leaf sheath base of the stem. Another potential caught my attention problem with measuring stalk tunneling (Fig. 1). Upon is the delay of 5-to-8 weeks between investigation, I found infestation and evaluation that allows that these were caused other biotic (predators, intraspecific by the SWCB larvae and cannibalism) and abiotic factors to that these feeding confound the insect/plant interaction. lesions were similar to Finally, splitting stalks and measuring those made by this tunnels is slow, boring, and costly. A problem which is not related to screening techniques, but can influence rate of progress in temperate zones is that germplasm can be screened only Figure 1. Lesions made by SWCB larvae feeding on the inner surface of the maize sheath. A NEW TECHNIQUE FOR EVALUATING SOUTHWESTERN CORN BORER DAMAGE TO POST-ANTHESIS MAIZE 191 Larvae up to 3 days old made only leaf sheath. These findings indicate that on husk–leaf tissue. The next preferred pinhole and small circular-to-elongated neonates begin feeding very near this tissue was the leaf sheath. The larvae (rectangular) lesions. By the time the release site and continue feeding on were found feeding on all ear tissues, larvae were 7 days old, they made these leaf sheath and ear tissues including kernels, cob, and shank at 14 elongated lesions of from 1.3 to 2.5 cm. through at least the third-instar. Seven– days after infestation. However, leaf Larvae 10 days old made elongated day–old larvae were feeding primarily sheaths and husk–leaves were still the preferred tissues. lesions that exceeded 2.5 cm, plus some rather small lesions that were wider A B C1 (>3mm) and which varied in shape from uniform (i.e., squares and rectangles) to irregular. By the time the The larvae feed on the inner surface of the leaf sheath below the collar. They C2 feed on all areas of the husk–leaves, especially in the lower portion where larvae were 14 days old, they made significantly larger, uniform–to– C3 irregular lesions. Additionally, the 10to-14-day-old larvae often ate through D1 the husk–leaves, leaving clean holes. Occasionally, larvae would eat a hole D2 the husk–leaf attaches to the ear shank. When the husk–leaves from top ears were compared for extent of damage, invariably the three outer husk–leaves suffered the most damage. The small outermost husk– leaf was a primary site through the leaf sheath. D3 for initiation of neonate feeding. From these observations, we classified From this baseline information, we lesions into four types by shape developed a new screening technique (pinhole, circular, elongated, and C1 C2 C3 uniform–to–irregular) and then by size (Fig. 2). Within the elongated and the uniform–to–irregular shaped lesions, we established three size groups (small, mid–sized, and large). For elongated lesions, size was based on length (small = <1.3 cm, mid–sized = 1.3–2.5 cm, and large = >2.5 cm), whereas for the Figure 2. Types of lesions made by SWCB larvae feeding on ear husk– leaves and leaf sheaths: A, pinhole; B, small circular; C1, small, elongated; C2, mid–sized, elongated; C3, large, elongated; D1, small, uniform–to– irregular; D2, mid–sized, uniform–to– irregular; and D3, large, uniform–to– irregular. utilizing rating scales for visually scoring larval feeding damage on the leaf sheath and husks (Tables 1 and 2, respectively). Rating scores are based on the type and number of lesions observed 14 days after infestation, and separate degrees of damage but also uniform–to–irregular lesions, size was based on diameter. Lesions 17 mm in diameter or smaller (about the size of a Table 1. Visual scale for rating the degree of damage caused by SWCB larvae to leaf sheaths of post-anthesis stage maize plants. US dime — the $0.10 coin) were Score considered “small;” lesions up to 22 mm in diameter, “mid–sized;” and those 23 mm (about the size of a U.S. quarter) in diameter or larger, “large”. Lesions at the base of the husk–leaves were considered as belonging to the elongated lesion group. Seven and 14 days after releasing neonates in the axil of the top ear leaf of anthesis stage plants, approximately 70% or more of the larvae recovered from these plants were feeding on tissues of the top ear and its associated 0 1 2 3 4 5 6 7 8 9 Description No visible damage. Only pinhole lesions. Pinholes plus a few small, circular lesions. Pinholes and small, circular lesions or a few small, elongated lesions, or both. Several to many small, elongated lesions or up to several mid-sized, elongated lesions, or both. Mid-sized, elongated lesions plus a few large, elongated lesions or small uniform-to-irregular lesions or a combination. Several large, elongated lesions or several small to a few mid-sized, uniformto- irregular lesions, or both. Many large, elongated lesions or small, uniform-to-irregular lesions, or several mid-sized to a few large, uniform-to-irregular lesions, or a combination. Elongated lesions of all sizes or small to mid-sized, uniform-to-irregular lesions, or several large, uniform-to-irregular lesions, or a combination. Many lesions of all types present. Lesion numbers: Few = 1 to 3; several = 4 to 6; many = 7 or more. Taken from Davis and Williams (1994). 192 F.M. DAVIS AND W.P. WILLIAMS reflect the plant’s effects on insect ratings, 0.63; survival and husk–leaf We conducted another experiment to survival and growth. The scales were ratings, 0.72; growth and leaf sheath determine the effect of the modeled on the 1–9 scale developed by ratings, 0.40; and growth and husk–leaf physiological age of the reproduction- Guthrie et al. (1960) for evaluating ratings, 0.56. Also, significant stage plant on larval survival and maize for leaf feeding resistance to the differences in larval survival and growth (Davis and Williams 1994). ECB. The rationale for having separate growth were found among larvae Significant differences were found in rating scales for leaf sheaths and husk– reared on some test hybrids. When this larval survival and growth when maize leaves was that the tissues are different occurred, differences in rating scores hybrids were infested on the same day, and that one tissue might possess among hybrids also were found to be but at different physiological stages. resistant factors while the other might significant. Therefore, we concluded We have decided to infest our test not. Also, the rating of husk–leaves that the leaf sheath and husk–leaf genotypes as each reaches a pre– involves multiple husk–leaves from an ratings were successfully measuring selected physiological stage. This ear. differences in damage as reflected by procedure of timing infestations is rates of larval survival and growth and especially important when genotype Because resistant genotypes were that this evaluation technique had maturities are unknown. unknown, we had to test the utility of potential. Rating leaf sheath and husk– the new rating scales for separating leaf damage on plants 2 weeks after Based on our experience in Mississippi, resistant from susceptible plants by infestation solves two of the our protocol for screening hybrid conducting experiments that simulated aforementioned problems. Evaluation genotypes using the new technique is different rates of larval survival and occurs sooner after infestation, thus as follows: growth. The methodology and results minimizing the confounding effects • of these experiments have been that abiotic and biotic factors may have published (Davis and Williams 1994). on larval numbers, growth, and Here is a brief summary of the results damage when evaluation occurs 5–7 and conclusions from these studies. weeks after infestation. Also, this neonates (preferably split Highly significant correlation’s were evaluation technique measures feeding applications of 30 neonates on found between larval survival and damage to sheath and husk–leaf tissue consecutive days) released in the growth (weights) and both rating by larvae without the influence of their axil of the top ear leaf using the diapause state. ‘bazooka’ method (Mihm 1983; scales. The r2 values were as follows: between survival and leaf sheath Genotypes are infested 7 days after 50% of the plants in a row reach anthesis. • Each plant is infested with 60 Davis et al. 1989). Table 2. Visual scale for rating the degree of damage caused by SWCB larvae to husk– leaves of the top ear. Larval damage on the three outermost Score associated leaf sheath of each plant is Description husk–leaves of the top ear and its evaluated 14 days after infestation by 0 1 2 3 4 5 6 7 8 9 No visible damage. Only pinhole lesions. Pinholes plus a few small, circular lesions. Pinholes and small, circular lesions common on husk-leaves or a few small, elongated lesions, or both. Several to many small, elongated lesions or up to several mid-sized, elongated lesions. Many mid-sized, elongated lesions or a few large, elongated lesions, or a few small, uniform-to-irregular lesions, or a combination. Several large, elongated lesions or a few small or mid-sized, uniform-toirregular lesions, or both. Many large, elongated lesions or small to mid-sized, uniform-to-irregular lesions, or a few large, uniform-to-irregular lesions, or a combination. Many lesions of all types on two of the three husk-leaves. Many lesions of all types on each of the husk-leaves. Lesion numbers: Few = 1 to 3; several = 4 to 6; many = 7 or more. Taken from Davis and Williams (1994). visual scoring using the rating scales presented in Tables 1 and 2. When the top ear is accompanied by small immature ears that also originate from the primary ear node, the rater must consider the extent of damage to them before arriving at a final score for the husk–leaves. Damage to these small ears is determined by counting entrance and exit holes and may or may not influence the final score. However, if the larvae preferred feeding within these ears instead of the 193 A NEW TECHNIQUE FOR EVALUATING SOUTHWESTERN CORN BORER DAMAGE TO POST-ANTHESIS MAIZE husk–leaves of the main ear, the score RCB with two or three replications. resistant genotypes have been reflects the extent of damage to these Each genotype is represented in each identified. If the potentially resistant ears. replication by a single row of 15 plants. genotypes are confirmed as having Rows are 5.08 m long with 0.97 m resistance, the breeding process for Evaluation data can be taken directly in between rows. Data on leaf sheath and developing germplasm for release will the field 14 DAI or delayed by husk–leaf ratings are taken from 10 continue using techniques appropriate collecting the leaf sheath, husk–leaves plants per row. These data are for that germplasm. and small inner ear samples from the analyzed using ANOVA and means are plants, and placing these tissues in pre– separated using the least significant labled plastic bags and freezing them. difference test (P=0.05). Acknow le dgm e nt s The samples are thawed, when convenient, and rated visually for The authors appreciate the technical assistance of Thomas Oswalt, Susan Progre ss damage by placing them on a light Wolf, and Paul Buckley in developing table similar to those used to view We are presently screening maize the new evaluation technique. Thanks photographic slides. The light table during post-anthesis using leaf sheath are also extended to Edna Carraway helps the rater see the larval feeding and husk–leaf ratings (Davis and for manuscript preparation assistance. signs clearly. When rating leaf sheaths Williams 1994). Additionally, we This article is a contribution of the or husk–leaves, the rater should first (primarily Williams) have developed a Crop Science Research Laboratory, identify the lesion types present and laboratory bioassay using lyophilized Agricultural Research Service, U.S. then consider lesion numbers. The husk diets for screening. These two Department of Agriculture in most severe lesion type(s) immediately techniques should complement each cooperation with the Mississippi indicates to the rater the approximate other. Agricultural and Forestry Experiment Station. It is published with approval score. A final score can be obtained quickly by estimating the numbers of A few inbred lines have been identified of both agencies as Journal no. PS-8636 each lesion type. The amount of time as consistently susceptible (e.g. GE333). of the Mississippi Agricultural and required for an experienced rater to Also, a few candidates (e.g. Mp89:5459) Forestry Experiment Station. score a plant in the field is ca. 30 have shown potential resistance. seconds and, in the laboratory, Ratings for test inbreds GE333 and approximately 1 minute (includes Mp89:5459 are presented in Table 3. removing tissues from plastic bags and These data indicate the range of arranging them on the light table). differences that we have observed among inbreds. Our protocol for screening inbred genotypes differs slightly from that We feel we are making progress since used for hybrids, primarily because the susceptible checks and potential Re fe re nce s Davis, F.M., and W.P. Williams. 1994. Evaluation of reproductive stage maize for resistance to the southwestern corn borer (Lepidoptera: Pyralidae) using visual rating scores of leaf sheath and husk damage. J. Econ. Entomol. 87: 1105–1112. inbreds senesce rapidly after anthesis. Inbreds are infested with 45 instead of 60 neonates per plant when most of the plants in a row are in the anthesis Table 3. Evaluation of effects of feeding by 45 SWCB neonate larvae on 2 maize inbred lines at anthesis , 14 days after infestation. stage. Normally, infestations are split Means Damage ratings (1-9) over 2 consecutive days (30 larvae the first day and 15 larvae the next day). This mediates the effects of unusual environmental stresses or unfavorable events. Our experimental design of choice for screening post-anthesis stage plants is a Inbred GE333 Mp89:5459 LSD (P=0.05) Survival/plant Larval weight (mg) Leaf sheath Husk–leaves 4.5 3.0 1.3 69.3 35.1 13.7 4.6 3.6 0.8 6.4 4.2 0.9 Experimental design: RCB with 3 replications. Data were taken on 10 plants per genotype per replicate. Plants of these inbreds were infested on the same day. 194 F.M. DAVIS AND W.P. WILLIAMS Davis, F.M., C.A. Henderson, and G.E. Scott. 1972. Movements and feeding of larvae of the southwestern corn borer on two stages of corn growth. J. Econ. Entomol. 65: 519–521. Davis, F.M., W.P. Williams, and B.R. Wiseman. 1989. Methods used in screening and determining mechanisms of resistance to the southwestern corn borer and fall armyworm. In Toward Insect Resistant Maize for the Third World: Proceedings of the International Symposium on Methodologies for Developing Host Plant Resistance to Maize Insects, 101–108. Mexico, D.F.: CIMMYT. Guthrie, W.D., F.F. Dicke, and C.R. Neiswander. 1960. Leaf and sheath feeding resistance to the European corn borer in eight lines of dent corn. Ohio Agric. Exp. Stn. Res. Bull. 860. Guthrie, W.D., W.A. Russell, G.L. Reed, A.R. Hallauer, and D.F. Cox. 1978. Methods of evaluating maize for sheath– collar feeding resistance to the European corn borer. Maydica 23: 45–53. Mihm, J.A. 1983. Techniques for efficient mass rearing and infestation in screening for plant resistance to Diatraea sp. maize borers. Technical Bulletin. Mexico D.F.: CIMMYT. Williams, W.P., and F.M. Davis. 1989. Breeding for resistance in maize to southwestern corn borer and fall armyworm. In Toward Insect Resistant Maize for the Third World: Proceedings of the International Symposium on Methodologies for Developing Host Plant Resistance to Maize Insects, 207–210. Mexico D.F.: CIMMYT. Williams, W.P., F.M. Davis, and G.L. Windham. 1990. Registration of Mp708 germplasm line of maize. Crop Sci. 30: 757. Assessing Damage by Second-Generation Southwestern Corn Borer and Sugarcane Borer and Development of Sources of Resistance in Tropical and Subtropical M aize H. Kumar, CIMMYT, Mexico J.A. Mihm, French Agricultural Research Inc., Lamberton. MN, USA. Abst r a c t In 1992, having achieved adequate levels of resistance to first-generation (whorl stage attack) Diatraea spp. borers, Population 391 was formed to attempt to identify sources of resistance to second-generation (post anthesis stage) attack. The ultimate objective is to develop complete cycle (planting to harvest) resistance to the two most important stem borer species that attack maize in the American subtropical and tropical growing areas. Plants were infested at anthesis +/- 1 week with Sugacane borer (SCB), Diatraea saccharalis Fabricius, at Poza Rica (CIMMYT’s tropical lowland station) or Southwestern Corn Borer (SWCB), Diatraea grandiosella Dyar, at Tlaltizapan (CIMMYT’s subtropical station) in the ear zone (Ear leaf, one leaf above and below the ear) with 60-65 larvae larvae per plant. Selection was carried out over the next three cycles using one or several criteria (ear damage, sheath damage, stalk damage - indicated by the number of internodes tunneled) and compared and correlated with data from sub-samples rated for sheath and husk damage. For SCB in tropical environments, there was a marked and obvious preference for the larvae to attack the developing ears. The correlations between sheath damage and stalk damage were not significant, but those between ear damage and stalk damage were significant. However, the relationships were highly genotype specific. For SWCB, in subtropical environments, damage directly to the ears, sheath and husks was not so striking, so selection was based on stalk damage. The best lines were recombined at S3 levels, and the second cycle of S1 recurrent selection has begun, while the elite fraction is now available as S4 lines for further testing. Our data show sufficient variability to forestall concluding that there is a single best method to select for multiple species, second-generation resistance in tropical maize. Int roduct ion have been developed through the joint parts of the plants (i.e., leaf sheath, efforts of breeders and entomologists stalk, husk, ear peduncle and ear). To Maize, Zea mays L., is an important (Williams and Davis 1989; Smith et screen maize for resistance to second- food and fodder crop throughout the al.1989). The International Maize and generation stem borers, we did not world. In several developing countries Wheat Improvement Center (CIMMYT) know whether damage to all parts of Africa and Asia, maize is a major has an active program which has attacked by stem borers should be staple food of millions of people. Of the developed maize germplasm with a assessed or whether the selections various insect pests attacking maize, desirable level of resistance, in whorl could be based solely on damage to the stem borers are the most important, stage maize, to first-generation stem most important part of the plant. Davis causing severe yield losses at the whorl borers. However, information on and Williams (1994) developed a rating (Sarup et al. 1977; Smith et al. 1989, resistance in maize to second- scale based on damage by stem borers Seshu Reddy and Sum 1991) and generation stem borers is limited, and to leaf sheath for selecting maize anthesis (Kumar and Asino 1994) sources are few. Stem borer attack at genotypes for resistance to second- stages of maize. Many maize genotypes anthesis is complicated, because generation stem borers. However, resistant to first-generation stem borers damage is caused to several different given the multi-faceted nature of stem 196 H. KUMAR AND J.A. MIHM borer attack it seems prudent to Experiment 1 colony to maintain the vigor of the determine, firstly, if there is any For this experiment, the two hybrids laboratory reared insects. The neonates correlation among different parts of the were planted in a split plot design with were mixed with maize cob grits and plant damaged by the borers and variety as the main plot and the placed in the axil of the EL, -EL and secondly, to assess which tissue, if treatment as the subplot, with three +EL of the plant with a mechanical damaged, leads to maximum loss of replicates. The treatment involved dispenser called a ‘bazooka’ (Fig. 2). At grain yield. The first objective of this infestations at three leaves, the ear leaf the time of harvest, 10 plants from each study was to examine the relationships (EL), the leaf below the ear (-EL) and plot were uprooted. The ear leaf, leaf between damage to different parts of the leaf above the ear (+EL) (Fig. 1). below the ear and leaf above the ear the plants by Southwestern corn borer Plots consisted of single row plots, 2.5 were removed from each plant. The (SWCB), Diatraea grandiosella (Dyar) m long with 12 plants. Row-to-row and damage caused by the stem borers to and sugarcane borer (SCB), Diatraea plant-to-plant spacing was 75 cm and saccharalis Fabricius. Recently, work 25 cm, respectively. Plots were was also initiated to identify sources of fertilized with phosphorous at the rate resistance to second-generation stem of 50 kg/ha before planting and borers and the second objective of this nitrogen at the rate of 150 kg/ha in study was to provide information on split doses of half before planting and the progress made in this area. half 6 weeks later. M aterials and M ethods When 50% plants of each hybrid had reached anthesis, each plant was Experiments for this study were infested with 60-65 neonate larvae per conducted at CIMMYT’s research plant. The plants of the two hybrids at stations at Tlaltizapan (18º 41’N; 940 m Tlaltizapan were infested with SWCB elevation) and Poza Rica (20º 30' N, 50 and those at Poza Rica were infested m elevation) in the summer and winter with SCB. The insects used in this cycles of 1993 and 1994. In order to study were obtained from laboratory examine relationships between cultures of SWCB and SCB maintained different types of damage caused by on artificial diets as described by Mihm stem borers, two single cross hybrids (1989). After every 10 generations, field Ki3 x CML131 (susceptible) and CML67 collected adults were infused into the x CML135 (resistant) with known level +EL EL -EL Figure 1. Maize plant showing three sites of infestation by the borers. of resistance to first-generation stem borers were used. Two experiments were conducted at each location. For each experiment, the seeds of each hybrid were treated with the insecticides, Carbofuradan 27.5% (FMC Agroquimica de Mexico), Semevin (a.i. Thiodicarb 31.5%, Rhone- Poulenc Agro. Mexico) and Gaucho (a.i. Imidacloprid 70%, Bayer, Mexico). The seeds were treated at the rate of 350 g. a.i./ ha to protect seedlings from the attack of soil insects. In all the experiments, “zero tillage” was used and the trials were planted with a ALMACO planter (Model CTS, EODF, Nevada, U.S.A). Figure 2. “Bazooka” used for infesting anthesis stage maize with stem borers. ASSESSING DAMAGE BY SECOND-GENERATION SOUTHWESTERN CORN BORER AND SUGARCANE BORER 197 the three leaf sheaths was assessed Experiment 2 Guatemalan hybrids, best hybrids from using sheath damage rating scale of 1-9 The objective of this experiment was to CIMMYT’s lowland hybrid program, modified from that devised by Davis examine whether damage caused by hybrids Ki3 x CML 131, CML67 x and Williams (1994) as follows: 1 = no the stem borers to two hybrids would CML135, CML135 x CML139, CML61 x visible damage; 2 = only pinhole vary under artificial infestation applied CML69, Pop. 590 (MBR) and Pop. 590B lesions; 3 = pinholes plus a few small at different silking stage or time of day. (MBR-MDR). These source materials circular lesions; 4 = small circular The hybrids Ki3 x CML131 and CML67 were planted at Poza Rica station in the lesions and a few elongated lesions (< 1 x CML135 were planted in a split-split summer cycle of 1992 in two cm in size); 5 = mid-sized elongated plot design in a randomized complete replications. Trials were planted in zero lesions plus a small irregular shaped block design. The variety was the main tillage plots, using a ALMACO planter lesions; 6 = few elongated lesions (1 cm plot, the silking stage was the sub plot (Model CTS, EODF, Nevada, USA.). long) with a few mid-sized irregular and the time of day was the sub-sub Single row plots were 2.5 m long The shaped lesions; 7 = several elongated plot. The three silking stages utilized plants were infested with 40-50 SCB (1 cm long) and several mid-sized for this experiment were pre-silk larvae at the time when 50% plants had irregular shaped lesions; 8 = elongated (emergence of ear shoots), green silk (a flowered. The larvae were placed in the lesions of all sizes and a few large week after the silk emergence) and the leaf axil with a bazooka as described irregular shaped lesions; 9 = elongated brown silk (the drying of the silks). above. About 5-6 plants from each row lesions of all sizes and large irregular Each silking stage of a hybrid was were selfed to generate S1 lines. At the shaped lesions spread on the whole leaf infested at 8:00 a.m., 12:00 noon and time of harvest, the stems of selected sheath. 4:00 p.m. The larvae of SCB and SWCB plants were split along their length and were used at Poza Rica and the number of internodes tunneled in The primary ear (counting from the Tlaltizapan, respectively. The plants each plant was recorded. The plants top) of each plant was removed and the were infested with 60-65 larvae per with less damage (< 4 internodes damage caused by the borers to the plant, as described above. At the time tunneled) were selected and planted in husks was assessed on the basis of a of harvest, the sheath damage, husk the subsequent planting cycle . The S2 rating scale of 1-9 as described above, damage, ear damage and stalk damage lines selected under insect infestation but the assessment of damage was was assessed for each plant separately, were then planted both at Tlaltizapan based on feeding lesions of the borers as described above. Data were and Poza Rica and infested with SWCB on 2-3 husk leaves rather than only one subjected to factorial analysis and and SCB, respectively. The S3 lines (Davis and Williams 1994). The ear correlation’s were calculated between selected at the two locations were damage was evaluated on the basis of a sheath damage and stalk damage, husk advanced to S4 and recombinations rating scale 0-10, with 0 indicating no damage and stalk damage, ear damage were also made among the S3 lines to damage to the ear by the insects and 10 and stalk damage. start another cycle of selection. The S3 lines were also evaluated for sheath indicating 100% of the grains damaged Breeding for resistance to second-generation stem borers damage, stalk damage, husk damage, was split and the length of the tunnels made by the borers was measured. The The source germplasm used for the correlation’s among the parameters. sheath damage, husk damage, ear development of resistance to second- damage and the stalk damage of each generation stem borers was genetically plant were measured together, so diverse, with known level of resistance keeping the data for each plant to first-generation stem borers and Experiment 1 separate from the others. Two years of good agronomic traits. The notable When the two hybrids were infested data were combined and subjected to sources used were the best lines from with SCB at Poza Rica with 60-65 larvae factorial analysis (MSTAT-C, 1989).The population 390 (MIRT), selections of per plant at the ear leaf (EL), leaf below correlation’s were calculated between the Antigua landrace from the the ear (-EL) and leaf above the ear sheath damage and stalk damage, ear germplasm bank, the variety Across (+EL), the ANOVA showed that damage vs. stalk damage and husk 90390, Pop. 8523, Dekalb hybrids 810, genotype x site of infestation damage vs. stalk damage. 830,833, 840, 844, 555, SMC-305, interaction was not significant. When by the borers. The stalk of each plant and ear damage to examine the Results and Discussion 198 H. KUMAR AND J.A. MIHM infested at three leaves, damage by SCB hybrids were infested at the three significant (Table 5). Genotypes to leaf sheath above the ear (+ EL) and leaves. The correlation’s between differed significantly in terms of ear the stalks of the two hybrids differed different parameters were again varied damage and stalk damage. Stalk significantly (Table 1). When infested at according to the parameters and the damage also differed according to the the axil of the ear leaf, the differences in hybrid (Table 4). These observations silking stage at infestation and time of damage by SCB to the three leaf indicate that assessment of damage on day. The correlations between the ear sheaths were significant. The damage different parts of the plants at anthesis damage and the stalk damage were by SCB to the leaf sheath of the infested is quite independent of one another. significant irrespective of the silking stage at infestation. (Table 6). leaf axil was always greater than that of the other two leaf sheaths. The husk Experiment 2 and ear damage also differed When the two hybrids were infested When the two hybrids were infested significantly when infested at three with SCB at different silking stage and with SWCB at the three silking stages leaves of the maize plant at anthesis. time of the day, genotype x silking and time of day, genotype x silking However, the stalk damage remained stage x time of day interaction was not stage x time of day interaction was not the same at each of the three infestations. These results indicate that the SCB larvae move to the leaf sheath Table 1. ANOVA for damage by D. saccharalis to maize hybrids, infestation on three leaves at anthesis. of the leaf where they hatch from the Mean squares for damage eggs laid by the females and feed therein. When infested at the ear leaf, Source Ear sheath the correlation between ear damage and the stalk damage was highly significant for the hybrid Ki3 x CML131, but not for CML67 x CML135 Genotype (A) Site of infestation (B) AB Error 0 6.38* 0.36 0.55 -Ear sheath 0.04NS 7.93* 0.77NS 0.66 +Ear sheath Husk 4.00* 19.62** 1.06NS 1.12 1.73* 1.82* 0.44 0.40 Ear 1.14NS 3.70* 1.03NS 0.87 Stalk 568.03** 11.13NS 48.77NS 29.78 (Table 2) indicating that ear damage can replace the tedious procedure of maize evaluation by assessing stalk damage. Also, infestations of the maize Table 2. Correlation matrices of damage by D. saccharalis on two hybrids, infestation on three leaves at anthesis. Correlation coefficients plants at the leaves below and above the primary ear did not give significant correlation’s between the sheath Site of infestation Ear sheath vs stalk -Ear sheath vs stalk + Ear sheath vs stalk Husk vs stalk Ear vs stalk damage and the stalk damage in any of Genotype the hybrids. There was a significant Ki3 x CML131 Ear leaf - Ear leaf + Ear leaf 0.016 0.041 -0.12 -0.16 0.15 0.08 0.27* 0.07 0.27* -0.12 0.18 0.29* 0.44** 0.08 0.21 CML67 x CML135 Ear leaf - Ear leaf + Ear leaf -0.14 0.14 0.19 -0.19 0.04 -0.14 0.04 0.05 0.09 0.15 0.12 -0.06 0.08 0.20 0.38** correlation between leaf sheath above the ear and the stalk, but this was also not consistent between the two hybrids (Table 2). When the two hybrids were infested at a n = 60 plants. EL, -EL and +EL with SWCB at Tlaltizapan, the factorial ANOVA did not show genotype x site of infestation Table 3. ANOVA for damage by D. grandiosella to maize hybrids, infestation on three leaves at anthesis. interaction for any of the damage Mean squares for damage to different parts parameters (Table 3). Genotypes differed significantly in terms of ear sheath damage, ear damage and stalk damage. The sheath damage and the stalk damage also differed when the Source Ear sheath -Ear sheath Genotype (A) Site of infestation (B) AB Error 0.04NS 2.27** 0.39NS 0.28 0.28NS 7.57** 0.28NS 0.43 +Ear sheath 1.69* 6.84** 0.29NS 0.18NS Husk 0.94NS 0.24NS 0.33NS 0.35NS Ear 1.44* 0.30NS 0.17NS 0.89NS Stalk 146.8* 111.7* 37.9NS 20.1 ASSESSING DAMAGE BY SECOND-GENERATION SOUTHWESTERN CORN BORER AND SUGARCANE BORER 199 significant (Table 7). The two hybrids Thus, in the absence of clear-cut, demonstrated to cause yield reductions differed significantly in terms of husk, consistent correlations between sheath in maize (Kumar 1988; Kumar and ear and stalk damage. The damage damage and stalk damage or between Asino 1994). Stalk damage due to stem caused by SWCB to the hybrids also ear damage and stalk damage, the borers has also been used to select differed according to the silking stage selection of maize genotypes for maize genotypes resistant to second- at infestation. Infestations at different resistance to second-generation stem generation European corn borer, times of day did not affect damage by borers should continue to be based on Ostrinia nubilalis Hübner (Guthrie and SWCB, except for sheath damage. stalk damage, which has been Russell 1989). However, for other damage parameters to be useful in the Table 4. Correlation matrices of damage by D. grandiosella on two hybrids, infestation on three leaves at anthesis. Genotype Ki3 x CML131 borers, their role in determining grain yield of the plant will have to be Correlation coefficients Ear Site of sheath infestation vs stalk selection of maize resistant to stem demonstrated. -Ear sheath vs stalk + Ear sheath vs stalk Husk vs stalk Ear vs stalk Ear leaf - Ear leaf + Ear leaf -0.04 0.04 -0.06 -0.13 0.28 0.09 0.14 0.28 -0.13 0.14 0.35 0.16 -0.22 0.41 -0.27 Ear leaf - Ear leaf + Ear leaf 0.14 0.24 -0.09 0.09 0.24 0.07 0.12 0.24 0.52 0.43 0.11 0.16 -0.21 0.032 0.20 Breeding for resistance to second-generation stem borers When the genetically diverse germplasm, with known resistance to CML67 x CML135 first-generation stem borers and good agronomic traits, was infested with SCB at Poza Rica, 259 S1 lines were Table 5. ANOVA for ear damage and stalk damage by D. saccharalis on two hybrids, infestation at three silking stages and three different times of day. Source df Genotype (A) Silking stage at infestation (B) Time of day (C) ABC Error Ear damage 1 2 2 4 85 Stalk damage 32.12** 0.03NS 0.35NS 0.85NS 2.28 1089.97** 179.26* 97.29 32.03NS 42.31 selected based on a low number of internodes damaged by the borers (Fig. 3). The S1 lines were planted at Poza Rica and infested at anthesis with SCB. At harvest, on the basis of a low number of internodes tunneled by the borers, 314 S2 lines were selected. These S2 lines were then planted at Poza Rica and Tlaltizapan and were infested with Table 6. Correlation matrix of ear damage and stalk damage by D. saccharalis. Genotype Ki3 x CML131 CML67 x CML135 Silking stage r Significance n Pre-silk Green-silk Brown silk Pre-silk Green silk Brown silk 0.42 0.25 0.49 0.29 0.24 0.57 ** * ** * * ** 45 73 84 71 88 66 SCB and SWCB, respectively. At Poza Rica 369 and at Tlaltizapan 360 S3 lines were selected on the basis of low number of internodes tunneled by the borers. These S3 lines, at both locations, were planted in two replicates and infested with neonate larvae at anthesis. In the first replicate, random crosses were made among the selected lines and in the second replicate, the Table 7. ANOVA for damage by D. grandiosella to maize infestation at three silking stages, different times of the day. plants were selfed to generate S4 lines. Almost 30 randomly selected S3 lines Mean squares for damage Source df Sheath Husk Genotype (A) Silking stage at infestation (B) Time of the day (C) AxBxC Error 1 2 2 4 34 0.02NS 8.97** 0.24** 0.23NS 0.145 2.85* 19.65** 0.48NS 0.09NS 0.31 Ear 8.80** 0.37* 0.14NS 0.10NS 0.13 Stalk 596.67** 116.90* 4.92NS 31.22NS 18.30 were also sampled at each location and were evaluated for sheath damage, ear damage and stalk damage. Correlations were then calculated between sheath damage and stalk damage, and ear damage and stalk damage for the 200 H. KUMAR AND J.A. MIHM plants infested with SCB. These data SCB and SWCB (Figs. 4 and 5). The lines. (Table 8). These data again could not be collected in the plants correlations of leaf sheath vs. stalk showed that damage caused by stem infested with SWCB due to poor grain damage, husk vs. stalk damage were borers to different reproductive parts of formation in the ears. There were generally not significant, but the maize is independent of damage to significant differences among the S3 correlation between ear damage and others and observed relationships are lines in sheath damage, stalk damage stalk damage were significant in some highly genotype-specific. and ear damage on plants infested with S3 lines, but non-significant in the other In Poza Rica, 283 full sib families and Selection for Resistance to Second Generation D. saccharalis Fabricius and D. grandiosella (Dyar) PR-92B Genetically diverse germplasm with known resistance to first generation stem borers and agronomic traits planted, infested and selected for resistance to stalk damage (MIRT, Antiguas de banco de germ., Across 90390 W and Y, Pop 8523, Dekalb 810, 830, and 833, 840, 844, 555, SMC - 305, Guatemalan Hybrids, Low Land Tropical Program, KI3XCML131‘, CML67XCML135, CML135XCML139, CMLM61XCML69 and several crosses From POP MBR AND MDR) 81 S4 lines were harvested, while at Tlaltizapan, 78 full sib families and 74 S4 lines were generated. In the summer planting cycle of 1994, 283 full sib families and 81 S4 lines were planted at Poza Rica station and infested with SCB at the whorl stage (6-7 leaf stage) and at anthesis in separate trials. Based on leaf feeding damage by the firstgeneration stem borers and stalk damage by the second-generation stem PR-93A 259 S-1 lines planted, infested and selections made based on stalk damage PR-93B TL-93B 314 S-2 lines planted, infested and selections made on the basis of stalk damage PR-94A TL-94A 369 S-3 lines planted in two replications 360 S-3 lines planted in two replications borers, 97 S1 lines and 18 S5 lines were selected having resistance to both generations of SCB. Also, 283 S1 lines were selected with resistance to only second-generation SCB. In Tlaltizapan, 78 full-sib families and 74 S4 lines were planted and infested with SWCB at whorl and anthesis stage maize. 137 S1 Rep. 1 Recombinations made among he S-3 lines Rep. 2 Selfed to generate S-4 lines lines and 13 S5 lines were selected on the basis of resistance to first-and second-generation stem borers. Also, 263 S1 lines and 72 S5 lines were also PR-94B 283 full sib families planted and infested at whorl and at anthesis stage; 97 S-1 lines selected based on resistance o first and second generation larvae generation larvae. 81 S-4 lines planted and infested; 18 lines selected with resistance to first and second. selected for resistance to secondgeneration SWCB. Thus, of the large amount of germplasm with known levels of resistance to first-generation stem borers, a very low number of lines continue to show resistance to firstgeneration stem borer. In the process of TL-94B 78 full sib families planted and infested at whorl and anthesis stage. 137 S-1 lines selected on the basis of resistance to first and second gen. and 263 S-1 lines selected on the basis of resistance to 2nd brood only. 74 S-4 lines planted and infested; 13 S-5 lines selected on the basis of resistance to 1st and 2nd brood and 72 S-5 lines for res. to 2nd brood only. selection for resistance to secondgeneration stem borer attacks, it seems that a large pool of genes was eliminated and that entirely different types of genes seem to control resistance to the two generations of the PR-95A Continue selection Continue selection Figure 3. Schematic diagram showing the operations and breeding methodology used in developing populations and inbred lines for resistance to second-generation stem borers. stem borers. ASSESSING DAMAGE BY SECOND-GENERATION SOUTHWESTERN CORN BORER AND SUGARCANE BORER 4 3.5 3 2.5 2 1.5 1 0.5 0 5 4 3 2 1 0 ; ; ; ; ; ; ; ; ;; ; ; ;; ; ; ;; ; ; ;; ; ; ;; ; ; ;; ; ; ;; ; ; ;; ; ;; ; ; ; ; ; ;; ; ; ; ;; ; ; ;; ; ;; ; ;; ;;; ;;;; ;;; ;;; ; Conclusions Sheath Damage Stalk Damage 2 high (> 60 larvae/plant) to get In view of the highly variable adequate establishment of the larvae in correlations among the the leaf sheaths and ear husks. the selections in maize for Thus, using stalk damage by the stem resistance to second- borers as a selection parameter, two generation stem borers will populations of maize have been continue to be made on the synthesized with genes resistant to basis of stalk damage by the second-generation SCB and SWCB, stem borers. The infestation of respectively. Preliminary results also maize with SCB revealed show that we are in the process of adequate establishment of the developing inbreds and populations larvae in the leaf sheath, ear which have high gene frequencies for husks and stems as indicated both types of resistance. by the damage to these parts F=0.99;P>0.05 Acknow le dgm e nt s of the plants .The establishment of the SWCB larvae in the leaf sheaths and The authors wish to thank the Director ear husks was low, but and Associate Director of the CIMMYT damage to the stems of the Maize Program for providing the plants was moderate, thus facilities to carry out this work. The facilitating the separation of financial support provided by the the resistant and susceptible UNDP Project no. GLO/90/003/A/01/ genotypes. It seems that the 42 is gratefully acknowledged. ;;; ; ;; ; ; ;;; ;; ; ; ;; ; ;; ; ; ;; ;;; ; ; ;; ; ;; ; ; ;; ;;;; ;;;;; ;;;;;; ; ;;; ; ; ;; ;; ; ;;; ; ; ;; ; ;; ; ;; ; ;; ;;;; ;;; ;;;; ; ;;; ;; ; ; ;; ; ;; ; ; ;; ;;; ; ; ;; ; ;; ; ; ;; ;;;; ;;; ;;;; ; Stalk 5 3 plants at Tlaltizapan will have to be different damage parameters, Figure 4. Sheath and stalk damage by SWCB to selected S3 lines of Population 391. 4 201 infestation level of SWCB on 1 0 Ear 4 Table 8. Correlation matrices of damage by D. saccharalis on selected S3 lines of population 391 at Poza Rica. Family n Sheath vs. stalk Husk vs. stalk Ear vs. stalk 1 10 20 30 40 49 70 80 89 120 130 140 150 180 200 210 223 230 250 260 271 280 291 299 18 10 18 20 20 20 20 20 20 17 16 20 20 17 20 18 20 20 19 20 20 17 10 14 -0.178 -0.352 0.229 -0.099 0.513* 0.086 0.395 0.29 0.44 0.23 0.12 -0.29 0.106 0.47 0.27 0.40 0.13 -0.129 0.197 -0.075 0.30 -0.021 -0.063 -0.158 0.308 -0.401 0.40 -0.134 0.414 -0.011 0.44 0.102 0.48 0.25 0.25 -0.13 -0.090 0.65 0.24 0.23 -0.035 0.35 0.145 0.381 0.20 0.172 -0.61 0.00 0.468* 0.532NS 0.48* 0.203 0.509* 0.281 0.304 -0.055 0.63** 0.56* 0.06 0.43 0.27 0.63 0.100 0.51* 0.166 0.61* 0.57* 0.37 0.25 0.184 -0.185 -0.104 F = 2.34; DF=21.21; P>0.05 3 2 1 0 Sheath 7 6 5 4 3 2 1 0 Family 1 F = 3.92; DF=21.21; P>0.01 Figure 5. Sheath, ear and stalk damage by SCB on selected S3 lines of Population 391. 202 H. KUMAR AND J.A. MIHM Re fe re nce s Davis, F.M., and W.P. Williams. 1994. Evaluation of reproductive stage maize for resistance to the Southwestern Corn Borer (Lepidoptera : Pyralidae) using visual rating scores of leaf sheath and husk damage. J. Econ. Entomol. 87: 11051112. Guthrie, W.D., and W.A. Russell. 1989. Breeding methodologies and genetic basis of resistance in maize to the European corn borer. In Toward Insect Resistant Maize for the Third World: Proceedings of the International Symposium on Methodologies for Developing Host Plant Resistance to Maize Insects, 192-202. Mexico, D.F.: CIMMYT. Kumar, H. 1988. Effects of stalk damage on growth and yield of certain maize cultivars by the maize stalk borer Chilo partellus. Entomol. Exp. Appl. 46: 149153. Kumar, H., and G.O. Asino. 1994. Grain yield losses in maize (Zea mays L.) genotypes in relation to their resistance against Chilo partellus (Swinhoe) infestation at anthesis. Crop Prot. 13: 136-141. Mihm, J.A. 1989. Mass rearing stem borers, fall armyworms and Corn ear worms at CIMMYT.. In Toward Insect Resistant Maize for the Third World: Proceedings of the International Symposium on Methodologies for Developing Host Plant Resistance to Maize Insects, 5-21. Mexico, D.F.: CIMMYT. MSTAT - C. 1989. A micro computer program for the design, management and analysis of agronomic research experiments. MSTAT Development Team, Michigan State University, East Lansing. Sarup, P., K.K.Marwaha, V.P.S. Panwar, and K.H. Siddiqui. 1977. Studies on insect plant relationship - evaluation of introduction nursery for resistance to the maize stalk borer, Chilo partellus (Swinhoe) under artificial infestation. J. ent. Res. 2: 98-105. Seshu Reddy, K.V.S., and K.O.S. Sum. 1991. Determination of economic injury level of the stem borer Chilo partellus (Swinhoe) in maize. Insect Sci. Applic.12: 269 - 274. Smith, M.E., J.A. Mihm, and D.C. Jewell. 1989. Breeding for multiple resistance to temprate, subtropical and tropical maize insect pests at CIMMYT. In. Toward Insect Resistant Maize for the Third World: Proceedings of the International Symposium on Methodologies for Developing Host Plant Resistance to Maize Insects, 222-234. Mexico D.F.: CIMMYT. Williams, W.P., and F.M. Davis.1989. Breeding for resistance in maize to Southwestern corn borer and Fall armyworm. In. Toward Insect Resistant Maize for the Third World: Proceedings of the International Symposium on Methodologies for Developing Host Plant Resistance to Maize Insects, 207 - 210. Mexico D.F.: CIMMYT. Advances in Rating and Phytochemical Screening for Corn Rootworm Resistance D.J. Moellenbeck, Pioneer Hi-Bred Intl., Johnston, IA, USA. D.J. Bergvinson, Agriculture Canada, Ottawa, Ontario, Canada. B.D. Barry and L.L. Darrah, USDA-ARS, Univ. of MO, Columbia, MO, USA Abst r a c t Evaluating and identifying sources of resistance to the corn rootworm, Diabrotica spp., continues to be a challenge due to subterranean feeding by the larvae and the destructive sampling to evaluate resistance. With the development of artificial infestation techniques, screening for resistance has progressed rapidly. However, evaluation of resistance continues to be labor intensive, with the most accurate rating system requiring root extraction, cleaning, and visual assessment of damage. Because field sampling and evaluation is costly, new evaluation techniques are constantly being evaluated. Refinement of field evaluation techniques using vertical root pulling resistance has increased the amount of corn germplasm that can be evaluated. In addition, consistent preliminary evaluations in the greenhouse and laboratory can reduce the amount of material screened in more costly field evaluations. Greenhouse evaluations have been used successfully to screen both maize germplasm and Tripsacum dactyloides L. for corn rootworm resistance. With the identification of DIMBOA as an antibiosis resistance mechanism, screening for elevated levels of DIMBOA in the roots can now be done on a large scale. Using a hydroponic system, over 100 genotypes a day can be evaluated for hydroxamic acid content and root mass. Genotypes with good root growth and high DIMBOA levels have shown field resistance to both artificial and natural infestations of Diabrotica spp. in sandy-loam and clay soil types. Bioassay systems are presently being developed to further large-scale screening efforts as well as our understanding of resistance mechanisms and feeding behavior of Diabrotica spp. Int roduct ion Barber and the banded cucumber rotation has traditionally been an beetle, D. balteata LeConte, are also effective control strategy. Females only The New World genus Diabrotica pests of several crops in addition to lay eggs near maize, thus, maize contains some of the world’s most maize. planted following a rotation crop will avoid larval feeding damage. However, damaging agricultural insect pests. Among the ten known pest species in Diabrotica beetles are most damaging in populations of NCRW have developed the genus, the western corn rootworm the immature stage. Larvae feed on the extended diapause in areas where a (WCRW), D. virgifera virgifera LeConte, root system of the maize plant. Their maize-soybean rotation is prevalent and the northern corn rootworm feeding activity reduces maize yield by (Krysan et al. 1986; Steffey et al. 1992). (NCRW), D. barberi Smith and interfering with water and nutrient In these populations, the eggs do not Lawrence, are the most important insect uptake. In addition, severe feeding hatch in the first spring following pests affecting maize, Zea mays L., damage often results in root lodging overwintering. Instead, they hatch after production in the United States Corn which can hinder mechanical two winters, thus damaging first year Belt. Metcalf (1986) calculated that these harvesting, further reducing yield. maize. This trait is becoming more widespread, making crop rotation a two corn rootworms (CRW) cost US farmers US$1 billion annually in Pest Diabrotica in the US Corn Belt are treatment expenses and crop losses. generally controlled by crop rotation or Other species, such as the southern corn soil insecticides. Because NCRW and In situations where it is not economical rootworm, D. undecimpunctata howardi WCRW larvae feed only on maize, crop for farmers to rotate crops, insecticides less useful control strategy. 204 D.J. MOELLENBECK, D.J. BERGVINSON, B.D. BARRY AND L.L. DARRAH Field Evaluations are widely used for CRW control. In after feeding damage has occurred, is some years, soil insecticides are applied the only mode of CRW resistance found to 50-60% of the total US maize acreage in commercial maize germplasm. Vertical root pulling strength has long (Metcalf 1986). These insecticide Evaluating maize germplasm for been used to evaluate maize for CRW treatments have generally been resistance to the CRW complex resistance (Ortman et al. 1968). Several effective in protecting maize roots from continues to be a challenge due to the researchers have modified the feeding damage; however, a growing subterranean feeding of the larvae and technique to increase the consistency of number of field reports suggest the destructive sampling methods the scores and reduce the amount of inconsistent performance of soil necessary for evaluations. The labor involved (Beck et al. 1987; insecticides. Problems with variable development of techniques to artificially Donovan et al. 1982; Penny 1981). insecticide degradation (Felsot 1989), infest field plots (Sutter and Branson Using hydraulic power, cable pullers, and insecticide resistance in CRW 1986) have enhanced CRW research and hand-held computers, the Plant (Chio et al. 1978), coupled with considerably; however, evaluations for Genetics Research Unit has taken increasing safety and environmental host plant resistance continue to be vertical root pulling strength on up to concerns of these soil insecticides, point labor intensive and costly. Because of 3,000 plants in one day. Vertical root to a need to reduce soil insecticide use. this, easier and more consistent field pulling strength can be used to To make this possible, host plant techniques are continually being measure maize resistance to CRW resistance will need to be more developed and refined. The most feeding; however, alone it does not predominant in CRW management reliable evaluations of CRW damage differentiate between antibiosis, non- strategies. entails digging plants from the soil, preference or tolerance. Moellenbeck et washing soil off of the root system, and al. (1994) evaluated using differences in Traditionally, host plant resistance has visually assessing damage using a vertical root pulling strength in not played an important role in CRW rating system. These techniques are infested rows compared to the strength management (Levine and Oloumi- widely used, however, because of the in uninfested rows to attempt to Sadeghi 1991), despite 40 years of effort labor and expense involved, they limit separate tolerance from antibiosis and to select for CRW resistance. Melhus et the amount of germplasm that can be non-preference. In that study, two al. (1954) conducted one of the first evaluated in a growing season. Vertical commercial maize hybrids, Pioneer evaluations of CRW resistance and root pulling strength, yield, and other Brand 3377 and Pioneer Brand 3184; found resistance in Guatemalan maize methods of evaluation can potentially two inbred lines, CI31A and SC41R; strains. This resistance was found to be increase the output of a CRW resistance and a B84/Iowa Stiff Stalk Synthetic heritable and transmittable to a screening program. Corresponding with breeding population selected for high susceptible US hybrid. Welch (1977) a field selection program, consistent vertical root pulling resistance, B84R, described a recurrent selection laboratory and greenhouse techniques were tested using paired row vertical program that enhanced CRW resistance can be used to reduce the amount of root pulling strength evaluations. by selecting for low damage ratings. material that is screened in more costly Kahler et al. (1985) released a field evaluations. The ability to rapidly An artificial infester based on the rootworm resistant synthetic selected and consistently evaluate maize model described by Sutter and Branson using row vertical root pulling germplasm before initiating field (1986) was used to distribute the eggs resistance. Unfortunately, the high evaluations can greatly increase the in the plots. Several slight costs of conducting a selection program amount of material that can be modifications were made to their for CRW resistance, inconsistent CRW evaluated. The following techniques, infester. First, two modified anhydrous infestations, difficulties in separating recently developed or refined at the fertilizer knives spaced 25.4 cm apart antibiosis from tolerance, and USDA-ARS Plant Genetics Research were used to ‘knife’ the egg/agar polygenic modes of inheritance have all Unit and the Agriculture Canada Plant suspension into the soil. Flow to each kept CRW resistance from reaching the Research Center, have been used to knife was controlled by an individual marketplace. screen and select maize and maize solenoid that could be activated by the relatives for host plant resistance to the operator. A rotary flow indicator was Currently, tolerance, in the form of WCRW in the field, greenhouse, and placed in the solution line immediately large root systems and root regrowth laboratory. above each knife to monitor solution ADVANCES IN RATING AND PHYTOCHEMICAL SCREENING FOR CORN ROOTWORM RESISTANCE 205 flow. A radar speed detector was also Wet conditions throughout July delayed revealed that depending on climatic and added to accurately monitor ground root pulling until the maize plants soil conditions, 600 eggs per 30.5 cm speed. reached the milk stage. Penney (1981) may not be adequate. The higher found that vertical root pulling strength infestation rate is now recommended to Ideally, infestations are made when differences are greatest when maize is ensure adequate feeding pressure. In plants reach the four-leaf stage to at the milk stage; however, during this test, cultivar ranks were similar at ensure adequate food supply for the pulling at both locations, heavy adult both infestation rates. hatching larvae (Branson and Sutter rootworm populations were noted. 1986); however, it is best to begin Kuhlman et al. (1970) found that the The five cultivars differed in vertical infesting when the plants are in the WCRW pupal stage lasts approximately root pulling resistance at both locations. two-leaf stage to ensure infestations are 10.5 d at 22 °C. Thus, assuming the Vertical root pulling resistance completed by the four-leaf stage. WCRW population was well differences (Table 1) between these Infestations later than the four-leaf synchronized, the cultivars had at least cultivars were expected, because of the stage often result in the plants having a 10 d to recover from any root damage inclusion of commercial hybrids, large root system before the larvae that had occurred. Differences in root inbreds, and a root-strength selected reach the more damaging late instars, pulling strength reductions among the population. At both locations, inbred reducing the amount of damage cultivars may have been caused by lines SC41R and CI31A had lower inflicted on the maize plant. One row differing levels of initial damage, measurements than the other cultivars. of each two-row plot was infested with recovery (regrowth), or both. Calculating the reduction of vertical root 600 or 1,200 eggs per 30.5 cm. The 1,200 eggs per 30.5 cm rate was implemented Combined vertical root pulling pulling resistance of the infested row by infesting 600 eggs per 30.5 cm on resistance averaged 217.7 ± 7.1, 181.1 ± from the control row assesses cultivar each side of the row. For 600 eggs per 9.5, and 163.9 ± 8.3 load-kg per plant for response to CRW infestations, 30.5 cm, only one knife was activated. 0, 600, and 1,200 eggs per 30.5 cm, accounting for differences in their initial The second row of the plot was used as respectively. WCRW infestations vertical root pulling resistance. Across an uninfested control. reduced vertical root pulling resistance all cultivars, rows infested with 1,200 at both locations. The interaction eggs per 30.5 cm had significantly Root damage was evaluated using the between infestation rate and cultivar greater vertical rootpull resistance vertical root pulling resistance (load-kg was not significant at either location. reductions than rows infested with 600 per plant) method described by Beck et The differences between uninfested eggs per 30.5 cm. The cultivars did not al. (1987); however, cable pullers have rows and rows infested with 600 eggs differ in root pulling strength reduction replaced the clamp to reduce stalk per 30.5 cm, and the lack of interactions (Table 1). In terms of percent reduction, breakage. Ten competitive plants between cultivar and infestation rate, the cultivars varied with inbred lines within each row were pulled where indicate that the lower infestation rate is SC41R and CI31A having larger percent possible. Noncompetitive plants or adequate for evaluations. However, reductions than B84R and Pioneer Brand plants adjacent to previously uprooted further studies (unpublished data) 3184. These differences are probably due plants were not used. Cultivar resistance to rootworm damage was evaluated by taking the mean of the ten vertical root pulling resistance observations within an infested row and subtracting it from the mean of the adjacent uninfested row. Percent root pulling resistance differences were calculated by dividing the difference by the root pulling resistance of the uninfested row. Table 1. Vertical root pulling resistance (load-kg per plant) for cultivars at two Missouri locations and combined vertical root pulling resistance reduction (loadkg per plant) due to corn rootworm infestations (from Moellenbeck et al. 1994). Cultivar Location 1 Location 2 Combined reduction a B84R CI31A b Pion. 3184 b Pion. 3377 SC41R 275.7 a 143.8 c 281.8 a 269.2 a 190.5 197.0 b 72.6 d 193.7 b 213.0 a 113.7 45.7 a 40.3 a 34.7 a 51.0 a 54.1 a Means (n=24) within a column followed by the same letter are not significantly different (P = 0.05) a Vertical root pulling resistance of the control row - infested row. Values shown are combined across locations and infestation rates. b Pioneer Brand 206 D.J. MOELLENBECK, D.J. BERGVINSON, B.D. BARRY AND L.L. DARRAH to the level of initial root strength and To combine root rating data with Stratified T. dactyloides seed (c.v. PMK do not correspond to differences in vertical root pulling strength, it is 24) was obtained from Shepherd Farms WCRW feeding. Thus, differences in possible to take root damage ratings of Clifton Hills, MO, and caryopses tolerance, based on vertical root pulling (Hill and Peters 1971; Welch 1977) and were germinated based on procedures resistance of uninfested plants, were secondary root developments ratings described by Kindiger (1994). Emerging found in these cultivars; however, (Rogers et al. 1977) from the pulled seedlings were transplanted into 10 cm differences in antibiotic or antixenotic plants. This allows the researcher to clay pots containing a sand:silt (1:1) resistance were not found. The selected determine if higher vertical root pulling mix and maintained in a greenhouse at breeding population, B84R, and Pioneer strength is caused by less feeding 25 ± 3 °C with a photoperiod of 14:10 Brand 3184 showed the greatest damage (antibiosis), larger root (L:D) h prior to use in two separate tolerance of the cultivars tested. systems (tolerance) or by root regrowth evaluations. (tolerance). Selections can then be The lack of interactions between location based on both favorable root rating One day prior to each infestation, and cultivar for root pulling resistance scores and low root pulling strength WCRW eggs were suspended in glass reduction indicates that cultivar reductions. centrifuge tubes containing 3 ml of a 1.5% agar solution. Each tube contained differences are repeatable. This agrees with the findings of Rogers et al. (1976) who showed repeatability across Greenhouse and Grow th Chamber Evaluations 50 counted WCRW eggs. Egg hatch was estimated at 80% prior to the evaluations. Pots containing 50-d old T. different environments. The LSD for percent root strength reduction was Evaluations of CRW resistance in dactyloides seedlings (n=40) were found to be 10.2%. Thus, the infestation greenhouses and growth chambers can infested with the egg/agar suspension and root pulling strength measurement decrease the cost of a CRW breeding using a pipetter on 4 May 1993. The procedures used in the study can detect program. Preliminary evaluations can suspension was placed 2.5 cm from the small differences among cultivars. be conducted to cull susceptible plant and 5.0 cm deep. Maize plants, material before it is planted in costly planted and infested on the same day, Paired-row evaluations for resistance to and labor intensive field plots. were used as susceptible checks. The the CRW based on vertical root pulling Greenhouse and growth chamber evaluation was conducted in a resistance differences could greatly evaluations have been used extensively Conviron E15 growth chamber at 25 °C increase the number of cultivars that can by the USDA-ARS Plant Genetics day and 20 °C night under a be evaluated in a growing season. Research Unit to evaluate maize and photoperiod of 14:10 (L:D) h. All plants Cultivars selected based upon paired- maize relatives for CRW resistance. The were fertilized until soil saturation with row evaluations could then be more following evaluation of Tripsacum a 250 ppm solution of 20-10-20 (N-P-K) closely evaluated using root damage dactyloides is an example of using a fertilizer every 14 d. ratings. Because larval movement into growth chamber to conduct initial the control rows could reduce the evaluations. A subset of plants was destructively sampled 3, 4, 5, and 6 wk post- differences between infested and uninfested rows, Sutter and Branson T. dactyloides has shown antibiosis or infestation (larval hatch occurred from (1986) recommended planting buffer extreme non-preference to the WCRW 14-18 d post-infestation). Ten T. rows between infested and uninfested as mature plants and cuttings from dactyloides plants and five maize plants rows to account for larval movement. mature plants (Branson 1971). If were evaluated at each sample date. Even in plots infested with 1200 eggs WCRW resistance from T. dactyloides is The number of live larvae and mean per 30.5 cm; however, significant root to be transferred into maize, and be larval weight were recorded for each pulling strength reductions were found, useful, it must be present in maize plant by removing the plants and soil indicating buffer rows may not be seedlings. In order to locate resistance from the pots and placing them in necessary. The artificial infestation in seedlings Moellenbeck et al. containers of water. After hand mixing, methods and paired-row evaluations (submitted to J. Econ. Entomol.) larvae that floated to the top were should be adequate for preliminary evaluated 50-day old T. dactyloides collected. The use of a sand:silt mixture evaluations of maize germplasm for seedlings for resistance to the WCRW. void of organic matter instead of a WCRW resistance. commercial growth mixture allows for ADVANCES IN RATING AND PHYTOCHEMICAL SCREENING FOR CORN ROOTWORM RESISTANCE 207 easier collection of the floating larvae. This difference in weight is consistent include 2,4-dihydroxy-7-methoxy-1,4- All of the larvae found in a single pot with antibiosis or non-preference in the benzoxazin-3-one (DIMBOA), 2,4- were weighed collectively. Mean larval T. dactyloides seedlings. dihydroxy-7,8-dimethoxy-1,4benzoxazin-3(4H)-one (DIM2BOA), one weight per plant was then calculated by dividing the total weight by the Resistance found in young T. dactyloides lactam, 2-hydroxy-7-methoxy-1,4- number of larvae. plants may be more useful for transfer benzoxazin-3(4H)-one (HMBOA) and into maize. The mechanism of one benzoxazolinone, 6- The number of larvae found on T. resistance in the seedlings has not been methoxybenzoxazolinone (MBOA). dactyloides and maize plants was not determined. A small percentage of the Screening roots for elevated levels of significantly different. The number of T. dactyloides seedlings did sustain hydroxamic acids may provide a larvae recovered peaked on the maize larval growth, indicating either method for reducing the number of plants 4 wk after infestation when 8.2 ± variation in T. dactyloides resistance to genotypes to be field evaluated. This 2.1 (mean ± SE) larvae per plant were the WCRW or variation in the approach has already been successfully recovered. Larval populations on maize rootworms’ susceptibility to the applied to leaf tissue for European corn dropped to 3.4 ± 0.7 per plant six weeks resistance factor(s). The T. dactyloides borer resistance screening (Russell et al. after infestation. Larval populations on cultivar ‘PMK 24’ is not a homozygous 1975). Once genotypes with elevated T. dactyloides reached 3.2 ± 0.4 at that breeding variety. Thus, the variation in levels of hydroxamic acids in the roots date. The decrease in the number of the ability of some larvae to survive on have been identified, field evaluations larvae on maize most likely was caused these seedlings may be due to genetic can then be conducted to confirm by larval competition. Infested maize variation among the seedlings. Because resistance. plants were heavily damaged at the of this variation, breeding programs final two sample dates and crowding in designed to transfer WCRW resistance Germplasm used for this study the small pots may have increased the from T. dactyloides into maize are included CRW resistant landraces competition for available feeding sites. advised to first evaluate the T. (Aguascalientes 6, Chiapas 41, Durango dactyloides. 25, Guanajuato 69, Guatemala 166, Larval weights on T. dactyloides were significantly less than larval weights on maize four, five, and six weeks after Guatemala 189, Guatemala 196, Laboratory and Bioche m ical Evaluat ions infestation (Table 2). Three weeks after Guatemala 489, Guatemala 633, Guatemala 757, Nayarit 203, Puebla 103, and San Luis Potosi 24) identified infestation, the larvae were still first- Recent studies on resistance by field evaluation at CIMMYT (Mihm instars and probably had not fed mechanisms of maize to CRW have personal communication). This enough on either plant type to see any identified hydroxamic acids as germplasm was crossed onto difference in weight. Six weeks after resistance factors (Xie et al. 1990; Agriculture Canada inbred lines infestation, the larvae were 3 times Arnason these Proceedings). The major (CO251, CO255, CO267, CO272, and heavier on maize than on T. dactyloides. secondary compounds in maize roots CO289) with good agronomic traits. Crosses were selfed to obtain Table 2. Mean weights of western corn rootworm larvae from corn (breeding population MoSQA) and T. dactyloides plants 3, 4, 5, and 6 weeks after infestation. Plant T. dactyloides Maize (MoSQA) T. dactyloides Maize (MoSQA) T. dactyloides Maize (MoSQA) T. dactyloides Maize (MoSQA) a Weeks after infestation Number of a larvae Mean larval a weight (mg) 3 3 4 4 5 5 6 6 2.3 ± 0.6a 5.4 ± 0.8a 6.9 ± 1.3a 8.2 ± 2.1a 4.8 ± 1.2a 3.8 ± 0.6a 3.2 ± 0.7a 3.4 ± 0.4a 0.1 ± 0.1a 0.3 ± 0.1a 0.5 ± 0.2b 1.1 ± 0.2a 0.5 ± 0.2b 1.6 ± 0.0a 2.3 ± 0.5b 7.4 ± 1.0a Means ± SE (N = 10 for T. dactyloides and N = 5 for maize) within a sample date followed by the same letter are not significantly different (P > 0.05) approximately 600 S1 individuals which were phytochemically screened using the hydroponic technique described below. Seed from individual ears with extremely high or extremely low DIMBOA levels in the root were advanced and the S2 generation was again evaluated for root DIMBOA content. Genotypes with extreme DIMBOA levels were considered for field evaluation. 208 D.J. MOELLENBECK, D.J. BERGVINSON, B.D. BARRY AND L.L. DARRAH Approximately 15 seeds from each approximately 0.5 g wet weight, but solutions and colorimetric reagents is genotype were germinated on wet filter samples as low as 0.05 g could be listed in Table 4. Root tissue was easily paper at 25 to 30 °C for 3 days until the analyzed. After recording the weight, ground by mortar and pedicel so that no radicle was approximately 2 cm long. the root sample was placed in a mortar large sections of root tissue were left Ten seedlings were then pinned to a and 3 ml of acidified 80% ethanol was intact. After homogenizing, the Styrofoam block as illustrated in Figure added. Preparation of extraction supernatant was decanted off into 1. The pin did not penetrate the seed, but supported the seed firmly against the wall of the Styrofoam block to hold the seed at the water line. Each block centrifuge tubes. An additional 2 ml of Table 3. Ingredients for Hoagland’s solution for growing maize seedlings hydroponically. Grams per 100 L of water held 50 seeds, allowing 5 genotypes to Ingredients be tested per block, with each row labeled to identify the genotype. After pinning, the block was immersed into nursery flats that were half full of Hoagland’s solution (Table 3). The trays were grown under optimal growing conditions (>80% RH, >25 °C, 16:8 (L:D)). After 14 d, the plants reached the 6 leaf stage and were removed from the trays. Tissue was stored at -20 °C for phytochemical analysis or used fresh for bioassays. Frozen root tissue was removed from the freezer and allowed to thaw for 5 minutes so individual roots could be handled easily. Individual roots were 1) Magnesium sulphate (MgSO4) 2) Potassium phosphate (KH2PO4) 3) Calcium nitrate (Ca(NO3)2)•24H2O 4) Fe Chelate 13% 5) Potassium nitrate (KNO3) 6) Minor Elements Solution acidified 80% ethanol was added to further grind the remaining pulp and rinse the mortar. The second volume was combined with the first, and the sample was centrifuged at 500 x g for 5 min. to provide a clear supernatant. 49.3 Two ml of the supernatant was added 13.6 to a spectrophotometer cuvette and an absorbance reading was taken at 520 or 118.1 0.11 50.6 590 nm. By taking the reading at 520 nm there is less interference by other root components that chelate with Fe3+. For leaf tissue, absorbance readings at 590 100 ml Minor Element Solution Grams per 10 L of water MnCl2•4H2O H3BO3 CuSO4•5H2O ZnSO4•7H2O H2MoO4•H2O KCl 18.1 28.6 0.8 2.2 0.2 63.0 weighed with a good sample size being nm are preferred due to chlorophyll interference. After recording the background reading, 50 ml of the dilute FeCl3 solution was added and the solution mixed by pipette. Immediately after mixing, the second absorbance reading was taken. The absorbance drops rapidly over time so readings should be taken immediately after the addition of FeCl3. The difference in absorbance before and after the addition of FeCl3 is calculated, multiplied by the 5 ml extraction volume, and divided by the weight of root tissue to give a Styrofoam 5 x 10 holes 3 Days incubation Tray placed in Hoagland’s Solution for Optimal growth Roofs frozen (-20º C) Thaw and homogenize Centrifuge Supernatant analized by Spectrophotometer Plants harvested at 14 days Bioassay using fresh root Figure 1. Phytochemical screening protocol for root tissue. Styrofoam trays are made from Stryofoam sheets cut to measure 25 x 50 cm (Dow SM, Dow Chemical Canada Inc., Weston, Ont. M9N 2M2). 50 1.2cm-holes were drilled using a high speed drill. Stryrofoam trays with seedlings pinned into holes were placed into heavy duty plastic nursery trays measuring 26 x 51 x 6 cm (model K10-20, Kord Inc., Toronto, Ont.). concentration in Abs520 per g wet tissue weight. A standard curve using authentic DIMBOA was generated to convert Abs520 into mg DIMBOA: Table 4. Preparation of solutions for FeCl3 screening for DIMBOA. 1. FeCl3 stock solution - store at <4 ∞C 50 g FeCl3 6 H20 in 495 ml H2O and 5 ml of 11 N HCL, final pH of 2. 2. FeCl3 screening solution - prepare as needed. Take 5 ml of FeCl3 stock solution and add 45 ml distilled water. 3. 0.1N HCl in 80% ethanol Add 50 ml of 1N HCl to 450 ml of 95% ethanol. 209 ADVANCES IN RATING AND PHYTOCHEMICAL SCREENING FOR CORN ROOTWORM RESISTANCE mg DIMBOA / ml = 0.1183 x (Abs520 root pruning than low DIMBOA of roots available for feeding. Despite with FeCl3 - Abs520 without FeCl3) genotypes, which is consistent with the higher damage ratings for plants earlier work (Xie et al. 1990). A recent with large, densely branched root For screening germplasm, only relative survey of DIMBOA content in root systems, this phenotype is often able to levels are required but the above tissue of commercial hybrids had regenerate roots readily, a reaction that equation should provide a reasonable demonstrated the low level of DIMBOA is considered an important component estimate of DIMBOA levels in the in the majority of hybrids, which may in resistance (Jenison et al. 1981). These tissue. Confirmation of DIMBOA levels in part explain the susceptibility of observations may provide an should be done using a water based commercial hybrids to root pruning by explanation for the poor correlation (Xie et al. 1991) or methanol based CRW larvae (Assabgui et al. 1993). between root lodging and the root damage rating (r = 0.3, P>0.1), as (Bergvinson et al. 1994) extraction method for quantification by high- During the course of DIMBOA genotypes with large root systems early performance liquid chromatography screening, root mass was also in development tended not to lodge. (HPLC). Genotypes with the highest considered as an important component relative levels of DIMBOA and large in root tolerance to CRW pruning and Screening root tissue for elevated levels healthy roots should be considered for was included in the selection process. of DIMBOA has enabled resistant field evaluation using standard field Despite the 10-fold difference observed genotypes to be identified and can screening techniques (these in root mass at the 6 leaf stage in the accelerate the development of resistant Proceedings; Branson and Sutter 1989). hydroponic system, field grown plants inbred lines as plants from both winter did not differ considerably in root mass and summer nurseries can be evaluated For the present field study, three at the time of field assessment (Table 5). in the laboratory. Using the FeCl3 genotypes were selected for each of It appears that poor root establishment screening technique, one person can four categories based on DIMBOA level early in plant development is process 150 samples per day. With this (high/low) and root mass at the 6 leaf compensated for during the growing processing capability, germplasm can stage (large/small). Plants had been season in the genotypes tested. be assessed after harvest and desirable evaluated at the S2 stage using the Reduced root growth early in plant ears identified before the next nursery above hydroponic system and seed development may be an avoidance for advancing another generation. from the same ear was used for field mechanism, as these plants had the evaluation. Field trials were conducted lowest root damage rating (Table 5). The potential danger of this screening in a clay soil with a high natural Given the nature of the damage rating method is only one phytochemical population of both NCRW and WCRW scale, plants with a small root system component is being assessed. Given the which had been maintained by planting early in development may have lower incomplete knowledge of root sweet maize and grain maize of ratings due to a lower probability of biochemistry as it relates to CRW different maturities for four consecutive root pruning given the reduced number resistance, other resistance mechanisms years. A complete randomized block design was used with three replicates and 12 plants per replicate. Ten weeks after planting, the plants were rated for lodging and the roots dug up, washed, and rated on a 9 point scale outlined by Branson and Sutter (1989). After rating, Table 5. Field evaluation of S2 genotypes selected by the iron chloride screening technique. Plant Attributes Background High DIMBOA Large root mass Durango 25 x CO255 S. Luis Potosi 24 x CO289 Guanajuato 69 x CO251 Durango 25 x CO255 Durango 25 x CO255 bxbx mutant (low DIMBOA) Guatemala 757 x CO289 Guanajuato 69 x CO251 Guanajuato 69 x CO251 MBR622 Lines developed MBR105 from MBR synthetic the roots were dried and weighed. Low DIMBOA Large root mass Field verification of the FeCl3 screening High DIMBOA Small root mass method indicated that DIMBOA content in root tissue is an important component in host plant resistance to Low DIMBOA Small root mass the CRW (Table 5). Genotypes with a elevated levels of DIMBOA had less Damage a rating 4.9 e 5.0 e 5.1 e 8.4 a 6.5 cd 5.7 de 3.3 f 5.3 ed 3.7 f 8.4 a 7.6 ab Root mass a dry wt. (g) 93 97 109 75 96 86 87 101 92 86 85 abc ab a cd ab bc bc ab abc bc bc Means followed by the same letter are not significantly different, Student-Neuman-Kuels test (P = 0.05). 210 D.J. MOELLENBECK, D.J. BERGVINSON, B.D. BARRY AND L.L. DARRAH would not be detected using this screening protocol. For this reason, further work is needed to better understand the biochemical mechanisms of host plant resistance to the CRW. Work is also needed on identifying the changes that occur in root chemistry for both resistant and susceptible genotypes. Having identified germplasm with a range of DIMBOA levels and root mass, we can now address questions regarding the relative importance of antibiosis and tolerance. By understanding the traits most desirable for host plant resistance and at what stage in plant development these resistance mechanisms are most important will accelerate the development of resistant inbred lines. Host plant resistance must play a more important role in future CRW management. As we learn more about mechanisms of CRW resistance, selection programs can continue to be refined. Evaluation of CRW resistance must always include field evaluations of feeding damage; however, techniques described here can reduce the amount of material that needs to evaluated in costly field testing by removing susceptible materials early in the screening process. Biotechnology and marker-assisted selection offer the opportunity to develop new ways to incorporate host plant resistance into commercial maize germplasm. Selection programs must continue to be refined in order to use these techniques efficiently. Re fe re nce s Assabgui, R.A., J.T. Arnason, and R.I. Hamilton. 1993. Hydroxamic acid content in maize (Zea mays) roots of 18 Ontario recommended hybrids and prediction of antibiosis to the western corn rootworm, Diabrotica virgifera virgifera LeConte [Coleoptera: Chyrsomelidae]. Can. J. Plant Sci. 73: 359363. Beck, D.L., L.L. Darrah, and M.S. Zuber. 1987. An improved technique for measuring resistance to root pulling in maize. Crop Sci. 27: 356-358. Bergvinson, D.J., J.T. Arnason, and L.N. Pietrzak. 1994. Localization and quantification of cell wall phenolics in European corn borer resistant and susceptible maize inbreds. Can. J. Bot. 72: 1243-1249. Branson, T.F. 1971. Resistance in the grass tribe Maydeae to larvae of the western corn rootworm. Ann. Entomol. Soc. Am. 64: 861-863. Branson, T.F., and G.R. Sutter. 1986. Influence of application date on damage resulting from controlled infestations with eggs of Diabrotica vergifera virgifera (Coleoptera: Chrysomelidae). J. Econ. Entomol. 79: 838-839. Branson, T.F., and G.R. Sutter. 1989. Evaluating and breeding for maize resistance to the rootworm complex. In Toward Insect Resistant Maize for the Third World: Proceedings of the International Symposium on Methodologies for Developing Host Plant Resistance to Maize Insects, 130139. Mexico D.F.: CIMMYT. Chio, H., C.S. Chiang, R.L. Metcalf, and J. Shaw. 1978. Susceptibility of four species of Diabrotica to insecticides. J. Econ. Entomol. 71: 389-393. Donovan, L.S., P. Jui, M. Kloek, and C.F. Nichols. 1982. An improved method for measuring root strength in corn (Zea mays L.). Can. J. Plant Sci. 62: 223- 227. Felsot, A.S. 1989. Enhanced biodegradation of insecticides in soil: Implications for agroecosystems. Annu. Rev. Entomol. 34: 453-476. Hills, T.M., and D.C. Peters. 1971. A method of evaluating postplanting insecticide treatments for control of western corn rootworm larvae. J. Econ. Entomol. 64: 764-765. Jenison, J.R., D.B. Shank, and L.H. Penny. 1981. Root characteristics of 44 maize inbreds evaluated in four environments. Crop Sci. 21: 233-237. Kahler, A.L., R.E. Telkamp, L.H. Penny, T.F. Branson, and P.J. Fitzgerald. 1985. Registration of NGSDCRW1(S2)C4 maize germplasm. Crop Sci. 21: 233- 237. Kindiger, B. 1994. A method to enhance germination of eastern gamagrass. Maydica. 39: 53-56. Krysan, J.L., D.E. Foster, T.F. Branson, K.R. Ostlie, and W.S. Cranshaw. 1986. Two years before the hatch: Rootworms adapt to crop rotation. Bull. Entomol. Soc. Am. 32: 250-253. Kuhlman, D.E., W.L. Howe, and W.H. Luckmann. 1970. Development of immature stages of the western corn rootworm at varied temperatures. Proc. N. Cent. Br. Entomol. Soc. Am. 25: 93-95. Levine, E., and H. Oloumi-Sadeghi. 1991. Management of Diabroticite rootworms in corn. Annu. Rev. Entomol. 36: 229-255. Melhus, I.E., R.H. Painter, and F.O. Smith. 1954. A search for resistance to the injury caused by species of Diabrotica in the corns of Guatemala. Ia. State Coll. J. Sci. 29: 75-94. Metcalf, R.L. 1986. Foreword, In Krysan, J.L., and T.A. Miller (eds.), Methods for the study of pest Diabrotica, vii-xv. New York: Springer-Verlag. Moellenbeck, D.J., B.D. Barry, and L.L. Darrah. 1994. The use of artificial infestations and vertical root pulling evaluations to screen for resistance to the western corn rootworm (Coleoptera: Chrysomelidae). J. Kansas Entomol. Soc. 67: 46-52. Ortman, E.E., D.C. Peters, and P.J. Fitzgerald. 1968. Vertical-pull technique for evaluating tolerance of corn root systems to northern and western corn rootworms. J. Econ. Entomol. 61: 373-375. Penny, L.H. 1981. Vertical-pull resistance of maize inbreds and their testcrosses. Crop Sci. 21: 237-240. Rogers, R.R., W.A. Russell, and J.C. Owens. 1976. Evaluation of a vertical-pull technique in population improvement of maize for corn rootworm tolerance. Crop Science. 16: 591-594. Rogers, R.R., W.A. Russell, and J.C. Owens. 1977. Expected gains from selection in maize for resistance to corn rootworms. Maydica. 22: 27-36. Russell, W.A., W.D. Guthrie, J.A. Klun, and R. Grindeland. 1975. Selection for resistance in maize to first-brood European corn borer by using leaf-feeding damage of the insect and chemical analysis for DIMBOA in the plant. J. Econ. Entomol. 68: 31-34. Steffey, K.L., M.E. Gray, and D.E. Kuhlman. 1992. Extent of corn rootworm (Coleoptera: Chrysomelidae) larval damage in corn after soybeans: Search for the expression of the prolonged diapause trait in Illinois. J. Econ. Entomol. 85: 268275. Sutter, G.R., and T.F. Branson. 1986. Artificial infestation of field plots. In J.L. Krysan, and T.A. Miller (eds.), Methods of Study of Pest Diabrotica, 147-158. New York: Springer-Verlag Welch, V.A. 1977. Breeding for corn rootworm resistance or tolerance. In Proc. 32nd Annual Corn Sorghum Research Conference, 131-142. Washington, D.C.: American Seed Trade Association. Xie, Y.S., J.T. Arnason, B.J.R. Philogène, J.D.H. Lambert, J. Atkinson, and P. Morand. 1990. Role of 2,4-dihydroxy-1,4benzoxazin-3-one (DIMBOA) in the resistance of maize to western corn rootworm, Diabrotica virgifera virgifera (Coleoptera: Chrysomelidae). Can. Entomol. 122: 1177-1186. Xie, Y.S., J. Atkinson, J.T. Arnason, P. Morand, and B.J.R. Philogène. 1991. Separation an quantification of 1,4-benzoxazin-3-ones and benzoxazolin-2- ones in maize root extract by high-performance liquid chromatography. J. Chromatography 543: 389-395. Factors Affecting a Laboratory Bioassay for Antibiosis: Influences of M aize Silks on the Corn Earworm and Fall Armyw orm Larvae B.R. Wiseman, USDA-ARS, Tifton, GA, USA Abst r a c t A useful laboratory bioassay has been developed to screen for resistance to lepidopterous insects attacking maize, Zea mays L., and for use in studying the antibiotic mechanism and bases of resistance to these insects. The bioassay may be used to detect minor as well as major differences between the resistant and susceptible maize cultivars. The bioassay has been used to study the influence of: the environment; pollinated vs. nonpollinated silks; ear position; age and type of silk; and callus tissue on expressions of antibiosis against the corn earworm (CEW), Helicoverpa zea (Boddie), or fall armyworm (FAW), Spodoptera frugiperda (J. E. Smith), larvae. Studies on some of the factors, such as temperature, diet and diet ingredients, and insect feeding responses, revealed interactions with the expressions of antibiosis. The bioassay has also been used in studies on the relationship between low larval weight with maysin content and the genetic and chemical bases of resistance in maize to CEW and FAW larvae. Int roduct ion pinto bean diet to characterize several Diets of 400-500 ml quantities with 20- factors of antibiosis to the corn earworm 25 g of dry silks were generally made Effective techniques are essential for (CEW), Helicoverpa zea (Boddie) and the using a standard household blender the identification of sources of plant fall armyworm (FAW), Spodoptera and dispensed into 30 ml plastic diet resistance to insect pests and, frugiperda (J.E. Smith). cups at a rate of 10 ml/cup even though frequently the amount of the especially, to characterize the mechanisms and the chemical and Much of the earlier work used large silk/diet mixture was expressed as 50 genetic bases of resistance. Wiseman et (several grams) amounts of plant mg/ml diet (Wiseman and Isenhour al. (1984) evaluated a series of material in 300-400 ml dilute pinto bean (1989). Later, Wiseman et al. (1986) substandard (incomplete) diets diet. Wilson et al. (1984) used 10 to 80 g developed a microassay that used 20 modified from the regular pinto bean of fresh maize silks in 300 ml of diet and ml pinto bean diet, 10 ml distilled diet (Perkins 1979). Two diets were 2 to 16 g of lyophilized silks in 300 ml of water and as little as 2 g of fresh, dried, acceptable for plant allelochemical diet to evaluate against the CEW. or equivalent extracted plant material investigations: the regular pinto bean Wiseman and Widstrom (1986) used 10 (Wiseman and Isenhour 1991) blended diet and the substandard pinto bean to 80 g of fresh silk in 300 ml diet to test in a 37 ml mini-blender and then diet without yeast. Since then the pinto against the FAW. Wiseman and Wilson aspirated into plastic soda straws. The bean diet bioassay has been modified (1987) were the first to use oven-dried final refinement came when Wiseman and has replaced the substandard diet silks in meridic diets against the CEW. and Isenhour (1991) described a and is now used to evaluate maize, Zea Then Wiseman and Isenhour (1988) microtechnique for evaluating mays L., and sorghum, Sorghum bicolor showed the importance of consistent antibiosis against the CEW. The (L.) Moench, for resistance to insects. handling of silks during the harvesting technique they developed used Various forms and amounts of maize and drying process. They reported that samples (0-100 mg dry weight) of silks silks (Wilson et al. 1984; Wiseman and silks harvested and immediately dried from individual ears. Since then, the Widstrom 1986; Wiseman and Wilson produced more consistent results than standard amount of silks/pipette bulb 1987) have been incorporated into the lyophilized or fresh silks in bioassay was increased to 150 mg. Dry silks diets. 212 B.R. WISEMAN were placed into a detached bulb of a Influences of the Plant originating in Georgia. The reverse was true for larvae which were fed 7.5 ml disposable pipette in which 2 cc of dilute pinto bean diet (3:2 pinto bean Planting date ‘Zapalote Chico 2451 # P (C3)’ (Z. diet:H2O) was mixed, at first using a 3/ Silks grown at two locations (Tifton, Chico) silks. Larvae fed on Z. Chico 8" reversible drill at 500-600 RPM. GA and Ames, IA) and on two planting silk-diets weighed significantly less Later, the mixing of the silks into the dates per location were fed in diets to than those fed on SEG silk-diets in dilute diet was accomplished by a CEW larvae (Wiseman and Wilson every case, even though the differences “Biovortexer” or a modified “Tooth 1987). Weight of larvae from test in weight between larvae on Z. Chico Polisher” (Fig. 1). The “Biovortexer” locations showed significant versus SEG ranged from 181 to 723 mg cost about $56 compared to $5.95 for differences between planting dates for on the 5 g silk-diets and 36 to 728 on the “Tooth Polisher”. The remaining the silks produced in Georgia, but not the 10 g silk-diets. The CEW larvae portion of this review will address the for those produced in Iowa. The tested at Tifton were generally larger influence of the plant and insect differences between cultivars occurred than those tested at Ames probably affecting this laboratory bioassay and irrespective of test location. Larvae fed because of the heterogeneity of the the expressions of maize silk antibiosis Iowa-produced ‘Stowell’s Evergreen’ Tifton colony. against the CEW larvae. (SEG) sweet maize silks weighed significantly more for each planting Wiseman and Isenhour (1992) studied date than those fed SEG silks environmental influences on silks resistant to the CEW. Environment had a greater influence on the response of CEW larvae fed silk-diets from an intermediate resistant or susceptible maize line, but had little influence on the feeding response of larvae on the highly resistant silk-diet of Z. Chico. In 8 of 12 tests using Z. Chico and 7 of 12 tests of 471-U6 X 81-1, no significant differences were found between planting dates for six characteristics of resistance. None of the intermediate or susceptible entries approached this level of consistency. Pollinated silks vs. nonpollinated silks Pollinated and nonpollinated silks from SEG and Z. Chico were incorporated, fresh and dried, in meridic diets and evaluated for their effects on the development of CEW larvae (Wiseman and Wilson 1987). Larvae weighed significantly less when fed fresh, pollinated silk-diets than when they were fed fresh, nonpollinated silk-diets. Differences between pollinated vs nonpollinated silks were not detected Figure 1. An Eppendorf repeater pipette was used to dispense 2 ml of dilute pinto bean-silk diet into a 7.5 ml detached bulb of a disposable pipette (top). The silk-diet mixture was mixed using a modified “Tooth Polisher” (bottom). for other insect developmental characters when either fed as fresh or FACTORS AFFECTING A LABORATORY BIOASSAY FOR ANTIBIOSIS 213 dried silks in diets. The larvae that for weight of larvae that were fed fresh noted for the dried silks. But, in fact the were fed fresh or dried Z. Chico silk- silk-diets or the 2 g and 4 g oven-dried amounts are much less; i.e., the mg/g of diets were significantly different for silk-diets were similar (Table 1). Lower maysin in fresh silks of Ab616 was 2.54 each developmental character than correlation coefficients occurred mg based on a wet weight basis, those fed on SEG silk-diets. between bioassay results for larvae that whereas the oven-dried silks of Ab616 were fed maysin deposited on celufil had 8.94 mg/g based on a dry weight First ear vs. second ear diets. This lower correlation coefficient basis. If the fresh weight were calculated Silks from first or second ears and silks was probably the result of having only on a dry weight basis there would be ca. regrown for one or two days after one chemical responsible for the silk 25.4 mg maysin/g of silk. The percent cutting were evaluated for antibiotic resistance when the chemical was moisture for each inbred silk would responses to CEW larvae (Wiseman et applied on the celufil, whereas the silk- need to be calculated. If the silks of al. 1993). Neonate CEW fed silk-diets diets, fresh or dried, had all Ab616 are assumed to be 90% water, from first ears weighed significantly phytochemicals present. The 4 g oven- then a loss of 16.46 mg/g of maysin to less than larvae fed silk-diets from dried silk-diets of Ab616, Ab618, GE37, undetected compounds is present in the second ears. Silks regrown for one or 8940C and 91201Y produced larvae with oven-dried silks. Even with the addition two days after initial cutting and much smaller weights than any other of the isomaysin, there is still ca. 10.9 incorporated into diet produced larger type of diets tested. By adding the mg/g of maysin or breakdown products larvae after eight days than those fed additional 2 g of oven-dried silk to the undetected in the dry silks. However, on silk-diets from the initial cutting. diets, a threshold was probably reached the biological activity was not lessened Weights of larvae were consistent for the expression of antibiosis in the dry silks, but was enhanced. If the among genotypes, whether the silks (Wiseman et al. 1992b). However, this amount of dry silks is doubled in the were from first or second ears. This was did not affect the rankings of the diets — from 2 g to 4 g oven dry silk/ especially true for silks of PI340856, inbreds in each test, hence the high 100 g of diet — then the amounts of which had a high level of antibiosis. correlation’s between the flavones and isomaysin and maysin available in the Larvae were quite small on silk-diets of weight of larvae among the four test fresh silks are more than accounted for both first and second ears of PI340856. diets. and the activity against the larvae appears to be enhanced. Isomaysin and It was concluded that silks could be sampled for chemical analysis and the Biological activity against CEW larvae apimaysin plus methoxymaysin were regrowth used to bioassay larvae with dry silks in diets appears to be only detected in the oven-dried silks. without risk of erroneous results, enhanced over that of fresh silks in providing that silks are used from the diets. The percent flavones (maysin) A highly significant (P < 0.01) negative same ear location. found in the fresh silk is based on the relationship was found between weight wet weight of the silks as compared to of larvae within each of the four diets Fresh vs oven dry silks those found in the dried silks (maysin, and maysin in fresh silks or maysin and Wiseman et al. (1995) evaluated silks of isomaysin, apimaysin and apimaysin isomaysin in dried silks (Table 2). No fifty field corn inbreds in four separate plus 3'-methoxymaysin) which were significant correlation was found bioassays (fresh silks, 2 g and 4 g oven- calculated on a dry weight basis; hence between weight of larvae and apimaysin dried silks/100 g diet and maysin the higher amounts of flavones are plus methoxymaysin. When isomaysin equivalent to 20 g fresh silks deposited on celufil and incorporated in 100 g diet) for growth responses of CEW larvae. Assays for maysin, isomaysin and apimaysin plus 3'-methoxymaysin Table 1. Pearson correlation coefficients (r) and levels of significance among eight-day weights of corn earworm larvae fed diets of fresh and oven-dried silks and maysin deposited on celufil. 1 Correlation coefficients content of silks were also made. Significant differences in growth of larvae were found among the silks of the fifty inbreds within each of the four bioassays. The correlation coefficients Fresh silk 2 g dry silk 4 g dry silk 1 2 g dry silks 4 g dry silks maysin + celufil 0.9193* ————- 0.9202* 0.9528* ——- 0.8058* 0.7990* 0.7783* Ho: Rho = 0. n = 52. * = significance at 0.0001. (From Wiseman et al. 1996) 214 B.R. WISEMAN was added to maysin, the level of the germplasm are not produced at the day pollinated silks, were found among relationship was only slightly enhanced same time but mature over an extended insect biological parameters measured. for larvae that fed on the oven-dried time period, making it extremely It appears that as age of silk increases, silk-diets and the maysin on celufil diet, difficult to use silks of the same age. maysin content decreases and growth of but not for those fed the fresh silk-diets. Fresh silk in the quantity necessary to CEW larvae often increases. It is not The highest correlation was found when achieve large differences among known if this phenomenon occurs in these two flavones were combined with weights of larvae are difficult to mix other cultivars, or if resistance decreases weight of larvae that fed on the maysin and/or dispense. Likewise, maysin in cultivars with chemicals other than on celufil diets. However, when deposited on celufil omits other maysin as the basis of resistance. isomaysin, apimaysin, and flavones or unidentified chemicals methoxymaysin found in the dry silks from the bioassay. Thus, bioassays with Callus tissue were added to maysin, the relationship oven-dried silks permits the use of The use of callus tissue to screen for between weight of larvae and the larger amounts, (4 g instead of 2 g of insect resistance has been suggested by flavones was enhanced significantly material), which should enhance the some as a substitute for plant tissue over maysin alone, i.e., 32.9% for the antibiotic effects. Similarly, germplasm (Williams et al. 1987; Isenhour and fresh silks and 43.7% for the oven-dried of varying maturities can be assayed Wiseman 1988). Callus is an silks (4 g). However, the lowest when the oven-dried silk bioassay is undifferentiated mass of living cells that correlation (-0.8542) in this group (all employed. can be grown on an agar-based nutrient medium under sterile conditions. Callus flavones combined) was found between all the flavones combined and the Age of silk growth is initiated by placing a piece of weight of larvae that fed on the maysin The effects of age of Zapalote chico and plant tissue (explant) on nutrient only in the celufil diet. Stowell’s Evergreen silk on medium, with both the explant type developmental characters of neonate and nutrient medium specific for a Although the results of the four CEW were studied by Wiseman and given plant species. Williams et al. bioassays compared favorably, those Snook (1995). Consistent significant (1985, 1987) proposed the use of fresh based on fresh silks or maysin differences between cultivars for each callus tissue as a method for screening deposited on celufil have limitations. In age group of silk (nonpollinated, two, maize genotypes for resistance to an evaluation, silks from inbreds or five, and ten day pollinated), except ten lepidopterous larvae. Isenhour and Wiseman (1988) tested fresh callus tissue incorporated into meridic diets Table 2. Pearson correlation coefficients (r) and significance levels among eight-day weights of corn earworm larvae and percent maysin, isomaysin, apimaysin plus methoxymaysin, maysin plus isomaysin and maysin plus isomaysin plus apimaysin plus methoxymaysin. Flavone Fresh silks 2 Prior maysin 1992 maysin Oven-dried silks Maysin Isomaysin Apimaysin + methoxymaysin Maysin + isomaysin Maysin + isomaysin + apimaysin + methoxymaysin 1 2 the FAW and CEW after feeding on calli-diets from resistant and susceptible genotypes. Callus-diet mixtures failed Correlation coefficients for1 Silks and compared biological differences of to confer the degree of resistance that Maysin fresh 2 g dried 4 g dried on celufil -0.5164* -0.6361* -0.5537* -0.7356* -0.5134* -0.6671* -0.6471* -0.8033* -0.5951* -0.5835* -0.5999* -0.6242* -0.5424* -0.5701* -0.6529* -0.6736* -0.0415 -0.0396 -0.0169 -0.0635 -0.5965* -0.6150* -0.5576* -0.6674* foliage-diet mixtures did. In cases where antibiotic resistance factors were present in the silk, the callus-diet mixtures failed to exhibit any evidence of resistance. Inse ct Influe nce s Temperature Isenhour et al. (1985) studied the effects -0.9656* -0.9762* -0.9793* -0.8542* Ho: Rho = 0. n = 52. * = significance at 0.0001. (From Wiseman et al. 1996). Prior maysin indicates the determinations of maysin made on the same inbred silks prior to 1992. of varying temperature on bioassay results of resistant versus susceptible plants. They found no differences in FACTORS AFFECTING A LABORATORY BIOASSAY FOR ANTIBIOSIS 215 weight of FAW larvae fed excised degradation of maysin in meridic diets. larvae and maysin content was cubic leaves of susceptible and resistant Increasing concentration of yeast (r2 = 0.893). A concentration of >0.2% genotypes at 25ºC, but differences were promoted growth of larvae that fed on maysin reduced CEW larval growth to found between weight of larvae that silk-diets. Diets, therefore, must be >50% of that of the control. Higher fed on genotypes at 30ºC and a fully characterized (i.e., components amounts of maysin, such as 0.4%, fluctuating temperature regime of 31/ identified) because small changes in reduced weight of CEW larvae to >70% 20ºC. This variation did not occur when diet components can affect the compared with the control. A stepwise comparisons were made between apparent levels of resistance. multiple regression analysis has shown susceptible and resistant genotypes Comparisons of data over more than that maysin was the major factor using a foliage-diet mixture. Wiseman one experiment should always be associated with resistance in silks of and Isenhour (1989) studied the effects carefully interpreted, especially if diet maize to both CEW and FAW larvae of interactions among temperature (20, components vary among experiments. (Wiseman et al. 1992b). The addition of 25, and 30ºC), resistant and susceptible apimaysin to the regression analysis genotypes, and concentration of silk/ Insect feeding only improved the r2 by about 10%. diet (0 and 18.75, 37.5 and 67.0 mg/ml Wiseman and Isenhour (1993) and Neither chlorogenic acid nor 3'- diet) on CEW developmental Wiseman and Hamm (1993) noted that methoxymaysin appeared to improve parameters. Significant differences young CEW larvae tended to bore the r2. However, when isomaysin, caused by the resistant silks compared directly through the diet surface when apimaysin and methoxymaysin found with the susceptible silks, were resistant silk-diets showed an increased in the dry silks (Table 2) were added to measured consistently at 25ºC for all oxidative process (turned dark brown), maysin, the relationship between four insect biological parameters. whereas larvae on susceptible diets weight of larvae and the flavones was tended to eat along the diet surface. enhanced significantly over maysin The meridic diet Wiseman and Carpenter (1995) studied alone, i. e. 32.9% for the fresh silks (t = Wiseman and Isenhour (1993) the growth inhibition factor of the 6.29; P = 0.001; n = 52) and 43.7% for evaluated the effects of the addition of antibiotic silks. They found using the oven-dried silks (4 g) (t = 8.279; P = varying levels of resistant silks, neonate, fourth and fifth instar CEW 0.001; n = 52). formalin, ascorbic acid, and yeast to the larvae that the antibiotic resistance was corn-soy-milk (CSM) diet (Burton and the result of an anti-nutritive factor that Perkins 1989) or modified pinto bean possibly binds the protein or that diet on weight of CEW larvae. results in degradation of essential A useful laboratory bioassay has been Interactions were found among weight amino acids, causing the larvae to developed for both routine screening of larvae that were fed on CSM or pinto excrete large amounts of protein. for resistance to CEW and FAW larvae attacking maize and to evaluate the bean diets with or without formalin, varying levels of resistance, and varying concentrations of ascorbic acid Summary and Conclusions Effectiveness of the Laborat ory Bioassay antibiotic mechanism of resistance. Evidence exists that the laboratory bioassay can detect large differences or yeast. In all cases larvae that were fed on regular diet with formalin The laboratory bioassay has been used between the resistant and susceptible weighed significantly more than those effectively in a number of studies to maize cultivars. The bioassay has been that fed on diets without formalin. The separate resistant and susceptible used to study the influence of: the oxidative process (top of diet turns genotypes, first ears from second ears, environment, pollinated vs. brown) of the resistant silks was and initial silks vs silks regrown for nonpollinated silks, ear position, age of enhanced in the silk-diets without one or two days (Wiseman et al. silk and callus tissue on expressions of formalin and delayed in silk diets as 1992a,b, 1993). Significant negative antibiosis against the CEW or FAW the concentration of ascorbic acid was relationships have been established for larvae. Some of the factors affecting the increased in the silk-diets. However, weight of CEW larvae and bioassay results were temperature, diet tests revealed that formalin did not concentration of maysin (r = -0.811 and and diet ingredients, and insect feeding react with maysin. Therefore, formalin -0.655) (Wiseman et al. 1992a,b). responses. The bioassay has also been would not cause any breakdown or Regression analysis of weight of CEW used to study the relationship between 216 B.R. WISEMAN low larval weight and maysin content (Wiseman et al. 1992a) and the genetic (Wiseman and Bondari 1992, 1995) and chemical (Snook et al. 1993) bases of resistance in maize to CEW and FAW larvae. Through technology transfer, the methodologies and procedures used in the laboratory bioassay have been imparted to a number of commercial companies as well as researchers in public institutions. Re fe re nce s Burton, R.L., and W.D. Perkins. 1989. Rearing the corn earworm and fall armyworm for maize resistance studies. In Toward Insect Resistant Maize for the Third World: Proceedings of the International Symposium on Methodologies for Developing Host Plant Resistance to Maize Insects, 37-45. Mexico D.F.: CIMMYT. Isenhour, D.J., and B.R. Wiseman. 1988. Incorporation of callus tissue into artificial diet as a means of screening corn genotypes for resistance to the fall armyworm and the corn earworm (Lep.: Noct.). J. Kansas Entomological Society 61: 303-307. Isenhour, D.J., B.R. Wiseman, and N.W. Widstrom. 1985. Fall armyworm (Lep.: Noct.) feeding responses on corn foliage and foliage/artificial diet medium mixtures at different temperatures. J. Econ. Entomol. 78: 328332. Perkins, W.D. 1979. Laboratory rearing of the fall armyworm. Florida Entomologist. 62: 87-91. Snook, M.E., R.C. Gueldner, N.W. Widstrom, B.R. Wiseman, D.S. Himmelsbach, J.S. Harwood, and C.E. Costello. 1993. Levels of maysin and maysin analogues in silks of maize germplasm. J. Agric. Food Chem. 41: 1481-1485. Williams, W.P., P.M. Buckley, and F.M. Davis. 1985. Larval growth and behavior of the fall armyworm on callus initiated from susceptible and resistant corn hybrids. J. Econ. Entomol. 78: 951-954. Williams, W.P., P.M. Buckley, and F.M. Davis. 1987. Tissue culture and its use in investigations of insect resistance of maize. Agric. Ecosys. Environ. 18: 185190. Wilson, R.L., B.R. Wiseman, and N.W. Widstrom. 1984. Growth responses of corn earworm (Lep.: Noct.) larvae on meridic diets containing fresh and lyophilized corn silks. J. Econ. Entomol. 77: 1159-1162. Wiseman, B.R., and K. Bondari. 1992. Genetics of antibiotic resistance in corn silks to the corn earworm. J. Econ. Entomol. 85: 289-298. Wiseman, B.R., and K. Bondari. 1995. Genetic resistance in corn silks to the corn earworm (Lep.: Noct.). Entomol. Exp. Appl. 77: 315-322. Wiseman, B.R., and J.E. Carpenter. 1995. Growth inhibition of corn earworm (Lepidoptera: Noctuidae) larvae on resistant corn silk-diets. J. Econ. Entomol. 88: 1037-1043. Wiseman, B.R., R.C. Gueldner, and R.E. Lynch. 1984. Fall armyworm (Lep.: Noct.) resistance bioassays using a modified pinto bean diet. J. Econ. Entomol. 77: 545-549. Wiseman, B.R., and J.J. Hamm. 1993. Nuclear polyhedrosis virus and resistant silks enhance mortality of corn earworm (Lep.: Noct.) larvae. Biological Control. 3: 337-342. Wiseman, B.R., and D.J. Isenhour. 1988. The effects of prebioassay treatment of resistant and susceptible corn silks on the development of the corn earworm and fall armyworm. J. Agric. Entomol. 5: 247-251. Wiseman, B.R., and D.J. Isenhour. 1989. Effects of temperature on development of corn earworm (Lep.: Noct.) on meridic diets of resistant and susceptible corn silks. Environ. Entomol. 18: 683-686. Wiseman, B.R., and D.J. Isenhour. 1991. A microtechnique for antibiosis evaluations against the corn earworm. J. Kansas Entomological Society. 64: 146153. Wiseman, B.R., and D.J. Isenhour. 1992. Relationship of planting dates and corn earworm developmental parameters and injury to selected maize entries. Maydica 37: 149-156. Wiseman, B.R., and D.J. Isenhour. 1993. Interaction of diet ingredients with levels of silk of a corn genotype resistant to corn earworm (Lep.: Noct.). J. Econ. Entomol. 86: 1291-1296. Wiseman, B.R., R.E. Lynch, K.L. Mikolajczak, and R.C. Gueldner. 1986. Advancements in the use of a laboratory bioassay for basic host plant resistance studies. Florida Entomologist. 69: 559-565. Wiseman, B.R., and M.E. Snook. 1995. Effect of corn silk age on flavone content and development of corn earworm (Lep.: Noct.) larvae. J. Econ. Entomol. 88: 1795-1800. Wiseman, B.R., M.E. Snook, and D.J. Isenhour. 1993. Maysin content and growth of corn earworm larvae (Lep.: Noct.) on silks from first and second ears of corn. J. Econ. Entomol. 86: 939944. Wiseman, B.R., M.E. Snook, D.J. Isenhour, J.A. Mihm, and N.W. Widstrom. 1992a. Relationship between growth of corn earworm and fall armyworm larvae (Lep.: Noct.) and maysin concentration in corn silks. J. Econ. Entomol. 85: 24732477. Wiseman, B.R., M.E. Snook, and N.W. Widstrom. 1996. Comparisons of methods and relationship of corn earworm (Lepidoptera: Noctuidae) feeding responses and flavone content of corn silk. J. Econ. Entomol. (In Press). Wiseman, B.R., M.E. Snook, R.L. Wilson, and D.J. Isenhour. 1992b. Allelochemical content of selected popcorn silks: Effects on growth of corn earworm larvae (Lepidoptera: Noctuidae). J. Econ Entomol. 85: 25002504. Wiseman, B.R., and N.W. Widstrom. 1986. Mechanisms of resistance in ‘Zapalote Chico’ corn silks to fall armyworm (Lep.: Noct.) larvae. J. Econ. Entomol. 79: 1390-1393. Wiseman, B.R., and R.L. Wilson. 1987. Corn earworm development on meridic diets containing pollinated and unpollinated silks from two planting dates at two locations. Maydica 32: 201220. Development of Germplasm w ith Resistance to the European Corn Borer B.D. Barry and L.L. Darrah, Plant Genetics Research Unit, University of Missouri, Columbia, MO, USA Abst r a c t The European corn borer (ECB), Ostrinia nubilalis (Hübner), is a primary economic pest of maize, Zea mays (L.), in the United States. It was introduced into this country from Europe prior to 1917 when it was first described as a maize pest. Host-plant resistance studies began in the United States during the 1920s. Considerable progress in developing maize cultivars with first-generation ECB resistance was made by the 1950s when several inbreds with resistance to first-generation ECB were available. Due to lack of domestic resistant germplasm and the intensive labor required for identification of second-generation ECB resistance, few resistant cultivars were identified. However, with more emphasis placed on second-generation ECB resistance, it has been successfully identified by Missouri and Iowa scientists and levels enhanced by recurrent selection. In Missouri, germplasms Mo-2ECB and Mo-2ECB-2 and inbreds Mo45, Mo46, and Mo47 have been released as sources of resistance to both generations of ECB. The European corn borer (ECB), resistance research were established, and W.D. Guthrie assisted in Ostrinia nubilalis (Hübner), is a and some varietal resistance was developing several inbred lines with significant economic pest of maize, Zea identified (Patch and Pierce 1933; Patch the antibiosis type of resistance for mays (L.), in the United States. Annual 1947; Patch and Everly 1948). However, first-generation ECB, but germplasm losses are estimated between 200 and this was for first-generation ECB, and for second-generation ECB was not 500 million dollars for the Corn Belt. at this time, it was not realized that readily available in Corn Belt The ECB was first described as a pest of resistance for second-generation ECB germplasm, and labor required for maize in the United States in 1917 was a different genetic trait. F.F. Dicke identification prevented screening (Vinal 1917), but it probably entered the country about 1914 in hemp, Cannabis sativa (L.), or hops, Humulus lupulus (L.). In 1918, devastation of maize production by ECB in Medford, MA, occurred and was recorded by B.E. Hodgson (Fig. 1). As early as the late 1920s, Huber (Huber et al. 1928) suggested plant resistance as a control method. Patch, Schlosberg, and Vance promoted the idea while working with sweet and field maize (personal communication, Orlo Vance 1994). During the 1930s and 1940s, initial techniques for host-plant Figure 1. European corn borer damage to maize in 1918, shortly after ECB was introduced in the United States (photograph by B. E. Hodgson, Medford, MA). 218 B.D. BARRY AND L.L. DARRAH many germplasm sources. Dicke (1954) The Iowa State team of entomologists Because second-generation ECB suggested that the way to manage the and breeders has successfully identified resistant germplasm was not readily second-generation of ECB was to inbreds, such as B52 and B86, and other identified in the Corn Belt, it appeared develop tolerant plants, and to a large germplasm sources with second- that the logical place to seek new degree, this has been done by the maize generation ECB resistance. In 1975, a sources was the tropics. The first hint of breeders in their stalk strength new team including the disciplines of a new source of resistance was in maize improvement programs. Figure 2, entomology, plant pathology, and populations developed by Dr. M.S. illustrating results of selection for high breeding was organized in Missouri. At Zuber, a USDA-ARS maize breeder at and low stalk rind strength, indicates a Columbia, this team could work with the University of Missouri, which he mechanism by which tolerance may be longer-season maize germplasm, called PR-Mo2, PR-Mo2 x MoSQA and achieved. Although Figure 2 shows the including some tropical material, which PR-Mo2 x MoSQB. The source of the efficacy of selection for rind strength, could not be done in Iowa. resistance (PR-Mo2, released by USDA- the biological response by ECB is yet to ARS and the University of Missouri in be determined. 1975) was Nigeria Composite B, also a Plate A valuable source of resistance for Puccinia polysora (Underw.), Bipolaris maydis [(Nisik.) Shoem.], and Ustilago maydis [(DC.) Cda.]. Nigeria Composite B source material included Nigeria NS1 (Caribbean origin); NS-5 (Local varieties, Mexico 5, EAAFRO 231, and Sicaragua); University of Ibadan FlintDent Composite; Pioneer Brand X301 and X306; Caribbean Composite; Jamaican Selected Yellow; Dahomy Jaune d’la Ina; EAAFRO 231 (Rocamex 520C); Mexico Hybrids H503, H504, and H507; Ivory Coast M.T.S.; Kenya Coast Composite (Local varieties, Plate B Caribbean, Mexican, and Colombian lowland germplasm); Nigeria Bida Yellow; South Africa Tsola; and selected Tuxpeño and Caribbean material from the International Maize and Wheat Improvement Center (CIMMYT). By 1976, we had determined that PRMo2, PR-Mo2 x MoSQA, and PR-Mo2 x MoSQB were more resistant to secondgeneration ECB than an intermediately resistant hybrid, Pioneer Brand 3369A. These three populations had been Figure 2. Cross sections of internodes below the top ear node from stalks of representative plants from cycle 0 and cycle 6 of bi-directional selection in the internode below the top ear node for rind penetrometer resistance in MoSCSSS and their respective rind penetrometer resistance readings (load-kg/plant) (Plate A). Cross sections of the internode used for selection and those below showing progressive changes in rind thickness, stalk diameter, and stalk morphology ) (Plate B). selected for adaptation and, in MoSQA and MoSQB, for increased stalk crushing strength for several years by Dr. M.S. Zuber before we started our program. Our ECB breeding program DEVELOPMENT OF GERMPLASM WITH RESISTANCE TO THE EUROPEAN CORN BORER 219 (USDA-ARS and University of made after three cycles of selection Since then, we have identified several Missouri) began in 1977 and has (Tables 1 [includes evaluations for C4, other resistant cultivars from these continued to the present time. C5, and C6], 2, and 3) (Barry 1989). The regional trials. We are working with 11 Throughout the program, additional selection program was continued (list follows) of these which were germplasm, principally from exotic through cycle 6 for Mo-2ECB and Mo- crossed to a resistant (first- and second- sources, has been incorporated as it was 2ECB-2, as well as PR-Mo2 x MoSQA generation ECB) hybrid, Pioneer Brand identified. (Table 1). Maize breeders had suggested 3184, and the crosses were used to that this would provide further develop a composite breeding In 1977, we planted 1000+ seeds of each improved populations with a more population that has been improved by of Zuber’s populations and infested all desirable level of ECB resistance. using a modified recurrent selection program. Three inbreds, Mo45 (Negro plants with ECB egg masses and selfed about 400 of these plants. From 400 We have also screened germplasm from de Tierra Caliente exotic source), Mo46 selfed plants in each population, about the Regional Maize Disease and Insect (Cravo Paulista exotic source), and 200 were harvested and dissected to Resistance Nurseries that originated Mo47 (Candela exotic source), have measure stalk tunneling, and 10% of from the North Carolina program. One been released (Barry et al. 1995) as S6 these with the least amount of of the early selections for second- lines with resistance to first- and tunneling provided seed for genetic generation ECB resistance from these second-generation ECB. Evaluations of recombination in our Puerto Rican materials was NC 4-275. It came from these inbreds for ECB resistance and winter nursery. Selected ears from Dr. M.M. Goodman’s collection PAG yield in testcrosses (as S3s and S4s) were Puerto Rico were used for insect VI-A, race Moroti Guapi; and had been made at Columbia and Novelty, MO, selection and selfing in Missouri for the crossed with Dr. C.W. Stuber’s “D-2” during 1992. Results from these next generation. tester. This germplasm source has been evaluations are presented in Table 3. included in an experimental maize After five cycles of selection, Mo-2ECB population that we refer to as The 11 sources (race and collection (PR-Mo2 x MoSQB source) was “Experiment 52.” This population was given) currently undergoing selection released in 1983 (Barry and Zuber primarily developed from domestic include: 1984), and following six cycles of inbreds that demonstrated high yield 1. Cuban Tuscon, ECU 542 selection, Mo-2ECB-2 (PR-Mo2 source) potential and some resistance to first- 2. Early Caribbean, MAR 2 was released in 1984 (Barry et al. 1985). and/or second-generation ECB (e.g., 3. Nal-Tel A.T.B., GUA III In order to determine if our modified B52, SC13, SC13R, SC213R, NC33, Oh43, 4. Negro de Tierra Caliente, GUA III recurrent selection program was CI31A, B73, and Mo17). 5. Moroti, PR II making progress, evaluations were Table 1. Mean stalk tunneling (cm) by larvae of secondgeneration ECB in three maize populations during six cycles of recurrent selection for resistance. Population Year / cycle 1977 / C0 1978 / C1 ‡ 1979 / C2 1980 / C3 1981 / C4 1982 / C5 1983 / C6 † ‡ Control 22.0 25.3 32.5 13.9 12.8 8.4 9.4 20.5 20.3 21.3 11.8 9.2 6.1 8.3 — 22.6 30.1 15.4 15.0 10.6 12.5 † Mean stalk tunnel length (cm) † PR-Mo2 x PR-Mo2 x InterPR-Mo2 MoSQA MoSQB mediate Resistant 22.0 22.6 22.5 9.9 9.4 7.3 6.7 Table 2. Stalk tunnel length and least-squares estimates of gain from selection in three maize composite populations (PR-Mo2, PR-Mo2 x MoSQA, and PR-Mo2 x MoSQB). — 15.3 20.3 10.4 7.0 6.2 16.1 The intermediately resistant control was Pioneer Brand 3369A, except for 1980, when a susceptible single cross (Wf9 x W182E) was used. The resistant control was Pioneer Brand 3184. Cycles of selection were conducted at two locations, Columbia and Portageville, MO, except in 1979, when drought destroyed the Portageville tests. Type of meaná PR-Mo2 C0 12.9ab 12.3bc C1 11.4bc C2 C3 10.0c Resistant control 9.6c Susceptible control 15.2a Gain/cycle±SE -0.96±0.41 Percent gain cycle, based 7.3 on predicted value of C0 † ‡ PR-Mo2 x MoSQA PR-Mo2 x MoSQB 16.4a 13.9ab 14.4ab 13.5b 9.6c 15.2ab -0.84±0.40 11.7b 10.2bc 8.6cd 7.3d 9.6bc 15.2a -1.48±0.34 5.3 12.7 Means followed by the same letter within a column are not significantly different at the 0.05 probability level. Resistant control = Pioneer Brand 3184, susceptible control = Wf9 x W182E; checks were grown in common plots for all three populations. 220 B.D. BARRY AND L.L. DARRAH 6. Chandelle, CUB 54 Re fe re nce s 7. Candela, ECU 344 8. Caingang, PR III 9. Cuban Flint, CUB 65 10. Avanti Moroti Mita, PAG 106 11. Cravo Paulista, SP II The two composite maize populations and three inbred lines which have been released as ECB resistance sources should soon contribute to resistance in commercial hybrids. The hybrids developed should reduce yield loss caused by ECB and at the same time reduce the need for insecticide applications for control of ECB. Barry, B.D. 1989. Host plant resistance: Maize resistance to the European corn borer (Lepidoptera: Pyralidae). Acta Phytopathologica and Entomologica Hungarica. 24(1-2): 43-47. Barry, D, A.Q. Antonio, and L.L. Darrah. 1995. Registration of Mo45, Mo46, and Mo47 germplasm lines with resistance to European corn borer (PI583350, PI583351 and PI583352). Crop Sci. In press. Barry, D., and M.S. Zuber. 1984. Registration of Mo 2ECB(S1)C5 maize germplasm. Crop Sci. 24: 231. Barry, D., M.S. Zuber, and L.L. Darrah. 1985. Registration of Mo-2 ECB-2 maize germplasm. Crop Sci. 25: 715-716. Dicke, F.F. 1954. Breeding for resistance to European corn borer. Proc. Ann. Hybrid Corn Industry Res. Conf. 9: 44-53. Table 3. Mean ECB responses and testcross yields for Mo45, Mo46, and Mo47 evaluated at Columbia and Novelty, MO. (This table is from information provided with the original release notice for the three inbreds dated 22 February 1994). Inbred and level of inbreeding First-generation ECB rating† (1-9) Mo45 as S3 Mo45 as S4 Mo45 as S6 Mo46 as S3 Mo46 as S4 Mo46 as S6 Mo47 as S3 Mo47 as S4 Mo47 as S6 Rest. ck. for S3 (Pioneer 3184) Rest. ck. for S4 (Pioneer 3184) Rest. ck. for S6 (CI31A) Susc. ck. for S3 (Wf9 x W182E) LSD 0.05 † Secondgeneration tunneling (cm) 2.4 9.4 1.9 3.2 3.9 8.4 1.9 3.2 2.6 7.4 1.8 4.0 1.8 4.6 1.0 4.6 6.0 16.5 Tester Mo17 7.70 5.77 7.24 7.64 1.49 MoSCSSS Oh43 (t/ha) CI31A 7.52 7.86 8.41 7.20 8.05 6.64 7.77 9.88 8.73 1.23 1.37 7.22 7.80 9.17 1.79 The first-generation rating was based on a 1 to 9 scale in which 1 represented resistance and 9 represented susceptibility (Guthrie et al. 1960). Guthrie, W.D., F.F. Dicke, and C.R. Neiswander. 1960. Leaf and sheath feeding resistance to the European corn borer in eight inbred lines of dent corn. Ohio Agric. Exp. Sta. Res. Bull. 860. Guthrie, W.D., E.S. Raun, F.F. Dicke, G.R. Pesho, and S.W. Carter. 1965. Laboratory production of European corn borer egg masses. Iowa State J. Sci. 40: 65-83. Huber, L.L., C.R. Neiswander, and R.M. Salter. 1928. The European corn borer and its environment. Ohio Agr. Expt. Sta. Bull. No. 429. Patch, L.H. 1947. Manual infestations of dent corn to study resistance to European corn borer. J. Econ. Entolmol. 40: 667-671. Patch, L.H., and R.T. Everly. 1948. Contribution of inbred lines to the resistance of hybrid dent corn to larvae of the early summer generation of the European corn borer. J. Agr. Res. 76: 257-63. Patch, L.H., and L.L. Pierce. 1933. Laboratory production of clusters of European corn borer eggs for use in hand infestation of corn. J. Econ. Entomol. 26: 196-204. Vinal, S.C. 1917. The European corn borer, Pyrausta nubilalis Hübner, a recently established pest in Massachusetts. Mass. Agric. Exp. Stn. Bull. 178. Variability for Resistance to Fall Armyworm in Guadeloupe among M aize Populations Improved for Resistance to Various Insects C. Welcker, D. Clavel, J.D. Gilet, F. Felicite, and I. Guinet, INRA, Pointe-a-Pitre Cedex, Guadeloupe, F.W.I Abst r a c t Insect pests are one of the main constraints to the development and farming of maize in the Caribbean. INRACIRAD breeding efforts for well adapted maize populations with effective levels of resistance should contribute to the improvement of yield and yield stability. Screening of various insect resistant improved materials for resistance to fall armyworm, Spodoptera frugiperda (J.E. Smith) and for other characters with agronomical interest was undertaken. Multiple resistance has been observed in introduced (MIRT, TZBR) and local (PopG, Spectral) populations. The results show the high level of resistance of MpSWCB4 and ANTIGUA Gpo2, but also their low productivity. Advanced cycles, obtained through a recurrent S1 selection scheme, of a local improved population (PopG) show an intermediate level of resistance similar to FAWCC’s, but are associated with high adaptability. A study of the variability within these populations and transfer of resistance to high yielding populations was initiated. The interest of this variability and its utilization in selection are discussed. Int roduct ion breeding for resistance to insects We report here the results on requires first an appropriate screening variability for insect resistance in Insects pests are one of the main methodology (Mihm 1983) plus an breeding populations. These constraints to the development and assessment of the available variability populations have shown different farming of maize in the Caribbean. In (Painter 1951). levels of adaptation to Caribbean conditions associated with their Guadeloupe, joint breeding efforts of the French National Institute of Our main objective was to identify and resistance level. In the future, Agricultural Research and the Center improve regional genotypes with host agronomic characters such as vigor, for International Cooperation in plant resistance to insects. The first step plant height, ear productivity should Agricultural Research for Development of the selection process was to be associated with insect resistance in a (INRA-CIRAD), France, for well introduce various insect resistant selection index (Overman 1989; Thome adapted maize populations with improved materials and to screen for et al. 1994). effective levels of resistance to leaf- FAW resistance under Caribbean feeding by fall armyworm (FAW), conditions. Then, we screened the best Spodoptera frugiperda (J.E. Smith), adapted resistance sources. Studies of should contribute to the improvement the variability within these populations Since 1989, a wide diversity of of yield and yield stability. and the transfer of resistance to high germplasm has been screened for M aterials and M ethods yielding populations were initiated. reaction to natural or artificial Initial breeding operations led to the The results have highlighted the infestation by FAW and CEW, creation in 1988 of a well-adapted potential of some populations for use in according to the artificial infestation variety, named ‘Spectral’, with medium a breeding program. methodology developed by Mihm susceptibility to insects. However, (1983). 222 C. WELCKER, D. CLAVEL, J.D. GILET, F. FELICITE, AND I. GUINET Previous host plant resistance results populations improved for resistance to demonstrated that controlled, uniform, Sesamia calamistis (PSB) or to Eldana artificial infestations are needed to sacharina (ASCB) from IITA (Kling et al. develop insect resistant germplasm 1994) (Fig. 1). Results and Discussion Formation of FAW resistant composite In 1989-90 advanced inbred lines from (Williams 1978; Mihm 1989). Since 1993, we have developed, in association All these sources have been compared Antigua germplasm selected at with French entomologists, efficient to local materials, such as PopG and CIMMYT for resistance to FAW and FAW mass rearing and screening pools of Guadeloupean ecotypes resistance to SWCB, plus full sib methodologies. The mass rearing (Welcker 1993; Welcker et al. this families of MBR selected for SWCB laboratory is based in France at Le review), and to INRA improved resistance, were tested (Clavel et al. Magneraud INRA Station (7000km populations (Spectral, PopA, CR01) 1993). Components of these from Guadeloupe). We have developed (Fig. 1). We have described this populations were evaluated in 1989 for an efficient egg transfer from France, germplasm in Guadeloupean their per se value and, in 1990, for the coordinating egg production and environments (particular climatic and best families from their S1 progenies. artificial infestations. soil conditions, under FAW pressure) We have selfed 98 plants within 24 for resistance parameters and other selected families of Antigua-FAW. On The first egg productions were used to agronomical characters. The following the other hand, we have selected less screen various lines and populations results on resistance parameters are families and plants of populations for resistance to FAW. Nowadays, the presented in chronological order, when previously selected for SWCB. Our laboratory produces 4 million eggs per breeding populations and screening results have shown that available year for the FAW resistance breeding methods were simultaneously variability existed between and within program and for developing biological enhanced. these populations. According to insecticides. Native strains of FAW are relative population levels, more reintroduced into the mass rearing families and individuals from Antigua- program every six generations in order FAW were kept in the formation of the to preserve insect diversity and vigor. Artificial infestations are made with 25 larvae per plant (5 leaves stage) and resistance evaluation is based on FAW FAWCC damage rating 14 days after infestation (DAI) using the Davis and Williams (1992) scale (0 to 9). USDA Plant materials chosen for studies were SWCB Diatraea grandiosella MpSWCB4 derived from populations improved by MIRT & MBR selection efforts in tropical and subtropical areas and introduced to Guadeloupe (Clavel et al. 1993). These FAW Spodoptera frugiperda AFAW included multiple resistance sources, populations developed by Mihm (Smith et al. 1989). We also screened more specific resistant germplasm from FAW & SWCB Antigua Gpo 2 CIMMYT like MIRT, MBR or Antigua INRA Guadaloupe FAW & CEW PopG Spectral Local pools SSB Eldana saccharina TZBR.E USDA such as FAWCC with resistance to FAW (Widstrom et al. 1992) and MpSWCB4 with resistance to SWCB (Scott et al. 1981), and TZBR IITA PSB Sesamia calamistis TZBR.S Figure 1. Maize germplasm with resistance to insects screened in Guadeloupe for fall armyworm resistance. VARIABILITY FOR RESISTANCE TO FALL ARMYWORM IN GUADELOUPE AMONG MAIZE POPULATIONS IMPROVED FOR RESISTANCE TO VARIOUS INSECTS 223 FAW resistant composite ‘SPODO’ (Fig. (extremely susceptible). A susceptible artificial infestation with 25 fall 2). The MBR population was not really check entry rated 3.8 and a resistant armyworm larvae per plant. Materials well adapted to lowland tropics, check entry rated 2.6. We selected ears included: affecting its resistance performance in from families rating 2.5 or less for • our conditions. After two intercrossing utilization as resistant sources. Most population known for its high level generations, the composite ‘SPODO’ families rated either as resistant or of resistance to SWCB and FAW. could be an interesting source of intermediate across sites (Smith et al. resistance to FAW. 1989). In Guadeloupe, the resistant category comprised no more than 10% • MpSWCB4 from USDA, a GT populations with resistance to CEW from USDA. • TZBR-E and TZBR-S populations Potential interest of MIRT of the families tested, but represents introduced from IITA and improved In 1991, 196 full sib families of MIRT useful levels of resistance with good respectively for resistance to ASCB were screened in an international potential for adaptation to the area. and to PSB. • testing trial proposed by John Mihm. Figure 3 illustrates the number of • intermediate, and susceptible to FAW in Guadeloupe. Ratings were done on a A wide range of germplasm was tested • scale of 1 (extremely resistant) to 5 in a replicated trial in 1993 under families classified as resistant, ANTIGUA FAW ANT. SWCB MBR. SWCB 65-S2 29-S4 47-FS 1989 per se test 24 selected 6 fam. families 15 families 100 1990 98 S3 S1 progenies test 12 S5 38 S1 50 16 sel. fam. 3f 2 fam. 0 6 Resistant Inter- Susceptible (1 - 2.5) mediate (3.6 - 5) (2.6 - 3.5) 2 1991 & 1992 Composite formation ;; ; ; ;;; Number of families 150 51 S3 Leaf-feeding ratings 35 days after sowing Figure 2. Formation of the fall armyworm resistant population ‘composite SPODO’. Figure 3. Damage ratings for fall armyworm on 196 full sib families of MIRT in Guadeloupe - 1991. Table 1. Populations tested for resistance to insects (infested trial with fall armyworm) and adaptation in Guadeloupe in 1993. Different breeding Antigua entries from CIMMYT. FAW resistance levels among various insect resistant populations Main sources of resistance to CEW i.e. Zapalote and Maïa. Local germplasm such as native populations, pools of ecotypes and selected varieties (IRAT340 as a susceptible check) (Table 1). The results underlined the good performance of MpSWCB4 and Antigua, an intermediate position of several populations including other Antigua materials and Guadeloupean materials (Fig. 4). PopG-C1 performed better than its pool of after two intercrossing generations and PopA — a result of selection between progenies of MpSWCB4, ETO and a recombined population of local material — presents an interesting level of insect resistance and lowland tropical adaptation. Populations improved for resistance to Sesamia calamistis in Africa perform better than those selected for Eldana sacharina. So, TZBR-S should present multiple resistance to PSB and FAW, although TZBR-E3, selected for Eldana calamistis, has shown a high level of resistance to CEW (Welcker, this Germplasm Origin Germplasm Origin Germplasm Origin review). Zapalote chico seems to be 1 MpSWCB4 2 GTRI4 3 GTRI9 4 TZBR-E1 5 TZBR-E2 6 TZBR-E3 7 TZBR-S1 8 TZBR-S3 USDA USDA USDA IITA IITA IITA IITA IITA 9 Antigua gpo2 10 Antigua 2D.118 11 A1-FAW-tux 12 A2-FAW-ntux 13 A3-FAWgca 14 Zapalote Chico 15 Zapalote Grande 16 Maïa XXIX CIMMYT CIMMYT CIMMYT CIMMYT CIMMYT CIMMYT CIMMYT CIMMYT 17 Fond’or 18 Desirade 19 Pop1/2 P 20 Pop T 21 PopG-C1a 22 PopA 23 Spectral 24 IRAT 340 INRA INRA INRA INRA INRA INRA INRA CIRAD better than Zapalote grande and similar to our local early population Desirade. 224 C. WELCKER, D. CLAVEL, J.D. GILET, F. FELICITE, AND I. GUINET Evidence of genotype-byenvironment interaction for FAW resistance Statistical analysis indicated the environment (GxE) interactions. Genetic variation for FAW resistance within breeding populations Based on these results, selected Extreme differences between resistant Although information on genetic populations were tested in 1994 in and susceptible checks appeared variation between and within these different environments to determine constant (Fig. 5). Response to FAW populations can contribute to choosing the stability of their response and between other populations varied an appropriate breeding strategy, plant respective interest. MpSWCB4 was the significantly from site to site, to plant variation within some resistant check and two high yielding suggesting that the effect of populations appears to be important, varieties (FWIP136 and PioneerX304C) environmental conditions on damage suggesting that potential variability were used as susceptible checks. Figure rating is generally high. Hence, it remains in these populations. Figure 6 5 shows the variability of response to should be integrated as a main factor in illustrates the results for five breeding FAW of these populations in tests in selection (Smith et al. 1989; Widstrom populations, with observations on one four environments (i.e., different dates et al. 1992). Stable performances of hundred plants per population of sowing and differing intensities of Spectral, selected in a multilocal trial screened in the most discriminant FAW infestation). for adaptation to environmental environment of the latest multilocal constraints of the area, could sustain experiment. presence of significant genotype by Axe 2 (32%) Resistance Morphology 9 7 10 22 11 13 16 20 8 24 21 19 12 4 2 23 3 30 20 10 0 6 15 14 30 20 10 0 Figure 4. Variation among maize populations in Guadeloupe - 1993. FWIP136 FAWCC4 Spectral Pop. G-C1a Pioneer X304C 5 CRO1 Antigua GPO2 MpSWCB 4 4 3 2 Env 2 environments / infestation intensity Env 1 Env 3 Env 4 Figure 5. Fall armyworm damage ratings of breeding populations grown in four environments. No. of plants 14 DAI ratings 6 No. of plants Axe 1 (52%) No. of plants 17 7 ;; ; ;; ;; ;; ; ;; ;; ; ; ;; ; ;;;; ; ;;;; ;; ;; ; ; ;;;;; ;; ;;; µ’= 3.1 µ= 3.51 30 20 10 0 30 20 10 0 30 20 10 0 MpSWCB4 σ◊w = 0.788 σ◊w: genetic variability µ= observed mean µ’= selected mean µ= 3.36 µ’= 2.45 5 No. of plants 1 16 No. of plants this approach. Antigua Gpo2 σ◊w = 1.425 µ= 3.79 CR01 σ◊w = 0.915 µ’= 2.71 µ’= 3.7 µ= 4.67 FAW CC σ◊w = 0.737 Observed plants Selected plants Spectral σ◊w = 1.387 µ= 5.48 µ’= 3.5 1 2 3 4 5 6 7 8 Leaf-feeding ratings 14DAI 9 Figure 6. Genotypic variation within breeding populations for feeding damage by fall armyworm. VARIABILITY FOR RESISTANCE TO FALL ARMYWORM IN GUADELOUPE AMONG MAIZE POPULATIONS IMPROVED FOR RESISTANCE TO VARIOUS INSECTS Compared to the mean value of continuing to place the greatest MpSWCB4, CRO1 and Antigua Gpo2 emphasis on developing insect resistant show a good level of resistance. source populations. Nevertheless, there are differences between the damage rating Ac k now le dgm e nt distributions of the populations studied (Fig. 6). Within genetic variation was Thanks to John Mihm for his great and estimated from residual variance of the helpful collaboration. hybrid check and residual variance of the model for each population. Re fe re nce s The results suggest that there remains Clavel, D., I. Guinet, and C. Welcker. (1993) Evaluacion de germoplasm de maize para la resistancia a Spodoptera frugiperda (Antillas Francescas), PCCMCA, Guatemala City. Davis, F.M., S.S. Ng, and W.P. Williams. (1992) Visual rating scale for screening whorl-stage corn for resistance to fall armyworm. Mississippi Agricultural and Forestry Experiment Station technical bulletin 186. Kling, J.G., and N.A. Bosque-Pérez. (1994) Progress in screening and breeding for resistance to the maize stem borers Eldana saccharina and Sesamia calamistis. In Proceedings of The Fourth Eastern and Southern Africa Regional Maize Conference, Maize Research for Stress Environments, Harare, Zimbabwe. Mihm, J.A. (1983) Efficient mass rearing and infestation techniques to screen for resistance to fall armyworm, Spodoptera frugiperda. In Maize program report, 1223. Mexico, D.F.: CIMMYT. Mihm, J.A. (1989) Evaluating maize for resistance to tropical stem borers, armyworms, and earworms. In Toward Insect Resistant Maize for the Third World: Proceedings of the International Symposium on Methodologies for Developing Resistance to Maize Insects, 109-121. Mexico, D.F.: CIMMYT. Overman, J.L.(1989) A maize breeding program for development of hybrids with resistance to multiple species of leaf-feeding and stalk-boring lepidoptera. In Toward Insect Resistant Maize for the Third World: Proceedings of the International Symposium on Methodologies for Developing Resistance to Maize Insects, 235-243. Mexico, D.F.: CIMMYT. sufficient variation within these populations to justify recurrent selection, especially in Antigua gpo2 (as J. Mihm proved), and in Spectral, the breeding population developed by INRA for adaptation to Caribbean conditions (Fig. 6). The best plants were selfed, and S1 progeny testing will provide useful information about genetic variability and expected selection response within each population. Conclusion The great variability and relatively good response observed in Antigua materials support their potential for use in a selection program and for crossing with other resistant sources and adapted populations to provide significant additive gain. The importance of GxE indicates the effectiveness of testing at more than one location and of enhancing international cooperation. Some attention will be given to agronomic characteristics in the future, while 225 Painter, R.H. (1951) Insect resistance in crop plants. The MacMillan Co., New York.. Scott, G.E., and F.M. Davis. (1981b) Registration of MpSWCB-4 population of maize. Crop Sci. 21: 148. Smith, M.E., J.A. Mihm, and D.C. Jewell. (1989) Breeding for multiple resistance to temperate, subtropical, and tropical maize insect pests at CIMMYT. In Toward Insect Resistant Maize for the Third World: Proceedings of the International Symposium on Methodologies for Developing Resistance to Maize Insects, 222-234. Mexico, D.F.: CIMMYT. Thome, C.R., M.E. Smith, and J.A. Mihm. (1994) Yield reduction in a maize diallel under infestation with southwestern corn borer. Crop Sci. 34 (6): 1431-1435. Welcker, C. (1993) Breeding for resistance in maize to fall armyworm in Caribbean region. Plant Resistance to Insects News Letter 20: 19-20. Welcker, C., J.D. Gilet, D. Clavel, and I. Guinet. (1997) Response to selection for resistance to leaf feeding by fall armyworm in PopG, Guadeloupean maize population. In Insect Resistant Maize - Recent Advances and Utilization. Proceedings of the Symposium held 27 November - 3 December, 1994. Mexico, D.F.: CIMMYT. Welcker, C., G. Febvay, and D. Clavel. (1997) Variability for maysin in maize germplasm developed for insect resistance. In Insect Resistant Maize Recent Advances and Utilization. Symposium of the 27 November - 3 December, Mexico, D.F.: CIMMYT.(in press) Widstrom, N.W., W.P. Williams, B.R. Wiseman, and F.M. Davis. (1992) Recurrent selection for resistance to leaf feeding by fall armyworm on maize. Crop Sci. 32: 1171-1174. Williams W.P., F.M. Davis, and G.E. Scott. (1978) Resistance of corn to leaf feeding damage by the Fall armyworm. Crop Sci. 18: 861-863. M aize Germplasm with Resistance to Southwestern Corn Borer and Fall Armyworm W.P. Williams and F.M. Davis, USDA-ARS, Mississippi State, MS, USA. Abst r a c t Leaf feeding by the Southwestern corn borer (SWCB), Diatraea grandiosella Dyar, and the fall armyworm (FAW), Spodoptera frugiperda (J.E. Smith), can result in substantial reductions in grain yield of maize, Zea mays L. Development and deployment of varieties and hybrids with resistance to these pests can greatly reduce these losses. Scientists working in Mississippi have developed and released nine maize germplasm lines and one population as sources of leaf feeding resistance to these pests. These lines were derived primarily from Antigua Gpo. 2 germplasm originally obtained from the International Maize and Wheat Improvement Center (CIMMYT). In developing the earlier released lines, selection was based entirely on visual ratings of leaf feeding damage; however, larval growth was also considered in the development and release of the newer lines. Analyses of diallel crosses among resistant and susceptible lines indicated that general combining ability was the primary source of variation in the inheritance of resistance to fall armyworm and southwestern corn borer whether resistance was measured as either reduced leaf feeding or reduced larval growth. In 1992, in cooperation with the United States Department of Agriculture, and Agricultural Research Service (USDA–ARS) scientists at Tifton, Georgia, GT–FAWCC(C5) maize germplasm population was released. This population was developed by five cycles of S1 progeny selection for resistance to leaf feeding by fall armyworm. research program have been: a reliable source of insects for infesting • Identification of maize germplasm plants; 2) techniques for evaluating Plant resistance is an attractive method with resistance to fall armyworm damage; and 3) a source of resistant of insect control. It provides farmers (FAW), Spodoptera frugiperda (J.E. germplasm. At the first CIMMYT with a means of preventing or reducing Smith), and southwestern corn borer symposium on insect resistance, Frank yield losses while avoiding the costs and (SWCB), Diatraea grandiosella Dyar. Davis described our insect rearing Int roduct ion • Development and release of breeding program (Davis 1989) and the methods insecticides. For plant resistance to be a lines and populations to maize we use for evaluating germplasm for viable alternative to chemical control of breeders with public or private resistance to FAW and SWCB (Davis insects in maize, Zea mays L., sources of institutions engaged in development and Williams 1989). resistant germplasm must be identified, of hybrids and varieties. hazards associated with chemical When screening maize for resistance to and agronomically acceptable hybrids and varieties deployed to farmers. For almost 30 years, scientists with the It is our expectation and desire that leaf feeding by FAW and SWCB, we use these breeders will use the germplasm similar procedures for the two insects. we release to develop superior hybrids Most germplasm is evaluated for United States Department of Agriculture with resistance to FAW and SWCB, reaction to both insects. Although it (USDA), Agricultural Research Service thereby ultimately making such hybrids depends somewhat on availability of (ARS), Corn Host Plant Resistance available to farmers. seed and heterogeneity of the material to be evaluated, we most frequently Research Unit at Mississippi State, Mississippi (USA) have conducted To successfully identify and develop evaluate breeding material in one row, research on insect and disease pests of maize with resistance to insects, a 20 plant plots with two or three maize. The primary objectives of our program such as ours must first have 1) replications per insect. Plants in the 8– MAIZE GERMPLASM WITH RESISTANCE TO SOUTHWESTERN CORN BORER AND FALL ARMYWORM 227 to 10–leaf stage of growth are infested heterogenous population as sources of We also cooperated with scientists in with 30 larvae/plant; leaf feeding is resistance to leaf feeding by FAW and the USDA–ARS Insect Biology and visually rated 14 days after infestation. SWCB (Table 1): Mp496 (Scott and Populations Management Research Davis 1981a); Mp701 and Mp702 (Scott Laboratory in a joint release of GT– et al. 1982); MpSWCB–4 population FAWCC(C5) maize germplasm (Scott and Davis 1981b); Mp703 population in 1992 (Widstrom et al. At the first CIMMYT symposium on (Williams and Davis 1980); Mp704 1993). This population was developed insect resistance in maize, the breeding (Williams and Davis 1982); Mp705, by five cycles of recurrent S1 progeny methods that we have used to develop Mp706, and Mp707 (Williams and selection at Tifton, GA and Mississippi maize germplasm lines with resistance Davis 1984a); and Mp708 (Williams et State, MS for resistance to FAW to leaf feeding by FAW and SWCB al. 1990). All of these were derived from damage. The original breeding were described (Williams and Davis germplasm initially obtained from population was created by combining 1989). The procedure that we have most CIMMYT. It is also evident (Table 1) three broadbased breeding populations: frequently followed has been to self– that Antigua Gpo. 1 and 2 and a bulk of more than 60 Mexican and pollinate plants in a source population; Republica Dominicana Gpo. 1 are the Caribbean collections, a bulk of six evaluate the S1 progeny rows in primary sources of this resistance. We collections with Antigua background, replicated experiments; select those have screened germplasm from other and a bulk of 100 Brazilian collections. genotypes showing the least damage; sources, but, unfortunately, we haven’t self–pollinate plants in uninfested found significant resistance in them. Inheritance of Resistance for approximately eight generations. At The lines that we have released We have conducted only limited studies times, the procedures have been varied generally exhibit an intermediate level on the inheritance of leaf feeding somewhat: plants in infested rows were of resistance in our tests at Mississippi resistance to either FAW or SWCB. The self–pollinated, or remnant seed of State (Table 2). Mp496, the first line resistance is not simply inherited. selected rows was grown in a winter released, frequently falls into the Although visual ratings of leaf feeding nursery. susceptible (7–9) rating category. The are extremely useful in a breeding Breeding for Resistance nursery rows; and repeat the process SWCB ratings in Table 2 are three–year program when the primary focus is on Our breeding program has relied averages. FAW damage was rather low eliminating susceptible germplasm as heavily on germplasm from CIMMYT. in 1994, so those data were not quickly and inexpensively as possible, We have released and registered nine combined with the 1992–93 data. they are less useful in differentiating highly inbred germplasm lines and one among genotypes that vary only slightly in level of resistance. Regrettably, the latter situation is the Table 1. Nine germplasms and one population with resistance to 1 southwestern corn borer and fall armyworm developed and released at Mississippi State, MS. Germplasm Year of release Mp496 1974 MpSWCB–1 (Mp701) 1975 MpSWCB–2 (Mp702) 1975 MpSWCB–4 population Mp703 Mp704 1979 1982 Mp705 Mp706 Mp707 Mp708 1984 1984 1984 1988 1 1976 Source Grain color Antigua Gpo. 2 Antigua Gpo. 1,2 Antigua Gpo. 2, Republica Dominicana Gpo. 1 Antigua Gpo. 1,2 Guadelupe Gpo. 1A, Republica Dominicana Gpo. 1A Antigua Gpo. 1,2 Mp496, Republica Dominicana Gpo. 1 MpSWCB–4 MpSWCB–4 MpSWCB–4 Mp704, Tx601 Orange Yellow Yellow Yellow–orange one we usually find ourselves in when conducting genetic studies. In an analysis of a diallel cross of nine inbred lines evaluated for FAW damage under natural infestation, both general and specific combining ability were found to be significant sources of variation in the inheritance of resistance to leaf feeding (Williams et al. 1978). In Pale yellow the analysis of a six–parent diallel Yellow Yellow Yellow Yellow–orange evaluated for SWCB leaf feeding Seed are available in limited quantities from the Department of Plant and Soil Sciences, Box 9555, Mississippi State, MS 39762 (USA). damage after infestation with 30 neonates per plant, general combining 228 W.P. WILLIAMS AND F.M. DAVIS ability was a significant source of feeding resistance. The germplasm base height of the susceptible hybrid was variation, but specific combining ability for these lines is, unfortunately, rather reduced 18%, and yield was reduced was not (Williams and Davis 1985). narrow. We would very much like to 39%. identify additional sources of resistance. More recently, we evaluated an eight– If other sources of resistance do not In Mississippi, FAW damage can be parent diallel cross for both FAW and occur naturally, we may have to rely on especially heavy on maize that is SCWB resistance (Williams et al. 1989). genetic engineering approaches to planted later than normal. Leaf–feeding We selected parental inbred lines that provide them. Also, we have not yet resistant and susceptible maize hybrids had previously exhibited a range of leaf identified germplasm that has resistance were planted after wheat, Triticum feeding damage by the two insects; to SWCB during the reproductive stage aestivum L., was harvested to compare however, we evaluated the diallel cross of growth. This could be due to either their yields in a double cropping for larval growth and survival rather unsatisfactory evaluation techniques or system (Sanford et al. 1988). The maize than using the more subjective leaf a lack of germplasm sources with hybrids were planted about two feeding ratings. The correlation between resistance at this stage of growth, or months later than usual for our area number of FAW and SWCB surviving both. and were subjected to large naturally occurring FAW populations. The leaf on the different crosses was highly significant (r = 0.74) as was the Although high resistance levels have feeding resistant hybrids yielded 62% correlation between larval weights of not yet been obtained, the resistant more grain and 53% more silage than the two species (r = 0.81). General and germplasm that we have identified can the susceptible hybrids. specific combining abilities were reduce damage from these insects. In significant sources of variation for both one experiment, leaf–feeding resistant larval number and larval weight for hybrid, Mp496 x Mp701, and a leaf– both insects. feeding susceptible hybrid, Ab24E x Contribution of the USDA, ARS Crop Mp305, were infested with SWCB larvae Science Research Laboratory in while plants were in the whorl stage of cooperation with the Mississippi growth (Williams and Davis 1984b). Agricultural and Forestry Experiment In our quest for maize germplasm with When infested with 40 larvae per plant, Station, Mississippi State, MS, USA. resistance to FAW and SWCB, we have neither the height nor yield of the Published as Paper no. P–8613 of the developed several lines with leaf resistant hybrid was reduced. The Mississippi Agric. and Forestry Exp. Effectiveness of Resistance Ac k now le dgm e nt Stn. Re fe re nce s Table 2. Mean ratings of leaf feeding damage sustained by released lines infested with southwestern corn borer (SWCB) and fall armyworm (FAW) larvae in 1992–1994 at Mississippi State, MS. 1 SWCB Line 1 FAW 1 1992–94 1992–93 1994 Mp496 Mp701 Mp702 Mp703 Mp704 Mp705 Mp706 Mp707 Mp708 Ab24E2 Tx6012 7.3 6.0 6.9 5.3 5.4 6.6 6.5 5.8 6.1 7.9 7.9 7.0 6.4 6.5 6.3 5.7 6.3 6.8 6.0 5.7 7.8 8.4 4.3 2.7 4.0 3.3 1.7 2.7 2.7 3.0 3.0 7.0 6.0 LSD (0.05) 0.7 0.9 1.6 Damage was visually rated 14 days after infestation with 30 neonates per plant on a scale of 0 (no damage) to 9 (extensive damage). Davis, F.M. 1989. Rearing the southwestern corn borer and fall armyworm at Mississippi State. In Toward Insect Resistant Maize for the Third World: Proceedings of the International Symposium on Methodologies for Developing Host Plant Resistance to Maize Insects, 27–36. Mexico, D.F.: CIMMYT. Davis, F.M., and W.P. Williams. 1989. Methods used to screen maize for and to determine mechanisms of resistance to the southwestern corn borer and fall armyworm. In Toward Insect Resistant Maize for the Third World: Proceedings of the International Symposium on Methodologies for Developing Host Plant Resistance to Maize Insects, 101–108. Mexico, D.F.: CIMMYT. MAIZE GERMPLASM WITH RESISTANCE TO SOUTHWESTERN CORN BORER AND FALL ARMYWORM Sanford, J.O., W.P. Williams, J.E. Hairston, and L.L. Reinschmiedt. 1988. Doublecropping insect and disease resistant corn with wheat. J. Prod. Agric. 1: 60–63. Scott, G.E., and F.M. Davis. 1981a. Registration of Mp496 inbred of maize. Crop Science 21: 353. Scott, G.E., and F.M. Davis. 1981b. Registration of MpSWCB–4 population of maize. Crop Science 21: 148. Scott, G.E., F.M. Davis, and W.P. Williams. 1982. Registration of Mp701 and Mp702 germplasm lines of maize. Crop Science 22: 1270. Widstrom, N.W., W.P. Williams, B.R. Wiseman, and F.M. Davis. 1993. Registration of GT–FAWCC(C5) maize germplasm. Crop Science 33: 1422. Williams, W.P., P.M. Buckley, and F.M. Davis. 1989. Combining ability for resistance in corn to fall armyworm and southwestern corn borer. Crop Science 29: 913–915. Williams, W.P., and F.M. Davis. 1980. Registration of Mp703 germplasm line of maize. Crop Science 20: 418. Williams, W.P., and F.M. Davis. 1982. Registration of Mp704 germplasm line of maize. Crop Science 22: 1270. Williams, W.P., and F.M. Davis. 1984a. Registration of Mp705, Mp706, and Mp707 germplasm lines of maize. Crop Science 24: 1217. Williams, W.P., and F.M. Davis. 1984b. Reaction of a resistant and a susceptible corn hybrid to various southwestern corn borer infestation levels. Agron. J. 76: 855–856. Williams, W.P., and F.M. Davis. 1985. Southwestern corn borer larval growth on corn callus and its relationship with leaf feeding resistance. Crop Science 25: 317–319. 229 Williams, W.P., and F.M. Davis. 1989. Breeding for resistance in maize to southwestern corn borer and fall armyworm. In Toward Insect Resistant Maize for the Third World: Proceedings of the International Symposium on Methodologies for Developing Host Plant Resistance to Maize Insects, 207–210. Mexico, D.F.: CIMMYT. Williams, W.P., F.M. Davis, and G.L. Windham. 1990. Registration of Mp708 germplasm line of maize. Crop Science 30: 707. Williams, W.P., F.M. Davis, and G.E. Scott. 1978. Resistance of corn to leaf–feeding damage by the fall armyworm. Crop Science 18: 861–863. M aintenance of, and Requests for, M aize Germplasm Having Resistance to Insect Pests R.L. Wilson, USDA-ARS, Iowa State University, Ames, IA, USA Abst r a c t There are 33,766 maize accessions in the US National Plant Germplasm System (NPGS). Just over 13,000 are in the active collection maintained at the North Central Regional Plant Introduction Station at Ames, IA, USA. Through extensive evaluation, many of these accessions have been identified as containing genes for host-plant resistance to several maize insect pests. This presentation provides a general discussion of how the insect-resistant maize germplasm is regenerated and stored. Accessions from low latitudes present problems for seed regeneration in Iowa because of their photoperiod sensitivity. An increased frequency of requests for insect resistant germplasm usually follows the publication of evaluation results. These requests for seed are documented in the NPGS’s Germplasm Resources Information Network (GRIN). Illustrations of the number of requests for insect resistant germplasm are presented. Requests, both foreign and domestic, originate mostly from private companies and public institutions. Researchers are asked at the time of seed request to provide the maize curator with a performance report of their evaluation results. Int roduct ion The US National Plant Germplasm for and the availability of insect Let’s first look at germination testing. resistant maize germplasm for This test is performed at least every 5 researchers are discussed. years. Four replications of 50 seeds are placed on wet paper toweling, rolled System (NPGS) includes a collection of 33,766 maize accessions. These accessions have been collected or M aintenance of M aize Ge rm plasm into a tube, and placed in a germination chamber set at 20º C with 12 hours of darkness and 30º C during donated from 127 countries around the world. When they are incorporated into The NCRPIS active maize collection is 12 hours of light. The number of seeds the NPGS, the NPGS accepts the stored in clear plastic, one gallon (3.78 germinating is counted after 7 days and responsibility of maintaining them. The liter) jars at 4º C and a relative then again at 10 days. The total percent active, working collection of 13,000+ humidity of 25-40% (Clark 1989). The germination is calculated and entered maize accessions is maintained at the maize curator, Mark Millard, manages into the computer records for that USDA/ARS North Central Regional the collection. He decides which accession. Plant Introduction Station (NCRPIS) at accessions need regeneration each year Ames, IA. Scientists needing maize based on seed availability and Regeneration of maize in the field is an germplasm for research should direct germination percentage. If a particular important function performed their requests to the NCRPIS in Ames. accession is requested frequently routinely at the NCRPIS. When (normal seed requests are for 100 regenerating Corn Belt adapted maize The essence of this talk is a description kernels) and the supply of seed on germplasm, 200 plants are planted in of how the NCRPIS maize germplasm hand is less than 2,500 kernels, then blocks of four rows each and pair- is stored, regenerated, and tested for that accession will be regenerated. If crossed by hand. Shoot bags are placed germination. In addition, my progress routine germination tests indicate that on the developing ears before silks with evaluating the collection for fewer than 85% of the kernels of a appear to prevent contamination by sources of host-plant resistance to particular accession germinate, then it extraneous pollen. Larger bags are insects will be reported. The requests will be regenerated. placed over the tassels to collect the MAINTENANCE OF, AND REQUESTS FOR, MAIZE GERMPLASM HAVING RESISTANCE TO INSECT PESTS pollen. The pollen is collected from one me in evaluating the large number of NCRPIS collection for all of these plant and placed on the silks of another accessions maintained in the collection. important maize pests. plant. Ideally, plants are used as either Usually, when I find a new source of a male or a female parent but host-plant resistance to insects, these sometimes a plant may be used for scientists will cooperate with me to both. This method helps maintain the confirm the resistance in other genetic integrity for each accession. locations. The NCRPIS also receives 231 Evaluation of M aize at the NCRPIS for Host-Plant Resistance to Insects requests for maize from scientists who European corn borer (ECB) Long-season, or day length sensitive are interested in evaluating the Evaluating for whorl leaf-feeding (in maize lines, are regenerated in a winter germplasm for new sources of host- the United States, this would be the nursery located near Isabela, Puerto plant resistance to insect pests which I plant growth stage susceptible to 1st Rico. Sometimes original accessions am not able to evaluate. generation attacks) resistance involves include few kernels and they must be increased in the greenhouse at Ames. Uses of M aize Germplasm a well established technique. Newly Maize seed is sent to researchers at no hatched ECB larvae (about 300) are cost. We ask that requesters send a placed in the whorl of six maize plants progress report detailing results of at the V4-V6 stage of development with their experiments. The information the “bazooka” applicator (Mihm 1983). One of the criticisms that has been received can then be entered into the Three weeks after infestation, the directed to the NPGS is that there is not Germplasm Resources Information plants are visually rated using the scale enough information available about its Network (GRIN) so that the evaluation developed by Guthrie et al. (1960). accessions. Chapman (1989) said “Until data are available to all. Any scientist Ratings of 1-3 are categorized as a collection has been evaluated and with a personal computer, a modem, resistant, 4-6 are intermediate, and 7-9 something is known about the material and communication software can are susceptible. Resistant inbred CI31A it contains, it has little practical value”. access GRIN. Login IDs can be and susceptible inbred WF9 are also Many plant scientists will not request obtained at no cost from the National planted as checks. Using this technique, maize accessions that are accompanied Germplasm Resources Laboratory I can evaluate 700-1,000 accessions per by little descriptive information. If a (Telephone No. 301-504-6235) in year depending on the availability of plant breeder or other scientist requests Beltsville, MD, USA. land and the number of other projects in progress. Ratings obtained are germplasm, they probably have a particular need in mind. For example, Previously, I have evaluated maize they may want maize with a certain germplasm for resistance to corn maturity, or a particular height, or with rootworms, Diabrotica spp., and black Evaluation for resistance to stalk boring host-plant resistance to a particular cutworm, Agrotis ipsilon (Hufnagel) (2nd generation) by the ECB requires a pest. Complete information is not (Wilson and Peters 1973; Wilson et al. more labor-intensive method. During available for all 13,000+ NPGS maize 1983). At present, I evaluate maize for maize anthesis, newly hatched larvae accessions. Much of the passport resistance to European corn borer (about 300) are placed in the leaf axils information (e.g. collection data, seed (ECB), Ostrinia nubilalis (Hübner), and of 10 plants per accession. The plants type, height, etc.) is available, but most corn earworm (CEW), Helicoverpa zea are rated for damage 30 days after accessions have not been evaluated for (Boddie). There are many other infesting by cutting them at soil level, host-plant resistance to insects and important maize insect pests in the splitting them with a band saw, and pathogens. United States (e.g., southwestern corn measuring the length of tunneling. At borer, Diatraea grandiosella Dyar, corn present, we evaluate about 300 As an entomologist in the NPGS, I rootworms, fall armyworm (FAW), accessions per year. evaluate NPGS accessions of maize and Spodoptera frugiperda (J.E. Smith), etc.) other species for new sources of host- for which resistant maize would be Corn earworm plant resistance to insects. There are useful, especially in pest management Evaluation for silk-feeding resistance to many domestic federal, state, and and sustainable agriculture systems. CEW also requires a rather labor private scientists who cooperate with Unfortunately, I do not have the intensive technique. We collect fresh financial resources to evaluate the entered into the GRIN system. 232 R.L. WILSON silks (1-3 days old) from field-grown kernel types, e.g., popcorn, flour, dent, insects were requested more often than plants, then freeze dry and mill them in etc. An evaluation of all the popcorns in were the non-resistant accessions, with the laboratory. The milled silks are the NPGS collection identified several a few exceptions. added to the standard laboratory diets accessions with silk-feeding resistance used to rear CEW. A single, newly to CEW and leaf-feeding resistance to The entire popcorn collection, of 299 hatched, larva is placed into a 30 ml ECB (Wilson et al. 1993). accessions, was evaluated for resistance to CEW and ECB between 1983 and plastic cup containing about 10 ml of test diet. A paper lid is placed on the Other criteria for selection might be 1990 (Wilson et al. 1993). This material cup and, after 8 d, the larva is weighed. specific races of maize or maize has not been requested as much as the The test accessions are compared to obtained from specific geographic areas material from the earlier publications. results obtained from diets prepared of the world. For example, the 1,600 For example, PI 245133 and PI 415283, with silks from a resistant check, available NCRPIS accessions from Peru rated as resistant to CEW, have been ‘Zapalote Chico’, and a susceptible were evaluated for leaf-feeding requested only 3 and 4 times, check, ‘Stowell’s Evergreen’. About 200 resistance to ECB. Eleven accessions respectively. accessions are evaluated annually. were found to have a unique leaffeeding resistance that was not based With so many maize accessions in the on the chemical 2,4-dihydroxy-7- NPGS collection and the few that we methoxy-1,4-benzoxazin-3-one can evaluate yearly, it is impossible to (DIMBOA) (Abel 1993; Abel et al. 1995). test them all (except perhaps for leaffeeding by ECB). At 200-300 accessions Table 1. Number of requests for J. C. Eldredge collection since 1991. Entry Requests for Resistant M aize Germplasm The best way to approach the dilemma ECB, CEW, and FAW had been of too many accessions and too little published. In 1987, I published a paper time and resources is to be more listing three PIs (PI 369361, PI 213705, selective in the material we evaluate. and PI 340856) that had silk-feeding One approach is to define an resistance to corn earworm (Wilson “evaluation subset” that is genetically 1987). Since then, there have been 10 representative of the whole maize requests for PI 369361, 13 requests for collection. Recently, such a maize PI 213705, and 43 requests for PI subset of about 1,500 entries has been 340856. PI 340856 is part of a popcorn developed. It is heavily weighted with collection donated in 1960 to the NPGS Latin American and North American by the late Dr. J. C. Eldredge, who was maize with the intent of containing a an Iowa State University plant breeder. maximum diversity of alleles. This This collection of 35 popcorns was evaluation subset can be requested from evaluated for resistance to CEW, ECB, the NCRPIS in Ames (Telephone No. and FAW (Wilson et al. 1991). The 515-294-6502). number of requests for this germplasm 340835 340836 340837 340839 340840 340841 340842 340843 340844 340845 340846 340847 340850 340851 340853 340854 340855 340856 340857 340858 340859 340860 340861 340862 340863 340865 340866 340867 340868 340869 340870 340871 340872 340873 since 1991 is listed in Table 1. The a per year, it will take from 45 to 65 years to evaluate the whole collection! And to further complicate the problem, the Since 1987, the GRIN system has collection is growing at about 5% per maintained a request history of year, with most of the accessions from germplasm orders. I thought it would low latitudes and, hence, difficult to be of interest to see how many requests manage in the Corn Belt. for seed were received after information detaining the resistance to Another aid for selecting germplasm to accessions that were noted in the evaluate is selecting specific maize publication as being resistant to these b c No. requests 1 8 7 11 18 9 7 6 13 6 9 10 9 9 15 9 10 25 13 5 16 7 10 7 11 11 16 7 8 16 14 21 7 13 Resistant to CEWa CEW CEW b ECB CEW CEW FAWc CEW CEW CEW CEW CEW Corn earworm, Helicoverpa zea (Boddie) European corn borer, Ostrinia nubilalis (Hübner) Fall armyworm, Spodoptera frugiperda (J.E. Smith) MAINTENANCE OF, AND REQUESTS FOR, MAIZE GERMPLASM HAVING RESISTANCE TO INSECT PESTS Since initiating this evaluation program determine the chemical(s) or other at the NCRPIS in 1980, I have been factors responsible for the resistance in evaluating maize for leaf-feeding the Peruvian maize. resistance to ECB (1st generation). The ratings obtained each year were Ac know le dgm e nt s entered into GRIN. The number of accessions in the NCRPIS collection The author wishes to thank Mark having a resistance rating of 3 or less is Millard, Linda Pollak, and W.B. 217. Table 2 lists the number of Showers for their constructive requests for these 217 accessions. More comments regarding this presentation. than half (121) have been requested from one to five times. A few accessions were requested more than 30 times. Of course, not all of the germplasm requested was necessarily requested just for the ECB resistance. We are not always aware of the rationale for requesting germplasm from the NCRPIS. The maize may have been requested because it possesses other characteristics of interest. There has been considerable interest in the Peruvian maize that Craig Abel evaluated for resistance to leaf-feeding by ECB as partial fulfillment of the requirements for his MSc degree (Abel 1993; Abel et al. 1995). I would anticipate that requests for this material will increase because the resistance apparently is not based on the chemical DIMBOA. At present, our resistant Corn Belt maize inbreds that possess resistance to ECB have DIMBOA based resistance. Testing is underway to Table 2. Number of requests for 217 maize accessions with European corn borer leaf-feeding resistance. No. of requests No. of accessions 1-5 6 - 10 11 - 15 16 - 20 21 - 25 26 - 30 31 - 35 36 - 40 41 - 45 46 - 50 121 36 19 12 15 6 4 3 0 1 Re fe re nce s Abel, C.A. 1993. Evaluation of Peruvian maize (Zea mays L.) for resistance to European corn borer [(Ostrinia nubilalis Hübner) Lepidoptera: Pyralidae] leaf feeding and ovipositional preference. M.S. Thesis, Iowa State University, Ames. Abel, C.A., R.L. Wilson, and J.C. Robbins. 1995. Evaluation of Peruvian maize for resistance to European corn borer (Lepidoptera: Pyralidae) leaf-feeding and ovipositional preference. J. Econ. Entomol. 88: 1044-1048. Chapman, C. 1989. Principles of germplasm evaluation. In H.T. Stalker, and C. Chapman (eds.), IBPGR Training Courses: Lecture Series. 2., Scientific Management of Germplasm: Characterization, Evaluation and Enhancement, 55-63. Rome: International Board for Plant Genetic Resources. Clark, R.L. 1989. Seed maintenance and storage. In J. Janick (ed.), Plant Breeding Reviews: The National Plant Germplasm System of the United States, Vol. 7, 95110. Portland: Timber Press. 233 Guthrie, W.D., F.F. Dicke, and C.R. Neiswander. 1960. Leaf and sheath feeding resistance to the European corn borer in eight inbred lines of dent corn. Ohio Agric. Exp. Stn. Res. Bull. 860. Mihm, J.A. 1983. Efficient mass rearing and infestation techniques to screen for host plant resistance to maize stem borers, Diatraea spp., Centro Internacional de Majoramiento de Maiz y Trigo (CIMMYT). El Batan, Mexico. Technical Bulletin. Wilson, R.L. 1987. Evaluation of selected corn plant introductions for silk-feeding resistance to corn earworm (Lepidoptera: Noctuidae). J. Econ. Entomol. 80: 1048-1050. Wilson, R.L., and D.C. Peters. 1973. Plant introductions of Zea mays as sources of corn rootworm tolerance. J. Econ. Entomol. 66: 101-104. Wilson, R.L., J.L. Jarvis, and W.D. Guthrie. 1983. Evaluation of maize for resistance to black cutworm. Maydica 28: 449-453. Wilson, R.L., L.M. Pollak, and K.E. Ziegler. 1993. Evaluation of the U.S. National Germplasm System popcorn collection for resistance to corn earworm (Lepidoptera: Noctuidae) and European corn borer (Lepidoptera: Pyralidae). J. Econ. Entomol. 86: 952-956. Wilson, R.L., B.R. Wiseman, and G.L. Reed. 1991. Evaluation of J. C. Eldredge popcorn collection for resistance to corn earworm, fall armyworm (Lepidoptera: Noctuidae), and European corn borer (Lepidoptera: Pyralidae). J. Econ. Entomol. 84: 693-698. Recent Advances in the Development of Sources of Resistance to Pink Stalk Borer and African Sugarcane Borer N.A. Bosque-Pérez, J.G. Kling, and S.I. Odubiyi , International Institute of Tropical Agriculture, Ibadan, Nigeria. Abst r a c t The lepidopterous stem borers Sesamia calamistis Hampson (Noctuidae) and Eldana saccharina (Walker) (Pyralidae) are among the most important insect pests of maize in West Africa. Efforts to breed for resistance to these two borer species are an integral part of a project to develop control practices for maize pests at IITA. Since 1985, a wide diversity of maize germplasm has been evaluated for resistance to either S. calamistis or E. saccharina. Three populations with moderate resistance to E. saccharina (TZBR Eldana 1, 2, and 3) and two with moderate resistance to S. calamistis (TZBR Sesamia 1 and 3) were formed in the late 1980’s and are being improved for adaptation and resistance levels primarily through S1 family testing. The populations are intended as sources of resistance to be used by African national breeding programs, as well as by colleagues in other parts of the world. TZBR Eldana 3 was developed from elite, adapted populations and has performed well in multilocational yield trials in Nigeria and Cote d’ Ivoire. TZBR Eldana 1 was derived from exotic germplasm and is less adapted to the lowland humid tropics. A selection index which combines agronomic characteristics and E. saccharina resistance, is used to improve the TZBR Eldana populations. Cycles of selection trials with these populations have shown continual progress in selecting for resistance to E. saccharina. Of the two Sesamia populations, TZBR Sesamia 3 appears to have higher levels of resistance than TZBR Sesamia 1. Future selection will be based on improved agronomic characteristics and disease resistance levels, concurrent with higher levels of resistance to S. calamistis. while C. partellus originated in Asia and indigenous grasses and sedges. was accidentally introduced to eastern Attempts to control indigenous insect Lepidopterous stem borers are among Africa approximately 60 years ago. In pests must take into consideration the the most damaging insect pests of West Africa, E. saccharina and S. close relationship between their maize in Africa (Appert 1970). Four calamistis are among the most ecology and that of the native grasses borer species are known to cause damaging and widespread stem borer (Bowden 1976; Shanower et al. 1993). significant yield loss: the maize stalk species of maize (Bosque-Pérez and Due to the complexity of these borer, Busseola fusca Fuller (Noctuidae); Mareck 1990a; Shanower et al. 1991; interactions, long-term control of stem the pink stalk borer, Sesamia calamistis Gounou et al. 1994). borers can only be achieved through Int roduct ion integration of various control practices Hampson (Noctuidae); the African sugar cane borer, Eldana saccharina Maize (Zea mays L.) is an exotic crop such as biological and cultural Walker (Pyralidae), and the spotted introduced to Africa in the 16th century methods, as well as host plant stalk borer, Chilo partellus Swinhoe by the Portuguese, from its native resistance. Breeding for resistance to (Pyralidae) (Bowden 1954; Harris 1962; homeland in the Americas (Miracle stem borers at the International Appert 1970; Brenière 1971). The first 1966). The most important insect pests Institute of Tropical Agriculture (IITA), three are African, and are present in of maize in the field are indigenous to is part of a strategy to develop most countries of sub-Saharan Africa, Africa and their natural hosts are integrated control of maize pests. RECENT ADVANCES IN THE DEVELOPMENT OF SOURCES OF RESISTANCE TO PINK STALK BORER AND AFRICAN SUGARCANE BORER Biology and Distribution of S. ca la m ist is 235 hosts of this borer in West Africa are with high levels of stalk lodging due to crop plants such as maize, sugarcane tunneling and the effect of stalk rots. (Saccharum officinarum L ), sorghum and S. calamistis is present in most countries millet. However, the original hosts of E. of sub-Saharan Africa, Madagascar and saccharina are sedges (Cyperus spp.) the Comores. The host range of this (Atkinson 1980). Formation of Stem Borer Re sist ant Populat ions Since 1985 a wide diversity of pest is reported to be limited to the family Gramineae and includes Infestations of maize plants by E. germplasm has been screened at IITA cultivated crops such as maize, saccharina usually start at anthesis for reaction to either S. calamistis or E. sorghum (Sorghum bicolor (L.) Moench) (Carter 1985). Females lay eggs on saccharina. This includes the BR (borer and millet (Pennisetum americanum (L.) debris on the soil (Atkinson 1980) or on resistant) population of IITA K. Schum.), as well as wild grasses like the hairy margins of maize leaf sheaths (developed by screening for S. calamistis P. purpureum Schum., Panicum (Cochereau 1985). Under field under natural infestations), and a wide maximum Jacq. and Setaria sp. (Harris conditions, eggs hatch in 5 to 6 days range of germplasm from North and 1962). and, after feeding on the leaf sheaths South America which has shown for a few days, larvae enter the stem resistance to other species of stem S. calamistis females lay their eggs where they continue to feed. Larvae borers (Mihm et al. 1988), including between the leaf sheaths of the host may eventually move into the ears and CIMMYT’s MBR (multiple borer plant. Under field conditions, eggs feed on the grain. Pupation occurs resistant) and MIRT (multiple borer hatch in 5 to 6 days and most larvae inside the stem and the pupa is covered resistant tropical) populations and a penetrate the stem shortly after egg by a cocoon made of silk and plant portion of the MIR (maize inbred hatch. Larval feeding might result in debris. Adults emerge 7 to 14 days after resistant) lines from Hawaii. Sources of the destruction of the growing point, pupation. resistance to S. calamistis or E. saccharina were found among some of these typically referred to as “deadheart”. At later stages, the tunneling and girdling Although infestations by this stem activities of the larvae often result in borer occur relatively late in the stalk breakage. In the field, larval development of the maize plants, Three TZBR (Tropical Zea Borer development takes 4 to 6 weeks and damage as a result of their feeding can Resistant) populations with moderate most larvae pupate within the stem or be severe, with yield losses of up to resistance to S. calamistis were formed cobs. S. calamistis breeds throughout 20% (Bosque-Pérez and Mareck 1991). between 1987 and 1988 (Table 1). TZBR- the year and has no resting stage Damage caused by E. saccharina Sesamia 1 was formed by recombining (Harris 1962). However, densities of provides access into the stem and cobs six introduced tropical inbred lines that this pest are low during the dry season for pathogens which cause rots. had shown resistance to S. calamistis in when its hosts are restricted to mature Infestations by this borer are associated our screening trials. TZBR Sesamia 2 germplasm groups. grasses and maize growing in hydromorphic soils. a Table 1. Genetic background of stem borer resistant populations Biology and Distribution of E. saccharina E. saccharina was first described from Sierra Leone and has been known as a pest of graminaceous crops in West Africa for more than a century (Appert 1970). It probably occurs in all suitable areas of sub-Saharan Africa from approximately latitude 15° N to 30° S (Girling 1978). The most important Population TZBR Eldana 1 TZBR Eldana 2 TZBR Eldana 3 Genetic background b 14 test crosses with hybrid 8338-1 TZi 2, 10, 12, 15 and ICAL 27 S1 lines from DMR-LSRW (33 lines), La Posta (15 lines) and TZSRW-1 (28 lines) TZBR Sesamia 1 CM 116, INV 575, Cateto Grande Mil, Cateto Assis Brazil RGS x IV, Costeño Mag. 350 and Cubano Cateto Ecuador 339 crossed to TZi 4 TZBR Sesamia 3 29 lines, mostly from the CIMMYT MBR population, crossed to TZi 4. a b TZBR Eldana 3 has white grain; all others are of mixed grain color; all populations are late maturing (115-120 days). Fourteen entries used for test crosses: MP496 x VG-ECB-24X, MP702 x ECB PI 3, PRMO2 x PRMOSQB 87-4-1, PRMO2 (S1) C6 88-3, PRMO2 (S1) C6 88-12, Pool 24 x (MP496 X MP706), PRMO2 (S1) C6 752X-2, PRMO2 (S1) C6 x (MP496 X MP701), PRMO2 (S1) C6 752-1, 100-5 x 44-6 (2), PRMO2 (S1) C6 752X-4, MP701, MP68, and MP704. 236 N.A. BOSQUE-PÉREZ, J.G. KLING, AND S.I. ODUBIYI was formed after recombination of five susceptible (TZi 19 or TZi 25) inbred simultaneously compare the resistance IITA-developed inbred lines which line checks (Mareck et al. 1989). To performance of S1 families from TZBR showed some resistance to this pest. screen for resistance to S. calamistis, Sesamia 1 Cycle 1, with test crosses This population was eventually plants are infested with 25-30 eggs derived from the same families (Kling discontinued as it did not show (black-head stage) obtained from a and Bosque-Pérez 1995). There was no adequate levels of resistance in laboratory colony. Eggs are placed difference in damage ratings between subsequent trials. TZBR-Sesamia 3 was between the leaf sheaths at the base of 176 S1 families and their test crosses, created by recombining 29 S1 lines, the plant. For trials conducted in the most likely because a highly derived mostly from the CIMMYT screenhouse, plants are infested 3 susceptible inbred was used as the MBR population, crossed to the IITA weeks after planting, for those in the tester, in order to maximize expression inbred TZi 4. field, infestation takes place 2 weeks of resistance among the test crosses. after planting. Damage ratings are Highly significant differences in Screening for resistance to E. saccharina taken 2 and 6 weeks after infestation resistance levels were found among has received major emphasis. After using a 1-9 rating scale (Bosque-Pérez families, but the family x type (S1 or intensive screening from 1985 to 1987, et al. 1989). test crosses) interaction was not significant. Analysis within types three populations with moderate resistance to E. saccharina were formed Resistance levels in the TZBR Sesamia showed that genetic differences were between 1988 and 1989 (Table 1). In populations are improved primarily significant among the S1 families but 1985, 102 accessions, most introduced through S1 family testing. Plant vigor not among the test crosses, implying from CIMMYT, were screened for influences the plants’ reaction to attack more replication would be required to resistance as test crosses with the by S. calamistis. The possibility that make comparable progress from hybrid 8338-1; superior materials were differences in inbreeding depression selection based on evaluation of test selected and backcrossed to their among S1 families could make it crosses (Kling and Bosque-Pérez 1995). original introduction. TZBR Eldana 1 difficult to detect resistance that would These results suggest that S1 family was formed from the best 14 of these be expressed in a non-inbred selection for S. calamistis resistance will backcrosses. Additionally, inbred lines background was of concern. be more effective than selection using developed at IITA were screened for Experiments were thus conducted to test crosses. resistance and the best five recombined to form the population TZBR Eldana 2. Tropically-adapted, early, intermediate and late-maturing open-pollinated populations were also screened for resistance in 1988-89 (Table 2). S1 lines from the three most resistant late populations (La Posta, DMR-LSRW and TZSR-W-1) were screened and superior lines were selected and recombined to form the TZBR Eldana 3 population. Improvement of Screening and Selection M ethods Sesamia calamistis Table 2. Performance of elite, late and intermediate maize varieties under E. saccharina infestation, Ibadan, Nigeria, 1989. Entry a b c Frass rating Penetrometer reading 8329-15 La Posta C8 DMR-LSRW LB 8227 IK 83 TZSR-W-1 Ferke 8223 PR 8326 8338-1 EV 8725-SR PR 8536 LB 8232 ACR 8224 2.28 2.39 2.61 2.67 2.89 3.06 3.17 3.28 3.33 3.34 3.39 3.86 2.33 1.67 2.17 2.17 1.33 2.00 2.33 1.33 1.50 2.33 2.67 2.00 9.54 11.87 9.27 8.87 11.30 6.31 6.42 11.72 8.48 8.22 7.49 8.38 LSD 5% Prob. of F CV % – 0.139 30.3 0.66 0.001 28.9 2.36 <0.001 22.7 The development of screening methods a and the selection of Sesamia resistant b materials was enhanced by the c identification of resistant (TZi 4) and Ear damage 1-5 rating scale to assess percentage of grain consumed or damaged by the borer (1= 0-5; 2 = 6-25; 3 = 26-50; 4 = 51-75 and 5 = 76-100%). Amount of frass in the leaf axils where: 1= very little frass; 5 = abundant frass. Rind puncture determined as the force in kilograms required to penetrate the second internode above the ground. Readings taken at flowering; larger values indicate that greater force was required to penetrate the stem. RECENT ADVANCES IN THE DEVELOPMENT OF SOURCES OF RESISTANCE TO PINK STALK BORER AND AFRICAN SUGARCANE BORER 237 Eldana saccharina collaborators is used in further selected for recombination to form the To increase the number of breeding improvement of the populations. next cycle of selection. This population appears to have the greatest borer materials that can be screened for resistance to E. saccharina, an TZBR Sesamia resistance of the two TZBR Sesamia augmented natural field infestation In addition to the evaluation of 176 S1 populations presently under method was developed (Bosque-Pérez families and their test crosses from the improvement. However, it is relatively and Mareck 1990b). Strips of a borer TZBR Sesamia 1 population, 26 superior susceptible to lowland rust, Puccinia susceptible maize variety are planted families were selected in 1993 for polysora, probably due to its temperate one month prior to planting test recombination to form the next cycle of background. Thus, more emphasis will materials to serve as spreader rows. selection. Recombination took place in be placed in the future to improving Test materials are planted 1994 and new S1 families will be disease resistance as well as agronomic perpendicular to the strips using 3 m evaluated in the near future. Although characteristics, while continuing to rows and 1 m alleys. Plants of the we believe S1 family selection for S. select for higher levels of resistance to spreader rows are infested at silking calamistis resistance will be more S. calamistis. with E. saccharina egg masses (65-75 effective than selection using test eggs per plant) obtained from a crosses, one cycle of selection will be TZBR Eldana laboratory colony. Adults which carried out separately for both types of In screening for resistance to E emerge from the spreader rows move families to determine actual progress saccharina, the following assessments to the test plants resulting in a ‘natural’ that can be obtained from the two are made: percentage of plants with infestation. Test materials are checked selection methods. broken stalks, plant aspect (plant and regularly to ensure a uniform level of infestation has been achieved. ear height, uniformity), ear aspect (size, Evaluation of Cycle 0 of the TZBR uniformity), quality of husk cover and Sesamia 3 population was conducted disease resistance (rust, blight, MSV), between 1991–92 by screening 204 S1 using 1-5 rating scales. Ear damage is families under artificial infestation in assessed using a 1-5 scale that estimates the screenhouse. Twenty families had the percentage of grain consumed or Borer resistant populations are being better resistance ratings than the damaged by the borer (1= 0-5; 2 = 6-25; improved for adaptation and resistance resistant check, TZi 4 (Fig. 1), and were 3 = 26-50; 4 = 51-75 and 5 = 76-100%). Measurements on agronomic levels primarily through S1 family takes into consideration agronomic characteristics and E. saccharina 30 resistance is used to improve the TZBR Eldana populations (Kling and Bosque- Eldana 3, the TZBR populations that we 20 Pérez 1995). The relative weights assigned to agronomic characteristics of resistance to be used by national breeding programs in Africa and and E. saccharina resistance vary 10 depending on the population and collaborators elsewhere, rather than as stresses in any given location. The populations have been made available to collaborators in various countries including Cameroon, Ghana, Mali, Senegal, Zaire, Kenya (ICIPE), Uganda, and Guadeloupe. Feedback from our -1.2 particular complex of biotic and abiotic severity of infestation in a particular -1.8 adaptation will be required to fit the 0 -2.4 final products. Selection for local 2.4 have developed are intended as sources 1.8 families. With the exception of TZBR 1.2 plants are selfed to make new S1 ;;; ; ;;; ;; ; ;;; ; ;;; ; ;; ; ;;; ;;; are also taken. A selection index which diseases is carried out when individual 0.6 maize streak virus (MSV) and other characteristics (days to silk, grain yield) Frequency 40 0 testing. Mass selection for resistance to -0.6 Improvement and Testing of TZBR Populations Deviation from set means in S. calamistis rating Figure 1. Distribution of deviations from set means in Sesamia calamistis resistance ratings of 204 S1 families from TZBR Sesamia 3 C0. Overall mean = 2.8; susceptible check = 2.12; resistant check = -0.91. Probability of F = 0.002; LSD (5%) = 0.27. year. To evaluate the progress achieved in selecting for resistance to E. saccharina, cycles of selection trials are periodically conducted. In 1991, Cycles 0 to 4 of TZBR-Eldana 1 and Cycles 0 to 2 of TZBR Eldana 2, along with a 238 N.A. BOSQUE-PÉREZ, J.G. KLING, AND S.I. ODUBIYI susceptible check, were evaluated New cycles of selection were evaluated under artificial infestation. Ear damage during 1994. Ratings for plant aspect 3 ratings in later cycles were significantly months after planting showed that Studies on mechanisms of resistance to lower (P<0.05) than on early ones, significant progress has been made in stem borers have been directed mainly showing increased levels of resistance improving this character in TZBR towards E. saccharina, as more progress in these populations (Table 3). Results Eldana 1, especially in the last cycle has been made in selecting for also showed that time to maturity (Table 4). The use of a selection index resistance to this pest. Recently tests increased in TZBR Eldana 1. The use of which heavily weights agronomic have been initiated on S. calamistis. a selection index should prevent characteristics has assisted us in further inadvertent increases in ensuring that agronomic improvement Elevated plant silica content has been maturity in the future. is also made. The population TZBR reported as a mechanism of resistance Eldana 3 was developed from elite, to various cereal stem borer species. adapted varieties, and plant aspect has This may be due to the important role always been superior in this population of silica in strengthening plant cell (Table 4). walls (Painter 1951). For example, high Table 3. Ear damage ratings for TZBR Eldana cycles of selection, Ibadan, 1991. Entry TZBR Eldana 1 Cycle 0 Cycle 1 Cycle 2 Cycle 3 Cycle 4 TZBR Eldana 2 Cycle 0 Cycle 1 Cycle 2 Susceptible check Prob. of F LSD 5% CV % a Ear damage ratinga larval mortality of C. supressalis Walker Since TZBR Eldana 3 is adapted to the (Pyralidae) has been detected on rice region, it may be more immediately varieties with high silica content 2.3 2.1 1.8 1.6 1.3 transferred to NARS. Cycle 2 of this (Djamin and Pathak 1967). In maize, population performed well in resistance to the second generation multilocational yield trials in Nigeria European corn borer (ECB) (Ostrinia and Cote d’ Ivoire in 1993 (Table 5). It nubilalis Hübner) (Lepidoptera: 1.7 1.7 1.3 2.1 was included in IITA’s International Pyralidae) has been found to be Variety Trials for the first time in 1994. significantly correlated with the silica TZBR Eldana 1 was derived from exotic content in the sheath and collar tissue germplasm and is less adapted to the (Rojanaridpiched et al. 1984). 0.004 0.54 27.2 lowland tropics. Because this 1-5 rating scale to assess percentage of grain consumed or damaged by the borer (1= 0-5; 2 = 6-25; 3 = 26-50; 4 = 51-75 and 5 = 76-100%). population is intended for use as a To determine if increased levels of source of E. saccharina resistance by resistance in the TZBR Eldana national breeding programs, agronomic populations are related to levels of characteristics are given less weight in silica, analysis of stem silica content the selection index. was carried out for the various cycles of selection. Plant stem samples (three Table 4. Plant aspect ratings for TZBR Eldana cycles of selection, Ikenne, 1994. Entry Plant aspect rating TZBR Eldana 1 Cycle 0 Cycle 1 Cycle 2 Cycle 3 Cycle 4 Cycle 5 TZBR Eldana 3 Cycle 1 Cycle 2 Cycle 3 8338-1 2.67 2.42 2.50 3.25 Prob. of F LSD 5% CV % 0.001 0.38 12.42 a M echanisms of Resistance plants per plot, six replicates) were taken shortly after anthesis and oven a a 3.75 4.00 3.50 3.67 3.50 3.17 1-5 rating scale, 1= good, 5 = poor. Table 5. Across site performance of selected entries from the preliminary late variety trial, 1993. Entry 8321-18 TZL Comp. 3 C1 TZL Comp. 4 C0 TZBR Eldana 3 C2 Acr 9022-SR Acr 90 DMR-LSRW Suwan 1-SR Acr 9028-DMRSR a b c Yield (t/ha) Ear rot ratingb Husk cover ratingc 5.6 5.1 5.0 4.8 4.6 4.4 4.1 4.1 2.5 2.5 2.2 2.7 2.3 2.9 3.1 3.0 2.9 2.9 2.6 3.1 3.0 2.1 3.1 3.1 Trials conducted in Ikenne, Mokwa, and Samaru, Nigeria and Sinemantiale, Cote d’ Ivoire. Means for Ikenne only; 1-5 rating scale, 1= resistant, 5 = susceptible. Means for Mokwa only; 1-5 rating scale, 1= very good, 5 = poor. RECENT ADVANCES IN THE DEVELOPMENT OF SOURCES OF RESISTANCE TO PINK STALK BORER AND AFRICAN SUGARCANE BORER 239 dried at 65°C for 4 days. Stem pieces been made in increasing stalk strength trial, the ability of the insect to feed and were then ground and silica content in the TZBR Eldana 1 population (Table survive in the stem was indirectly determined using an atomic absorption 7). In contrast, no progress was measured by taking ratings of the spectrophotometer after extraction with observed in the TZBR Eldana 2 amount of frass in the leaf axils using a an acid mixture, using the method population (Table 8). This is consistent 1 to 5 rating scale (1 = very little frass, 5 described by Novosamsky et al. (1984). with the notion that greater genetic = abundant frass). A significant No significant differences in stem silica variability, and thus potential for correlation between the penetrometer content were detected among the cycles progress in selection, exists in the reading and frass rating (r = -0.66, of selection (Table 6), suggesting that former population. p<0.05) was detected. Extent of ear damage was also recorded. While there other mechanisms of resistance are Results of similar tests on cycles of was an indication of a possible selection of the TZBR Eldana 3 relationship between ear damage and Stalk strength has also been reported as population were erratic. Stalk strength penetrometer reading (r = -0.40, ns), the a mechanism of resistance to stem (as measured by penetrometer estimate of the correlation between borers. In our trials, stalk rind puncture readings) increased significantly from frass and ear damage rating was close is measured using a hand-held Cycle 1 to 2, but no progress was made to zero. This suggests that different penetrometer with a spring resistance in the next cycle of selection (Table 8). mechanisms may be involved in plunger (Thompson 1972) (Supplier: Additional tests are required to clarify determining E. saccharina resistance in Cert Instrument Corporation, these findings. the stalks and ears. determined as the force in kilograms Significant differences in penetrometer Re fe re nce s required to penetrate the second readings had earlier been detected in a internode above the ground (Twumasi- trial to evaluate the performance of Afriyie and Hunter 1982). Readings are tropically adapted intermediate and taken at flowering; larger values late maize populations under E. indicate that greater force is required to saccharina infestations (Table 2). In this probably involved. Oceanside, NY). Rind puncture is penetrate the stem. Penetrometer readings were taken on a cycles of Table 7. Stalk penetrometer a readings on cycles of selection of the stem borer resistant population TZBR Eldana 1 and selected checks, Ibadan, Nigeria, 1994. selection trial in 1994 and results showed that significant progress has Table 6. Percentage silica content in stalks of stem borer resistant populations and selected checks, Ibadan, Nigeria, 1990. Maize entry TZBR Eldana 1 Cycle 0 Cycle 1 Cycle 2 Cycle 3 TZBR Eldana 2 Cycle 0 Cycle 1 Susceptible synthetic 8338-1 8329-15 LSD 5% Prob. of F CV % a a Silica content (%) 0.53 0.61 0.47 0.54 0.43 0.48 0.53 0.51 0.48 Maize entry TZBR Eldana 1 Cycle 0 Cycle 1 Cycle 2 Cycle 3 Cycle 4 Cycle 5 Susceptible synthetic 8338-1 8329-15 LSD 5% Prob. of F CV % a 0.117 0.136 19.77 Means of three plants per replication per treatment, 6 replications. Penetrometer readingb b 6.64 6.94 6.93 7.30 8.92 8.54 7.01 7.21 8.30 1.467 0.001 18.83 Rind puncture determined as the force in kilograms required to penetrate the second internode above the ground. Readings taken at flowering; larger values indicate that greater force was required to penetrate the stem. Means of five plants per replication per treatment, 6 replications. Appert, J. 1970. Insects harmful to maize in Africa and Madagascar. Madagascar, Madagascar Institute of Agronomic Research. Table 8. Stalk penetrometer readingsa on cycles of selection of stem borer resistant populations and selected checks, Ibadan, Nigeria, 1994. Maize entry Penetrometer reading TZBR Eldana 2 Cycle 0 Cycle 1 Cycle 2 Cycle 3 TZBR Eldana 3 Cycle 1 Cycle 2 Cycle 3 Susceptible synthetic 8338-1 8329-15 8.71 10.22 9.02 7.01 7.21 8.30 LSD 5% Prob. of F CV % 1.467 0.001 18.83 a b b 9.24 8.81 7.47 8.34 Rind puncture determined as the force in kilograms required to penetrate the second internode above the ground. Readings taken at flowering; larger values indicate that greater force was required to penetrate the stem. Means of five plants per replication per treatment, 6 replications. 240 N.A. BOSQUE-PÉREZ, J.G. KLING, AND S.I. ODUBIYI Atkinson, P.R. 1980. On the biology and natural host plants of Eldana saccharina Walker (Lepidoptera: Pyralidae). J. Entomol. Soc. So. Africa 43: 171-194. Bosque-Pérez, N.A., and J.H. Mareck. 1990a. Distribution and species composition of lepidopterous maize borers in southern Nigeria. Bull. Entomol. Res. 80: 363-368. Bosque-Pérez, N.A., and J.H. Mareck. 1990b. Screening and breeding for resistance to the maize stem borers Eldana saccharina and Sesamia calamistis. Pl. Res. to Ins. Newsl. 16: 119-120. Bosque-Pérez, N.A., and J.H. Mareck. 1991. Effect of the stem borer Eldana saccharina (Lepidoptera: Pyralidae) on the yield of maize. Bull. Entomol. Res. 81: 243-247. Bosque-Pérez, N.A., J.H. Mareck, Z.T. Dabrowski, L. Everett, S.K. Kim, and Y. Efron. 1989. Screening and breeding for resistance to Sesamia calamistis and Eldana saccharina. In Toward Insect Resistant Maize for the Third World: Proc. Intl . Symp. on Methodol. for Developing Host Plant Resistance to Maize Insects, 163-169. Mexico, D.F. CIMMYT. Bowden, J. 1954. The stem-borer problem in tropical cereal crops. Report 6th Commonwealth Entomological Conference, 104-107. U. K. Bowden, J. 1976. Stem-borer ecology and strategy for control. Annl. Appl. Biol. 84: 107-134. Brenière, J. 1971. Les problèmes des lepidoptères foreurs des graminées en Afrique de l’Ouest. Annl. Zool. Ecol. Anim. 3: 287-296. Carter, A.O. 1985. An evaluation of the importance of stem borers and their control on maize grown in south eastern Nigeria. M Agr Sc Thesis, Reading University, UK. Cochereau, P. 1985. Ecologie des populations, en Côte d’Ivoire, du foreur africain de la canne ‘a sucre et du maïs Eldana saccharina Walker. Côte d’Ivoire, Institut des Savanes- ORSTOM. Djamin A., and M.D. Pathak. 1967. Role of silica in resistance to Asian rice borer, Chilo supressalis Walker, in rice varieties. J. Econ. Entomol. 60: 347-351. Girling, D.J. 1978. The distribution and biology of Eldana saccharina Walker (Lepidoptera: Pyralidae) and its relationship to other stem borers in Uganda. Bull. Entomol. Res. 68: 471-488. Gounou, S., F. Schulthess, T. Shanower, W.N.O. Hammond, H. Braima, A.R. Cudjoe, R. Adjakloe, and K.K. Antwi with I. Olaleye. 1994. Stem and ear borers of maize in Ghana. Plant Health Management Research Monograph No. 4. International Institute of Tropical Agriculture, Ibadan, Nigeria. Harris, K.M. 1962. Lepidopterous stem borers of cereals in Nigeria. Bull. Entomol. Res. 53: 139-171. Kling, J.G., and N.A. Bosque-Pérez. 1995. Progress in screening and breeding for resistance to the maize stem borers Eldana saccharina and Sesamia calamistis. In D.C. Jewell, S.R. Waddington, J.K. Ransom and K.V. Pixley (eds.). Maize Research for Stress Environments: Proceedings of The Fourth Eastern and Southern Africa Regional Maize Conference, 182-186. Harare, Zimbabwe. Mexico D.F. CIMMYT . Mareck, J.H., N.A. Bosque-Pérez, and M.S. Alam 1989. Screening and breeding for resistance to African maize borers. Pl. Res. to Ins. Newsl. 15: 58-59. Mihm, J.A., M.E. Smith, and J.A. Deutsch. 1988. Development of open- pollinated varieties, non-conventional hybrids and inbred lines of tropical maize with resistance to fall armyworm, Spodoptera frugiperda (Lepidoptera: Noctuidae) at CIMMYT. Florida Entomologist 71: 262268. Miracle, M.P. 1966. Maize in Tropical Africa. Madison, Wisconsin, University of Wisconsin Press. Novozamsky, I., R. van Eck, and V.J.G. Houba. 1984. A rapid determination of silicon in plant material. Comm. Soil Sci. Pl. Anal. 15: 205-211. Painter, R.H. 1951. Insect resistance in crop plants. New York, Macmillan. Rojanaridpiched, C., V.E. Gracen, H.L. Everett, J.G. Coors, B.F. Pugh, and P. Bouthyette. 1984. Multiple factor resistance in maize to European corn borer. Maydica 29: 305-315. Shanower, T., F. Schulthess, and S. Gounou. 1991. Distribution and abundance of some stem and cob borers in Benin. Plant Health Management Research Monograph No. 1, International Institute of Tropical Agriculture, Ibadan, Nigeria. Shanower, T.G., F. Schulthess, and N. Bosque-Pérez. 1993. The effect of larval diet on the growth and development of Sesamia calamistis Hampson (Lepidoptera: Noctuidae) and Eldana saccharina Walker (Lepidoptera: Pyralidae). Ins. Sci. & its Appl. 14: 681685. Thompson, D.L. 1972. Recurrent selection for lodging susceptibility and resistance in corn. Crop Sci. 12: 631-634. Twumasi-Afriyie, S., and R.B. Hunter. 1982. Evaluation of quantitative methods for determining stalk quality in shortseason corn genotypes. Can. J. Pl. Sci. 62: 55-60. The Importance of Institutional Linkages for the Development of M ultiple Borer Resistant M aize Hybrids J.L. Overman, DEKALB Genetics Corporation, Union City, TN, USA Abst r a c t Stalk-boring and leaf feeding lepidoptera are major pests of maize worldwide. Improvement of plant resistance to these pests is an objective of public maize research groups at international, federal, and state institutions. These institutions have played important and unique roles in the development of insect rearing techniques, efficient methods for infesting and evaluating germplasm for resistance, screening germplasm to identify sources of resistance, and the release of resistant germplasm to the public. Commercial seed companies have become the primary institution for developing new lines and hybrids in the USA and Europe. Private seed companies, both international and domestic, are also becoming important seed suppliers for the rest of the world. For these reasons, the introgression of insect resistant sources into elite germplasm has required the transfer of knowledge and resistant sources from public to private institutions. The development of multiple borer resistant hybrids illustrates the value of good institutional linkages in the improvement of maize. Int roduct ion countries, and various universities and has required the transfer of knowledge colleges in the USA and internationally. between the public (international, Maize, Zea mays, L., ranks third in These institutions have played and federal, and state agencies) and the world production among the major should continue to play important and private (seed companies) institutions. food grains. The genetic improvement unique roles in the development of and protection of this crop is of insect rearing techniques, methods for national and international importance infesting and evaluating material, and the reliability of grain production training of personnel, screening of globally is of concern to both exporting germplasm for pest resistance, and and importing countries. Public and preserving maize germplasm and private maize research institutions are related species. Historical Perspective of the M ultiple Borer Resistance Program at De kalb Ge ne t ics Corporat ion The evolution of the multiple borer established in many maize producing countries and have as their mandate With the advent of hybrid maize, resistance (MBR) program at DEKALB the agronomic and/or genetic commercial seed companies evolved in Genetics Corp. provides a historical improvement of the crop for yield and the USA, Western Europe, Africa, and perspective of the importance of control of maize pests. South America and have become the institutional linkages and the unique primary institutions for the contributions that international, Stalk-boring and leaf feeding development of new lines and hybrids. federal, state, and private agencies lepidoptera are major maize pests in Private seed companies are also make in the improvement of maize. essentially all maize growing regions of becoming important seed suppliers for DEKALB is an international seed the world. Development of plant the rest of the world. The introgression company with that supplies improved resistance to these pests is an objective of insect resistance for the major maize maize hybrids for both US and of maize research groups at CIMMYT, pests is an objective of many seed international markets. In support of our state and federal agencies in many companies and success in this venture domestic objectives, we have 242 J.L. OVERMAN established research stations in 16 The seed industry benefited from evaluations the leaf feeding rating states. The international markets are research begun in the mid-1960s by the system to identify whorl stage supported by research programs in 10 USDA/ARS team at Mississippi State. resistance was used and no resistance foreign countries. By the mid-1970s the SWCB rearing was found in elite cornbelt lines, old methods developed by Dr. Frank Davis open-pollinated varieties, Indian Stalk-boring and leaf feeding were being used by seed companies. maize, cornbelt composites, southern lepidoptera are major pests of the DEKALB responded by establishing a US composites, teosinte, or tropical maize plant and plant resistance is facility at Union City, TN, in 1976, to populations. Resistance was observed viewed as an opportunity to add value work on SWCB and other insect in Tripsacum, but not in tripsacoid to those hybrids we market. Thus many problems for the southern USA. maize. The absence of SWCB resistance in ECB resistant and high DIMBOA of our breeding locations have selection for pest resistance as an objective. The By 1977 DEKALB had implemented lines seemed to confirm Painter’s European corn borer (ECB) Ostrinia Davis’ SWCB rearing techniques and axiom that resistance is species specific nubilalis (Hübner), is the pest with the by 1979 had converted to using and not for an entire group of species broadest distribution over our CIMMYT’s bazooka device for such as the leaf feeding lepidoptera. domestic, Canadian, and European infesting plants with 1st instar larvae. markets and has received the most The development of the bazooka During this period SWCB resistance attention. However, many other species greatly increased the efficiency in was observed in germplasm from the are of regional or international infesting plants and allowed DEKALB Davis, Williams, and Scott program importance. to redesign its insect rearing lab to (USDA/ARS). From 1974 to 1984 this reduce labor and rearing costs. program released one SWCB resistant population and eight resistant lines. Early on investigators had observed genetic resistance to the ECB and From 1977 to 1980 DEKALB conducted This germplasm was subsequently USDA/ARS and university research extensive evaluations of maize shown not to be species specific. In groups were active in developing ECB germplasm and related species for 1974 Davis and Scott observed fall mass rearing and screening techniques. resistance to SWCB (Table 1). For these armyworm (FAW) Spodoptera frugiperda Through the efforts of Dr. W.D. (“Bud”) Guthrie and his staff at Ankeny, Iowa (USDA/ARS), this knowledge had been transferred to seed companies in the private seed industry and by the 1970s research was underway to commercialize resistance to ECB. Since the late 1960s corn hybrids marketed in the southwest encountered the southwestern cornborer (SWCB) Diatraea grandiosella Dyar, which had moved in from Mexico up through Texas to southern Kansas and east as far as middle Tennessee. This pest was devastating to corn production, with losses coming from both physiological yield reduction and increased harvest losses from the insect’s girdling habit. Table 1. Germplasm evaluated for SWCB resistance Germplasm Source Elite Inbreds International Inbreds Cornbelt Composites ECB Resistant Lines Southern Composites Popcorn Old Open-pollinated Varieties Sweet Maize Collections South American Maize Collection Indian Maize Collection South American Maize Collection African Collection Asian Collection Popcorn Collection European Collection U.S. Varietal Collection Trypsacum Trypsacum Collection Trypsacoid Maize Trypsacoid Maize Teosinte x Maize Crosses CIMMYT x Temperate Maize Teosinte SWCB Populations SWCB Lines DEKALB Genetics Corporation “ “ “ “ “ “ U.S. Plant Intro. Sta., Ames, Iowa “ “ “ “ “ “ “ “ “ D.H. Timothy, N.C. State Univ. J. Harlan, Univ. of Illinois V.E. Gracen, Cornell Univ. “ “ G.W. Beadle, Univ. of Chicago Davis, Williams, & Scott (USDA/ARS) “ THE IMPORTANCE OF INSTITUTIONAL LINKAGES FOR THE EVELOPMENT OF MULTIPLE BORER RESISTANT MAIZE HYBRIDS 243 MBR is expressed from seedling to pretassel pests. For example, we expect germplasm and, in DEKALB trials at Union City, 1st brood ECB resistance Many cornbelt sources of 1st brood be effective against the southern was observed. Over the next several ECB resistance exhibit high levels of cornstalk borer Diatraea crambidoides, a years this germplasm was evaluated resistance only in the seedling and problem in North Carolina and South against a broad range of lepidopterous early whorl stages and only low levels Carolina. species that feed in the maize whorl. as the plant approaches pretassel. (J.E. Smith) resistance in this Studies of MBR germplasm by the resistance developed for SWCB would A cooperative study by Davis et al. Davis and Williams group (USDA/ (1988) showed multiple borer ARS) under FAW infestation and by resistance (MBR) functioned against the DEKALB using SWCB show increased sugarcane borer (SB), Diatraea levels of the resistance from seedling to MBR allows the breeder/ entomologist to use the species best adapted to their environment as the selective organism saccharalis (Fabricius), in Mexico and pretassel. Resistance is highest at the Too often breeding locations attempt to Louisiana; ECB in Tennessee and late-whorl-to-pretassel stage, when the select for pest resistance in Missouri; SWCB in Mexico; Missouri plant is most subject to physiological environments that are not favorable for and Mississippi; and FAW in Georgia, loss from insect tunneling. these evaluations, or a particular pest Mississippi, and Mexico. Upon testing MBR germplasm, Ampofo et al. (1987) cannot be used because it is not endemic to the test region. For reported high levels of resistance to Winter nurseries can be used to select for MBR Chilo partellus in East Africa. Van DEKALB has been conducting a getting good 1st brood ECB Rensburg et al. (personal recurrent selection program to establishment at Union City, but in 17 communication 1990) observed incorporate MBR into elite lines years of testing at that location the example, we often have difficulty in resistance to Busseola fusca in South (Overman 1987). No SWCB or FAW SWCB have never failed to achieve Africa. Bato et al. (1983) in the resistant segregates have been good survival. However, SWCB cannot Phillipines reported high levels of observed that are not also resistant to be used at DEKALB’s other US resistance to the Asian cornborer 1st brood ECB. In a cooperative study breeding locations where it is not Ostrinia furnacalis. Bosque-Perez et al. in 1993 with Dr. Meagher of Texas endemic. (1987) observed resistance to Eldana A&M, comparable levels of resistance saccharina in Nigerian tests. J. Reese to the sugarcane borer (SB) and SWCB (1987) noted black cutworm Agrotis were found in DEKALB’s MBR MBR is the only known source of resistance to many species ipsilon (Hufnagel) resistance. At this hybrids. It is intuitive that both FAW For many species the MBR system is point there was little doubt that we and/or SB could be used for selecting the only resistant source available to were working with a defensive system for MBR in winter nurseries in south the breeder. MBR hybrids could be with a broad spectrum of activity. Florida, south Texas, Puerto Rico, deployed in the geographical areas Mexico, or Argentina. listed in Table 2 for reducing the Advantages and Problems Associated w ith the Use of M BR damage to a variety of pests. Resistance can be developed to secondary or regional pests through surrogate selection The MBR system exhibits joint action with chemical controls Multiple borer resistance provides It is not economical or practical to Larvae that survive on MBR plants breeding options that are not present incorporate species specific resistance grow at a slower rate and feed in the with species specific sources of into hybrids for all leaf feeding whorl for a longer period of time and resistance, but also presents difficulties lepidoptera of maize. Many of these are therefore more susceptible to in introgressing it into elite germplasm. pests are regional in importance or pesticide control for a longer period of The following advantages are affect only small maize markets. time. associated with MBR or can be inferred However, MBR provides an from the reaction of various pests to the opportunity to improve resistance MBR trait. towards these pests through surrogate selection for resistance to other major 244 J.L. OVERMAN Table 2. Geographical regions, and associated insect pests, where MBR hybrids could be deployed. Species Common Name Region Ostrinia furnicalis O. nubilalis (Hubner) Diatraea lineolata D. grandiosella Dyar D. saccharalis(Fabricius) D. crambidoides(Grote) Chilo partellus Bussiola fusca Sesamia spp. Spodoptera spp. Asiatic stalk-borer European cornborer Neotropical stalk-borer Southwestern cornborer Sugarcane borer Southern cornstalk borer Asian maize borer African maize borer Pink stem borers Armyworms China, Phillipines U.S., Canada, Europe Mexico, Central America U.S., Mexico Mexico, Argentina U.S. Africa, Asia Africa Africa, Middle East The Americas Infestations were made with 30 SWCB, 40 FAW, or 100 ECB larvae. Inbreds were grown in randomized complete block designs of two to four replicates. Entries were planted in single row plots four meters long and thinned to 15 plants. For 2nd brood ECB evaluations, each plant was infested at anthesis with 100 ECB larvae. The 2nd brood test was dissected 40 days after infestation and the length of tunneling/ plant recorded. SWCB and ECB hybrid yield trials - A independently from and used in The MBR system comes from a narrow germplasm base, has high ear placement, small ears and severe root lodging problems combination with other ECB resistance This resistance is not simply inherited infested and whorl stage infestations genes to enhance the level and/or and some form of recurrent selection with 100 ECB or 30 SWCB larvae per stability of pest resistance. and usually several cycles of selection plant. The hybrids were grown in 2- are needed to break linkages with row plots 4 meters long and thinned to unwanted genes. 30 plants/plot. The experimental The MBR system can be used with other sources of ECB resistance The MBR system can be selected The MBR system is complementary with biological control DEKALB MBR hybrid (FMBR1 x FMBR2 / MMBR1) was compared for yield against the commercial hybrids DK683, DK714, and P3245 in non- design was a randomized split-block The DEKALB/ Union City M BR Program with whole plots as infestation Materials and methods FAW hybrid yield trial - The MBR We introgressed MBR into both sides (F hybrid and the commercial hybrid Deployment of MBR system should reduce the population buildup of some migratory lepidoptera and M) of a heterotic pattern through DK626 were tested in FAW infested recurrent selection (Overman 1987). and non-infested single row plots 4 Three inbreds from this program have meters long and thinned to 15 plants been evaluated for MBR as lines and in per row. Plants were infested at mid- The deployment of MBR hybrids in the hybrid combination for yield whorl with 40 FAW larvae/plant and southern USA, northern Mexico, and performance under ECB, SWCB, or the leaf feeding rating taken 10 days the Carribean should slow the FAW infestation. Whorl- or tassel-stage later. Yields and moisture were development and size of migratory plants were infested by bazooka with recorded at harvest on the 1st 10 plants populations of FAW. laboratory reared neonate larvae. in each row. MBR hybrids are more likely to be compatible with other crops that are attacked by maize pests Inbred test - Three DEKALB MBR lines Results and Discussion The slower growing larvae in the MBR treatment and hybrids as subplots. plants are more subject to predation and parasitism. (FMBR1, FMBR2, MMBR1), a CIMMYT MBR line (CML67), two USDA/ARS DEKALB has utilized public MBR ECB resistant lines (Mo45, Mo47) and germplasm, insect rearing FAW and/or ECB susceptible maize two elite checks (B73Ht, Mo17Ht) were methodologies, and field infestation supports large populations of pests that evaluated against SWCB, FAW, and/or and evaluation techniques to develop a can attack a wide variety of other 1st and 2nd brood ECB. Leaf feeding commercial program for introgressing crops. ratings (1-9 scale) were determined 10- MBR into elite germplasm. These 14 days following whorl stage. improved lines have better agronomic THE IMPORTANCE OF INSTITUTIONAL LINKAGES FOR THE EVELOPMENT OF MULTIPLE BORER RESISTANT MAIZE HYBRIDS 245 attributes while maintaining good This progress would not have occurred There is a need for the continued (if not levels of resistance to ECB, SWCB, and without the work of public institutions expanded) involvement of public FAW (Table 3). in collecting and preserving institutions to develop resistance in germplasm, developing insect rearing maize to arthropod pests. State, federal, In the company’s single-location yield methods, perfecting field methods and and international institutions should be trials, the MBR hybrid was competitive laboratory techniques to evaluate cautious about reducing support for in non-infested plots with the most resistance, training scientists and pest resistance research on the competitive commercial hybrids and technicians, and testing resistant assumption that private seed showed a yield advantage under ECB, germplasm products. companies or other institutions can or will assume those responsibilities. SWCB, or FAW infestation (Tables 4 and 5). Table 3. Comparison of whorl stage resistance to ECB, SWCB, and FAW; and tassel stage ECB resistance in DEKALB MBR lines, elite public lines and resistant public lines. Inbred ECB Leaf Feeding ECB Tunnel Index SWCB Leaf Feeding FAW Leaf Feeding 2 3 2 6 4 3 5 5 3 6 6 6 9 8 7 7 9 9 9 9 1 2 1 6 5 2 - 6 - 0.9 3.1 1.5 2.1 DEKALB Lines FMBR1 FMBR2 MMBR1 Elite Checks B73Ht Mo17Ht Resistant Checks CML67 (CIMMYT) Mo45 (USDA/ARS) Mo47 (USDA/ARS) LSD (.05) (Rating of 1 = most resistant; rating of 9 = most susceptible) Table 4. 1994 Yield comparison of MBR and commercial hybrids under late whorl stage infestations with SWCB, ECB, or no infestation. Hybrid Non-Infested t/ha MST SWCB t/ha MST t/ha MST FMBR1*FMBR2/MMBR1 DK683 DK714 P3245 11.9 12.2 11.8 11.9 29 25 27 22 11.8 11.1 10.9 11.2 11.4 10.1 11.0 9.8 27 23 25 20 1.0 1.0 1.0 LSD (.05) ECB 27 24 25 22 Table 5. 1994 Yield comparison of MBR and commercial hybrid under whorl stage infestation and non-infested FAW plots. Hybrid Non-Infested t/ha MST FAW Infested t/ha MST FMBR1 * FMBR2 / MMBR1 DK626 13.0 12.5 12.1 10.4 25 21 27 21 FAW Leaf Feeding 5 9 Re fe re nce s Ampofo, J.K.O., and K.N. Saxena. 1987. Screening methodologies for maize resistance to Chilo partellus (Lepidoptera: Pyralidae). In Toward Insect Resistant Maize for the Third World: Proceedings of the International Symposium on Methodologies for Developing Host Plant Resistance to Maize Insects, 170-177. Mexico, D.F.: CIMMYT. Bosque-Perez, N.A., J.H. Mareck, Z.T. Dabrowski, L. Everett, S.K. Kim, and Y. Efron. 1987. Screening and breeding maize for resistance to Sesamia calamistis and Eldana sacharina. In Toward Insect Resistant Maize for the Third World: Proceedings of the International Symposium on Methodologies for Developing Host Plant Resistance to Maize Insects, 163-169. Mexico, D.F.: CIMMYT. Davis, F.M., W.P. Williams, J.A. Mihm, B.D. Barry, J.L. Overman, B.R. Wiseman, and T.J. Riley. 1988. Resistance to multiple lepidopterous species in tropical derived corn germplasm. MAFES Technical Bulletin 157. Reese, J.C., H.C. Waiss, Jr., and D.M. Legacion, 1987. Methodologies for determining mechanisms and factors of resistance. In Toward Insect Resistant Maize for the Third World: Proceedings of the International Symposium on Methodologies for Developing Host Plant Resistance to Maize Insects, 244-252. Mexico, D.F.: CIMMYT. Overman, J.L. 1987. A maize breeding program for development of hybrids with resistance to multiple species of leaf-feeding and stalk-boring lepidoptera. In Toward Insect Resistant Maize for the Third World: Proceedings of the International Symposium on Methodologies for Developing Host Plant Resistance to Maize Insects, 235-243. Mexico, D.F.: CIMMYT. Evaluation and Development of M aize Germplasm for Resistance to Spotted Stem Borer U. Kanta, B.S. Dhillon and S.S. Sekhon, Punjab Agricultural University, Ludhiana, India Abst r a c t Chilo partellus (Swinhoe), commonly known as the spotted stem borer, is the most serious pest of maize (Zea mays L.) in India. The best approach to manage this pest is the development and use of maize cultivars having genetic resistance. In the cultivar development process, germplasm needs to be precisely evaluated on a large scale utilizing insect mass rearing techniques, synthetic diets, and artificial infestation of plants. Insect rearing laboratories have been set up and synthetic diets developed and improved. Extensive evaluation of germplasm by Punjab Agricultural University, Directorate of Maize Research and other institutes in India led to the identification of some relatively resistant materials. The more promising ones are populations Antigua Gr. 1, Arun, D 791, J 22, J 3022, Pool 27 and Tarun, and inbred lines CML 67, CML 71, CML 72, (Partap x Mo17.B57)-17(S6), Suwan 1(S) C6-40(S5) and Suwan 1(S) C6-53(S5). Further, MBR-SCB Res. EV (Y), MBR 86-Stars and Diamonds and Pop. 24 Bulk were identified to be resistant to C. partellus and Ostrinia furnacalis Guenee. Populations Parbhat and Navjot, and inbred lines CM110L, CM 201, J101(S2), J663(S7) and Vijay 444(S2) showed resistance to C. partellus, maydis leaf blight [Drechslera maydis (Drechsl.) Nisikado and Miyaki] and brown stripe downy mildew (Sclerophthora rayssiae var. zeae Payak and Renfro). Many of these materials have been used to develop open pollinated and hybrid cultivars and to derive inbred lines. In Ageti 76, Navjot and Kiran, two to three cycles of recurrent selection for resistance to C. partellus under natural conditions led to appreciable gains. In Ageti 76, selection was carried out only for insect resistance, whereas, in Navjot and Kiran, selection criteria were based on grain yield and other traits including insect resistance. In J 22, four cycles of recurrent selection for borer resistance under artificial infestation resulted in a significant improvement of this trait. (Mathur 1991). About two dozen are often not adopted by the farmers to insects are known to cause moderate- the desired extent for various reasons. Maize is the third most important to-heavy damage to this crop (Sekhon Furthermore, insecticide use has many cereal crop, next to wheat and rice, in et al. 1993). Some of these pests are ill effects, such as environmental the world (FAO 1993). It is extensively major constraints to maize cultivation, pollution, residue problems and used as food, feed and fodder, and in with the maize spotted stem borer, C. destruction of useful insects. Thus, the the production of starch, oil, liquor, partellus being the most serious pest. development and use of insect resistant dextrose, dyes, etc. The average world The yield losses due to this pest were cultivars by exploiting host plant maize yield is 3.7 t/ha, whereas in estimated to be 26.7 to 80.4% in resistance offers a better alternative. In India it is only 1.6 t/ha (FAO 1993), different agro-climatic regions of the resistant cultivars pest control is despite India ranking fifth in the world country (Sarup 1980). ensured, along with the seed, without Int roduct ion incurring any extra expenditure. In in terms of acreage. Maize is an important crop in the Indian State of Various methods of pest control — addition, the control is non-polluting, Punjab, particularly in the rainy season, namely mechanical, cultural, biological stable and durable both through time but it is also grown during winter and and chemical — have been developed and environments. Resistant cultivars spring. to check the damage due to different can also be successfully incorporated insects in maize. Historically, most into an integrated pest management The number of insect and mite pests emphasis was placed on chemical strategy. In a resistance breeding attacking maize exceeds 250 in India control. Chemical measures, however, program, a wide spectrum of EVALUATION AND DEVELOPMENT OF MAIZE GERMPLASM FOR RESISTANCE TO SPOTTED STEM BORER 247 germplasm is evaluated for reaction to rajmah, green gram (Vigna radiata L.), based diets, Diet III and Diet IV, pests and the best is used in and sprouted legumes and cereals, proved to be better still. These diets appropriate breeding programs to namely green gram, maize and wheat reduced the period of insect develop resistant cultivars possessing (Triticum aestivum L.) (Tables 1 and 2). development and increased the other desirable traits. This approach They observed that Diet I and Diet II of number of larvae per generation (Table involves mass rearing of insects in the the nitrogen based diets gave rapid 3). Hence, Diet III is now being used for laboratory and germplasm evaluation multiplication of C. partellus in the mass rearing of C. partellus at under artificial infestation. This paper comparison to the earlier diets Punjab Agricultural University (PAU), presents results of research during the developed by Siddiqui et al. (1977). Ludhiana. last two decades on the standardization Among the other diets, two green gram of mass insect rearing and germplasm evaluation techniques, and the identification and development of germplasm Table 1. Artificial diets developed for the mass rearing of C. partellus. resistant to C. partellus. Siddiqui and M ass Rearing Siddiqui et al. (1977) Chatterji Uma Kanta and Sajjan (1989) Uma Kanta and Sajjan (1991) Diet II Diet I Diet II Diet III Treatment (1977) Diet I The availability of many eggs Red rajmah powder (g) 74.8 75.0 - 90.0 105.0 - - or neonate larvae of C. Green gram powder (g) - - 75.0 - - 90.0 75.0 partellus is a pre-requisite in Wheat powder (g) 20.0 20.0 20.0 20.0 20.0 20.0 - investigations on host plant resistance. Since it is not Sprouted wheat powder (g) Yeast (g) 4.0 5.0 5.0 5.0 5.0 5.0 20.0 5.0 possible to collect the required Ascorbic acid (g) 1.3 1.7 1.7 1.7 1.7 1.7 1.7 number of naturally occurring Vitamin E (g) 0.1 0.1 0.1 0.1 0.1 0.1 0.1 insects, these have to be reared Methyl Paraben (g) 0.8 0.8 0.8 0.8 0.8 0.8 0.8 on artificial diets. Thus, an Sorbic Acid (g) 0.4 0.4 0.4 0.4 0.4 0.4 0.4 insect rearing laboratory was Agar-Agar (g) Formaldehyde 40% (ml) 5.1 1.0 6.0 1.0 6.0 1.0 6.0 1.0 6.0 1.0 6.0 1.0 6.0 1.0 380.0 487.5 390.0 500.0 390.0 500.0 400.0 525.0 410.0 550.0 400.0 525.0 390.0 500.0 established to provide congenial temperatures, relative humidities, light Water (ml) Total diet (g) Diet IV intensities, and improved artificial diets for extensive multiplication. Table 2. Nitrogen concentration of plants of maize populations and of Rajmah diet. Initially, the artificial diet containing Nitrogen (%) rajmah (Phaseolus vulgaris L.), developed by Siddiqui and Chatterji (1972) and Siddiqui et al. (1977), was used at Treatment different centers of the All India Antigua Gr. 1 Ganga 5 JML 22 Vijay Ageti 76 Basi Local Makki Safed 1 Rajmah diet C.D. (0.05) Coordinated Maize Improvement Programme. Then, Uma Kanta (1985) and Uma Kanta and Sajjan (1989, 1994) formulated 26 different diets. Some of these diets were based on the comparative nitrogen concentration of susceptible varieties, the diets under current use, and others on variable contributions of legumes, mainly a Whole plant/diet 12 DAGa Stem 24 DAG 2.27 2.45 2.55 2.97 3.08 3.18 3.15 2.66 b 0.21 (0.42) 1.96 2.03 2.13 2.10 2.24 2.31 2.31 0.27 Leaf 36 DAG 24 DAG 36 DAG 1.56 1.76 1.68 1.12 0.84 0.79 0.80 0.14 2.13 2.10 2.24 2.20 2.24 2.45 2.24 0.20 1.55 1.02 0.84 0.77 0.79 0.73 0.73 0.14 DAG = days after germination. Includes Rajmah diet as a treatment for analyses. Source: Uma Kanta and Sajjan (1989). b Mean 1.95 1.95 1.99 2.02 2.04 2.06 2.06 - 248 U. KANTA Artificial Infestation Table 3. Relative performance of artificial diets based on two generations of mass rearing of C. partellus. Artificial infestation was carried out either by releasing ten larvae per plant- Artificial dieta whorl 16 to 18 days after emergence, or Diets based on nitrogen concentration d Diet I 34.2 20.2 Diet IId 33.4 20.8 e Diet I 35.2 13.4 by pinning tissue paper containing 25 to 30 black headed eggs onto each plant. The tissue papers were examined at random, a day after infestation, for Diets based on green gram Diet IIIf 29.1 49.9 f 29.7 51.9 Diet IV Diet IIe 29.5 45.6 hatching of eggs. A second release of eggs was carried out if infestation was low. a b Grading Plant Damage c d e Insect damage was expressed as leaf Period of Moth Pairs Progeny produced developmentb emergence of moth Eggs Larvae (days) (%) (no.) (no.) (no.) f Increase in number of larvaec (%) 9 8 6 2340 2240 1296 2048 1836 1064 92 72 - 23 24 19 9039 8976 5377 6645 7524 3185 109 136 - Details of the diet ingredients are given in Table 1. Period of development from larval hatching to adult formation. Increase over the diets developed by the Siddiqui et al. (1977). Uma Kanta and Sajjan (1989). Siddiqui et al. (1977). Uma Kanta and Sajjan (1991). scraping, small pin holes, or slit holes in the whorl leaves. Severe attack results in stunted growth, dead heart Table 4. Maize germplasm showing a relatively resistant reaction to C. partellus. and stem breakage. A nine-class rating scale (1 = healthy, 9 = dead heart) was Germplasm Location Reference used. This was developed by Chatterji Ludhiana Anonymous (1973) et al. (1970) and Sarup et al. (1974) by Comp. A 53 (SA) x Comp A54 (EV) RU 21, EH 2230, EH 3136, J22, Opaque B-15 modifying the scale of Starks and (Dcota x GCC) br2-## Pantnagar Sharma and Singh (1975) A6, A21, Amarillo Cristalino-1, Antigua Gr 1, Antigua Gr 2 Sel. Blanco, Antigua 7D, Antigua 8D, (Ant. x Cubans 157), British Virgin Island 117, Caribbean Flint Comp., Cuba 9, Cuba 12, Cuba 40, Dneproaskaja 200, Guatemala 257, M 512, MCPD(MS)6, Mezcla Amarilla Baja, Serie S3, R2CII, Thai DMR Comp. 17, V520CA, (Ver 181 x Ant GPO2) 02, CISTRON, EA1712 (late) FV 147 x BUP 116, FV147 x ZP 2077, K10 x 2 PR 588, LP 1712 x ZPR 588, MR 21 x R 588, MR 21 x SD 10, 0 118a x BUP 43, SD 10 x BUP 116 Syn 60J, T146 x BUP 116, T 146 x SD10, T166 x ZP 2077/54, T 116 x ZPR 588, T 169, T 341 x WF 9, U 221 x ZPR 588, VTR 116 x ZPR 588, YUZP 2077/54 New Delhi Sarup et al. (1978) Sarup et al. (1979) Dogget (1970). Singh and Sajjan (1983) evaluated row grading methods, and found that recording a single observation on 5 to 10 plants in a row may be as efficient as the gradation of an individual plant in a row. They also compared different class rating scales, namely the 1-9 scale (Chatterji et al. 1970) and a 1-5 scale (Kandoria 1975), using leaf injury and dead heart, percent infestation, percent dead heart, tunnel length and number of borers, as their criteria. On the basis of a variance ratio test, coefficient of variation and relative ranking of genotype, the 1-9 scale was considered to be better than Amber, Deccan 103, Sona, Vikram Pantnagar the others because it covers a wide Antigua Gr 2, (CM 201) 5 br2#, IACP Comp.1, J22, Syn P 203 x Kisan)## New Delhi and Ludhiana Harnampur Local, Kesari Local New Delhi Sarup et al. (1981) BS20, Iowa Long Ear Syn, Honey June, NC 59663, Pool 15 Pool 16, Pool 17, Pool 19, Pool 24, Pool 25, Pool 26, Pool 27, Pool 28, Pool 29, Pool 30, Pool 32, Pool 33, Pool 33 QPM, Tuxperate x Tropical QPM (Dent) Ludhiana Anonymous (1984, 1985) Tuxpeno QPM Antigua Gr 1, Mex 17 New Delhi Durbey and Sarup (1985) range of leaf injury, including dead heart. Identification of Resistant Ge rm plasm Extensive studies to evaluate exotic and indigenous germplasm under artificial EVALUATION AND DEVELOPMENT OF MAIZE GERMPLASM FOR RESISTANCE TO SPOTTED STEM BORER 249 infestation have been conducted. The cultivation in India, namely Hybrids early maturing, widely adapted and materials identified to be relatively Deccan, Deccan 103, Ganga 2, Ganga 4, relatively good yielders. Sartaj, Parbhat resistant are listed in Table 4. These Ganga 5 and Sartaj, and composites and Navjot also possess resistance to include indigenous collections from Ageti 76 (J 603), Amber, Arun (A 68), one or more diseases. Punjab and Uttar Pradesh, Chandan, Dhawal, Hunius, Jawahar indigenously developed hybrids, (A1 x Antigua Gr. 1), Kiran (J 660), Mean damage grade (m.d.g.) of some composites and synthetics, and exotic Kisan, Kundan, Navjot (J 684), Parbhat promising inbred lines and early germplasm from the International (J 115), Partap (J 54), Sona, Tarun (Syn maturing composites are presented in Maize and Wheat Improvement Center PK), Vijay and Vikram. Sartaj, Ganga 2, Tables 5 and 6, respectively. Three (CIMMYT, Mexico), Caribbean Islands, Ganga 5, Deccan 103, Parbhat, Vijay inbred lines showed a m.d.g. of 2.4 to Colombia, Guatemala, USA, Thailand and Jawahar possess both high yield 3.0 in comparison to 8.2 of the most and Pakistan. Some hybrids and and wide adaptation, whereas Ageti 76, susceptible inbred, CM 400 (Uma Kanta composites released for commercial Arun, Kiran, Navjot and Tarun are and Sekhon 1994). Five composites had a m.d.g. of 2.6 to 3.0 whereas the m.d.g. of susceptible material, D 741 EV 81 Table 4. cont’d (Ranchi) was 4.8. Germplasm Location Reference Hunius, BS 7, BS 8, BS 14 Cooks Early Yellow Dent New Delhi Panwar and Sarup (1985) Ganga 5, Antigua Gr 1, J 22, J 605 Ludhiana Sekhon (1985) Comp.217, Comp.218, Comp.219, Comp.222 Comp.223, Int. Comp.202, Int.Comp. 210, Int. Comp.214, Int. Comp.216, Int. Comp.217 AR 76, Comp.217, EVA 64-mst-80 New Delhi Siddiqui et al. (1986) Chandan, Deccan 103, Ganga 5, Jawahar (A1 x Antigua Gr. 1) Comp, Kundan Local Haryana-Hoshiarpur Local Gidderpindi, Lopon Yellow New Delhi CM 110L, CM 201, J 101(S2), J663(S6), J663(S7), Vijay (S3), Ludhiana Table 5. Reaction of promising inbred lines of maize to C. partellus. Sarup et al. (1987) Damage grade (1-9)a 1985 1986 Mean Inbred Dey et al. (1987) Ageti 76, Deccan Ganga 5, Pantnagar Ganga 2, Jawahar, Kisan, Tarun, Vijay, Vikram, Amarillo Pak, Caribbean Flint Comp, Cuba 11J, D 818, Golden Crystal, H 207, Hybrid Vanzyl, Mo x 117, Mo x 57, N 21, N 22, Pop. 31, PR 7921, Suwan 7528 Bulandshahar Local, Meerut Yellow Local, Saharanpur Local, Monghia Local, Gore Local and Dewarika Local Sharma (1987) Ganga 4, Dhawal, Hunius, Jawahar x Thai New Delhi Singh (1988) Comp 217 New Delhi Siddiqui et al. (1988) Comp. A-214, EA-82-4-87 New Delhi Marwaha et al. (1990) Arun Chindwara Sharma and Sharma (1992) Ageti 76, J2012, J3022, Kiran, Navjot, Navjot (HS) C3, Parbhat, Sartaj Ludhiana Dey et al. (1993) (J54xMo17.B57)-17-1-2-2-1-1-1#, Suwan 1(S) C6-40-1-1-1-2-1#, Suwan 1(S) C6-53-1-1-1-2-2#, Arun, D791, Kiran, Pool 17, Pool 27 Ludhiana Uma Kanta and Sekhon (1994) CML 67, CML 71, CML 72, MBR SCB Res.EV(Y), MBR 86 Stars and Diamond, Pop. 24 Bulk, Across 90390-W(IR), SCB(GCA) FAW (GCA), EEY DMR POOL (FS), EY Takfa (HS), Pop. 31 DMR C5 (S2 bulk) Ludhiana Uma Kanta et al. (Present publications) (J54 x Mo17.B57)-171-2-2-1-1-1# Suwan 1 (S) C6-40-1-1-1-2-1# Suwan 1 (S) C6-53-1-1-12-2# CM 400 (Susceptible) 3.9 2.1 3.0 2.6 2.2 2.4 3.9 2.0 3.0 7.4 9.0 8.2 a 1 = healthy; 9 = dead heart. Source: Uma Kanta and Sekhon (1994). Table 6. Reaction of promising early maturing populations of maize to C. partellus. Germplasm Pool 17 Pool 27 Tarun J 660 (Kiran) A 68 (Arun) D 791 D 741 EV81 (Ranchi) (Susc.) a Damage grade (1-9)a 1983 1984 Mean 2.6 2.6 2.6 2.2 2.6 2.8 4.2 3.4 2.7 2.0 3.3 2.8 2.3 5.5 1 = healthy; 9 = dead heart. Source: Uma Kanta and Sekhon (1994). 3.0 2.6 2.3 2.8 2.7 2.6 4.8 250 U. KANTA Dey et al. (1987, 1993) evaluated 70 500), a Caribbean introduction. It has resistant (MIR) and downy mildew advanced inbred lines, 11 composites been used as a parent of the widely (DMR) populations have been and 7 hybrids for multiple resistance to adapted, high yielding double top- evaluated for reaction to C. partellus in C. partellus, D. maydis and S. rayssiae cross hybrid, Ganga 5, and of the India and O. furnacalis in the var. zeae. The parameters of multiple varietal hybrid used to develop Comp. Philippines, and now efforts are being resistance, namely mean and standard Jawahar. Inbreeding in Antigua Gr. 1, made to develop DMR-borer resistant deviation were estimated following however, did not yield good inbred germplasm. Dhillon et al. (1984). Low values of lines. There are many other resistant these parameters indicated uniform germplasm sources that have been Among the CIMMYT maize lines multiple resistance. Six inbred lines utilized in the development of evaluated, three (CML 67, CML 71, (Table 7), four composites and one promising composites and hybrids CML 72) have shown a promising hybrid (Table 8) showed multiple (Table 9). reaction to C. partellus. Their m.d.g. varied from 2.6 to 3.0 in comparison to resistance. All five composites and one hybrid are released cultivars. CIM M YT’s Asian Regional Collaborat ive Proje ct Utilization of Resistant Ge rm plasm 6.5 for the susceptible check (Table 10). However, these lines per se, as well as in cross combinations, did not show Given the serious damage due to stem agronomically good performance borers in South-East Asia, CIMMYT’s under our conditions. We have planned The germplasm that has consistently Asian Region Maize Program initiated to evaluate their heterotic relationships shown resistance is Antigua Gr. 1 (CM collaborative research on the with our elite materials so as to utilize evaluation and improvement of the inbred lines in second cycle germplasm for resistance to C. partellus breeding. The inbred lines CML 123, and O. furnacalis in 1990. Since then, CML 126, CML 127 and CML 131 all inbred lines and multiple borer showed susceptible reactions. Table 7. Parameters of multiple resistance of promising inbred lines of maize to C. partellus, Drechslera maydis and Sclerophthora rayssiae var. zeae. Inbred CML 110 L CM 201 J 101 (S2)b J 663 (S7) J 663 (S6) Vijay (S2) a b Multiple resistance Standard Mean (1-5)a deviation 1.7 2.0 1.9 2.0 1.8 1.9 resistant (MBR), multiple insect Table 9. Sources of resistant germplasm used in the development of promising composites and hybrids. Source germplasm 0.361 0.874 0.513 0.681 0.577 0.513 Population Antigua Gr. 1 Arun and J 3022 Tarun 1 = healthy; 5 = susceptible. Generation of selfing. Inbred(s) derived from Arun Table 8. Parameters of multiple resistance to C. partellus, D. maydis and S. rayssiae var. zeae of some promising maize cultivars and local. Genotype Hyb. Sartaj Comp. Kiran Comp. Navjot Comp. Navjot (HSC3) Comp. Parbhat Local a J 3022 Suwan 1 Tarun Vijay J 3022 and Navjot Multiple resistance Standard Mean (1-5)a deviation 2.5 2.5 2.4 2.3 0.374 0.458 0.600 1.153 2.3 4.2 1.079 0.200 1 = healthy; 5 = susceptible. Tarun Ageti 76, Arun J 101, J660, J663, Kiran, Navjot, Partap, Tarun and Vijay Cuba 11J and Suwan 1 a Population or hybrid developed Status Hyb. Ganga 5 Comp. Jawahar Comp. Megha Comp. Navjot Released at the national level Released at the national level Released at the national level Released at the national level EH 2420 EH 3021 EH 2420 EH 21058 EH 3189 EH 200174 Indigenous early heterotic pool Semi-exotic early heterotic pool Makki Safed heterotic pool Evaluation in FYTa in 1994 a Evaluation in FYT in 1994 As explained above Evaluated in FYT in 1993 Evaluation in SYYTa in 1994 Evaluation in FYT in 1979 Tuxpeno heterotic pool FYT = final yield trial, SYYT = second year yield trial EVALUATION AND DEVELOPMENT OF MAIZE GERMPLASM FOR RESISTANCE TO SPOTTED STEM BORER 251 Three MBR populations, MBR SCB Res. also (Table 13). Inbreeding was In view of losses to downy mildew EV (Y), MBR Stars and Diamonds and initiated in these three populations, but (Sclerospora spp. and Sclerosphthora Pop. 24 Bulk, and three MIR they showed intense depression for spp.) in Asia, the collaborative project populations, Across 90390-W (IR), SCB grain yield and agronomic traits. The adopted DMR germplasm in 1993. Ten (GCA) and FAW (GCA), showed MBR and MIR germplasm that showed DMR populations were evaluated. The relatively good reaction to C. partellus a susceptible reaction to C. partellus populations that showed a relatively (Tables 11 and 12). The MBR included Phil. 05, Phil. DMR Comp. 1, resistant reaction were EY Takfa (HS), populations were also evaluated for TLY-DMR Pool C3 (HS), and Across EEY DMR Pool (FS) and Pop. 31 DMR reaction to O. furnacalis. The three 90390-Y(IR). C5 (S2 Bulk) (Table 14). The plants populations mentioned above showed resistant (m.d.f.> 4.0) to C. partellus at a good level of resistance to this pest Ludhiana and Hyderabad in India and to O. furnacalis at Los Banos, the Table 13. Reaction of multiple borer resistance populations of maize. Table 10. Reaction of promising inbred lines of maize to C. partellus. Damage grade (1-9) 1992 1993 1994 Mean CML 67 CML 71 CML 72 Basi Local (Susc. check) a 2.5 3.9 3.3 4.9 2.2 2.8 2.4 6.8 3.1 2.4 3.2 6.2 2.6 3.0 3.0 6.5 1 = healthy; 9 = dead heart. MBR - SCB Res. EV(Y) MBR 86- Stars and Diamonds Pop. 24 Bulk Susceptible Checkb a b Table 11. Reaction of promising multiple borer resistance (MBR) populations of maize to C. partellus. Germplasm MBR SCB Res. EV (Y) MBR 86 Stars and Diamonds Pop. 24 Bulk Basi Local (Susc. check) Damage grade (1-9)a 1990 1991 1992b Mean 3.8 5.0 4.0 5.2 3.8 4.3 3.3 6.4 3.9 6.0 3.4 5.0 3.5 5.8 1 = healthy; 9 = dead heart. Based on S1 lines, developed from resistant plants during 1991. Source: Sekhon et al. (1992). b Table 12. Reaction of promising multiple insect resistance (MIR) populations of maize to C. partellus. Across 90390W (IR) SCB (GCA) FAW (GCA) Basi Local (Susc. check) a 1.8 3.8 2.6 3.3 6.4 2.8 5.2 Damage grade (1-9)a 1992 1993 Mean 4.1 3.3 3.7 3.3 3.8 5.1 3.5 4.5 5.9 3.4 4.2 5.5 1 = healthy; 9 = dead heart. constitute three populations, namely Early Yellow, Early White and Late Yellow. The materials developed at one center were exchanged with others. PAU was the primary location to form the Early Yellow population and contributed 110 selfed lines to the total of 231 lines used to develop this population. The number of S1-S3 lines 1 = healthy; 9 = dead heart. Basi Local and Philippine Supersweet for C. partellus and O. furnacallis, respectively. contributed by different centers to develop the three populations are given in Table 15. As per the program Table 14. Reaction of promising downy mildew resistance (DMR) populations of maize to C. partellus. of the collaborative project, resistant plants in resistant lines were recombined to reconstitute the a Pedigree a Population 3.8 4.3 3.8 to S3 lines in these materials and to Damage grade (1-9)a C. partellus O. furnacalis Ludhiana, Los Banos, Germplasm India Philippines a Inbred Philippines, were selfed to develop S1 Early Yellow EEY DMR Pool (FS) EY TakFa (HS) Pop. 31 DMR C5 (S2 bulk) Pop. 145 EY DMR Pool (S2 bulk) Viemyt 49-Y (S2 bulk) Early White EEY DMR Pool (FS) Pop. 100 EW DMR (S2 bulk) Late Yellow LY Takfa (HS) Pop 28 EMR C6 (S2 bulk) Pop. 345 LY DMR (S2 bulk) Basi Local (Susc. Check) a b Damage Grade (1-9) 1993 1994b Mean 4.9 3.6 5.5 4.2 3.9 3.4 4.6 3.8 4.4 5.5 5.8 5.7 5.9 4.1 5.0 5.1 5.5 5.1 6.3 5.1 5.9 4.3 5.4 5.7 7.2 5.0 6.3 5.7 7.1 6.4 5.9 5.9 5.9 population. These will be sent to various collaborators. In addition, we have continued selfing in selected lines. Populat ion Im prove m e nt In the population improvement program for grain yield and other traits at PAU the families were also evaluated for resistance to C. partellus, D. maydis, and S. rayssiae var. zeae depending on the resources available. A number of composites, namely Ageti 76, Navjot, Parbhat, Partap, Vijay, Kiran and J 663 were subjected to population improvement for C. 1 = healthy; 9 = dead heart. based on S1 lines developed from resistant plants identified during 1993. partellus under natural conditions using square planting. Depending on grain yield and other traits including pest and disease resistance, the populations 252 U. KANTA were reconstituted. The result was that the performance has been evaluated families and the second cycle on S1 most of the cultivars developed at PAU under artificial infestation. There was families. The reconstituted and original — namely Ageti 76, Navjot, Kiran, gain for resistance to C. partellus and D. populations were evaluated under Parbhat, Partap and Sartaj during the maydis. However, no gain was artificial infestation, wherein the late 1970s and 80s — combine high observed in some other populations former showed a lower m.d.g. than the yield, wide adaptation and other (Dey et al. 1988). later, indicating improvement for resistance to C. partellus (Table 17). desirable traits including disease and pest resistance. Recurrent selection for resistance only to C. partellus was carried out in two Four cycles of selection were carried The performance of Kiran (J 660) and populations, Ageti 76 (J 603) and J 22. out in Composite J 22 for resistance to Navjot (J 684) after two and three cycles Ageti 76, an early maturing and high C. partellus under artificial infestation of selection for various traits, including yielding cultivar, was subjected to two (Dhillon et al. 1987). This population the reaction to C. partellus, is presented cycles of improvement under natural had high yield potential with good in Table 16. The selection was carried infestation by Singh et al. (1982). The agronomic traits, resistance to C. out under natural conditions, whereas first cycle was based on half-sib partellus and Atherigona spp. and tolerance to zinc deficiency. The Table 15. Number of selfed lines developed in downy mildew resistant (DMR) populations of maize during 1993 and evaluated during 1994 in a collaborative program on multiple borer resistance. Population DMR lines developed at different locations (no.) O. furnacallis C. partellus Los Banos Hyderabad Ludhiana Philippines India India Total Source germplasm Early Yellow 90 Early White 31 39 Late Yellow 110 13 21 36 - 22 231 88 43 EEY DMR Pool (FS), EY Takfa (HS), Pop. 31, DMR C5 (S2 bulk), Pop. 145 EY DMR Pool (S2 bulk) and Viemyt 49 Y (S2 bulk) EEW DMR Pool (FS) and Pop. 100 EW DMR (S2 bulk) LY Takfa (HS), Pop. 28 EMR C6 (S2 bulk) and Pop. 345 LY DMR (S2 bulk) selection comprised one cycle of halfsib, one cycle of full-sib and two cycles of S1 family selection, in that order. J 22 and the strains developed after secondto-fourth cycles of selection were evaluated under artificial infestation. The difference between J 22 C0 and the strains developed after four cycles of selection, J 22 C4, was significant (Table 18). The improved population is being used as a source germplasm to derive inbreds. Recurrent Selection and Hybrid Breeding At PAU, Ludhiana, major research efforts are now being devoted to hybrid breeding. Therefore, we have initiated recurrent selection and inbred line Table 16. Reaction of the original and improved versions of maize populations to C. partellus and D. maydis. Population C. partellus (1-9)a J 660 C0 J 660 HS (MER) C2 J 660 HS C2 J 684 C0 J 684 HS (MER) C2 J 684 HSC3 a 6.0 5.0 5.4 5.9 6.9 4.8 1 = healthy; 9 = dead heart. b 1 = healthy; 5 = susceptible. Source: Dey et al. (1988). D. maydis (1-5)b 2.3 2.0 1.8 2.5 2.1 1.9 Table 17. Reaction of original and improved versions of Composite J 603 after two cycles of selection for resistance to C. partellus. Damage grade (1-9)a Population J 603 C2 (B+W) J 603 C2 (B+W)# J 603 C2 (B) J 603 C0 a Natural infestation Artificial infestation 2.6 2.9 2.7 3.2 4.6 5.0 4.9 5.1 1 = healthy; 9 = dead heart. B = between family selection; W = within family selection. Source: Singh et al. (1982). b development in two heterotic pools, Table 18. Reaction of original and improved versions of Comp. J 22 after two to four cycles of selection for resistance to C. partellus. Population Damage grade (1-9)a J 22 C0 J 22 C2 J 22 C3 J 22 C4 Basi Local (Susc. check) a 4.8 5.0 4.0 3.5 5.6 1 = healthy; 9 = dead heart. Source: Dhillon et al. (1987). EVALUATION AND DEVELOPMENT OF MAIZE GERMPLASM FOR RESISTANCE TO SPOTTED STEM BORER Makki Safed and Tuxpeño developed by Khehra et al. (1986). Recurrent selection based on half-sib and selfed families (Dhillon et al. 1994) is being pursued, but selfing has been extended to the S2 generation in view of the greater emphasis on hybrid breeding. In each pool 600 plants were artificially infested and the most promising 100 were selected. The S1 lines of these plants were grown and subjected to among- and within-family selection. In the S2 generation 91 lines of each pool were evaluated. Selected plants within selected S2 families are being recombined and selfed to develop improved pools and inbred lines, respectively. Re fe re nce s Anonymous. 1973. Final Technical Report PL-480 Project, Investigation of the Major Insect Pest of Maize with Special Reference to Insect-Plant Relationship, Department of Entomology, Punjab Agricultural University, Ludhiana. Anonymous. 1984. Twenty-seventh Annual Progress Report. All India Coordinated Maize Improvement Project, Indian Agricultural Research Institute, New Delhi. Anonymous. 1985. Twenty-eighth Annual Progress Report. All India Coordinated Maize Improvement Project, Indian Agricultural Research Institute, New Delhi. Chatterji, S.M., V.P.S. Panwar, K.H. Siddiqui, W.R. Young, and K. Marwaha. 1970. Field screening of some promising maize germplasm against Chilo zonellus Swinhoe under artificial infestation. Indian J. Ent. 32: 167-170. Dey, S.K., B.S. Dhillon, V.K. Saxena, Uma Kanta, and A.S. Khehra. 1988. Improvement for resistance to maydis leaf blight (Drechslera maydis) and maize borer (Chilo partellus) on maize (Zea mays). Indian J. Agric. Sci. 58: 837839. Dey, S.K., B.S. Dhillon, Uma Kanta, S.S. Sekhon, V.K. Saxena, N.S. Malhi, and A.S. Khehra. 1993. Resistance to multibiotic stresses in maize. J. Ent. Res. 17: 75-80. Dey, S.K., N.S. Malhi, S.S. Sekhon, B.S. Dhillon, and A.S. Khehra. 1987. Multiple disease-insect pest resistance in maize. Proc. First Symp. Crop Improvement. Vol. II. 361-365. Dhillon, B.S., G. Granados, R., and A.S. Khehra. 1994. Recurrent selection for intrapopulation improvement for insect resistance. Cereal Res. Commun. 21: 331335. Dhillon, B.S., A.S. Khehra, S.K. Dey, S.C. Sharma, and V.K. Saxena. 1984. Multiple disease resistance in maize. SABRAO J. 16: 69-72. Dhillon, B.S., A.S. Khehra, V.V. Malhotra, S.S. Sajjan, S.S. Sekhon, and Uma Kanta. 1987. Intrapopulation improvement for resistance to Chilo partellus in Zea mays. Proc. Int. Symp. Maize Arthropods. Budapest, Hungary. Durbey, S.L., and P.Sarup. 1985. Antibiosis due to powdered dry plant material of maize varieties incorporated in artificial diet for rearing of the stalk borer, Chilo partellus (Swinhoe). J. Ent. Res. 9: 201-206. FAO (Food and Agriculture Organization). 1993. FAO Production Year Book, 1993, Rome, Italy. Kandoria, J.L. 1975. Chemical control and biology of the maize stem borer. Chilo partellus (Swinhoe). M.Sc. Thesis, G.B. Pant University of Agriculture and Technology, Pantnagar, India. Khehra, A.S., B.S. Dhillon, N.S. Malhi, V.K. Saxena, V.V. Malhotra, S.K. Dey, S.S. Pal, and W.R. Kapoor. 1986. Systematic introgression of the Corn Belt germplasm of maize. In: B. Napompeth and S. Subhadrabandhu (ed.) New Frontiers in Breeding Researches, 291-302. Bangkok, Thailand: Kasetsart Univ. Marwaha, K.K., K.H. Siddiqui, and P. Sarup. 1990. Location of multiple pest resistant sources amongst maize germplasms evaluated against tissue borers. J. Ent. Res. 14: 1-4. Mathur, L.M.L. 1991. Genetics of insect resistance in maize. In K.R. Sarkar, N.N. Singh, and J.K.S. Sachan (Eds.) Maize Genetics Perspectives, 238-259. New Delhi, India: Indian Society of Genetics and Plant Breeding. Panwar, V.P.S., and P. Sarup. 1985. Significance of extreme conditions for screening of maize germplasm for resistance to the stalk borer, Chilo partellus (Swinhoe). J. Ent. Res. 9: 237240. Sarup, P. 1980. Significant results of investigations carried out under maize improvement project in recent years. In: Joginder Singh (ed.) Breeding, Production and Protection Methodologies of Maize in India. 193-197. New Delhi, India: All India Coordinated Maize Improvement Project, Indian Agricultural Research Institute. 253 Sarup, P., K.K. Marwaha, V.P.S. Panwar, and K.H. Siddiqui. 1978. Identification of sources of resistance to the maize stalk borer, Chilo partellus (Swinhoe) amongst world maize germplasms comprising ‘international nursery’, J. Ent. Res. 2: 154-159. Sarup, P., K.K. Marwaha, K.H. Siddiqui, and V.P.S. Panwar. 1979. Investigations on Major Insect Pests of Maize with Special Reference to Insect-Plant Relationship. Res. Bull. No. 3, Div. Ent. Indian Agricultural Research Institute, New Delhi, India. Sarup, P., B.K. Mukherjee, K.K. Marwaha, V.P.S. Panwar, K.H. Siddiqui, and N.N. Singh. 1974. Identification of a source of resistance to Chilo partellus (Swinhoe) in Colombian maize hybrid H. 207 and formulation of a suitable breeding procedure for its utilization. Indian J. Ent. 36: 1-5. Sarup, P., K.H. Siddiqui, and K.K. Marwaha. 1987. Trends in maize pest management research in India together with bibliography. J. Ent. Res. 11: 19-68. Sarup, P., K.H. Siddiqui, V.P.S. Panwar, and K.K. Marwaha. 1981. Response of diverse maize germplasms to artificial infestation of the stalk borer, Chilo partellus (Swinhoe). J. Ent. Res. 5: 70-75. Sekhon, S.S., B.S. Dhillon, and Uma Kanta. 1993. Host plant resistance in maize to insect pests. In: G.S. Dhaliwal, and V.K. Dilawari (ed.) Advances in Host-Plant Resistance to Insects, 79-109, New Delhi, India: Kalyani Publ. Sekhon, S.S., and S.S. Sajjan. 1985. Antixenosis (non-preference) mechanism of resistance in maize against oviposition by maize borer, Chilo partellus (Swinhoe). Indian J. Ent. 47: 427-432. Sekhon, S.S., Uma Kanta, and G.R. Granados. 1992. Identification of multiple insect pest resistance sources in maize. In Recent Advances in Integrated Pest Management (Abstr.), 142-143. Ludhiana: Indian Society for the Advancement of Insect Science, Punjab Agricultural University. Sharma, M.L., and A.K. Sharma. 1992. Comparative resistance of maize cultivars/inbreds to stem borer, Chilo partellus (Swinhoe) J. Insect Sci. 5: 18384. Sharma, V.K. 1987. Maize insect pest problems, present status and future of host plant resistance in India. In Toward Insect Resistance Maize for the Third World: Proc. of the International Symposium on Methodologies for Developing Host Plant Resistance to Maize Insects, 281-285. Mexico, D.F.: CIMMYT. Sharma, V.K., and J.M. Singh. 1975. Screening for resistance to Atherigona spp. in spring sown maize. Indian J. Ent. 33: 419-424. 254 U. KANTA Siddiqui, K.H., and S.M. Chatterji. 1972. Laboratory rearing of the maize stem borer. Chilo zonellus Swinhoe (Cambridae:Lepidoptera) on a semisynthetic diet using indigenous ingredients. Indian J. Ent. 34: 183-185. Siddiqui, K.H., K.K. Marwaha, and P. Sarup. 1988. Performance of early maturing composites to locate sources of multiple pest resistance in monsoon (kharif) and spring sown maize. J. Ent. Res. 2: 1-3. Siddiqui, K.H., K.K. Marwaha, P. Sarup, and J.P. Singh. 1986. Search of sources of resistance amongst newly developed early and medium maturing maize composite subjected to manual infestation of the stalk borer,. Chilo partellus. J. Ent. Res. 10: 155-160. Siddiqui, K.H., P. Sarup, V.P.S. Panwar, and K.K. Marwaha. 1977. Evolution of base-ingredients to formulate artificial diets for the mass rearing of Chilo partellus (Swinhoe). J. Ent. Res. 1: 117131. Singh, J., and S.S. Sajjan. 1983. Evaluation of different techniques for screening maize germplasms for resistance to maize borer. Chilo partellus (Swinhoe). Indian J. Ent. 45: 424-430. Singh, J., S.S. Sajjan, and A.S. Khehra. 1982. Population improvement in maize composite J 603 for resistance to maize borer, Chilo partellus (Swinhoe). Crop Improv. 9: 106-110. Singh, N.N. 1988. Maize Improvement in India - Problems and prospects. In Proc. Third Asian Regional Maize Workshop, 4651. Kunming and Nanning, China: Chinese Academy of Agricultural Sciences and the CIMMYT-Asian Regional Maize Program. Starks, K.J., and H. Doggett. 1970. Resistance to a spotted stem borer in sorghum and maize. J. Econ. Ent. 62: 1790-1795. Uma Kanta, 1985. Studies on the formulation of diets for mass rearing of maize borer, Chilo partellus (Swinhoe) (Lepidoptera:Pyralidae). Ph.D. Thesis, Punjab Agricultural University, Ludhiana, India. Uma Kanta, and S.S. Sajjan. 1989. Formulation of improved artificial diet for the mass rearing of Chilo partellus (Swinhoe). J. Insect Sci. 2: 98-102. Uma Kanta, and S.S. Sajjan. 1994. Studies on improvement in artificial diets for the mass rearing of maize borer, Chilo partellus. Indian J. Ent. (In Press). Uma Kanta, and S.S. Sekhon. 1994. Location of sources of resistance amongst different varieties and inbreds of Zea mays to Chilo partellus. J. Ent. Res. 18: 1-6. Verification and Pre-Commercial Testing of European Corn Borer and Gibberella Ear Rot Resistant Varieties R.I. Hamilton, L.M. Reid, and F. Meloche, Agriculture Canada, Ottawa, Ontario, Canada. Abst r a c t Adapted cultivars must have an acceptable level of tolerance or resistance to major insect and disease pests. The European corn borer, ECB, (Ostrinia nubilalis, Hübner) and ear molds (Fusarium spps. in particular F. graminearum Schwabe) are important pests throughout the Northern corn belt of North America. An understanding of the insect, disease and genetic mechanisms of tolerance or resistance have led to the useful development and application of new and established techniques for developing improved cultivars. Modes(s) of entry, mechanisms of tolerance or resistance, degree of reasonable tolerance vis a vis effects on yield, lodging, grain quality, and source of genetic variability are key long-term steps towards a satisfactory solution. Int roduct ion RM or FAO 130. The two major pests of followed by 2nd generation tunneling maize are the European corn borer below the ear). Generally there are In Eastern Canada, agriculture is (ECB), Ostrinia nubilalis (Hubner), and three adult flights per year, with limited to the north by the Canadian ear rots caused by Fusarium spps. offspring reaching the mature larval Shield of rocks and forest. The soils Building tolerance/resistance to both stage by fall harvest. range from glacial till, lake bottoms, pests is a major goal of Canadian forest podzols to beach sands; and breeding programs. generation (i.e. leaf feeding) pests is not farming is restricted to river valleys surrounded by forest and hardwood Today, genotypic resistance to first European Corn Borer as important an objective, because plant tolerance is sufficient. Second bluffs. The development of maize germplasm generation damage (i.e., tunneling), Agriculture includes cereals, forages tolerant to the European corn borer has however, is certainly present, and plant and corn/soybean crops together with been in progress for many years, both dissection is the normal screening dairy, beef and some intensive pig/ nationally and worldwide. During the method. Limited resources require the poultry enterprises. The studies early history of corn breeding and the development of an improved screening reported occurred in the valleys of the move toward early maturing hybrids, method for direct field evaluation in Ottawa and St. Lawrence rivers, (Lat. N frequent devastation of farm fields and large plant breeding programs. 44°43'- 45°40'; Long. W 75° 31'-76°45') plant breeding nurseries occurred (Agr. similar perhaps to a region Can. Ann. Reports 1923-27). In Canada, Cultural practices such as conventional representing 55 RM to 80 RM using the ECB continues to account for annual ploughing and discing remain an Minnesota maturity rating system, or stalk breakage and loss of yield and effective control, but with the growing FAO 130-300 in Europe. The growing quality. popularity of conservation tillage, other management alternatives are under season begins after the last spring frosts in early May and is arrested by frost Canadian research followed the work investigation. This new environment from mid-to-late September. Corn described by Dicke and Guthrie (1988) has led to cool soil temperatures longer production is limited by the maturity of and Hudon et al. (1989), leading to the into the spring growing season, and cultivars recommended in regional use of artificial infestation screening necessitates a new look at corn borer trials. The earliest cultivars approach 55 (1st generation leaf feeding damage, behavior and methods of control. 256 R.I. HAMILTON, L.M. REID, AND F. MELOCHE The Canadian plant breeding effort background and their differing plant) at the whorl stage of maize does not use infestations as much as in susceptibility to ECB. development. Leaf feeding ratings were obtained at tassel elongation and the past. Certainly, ECB tolerance is observed and major companies have Genotype group (inbred, synthetic, tunneling measurements at grain entomological input to complement hybrid) were the main plot units, and harvest. development of stress tolerant inbred genotypes were randomized within lines. Selection of tolerance at all stages blocks of the four replicate split plot Both 1990 and 1991 were above average of inbreeding is routinely practiced, design. Rows were 8m long and 0.9m heat unit accumulation years. The and new line development evolves wide, with approximately 50 plants per maturity attained at Prescott, located largely from elite commercial hybrids. plot (55,000/ha). Each experimental site on the St. Lawrence river, showed the Final evaluation of potential was surrounded by four border rows of more favorable environment vis a vis commercial hybrids occurs across a susceptible commercial hybrid. Data the maturity attained at Ottawa (90 km many environments and the high on plant damage was obtained at grain north) as measured by grain moisture natural population of ECB/stress harvest (i.e. late October). In each row, (Tables 1 and 2). A larger population of provides a good measure of hybrid the four end plants were discarded and corn borer was observed in the rural tolerance. In Canada, a new hybrid every third plant dissected. Prescott region, largely attributable to the cultural practices of minimum requires licensing through a provincial committee from data where the hybrid ECB egg masses were produced at the tillage and leaving abundant crop is adapted. The hybrid must Ridgetown College of Agricultural residues. In contrast, the fall demonstrate superior yield/moisture Technology RM90 zone, Ridgetown, management at Ottawa, where fields plus stalk quality at harvest. The Ontario using the rearing techniques of are located in an urban environment, average commercial life of a hybrid is Guthrie (1989). Egg masses were sent to together with flailing of the stubble and less than 5 years. Host plant resistance Ottawa, incubated till the black head fall ploughing, reduced the natural requires continual research and has stage and two masses deposited on population (Fig. 1). produced and continues to produce each of two days (approx. 100 eggs/ improved yields plus satisfactory tolerance to ECB. Studies were conducted to investigate the present status of genetic tolerance to ECB. Further studies were made on Table 1. Average number of days to silking, grain moisture at harvest, and European corn borer leaf feeding, stalk damage and larval recovery for ten genotypes at Prescott (natural population) in 1990 and 1991. the biology of the insect/plant behavior to develop a technique that would Genotype allow rapid monitoring of plant tolerance. The study was conducted during the 1990 and 1991 growing seasons at two locations: Ottawa, 90RM zone, with artificial infestation; and Prescott, 95RM zone, with a natural population. Ten genotypes representing three maturity groups — early (inbreds CM7, CK44, and INRA synthetic SFP-1); medium (inbreds A619, DE811, hybrids Pickseed 4533 and Dekalb 435); and late Inbreds Early CK44 CM7 Medium A619 DE811 Late B73 CI31A Hybrids 4533 DK435 Synthetics SFP-1 BS9C0 (inbreds B73, CI31A and synthetic BS9 1 C0) — were selected for a wide genetic 2 3 4 RM1 Tunnel2 (cm) Number Number of of Leaf tunnels2 larvae2 feeding3 Number of days Grain silking moisture4 60 65 863 1218 133 184 71 108 1.7 1.7 71 65 7.8% 11.8% 95 100 1087 180 157 37 75 20 1.4 1.3 78 76 15.8% 28.0% 103 110 1165 500 177 90 113 63 2.3 1.0 82 89 21.1% 32.1% 90 95 858 286 94 40 44 16 2.0 1.1 70 71 11.3% 12.1% 65 105 642 338 86 57 26 31 1.3 1.2 67 80 9.6% 17.7% Relative maturity. Total for 40 plants. Average leaf feeding of 160 plants using Guthrie et al. (1960) rating. Average for 40 plants. VERIFICATION AND PRE-COMMERCIAL TESTING OF EUROPEAN CORN BORER AND GIBBERELLA EAR ROT RESISTANT VARIETIES Table 2. Average number of days to silking, grain moisture at harvest, and European corn borer leaf feeding, stalk damage and larval recovery for ten genotypes at Ottawa (artificial infestation) in 1990 and 1991. 2 Genotype Inbreds Early CK44 CM7 Medium A619 DE811 Late B73 CI31A Hybrids 4533 DK435 Synthetics SFP-1 BS9C0 2 3 RM1 60 65 1153 1373 179 183 106 68 1.1 1.2 63 63 11.7% 17.0% 95 100 500 224 85 38 52 10 1.1 1.3 85 91 38.8% 45.9% 103 110 1011 785 161 135 95 89 2.2 1.0 85 97 36.6% 64.1% 90 95 1140 844 134 103 37 23 1.5 1.2 72 74 18.4% 16.2% 65 105 894 434 120 75 0.4 0.3 0.2 0.1 0 Before Harvest After Flailing 1.6 1.3 69 92 15.6% 35.7% Percentage 90 70 50 40 30 20 10 0 60 50 1992 1993 1994 40 30 20 10 ; ; ; ; ; ; ; ;; ; 1988 1989 1990 1991 1988 1989 1990 1991 80 ; ; ; ;; ; ; ; ; ; ;; ; ; ;; ; ; ; ; ;; ; ; ;; ;; ; ; ; ;; ;; ; ; ;; ;; ; ;; ; ;; ; ; ;; 60 0 ;;; ; ;; ; 0-30 31-60 Height (cm) 61-122 Figure 3. European corn borer larval recovery below 30 cm, between 30-60 cm and below the ear, from 1988 to 1994. 1992 1993 1994 observed in the large nursery across many genotypes in this environment. There was a wide range in maturity as shown in Tables 1 and 2. Grain moisture ranged form 7.8 to 32.1% at Prescott compared with 11.7 to 64.1% in Ottawa. There was no significant difference in damage within locations 31-60 Height (cm) 61-122 Figure 2. Percentage of European corn borer tunnel length below 30 cm, between 31-60 cm, and below the ear, from 1988 and 1994. and CK44, used widely in the shortest season areas of Canada and Europe, are very susceptible. Similarly, B73, A619, Pickseed 4533 and the synthetic SFP-1 were also considered susceptible. However, in contrast to the early cultivars, medium and late inbreds DE811, CI31A, together with the resistant hybrid DK435 and SYN. BS9C0, showed good levels of tolerance. A rapid screening technique Since 1988, the vertical distribution of ECB within a plant has been monitored in several fields with corn hybrids of 70-90 RM maturity. Plants were dissected longitudinally at harvest (late October) and the presence/location of larvae and tunnel length was recorded. There were two important implications: • Observations of tunnel length would be of most interest and most Leaf feeding cost effective in the lower 30cm of There was no significant difference in the stalk since this is where more leaf feeding within locations and than 60% of tunnel damage occurs between years. All genotypes showed (Fig.2). minimal leaf feeding damage. First 0-30 Nevertheless, little damage has been ;;; ;;; ; ; ;;;;;; ;; ;; ;;; 22 14 ;; ;; ;; ;; ;; ;; ;; ; ;; ; ;; ;; ; ;; ; ;; ; ; ; ; ;; ; ;;;; ;; ;; ; ; ;;;;;; ;;; ; ; ;; ; ;; ; ;; ; ;; ;; ; ; ;; ;; ; ; ; ;; ;; ; ;;; ;;; ; ; Figure 1. Average European corn borer larval population before harvest (0-30 cm) and after flailing the field (0-7.5 cm) at grain harvest from 1988-1994 (except 1993). 70 1992 (Bergvinson et al. 1994). between years. The early cultivars CM7 1988 1989 1990 1991 1992 1994 Year 80 artificial infestation during 1991 and Genotype-ECB damage Relative maturity. Total for 40 plants. Average leaf feeding of 160 plants using Guthrie et al. (1960) rating. Average for 40 plants. Mean larva per plant 4 Number of days Grain silking moisture4 Tunnel (cm) Percentage 1 Number Number of of Leaf tunnels2 larvae2 feeding3 257 • Fall management of stubble to generation resistance appeared to be control ECB populations must satisfactory for this wide array of include management of the lower inbreds, hybrids and synthetics. stalk (Fig. 3). However, these data contrast with studies at Ottawa which demonstrated a range in leaf feeding response under 258 R.I. HAMILTON, L.M. REID, AND F. MELOCHE Ear Rots infection even in lines with high kernel of inoculum (spore suspension) are resistance. injected into the silk channel of each primary ear using a self-refilling cattle Fusarium graminearum Schwabe, the asexual state of Gibberella zeae (Schw.) vaccinator attached to a 2 L backpack pathogen of corn in Canada, the US, Screening for silk resistance usually involves one of three techniques: Europe, and other countries (Sutton • Insertion of a colonized substrate (Fig. 5) so that inoculum is not forced Petch, is an important ear-rotting 1982). Infected host debris is believed to be the major source of inoculum, with inoculum being dispersed via wind, rain, insects and birds. Spore entry into corn ears can occur through wounds (e.g. insects or birds) or by (Fig. 4). Care must be taken to ensure that the needle is held horizontally (e.g. toothpick or kernel) into the down the silk channel onto the kernels. silk channel. Higher volumes of inoculum • Spraying a spore suspension on the significantly increase the amount of infection in more susceptible hybrids exposed silks. • Injection of a spore suspension into (Reid et al. 1994). A single individual can inoculate an average of 400-500 the silk channel. growth of mycelium down the silks to the kernels and cob from spores Screening for kernel or wound germinating on the silks (Hesseltine resistance usually involves wounding and Bothast 1977; Koehler 1942; Sutton through the husk, kernels, and cob 1982). Mycelial growth on the kernels followed by insertion of a colonized has a characteristic pinkish colour and substrate (toothpick) or spores cobs become soft and spongy with rot. (saturated pipecleaner) into the wound. More recently, methods are being Although F. graminearum ear rot occurs developed to avoid wounding the cob sporadically, it can represent a serious by just puncturing the husk and problem due to mycotoxins with are kernels followed by application of a produced by this pathogen (Vesonder spore suspension. et al. 1981). This is of considerable concern to livestock producers. Swine Screening techniques are the most sensitive to F. graminearum We have developed a technique to mycotoxins. Two major mycotoxins are screen for infection via the silk. This produced by this pathogen: technique involves the injection of a zearalenone and deoxynivalenol (DON, spore suspension of F. graminearum into vomitoxin). the silk channel, inside the husk and above the cob. A concentration of 5 x The most satisfactory solution to 105 spores/ml has been control the disease is the development found to give maximum of resistant corn hybrids. Due to the differentiation between sporadic nature of the pathogen, genotypes (Reid et al. artificial inoculation must be used to 1994). Higher screen germplasm for resistance. concentrations Inoculation techniques are needed to significantly increase the test for resistance to both modes of amount of infection in fungal entry, i.e. growth down the silks more susceptible vs. kernel wounding. We have found hybrids. Although no inbreds and hybrids with resistance to significant isolate effects one, but not both modes of entry. have been found with Kernel resistance alone is not sufficient the use of this technique since earlier infections through the silk, (Reid et al. 1993), a when kernels are not yet fully mixture of two to three developed, can result in extensive isolates is used. Two ml Figure 4. A self-refilling cattle vaccinator attached to a 2 L backpack is used to inoculate corn ears with 2 ml of F. graminearum spore suspension. Figure 5. Injection of spore suspension into silk channel. Needle must be at right angles to ensure proper placement of inoculum. VERIFICATION AND PRE-COMMERCIAL TESTING OF EUROPEAN CORN BORER AND GIBBERELLA EAR ROT RESISTANT VARIETIES 259 ears per hour. Inoculations must be mm high). These nails have been sucrose or 1.0 g of dextrose. The made 2-6 days post-silk emergence. driven into a 50 cm long wooden medium is dispensed in 150 ml aliquots Insufficient infection is obtained when handle fabricated from a broomstick. into 500 ml erlenmeyer flasks, inoculations are made later and Prior to wounding, the nails are dipped autoclaved, then a 1 cm square piece of incorrect assessments or no in a spore suspension. Inoculations are PDA with mycelium and spores is differentiation occurs (Reid et al. 1993). made 10-15 days post-silk emergence. added. Cultures are shaken for 1 hr at 4 A humid environment should be We are currently investigating some of hr intervals under natural light maintained using irrigation, 2-5 mm the parameters involved in this supplemented with cool white daily, for the four-week period after technique such as: time of inoculation, fluorescent lights. Spore concentrations inoculation. This technique has been spore concentration, and position of can reach 2 x 106 spores/ml in one used since 1987. It has allowed for good wound. week depending on isolate. Prepared differentiation between inbreds and inoculum can be stored at 2-4∞ C for a hybrids, ranging from very susceptible For both techniques, a modified Bilay’s maximum of four weeks. Prior to to highly resistant. liquid medium is used to produce inoculation, the mixture is diluted and inoculum: 2.0 g potassium dihydrogen filtered through two layers of cheese We have also been developing a phosphate; 2.0 g potassium nitrate; 1.0 cloth. technique to screen for kernel g potassium chloride; 1.0 g magnesium resistance, which involves wounding of sulphate; 0.0002 g/L each of the minor A minimum of four replicates should the husk and kernels with four small (3 elements: ferric sulphate, manganese be used for each genotype mm dia.) nails spaced in the four sulphate, and zinc sulphate; 1.0 L (approximately 40 treated plants). Each corners of a rectangle (7 mm long, 5 distilled water; 2.0 g soluble starch or genotype can be planted in single row plots of 12-14 plants each, of A RATING B 0% 1 RATING 0% center 10 plants are inoculated. 1 1-3% 2 Ears are harvested at normal grain harvesting moisture in mid- 1-3% late October. Visual rating scales 2 4-10% which the primary ears of the (Fig. 6) have been developed for both techniques and correlated 4-10% with actual numbers of infected 3 3 11-25% Gridled 11-25% 4 Not gridled kernels. The number of infected kernels have been correlated with toxin (DON) level in the grain. 4 Randomized complete block 26-50% 5 26-50% are analyzed and presented as a 5 51-75% designs are usually used and data range in resistance. Relatively 51-75% good reproduction of infection ratings has been obtained across 6 6 76-100% 7 years. Check hybrids for different 76-100% 7 levels of resistance have been identified and correlate well with natural infection from field observations. Figure 6. Disease severity rating scale for (A) silk channel inoculations and (B) kernel wound inoculations with F. graminearum. 260 R.I. HAMILTON, L.M. REID, AND F. MELOCHE Both of the techniques described above Re fe re nce s have been standardized and are suitable for routine use in breeding programs. A wide range in resistance ratings can be obtained, so that genotypic differences are easily observed (Fig. 7). We are currently testing the use of these techniques with other Fusarium species such as F. moniliforme, F. subglutinans, and F. Bergvinson, D.J., J.T. Arnason, R.I. Hamilton, J.A. Mihm, and D.C. Jewell. 1994. Determining leaf toughness and its role in maize resistance to the European corn borer (Lepidoptera: Pyralidae). J. Econom. Entomol. 87(6): 1743-1748. Caffrey, D.J., and L.H. Worthley. 1927. A progress report on the investigations of the European corn borer. U.S.D.A. Bull. 1476. culmorum. 47 44 73 78 109 71 79 82 103 72 76 46 106 107 101 108 104 102 69 81 105 74 55 110 50 100 83 77 84 23 91 97 42 25 61 32 30 88 86 56 43 85 57 51 1 58 10 52 70 96 22 18 62 41 12 89 48 95 17 87 14 57 27 75 39 64 35 21 93 26 92 13 28 49 90 60 2 24 45 29 94 5 36 19 7 3 59 67 54 8 63 33 40 66 9 65 4 34 1 1.5 2 2.5 3 3.5 4 4.5 5 Figure 7. The range in resistance ratings for 98 Ontario hybrids inoculated with F. graminearum by silk channel injection (hybrids are coded to protect company confidentiality). Means followed by the same vertical bar are not significantly different at the 0.05 probability level by the Duncan’s multiple range test. Canada Department of Agriculture, Report to the Minister of Agriculture for the Dominion of Canada for the year ending March 31, 1923; 1924; 1925; 1926; 1927. Dicke, F.F., and W.D. Guthrie. 1988. The most important corn insects. In G.F. Sprague, and J.W. Dudley (Eds.) Corn and Corn Improvement, third edition, 767863. Amer. Soc. Agron. Guthrie, W.D. 1989. Advances in rearing the European corn borer on a meridic diet. In Toward Insect Resistant Maize for the Third World: Proceedings of the International Symposium on Methodologies for Developing Host Plant Resistance to Maize Insects, 46-59. Mexico, D.F.: CIMMYT. Guthrie, W.D., F.F. Dicke, and C.R. Neiswander. 1960. Leaf and sheath feeding resistance to the European corn borer in eight inbred lines of dent corn. Ohio Agric. Stn. Res. Bull. 860. Hesseltine, C.W., and R.J. Bothast. 1977. Mold development in ears of corn from tasseling to harvest. Mycologia 69: 328340. Hudon, M., E.J. LeRoux, and D.G. Harcourt. 1989. Seventy years of European corn borer (Ostrinia nubilalis) research in North America. Agric. Zool. Rev. 3: 53-96. Koehler, B. 1942. Natural mode of entrance of fungi into corn ears and some symptoms that indicate infection. J. Agric. Res. 64: 421-442. Reid, L.M., A.T. Bolton, R.I. Hamilton, T. Woldemariam, and D.E. Mather. 1992. Effect of silk age on resistance of maize to Fusarium graminearum. Can. J. Plant Pathol. 14: 293-298. Reid, L.M., D. Spaner, D.E. Mather, A.T. Bolton, and R.I. Hamilton. 1993. Resistance of maize hybrids and inbreds following silk inoculation with three isolates of Fusarium graminearum. Plant Dis. 77: 1248-1251. Reid, L.M., R.I. Hamilton, and D.E. Mather. 1994. Effect of macroconidial suspension volume and concentration on expression of resistance to Fusarium graminearum in maize. Plant Dis. 79(5): 461-466. Sutton, J.C. 1982. Epidemiology of wheat head blight and maize ear rot caused by Fusarium graminearum. Can. J. Plant Pathol. 4: 195-209. Vesonder, R.F., J.J. Ellis, and W.K. Rohwedder. 1981. Elaboration of vomitoxin and zearalenone by Fusarium isolates and the biological activity of Fusarium-produced toxins. Appl. Environ. Microbiol. 42: 1132-1134. Introducing Unadapted, Insect–Resistant M aize Germplasm in Three–Way Hybrid Combinations for Resistance to the M aize Stalk Borer, Busseola fusca (Fuller) (Lepidoptera: Noctuidae) J.B.J. van Rensburg, Summer Grain Center, Potchefstroom 2520, South Africa Abst r a c t The potential value of various levels of resistance to the maize stalk borer was evaluated by crossing three unadapted, resistant inbreds and three local elite inbreds in various combinations. The unadapted, resistant germplasm could be employed directly to introduce resistance, provided that undesirable traits inherent to the unadapted parents were sufficiently diminished by the genetic contribution of the adapted germplasm. The use of a single resistant parent in a three–way hybrid to increase the resistance level to 25% was sufficient to eliminate the need for chemical control at moderate levels of infestation. The use of two resistant parents to obtain a level of 50% resistance in the resultant three–way cross posed an unacceptable risk, due to an increased incidence of ear rot and lodging. resistance, the use of resistant exotic genetic diversity of adapted maize germplasm to introgress resistance into populations from which improved Breeding for resistance to the African locally adapted materials is necessarily inbreds were to be extracted (Albrecht corn borer, Busseola fusca (Fuller), at the time consuming. Since useful genes and Dudley 1987; Crossa and Gardner Grain Crops Institute was prompted found at low frequencies in the exotic 1987; Michelini and Hallauer 1993). The when high levels of resistance to this source and absent in the adapted direct use of unadapted inbred lines as species were observed in the source are more likely to be lost when parental sources in two–way hybrids Mississippi inbreds Mp705, Mp706 and selecting in the backcross than in the was however not contemplated, since a Mp707 (Van Rensburg and Malan cross (Crossa and Gardner 1987), delicate genetic balance for adaptability 1990). New sources of resistance have several cycles of recurrent selection are may easily be destroyed by genetic since been obtained in breeding required before backcrossing can be recombination in a two–parent cross material developed by CIMMYT, of attempted. Furthermore, at least one between an adapted, insect–susceptible which CML139 (yellow kernel type) backcross to the adapted parent would genotype and a non–adapted, insect– and CML123 (white) proved to be be required to ensure adaptation, resistant genotype. But the introduction particularly promising (Van Rensburg adding to the time required to develop of unadapted breeding material can and Van den Berg 1995). a resistant adapted population also be accomplished by employing (Albrecht and Dudley 1987). three–way and four–way crosses Int roduct ion (Gallun 1980). In this way a single Antibiosis observed in the Mp–inbreds was shown to be 35% heritable. The The question has arisen as to whether resistant parent may serve to improve gene action was largely additive, while unadapted, resistant inbred lines can be the resistance level in hybrids, whereas the dominance and epistasis utilized directly in hybrid undesirable traits may be diminished components of genetic variation were development. Previous research of this by the contribution of the adapted found to be negligible (Van Rensburg nature dealt with methods and the parents. The viability of such a strategy and Gevers 1993). As a result of the possible consequences when exotic to develop improved maize hybrids quantitative nature of the inheritance of germplasm is used to increase the resistant to B. fusca was therefore 262 J.B.J. VAN RENSBURG investigated, since local maize hybrids, rows of 10 m, with a row width of 1.5 In trial 2 only the four crosses (SS)S, until recently, were predominantly m to avoid larval migration between (SS)R, S(RR) and (RR)R were evaluated. four–way crosses. Emphasis is now rows. The trial was planted by hand The general trial procedure was similar being placed on the development of using two seeds per hill and thinned to trial 1, but plot size was increased to three–way and modified single crosses, one week after plant emergence to a six rows of 10 m per genotype. These all of which involve more than two uniform stand of 28 plants per 10 m. served as sub–treatments in which inbred parents. All plants in one row of each plot were different levels of artificial infestation artificially infested four weeks after were applied five weeks after plant The objective of the present emergence with 10 neonate larvae per emergence, namely 0, 3, 4, 6, 7 and 10 investigation was to assess the levels of plant, using techniques described for B. plants infested per 10 m. The same resistance obtained when utilizing one, fusca (Van Rensburg and Van Rensburg variables as in trial 1 were assessed at two and three resistant inbreds in 1993). Grain yield, number of damaged harvest. Yield data (square root three–parent crosses, at the same time internodes in 20 stalks per row, transformed) and percentage damaged evaluating the direct use of exotic percentage damaged ears, percentage ears were regressed on levels of germplasm for other characteristics. It lodging and percentage rotted ears infestation as the independent variable, was deemed that the improvement of (Stenocarpella (Diplodia) maydis) were using the model Y = aXb. A non–linear resistance in a hybrid combination to a determined at harvest. Yield data model Y = a + b * Hyptan (x – x) was level that would warrant an increase in (converted to t/ha), percentage lodging applied to the number of damaged the economic threshold for chemical and percentage diseased ears were internodes per 20 plants. Data on control would be of considerable subjected to factorial analyses, using lodged plants and rotted ears were significance in practice, as opposed to genotypes as factor 1 and infestation subjected to analyses of variance. striving for ultimate resistance levels. (infested vs uninfested) as factor 2. M aterial and M ethods Since no plant damage was recorded in Another experimental hybrid was the uninfested rows, data on ear and developed for evaluation at the internode damage were subjected to commercial level under conditions of Two susceptible elite inbreds (S) and analyses of variance aimed at genotype natural infestation. The single cross two resistant exotic inbreds (R) were differences only. All percentage values P150 x Mp706, (SR) which previously crossed to obtain four single crosses SS, were arcsin transformed before proved to be drought tolerant (Van SR, RS and RR. These served as parents analyses. Rensburg and Gevers 1993), was in crosses with two other inbred lines (one susceptible, one resistant) to obtain six three–way crosses ranging in susceptibility from SSS to RRR. The relative level of resistance of the Table 1. Experimental hybrid combinations derived from crosses between adapted, insect–susceptible (S) and unadapted, insect–resistant (R) inbred lines. combination RRR was assumed to be 100% and that of SSS to be nil. The hybrid combinations and their assumed resistance levels are provided in Table 1. These were evaluated in two field trials during 1993/94, conducted in the same field at Potchefstroom (26∞43’S, 27∞06’E), with a planting date of mid–November to avoid natural infestation. In trial 1 the single and three–way crosses were evaluated in a randomized block design with six replications. The plot size was two Genotype Assumed Resistance resistance designation level (%) (F2834t x M37W) x KO315Y (F2834t x M37W) x Mp706 M37W x (Mp706 x F2834t) M37W x (Mp706 x Mp707) (Mp706 x Mp707) x M37W (Mp706 x Mp707) x CML139 M37W x F2834t M37W x Mp706 Mp706 x M37W Mp706 x Mp707 (SS)S 0 (SS)R 50 S(RS) 25 S(RR) 50 (RR)S 50 (RR)R 100 SS SR RS RR 0 50 50 100 crossed to the locally prominent inbred line I137TN (S) as a pollen parent. The three–way cross was tested during the 1993-94 season in commercial plantings at two sites in the Northwest Province, Rysmierbult (26º21’S, 27º08’E) and Ottosdal (26º52’S, 25º47’E). The seed was planted mechanically in 20 alternate rows with a different commercial hybrid as the standard treatment at each site. The row width was 1.5 and 2.2 m respectively, and within–row plant spacing equivalent to 20,000 and 18,000 plants per ha, in accordance with local practice. A late– November planting date resulted in both trials being subjected to natural infestation. No chemical control or irrigation was provided. Yield, damaged ears, damaged internodes INTRODUCING UNADAPTED, INSECT–RESISTANT MAIZE GERMPLASM IN THREE–WAY HYBRID COMBINATIONS FOR RESISTANCE TO THE MAIZE STALK BORER 263 and rotted ears were determined at close genetic relationship between the contributed 75% in a three–way cross harvest. Plots of 20 adjacent plants inbreds Mp706 and Mp707. These S(RS). It is noteworthy that neither the were randomly taken from each row of differences are reflected in yield incidence of ear rot nor lodging was the experimental hybrid, as well as potential as indicated by the yields of affected significantly by stalk borer from the commercial standard in 20 of the uninfected sub–plots. infestation, indicating both traits to be genetically inherent to the unadapted, the adjacent rows. Mean values for resistant inbreds. each variable were calculated over the Susceptibility to both ear rot and 20 replicates per genotype and lodging in the unadapted, resistant compared by means of confidence parental lines is indicated by the results The resistance assessment of selected intervals. presented in Table 3. Lodging was three–way crosses at various levels of largely diminished by the use of a infestation (trial 2) is provided in single adapted parent in any hybrid Figure 1. Regression analyses provided Results and Discussion combination, but susceptibility to ear a significant fit for all hybrid Resistance assessments on single and rot seemed to be reduced significantly combinations with regard to yield (R2 three–way crosses (trial 1) are only when two adapted parents values from 70.9 to 96.4) and stalk presented in Table 2. Yield responses to infestation were closely correlated with both the incidence of damaged ears (r = Table 2. Evaluation of experimental single and three–way crosses for stalk borer resistance (Trial 1). 0.83) and damaged internodes (r = 0.82). Yield losses due to infestation of all plants ranged from more than one t/ha in the susceptible three–way cross (SS)S to virtually no loss in the fully resistant three–way cross (RR)R. With the exception of the combination (SS)R, the use of one and two resistant parents in a three–way cross reduced yield losses in accordance with the assumed resistance level of the hybrid (approximately 25% reduction in loss for each resistant parent included). The use of one resistant inbred as pollen Yield (t/ha) Resistance Resistance designation level (%) Infested Uninfested (SS)S (SS)R S(RS) S(RR) (RR)S (RR)R SS SR RS RR Mean 0 50 25 50 50 100 0 50 50 100 5.622 5.288 6.728 5.883 6.214 6.270 5.893 6.133 6.448 6.116 1.246 6.111 6.387 6.910 6.699 6.750 6.842 5.895 6.850 6.802 6.507 1.465 0.489 Yield loss (t/ha) % Damaged Damaged internodes ears /20 plants 1.099 0.182 0.816 0.536 0.572 0.002 0.717 0.354 0.391 0.219 5.5 12.5 2.8 9.1 6.5 4.8 1.0 14.2 8.2 7.8 0.4 17.6 36.7 13.8 23.3 10.5 11.6 6.7 32.6 18.4 18.8 3.7 Significance for yield: Genotypes F = 119.3, P<0.001; Infestation F = 27.3, P<0.001; Interaction F = 1.2, P = 0.283. Damaged ears F = 14.09, P<0.001, Damaged internodes F = 11.86, P<0.001. parent in the second cycle of producing a three–way cross (SS)R was more beneficial in enhancing resistance than when used as one parent in the Table 3. Evaluation of experimental single and three–way crosses for ear rot (Stenocarpella maydis) susceptibility and lodging. (Trial 1). preceding single cross S(RS). Resistance designation Compared with those for three–way (SS)S (SS)R S(RS) S(RR) (RR)S (RR)R SS SR RS RR crosses, yield losses for the single crosses were less severe in the susceptible hybrid (SS) but more pronounced in the fully resistant hybrid (RR). This may be attributed to differences in crop vigor, since the single cross SS was more vigorous than the three–way counterpart (SS)S, whereas hybrid vigor was largely absent in the single cross RR due to the Significance Genotypes Infestation Interaction % Diseased ears Infested % Lodging Infested Uninfested 0.3 3.9 0.1 4.3 4.1 5.5 0.2 5.1 3.7 7.7 Uninfested 1.3 1.9 0.2 3.4 4.3 3.0 1.2 5.0 2.6 6.8 1.0 3.0 2.3 6.5 4.3 32.1 4.3 4.3 5.6 26.7 3.7 2.3 0.9 9.1 3.0 36.6 3.2 1.7 2.5 23.4 F P F P 11.08 0.12 0.92 <0.001 0.728 0.515 17.5 0.34 0.53 <0.001 0.564 0.835 264 J.B.J. VAN RENSBURG damage (R2 = 96.7 to 99.6). A significant combinations. The same result is also The results obtained with an fit for ear damage was only obtained observed in the incidence of damaged experimental three–way cross under (R2 (R2 internodes (Fig. 1B) and damaged ears commercial conditions are provided in 97.7). All four hybrids displayed an (Fig. 1C). It is important to note stem Table 4. These results indicate the initial reduction in yield at only three damage by B. fusca. The accepted possible value that a level of only 25% infested plants/10 m, after which losses economic injury level of 10% infested resistance in a hybrid combination may were less pronounced in all the hybrid plants (Van Rensburg et al. 1988) have under practical conditions. At combinations containing at least one equates to three infested plants/10 m Rysmierbult the experimental hybrid resistant parent in the genetic in this study. At this level an average of suffered significantly less injury from composition (Fig. 1A). Based on the less than one internode per plant was stalk borer infestation than the amount of yield reduction at increasing damaged, yet notable yield losses were commercial standard, resulting in a levels of infestation, the fully resistant observed in all hybrid combinations. significant difference in yield of for S(RR) = 51.1) and (SS)S = approximately 200 kg/ha. This yield hybrid (RR)R suffered notably less, and the susceptible hybrid (SS)S more yield The estimated yield losses derived by difference is one which would loss than the other two hybrid equation at the economic injury level normally justify the expense of were 5.8% (SS)S, 2.7% (SS)R, 4.0% S(RR) chemical control of stalk borer. At and 0.9% (RR)R. At a level of 35% Ottosdal the incidence of stalk and ear infestation (10 infested plants/10 m) damage was significantly greater in the the estimated yield losses were 11.5% commercial standard than in the (SS)S, 5.6% (SS)R, 8.3% S(RR) and 1.8% experimental hybrid, although yields (RR)R. In spite of both (SS)R and S(RR) did not differ significantly. The being 50% resistant, a greater level of incidence of ear rot in the two hybrids resistance was achieved with the use of was similar at Ottosdal, but a single resistant parent than with two significantly greater in the resistant parents in a three–way cross, experimental hybrid at Rysmierbult. At illustrating the importance of the both localities the level of infestation choice of parents in employing exotic, was moderate, whereas mid–summer non–adapted germplasm in a hybrid drought conditions which often occur combination (Gallun 1980). throughout the western production 20 A % Damaged ears 19.5 RRR SRR SSR SSS 19 18.5 18 17.5 Damaged internodes/20 plants 17 16.5 40 RRR SRR SSR SSS 30 area were not experienced. Further 20 10 0 40 Yield (SQRT G/plant) B C RRR SRR SSR SSS The incidence of ear rot in trial 2 was testing under more typical conditions 6.9% (RR)R, 4.5% S(RR), 2.9% (SS)R and to assess agronomic acceptability of the 1.6% (SS)S. Lodging amounted to 41.4% experimental hybrid is therefore (RR)R, 7.4% S(RR), 4.5% (SS)R and 4.3% suggested. This also needs to be done (SS)S, confirming the susceptibility in in the absence of stalk borer infestation insect resistant germplasm observed in in order to ascertain comparative yield trial 1. potential. 30 Table 4. Evaluation of an experimental three–way cross under commercial conditions at two localities. (Mean values followed by standard errors, n = 20 for all variables assessed). 20 10 0 0 1 2 3 4 5 6 7 8 9 10 Infested plants/10m Figure 1. Yield responses and plant damage observed at increasing levels of infestation in three–way crosses with various levels of resistance. Locality Hybrid Damaged internodes /plant Rysmier bult Ottosdal Experimental PAN 3614 Experimental A 1650 0.84"0.17a 2.10"0.30b 0.72"0.13a 2.08"0.23b Damaged ears(%) Diseased ears(%) Yield (g/plant) 4.5"0.7a 11.2"1.7b 12.3"1.3a 20.6"1.3b 17.0"2.4a 10.2"1.1b 3.6"0.6a 3.5"0.5a 207.7"7.1a 182.8"5.5b 191.9"5.2a 192.3"5.9a Means within columns for each locality followed by different letters differed significantly at P=0.05 according to confidence intervals. INTRODUCING UNADAPTED, INSECT–RESISTANT MAIZE GERMPLASM IN THREE–WAY HYBRID COMBINATIONS FOR RESISTANCE TO THE MAIZE STALK BORER It can be concluded that unadapted From the agronomic viewpoint ear germplasm may potentially be prolificacy is a prerequisite of local employed directly in three–way crosses maize hybrids. In this study the mean in order to introduce resistance to B. ear numbers per plant were recorded fusca. This seems to be possible by as 1.96 (RR)R, 1.81 S(RR), 1.76 (SS)R using one resistant parent in the first and 1.9 (SS)S, indicating prolificacy to cycle two–parent cross (RS). The be a positive trait of the unadapted expected increase in the resistance level germplasm used. Future evaluation is of 25% of the resultant three–way cross required, therefore, for other (RS)S might be sufficient to eliminate characteristics of local importance such the need for chemical control at as drought tolerance and kernel moderate levels of infestation. This hardness. could be of considerable practical value, especially during years with Ac know le dgm e nt s reduced stalk borer populations. Since the seasonal abundance of stalk borers Mr. J. Klopper, Summer Grain Center, is linked to the rainfall cycle (Van provided technical assistance. Mr. P.J. Rensburg et al. 1987), comparatively van Rooyen gave invaluable assistance low levels of infestation often occur with statistical analyses. Dr W Wenzel over several years in a major part of the assisted with the preparation of the maize production area, resulting in manuscript. significant yield losses but which cannot economically justify control by Re fe re nce s means of insecticides. The use of either one or two unadapted parents to obtain 50% resistance in a three–way cross seems risky. In this study susceptibility to ear rot emerged at a low disease potential, suggesting an unacceptable risk of ear rot at higher disease potentials. This will also apply to other locally prominent diseases of which maize streak virus poses a particular hazard. Albrecht, B., and J.W. Dudley. 1987. Evaluation of four maize populations containing different proportions of exotic germplasm. Crop Science 27: 480– 486. Crossa, J., and C.O. Gardner. 1987. Introgression of an exotic germplasm for improving an adapted maize population. Crop Science 27: 187–190. Gallun, R.L. 1980. Breeding for resistance to insects in wheat. In M.K. Harris (ed.), Biology and breeding for resistance to arthropods and pathogens in agricultural plants: Proceedings of an International Short Course in Host Plant Resistance, 245–262. Texas: Texas A & M University, College Station. 265 Michelini, L.A., and A.R. Hallauer. 1993. Evaluation of exotic and adapted maize (Zea mays L.) germplasm crosses. Maydica 38: 275–282. Van Rensburg, J.B.J., and H.O. Gevers. 1993. Inheritance of antibiosis to the maize stalk borer, Busseola fusca (Fuller) (Lepidoptera : Noctuidae) and the combining ability for yield in resistant maize genotypes. South African Journal of Plant and Soil 10(1): 35–40. Van Rensburg, J.B.J., and C. Malan. 1990. Resistance of maize genotypes to the maize stalk borer, Busseola fusca (Fuller) (Lepidoptera : Noctuidae). Journal of the Entomological Society of Southern Africa 53(1): 49–55. Van Rensburg, J.B.J., and J. van den Berg. 1995. New sources of resistance to the stalkborers Busseola fusca (Fuller) and Chilo partellus Swinhoe in maize. South African Journal of Plant and Soil 12(2): 9193. Van Rensburg, J.B.J., and G.D.J. van Rensburg. 1993. Laboratory production of Busseola fusca (Fuller) (Lepidoptera : Noctuidae) and techniques for the detection of resistance in maize plants. African Entomology 1(1): 25–28. Van Rensburg, J.B.J., G.D.J. van Rensburg, J.H. Giliomee, and M.C. Walters. 1987. The influence of rainfall on the seasonal abundance and flight activity of the maize stalk borer, Busseola fusca in South Africa. South African Journal of Plant and Soil (4)4: 183–188. Van Rensburg, J.B.J., M.C. Walters, and J.H. Giliomee. 1988. Response of maize to levels and times of infestation by Busseola fusca (Fuller) (Lepidoptera : Noctuidae). Journal of the Entomological Society of Southern Africa 51(2): 283–291. European Corn Borer Resistance: Evaluation of Commercial M aize Hybrids and Transgenic M aize Cultivars B.D. Barry and L.L. Darrah, University of Missouri, Columbia, MO, USA Abst r a c t Annual economic losses to producers because of European corn borer (ECB), Ostrinia nubilalis (Hübner), damage to maize, Zea mays (L.), amount to several million dollars. This would be even greater if not for longterm host-plant resistance plant breeding programs in public and private organizations. To determine the degree of ECB resistance in commercial maize hybrids and the efficacy of transgenic plants to control ECB, experiments were conducted by manually infesting the plants in the research plots with neonate ECB larvae. Over a four-year period, 400 maize hybrids were evaluated. About 90% of the hybrids had some resistance to whorl-leaf feeding (first-generation ECB) and 75% had some resistance to sheath and sheath-collar feeding (second-generation ECB). In approximately two-thirds of these 400 hybrids, ECB resistance could be enhanced. Maize plants genetically transformed by using a gene(s) from Bacillus thuringiensis are effective in controlling the ECB throughout the life of the plant. As transgenic cultivars are developed and released, it will be necessary to have comparative, unbiased evaluations of performance from public institutions. If the contribution of host-plant Patch 1929, 1937, 1947; Patch et al. 1938, Maize plant resistance to European corn resistance to crop production is 1941). Many biological and ecological borer embodies two distinct traits. One recognized by producers and facts (Showers et al. 1989) were proven is resistance to whorl leaf feeding and consumers, support for a practical, over the years, such as the existence of the other is resistance to sheath collar environmentally friendly means of single or multiple generation strains of feeding during flowering. These are control will be easier to obtain. The borer populations and of genetic quantitatively inherited traits, and if European corn borer (ECB), Ostrinia differences among maize cultivars in there are any common genes for nubilalis (Hübner), is a major pest of susceptibility to ECB damage. Progress resistance, they have not been maize, Zea mays (L.), throughout the in developing first-generation, ECB- identified. These traits, in the literature maize growing areas of most of North resistant inbreds was given a and in practice, are referred to as “first- America, Europe, and North Africa. In tremendous boost after artificial generation European corn borer the US Corn Belt, estimated annual rearing techniques were developed resistance,” which is associated with at losses due to ECB range from $200-500 (Beck et al. 1949; Bottger 1942; and least six genes (Scott et al. 1964, 1966) million. This loss would be much Guthrie 1965). Two other significant and “second-generation European corn greater (Fig. 1, photo by B.E. Hodgson, contributions towards selection for ECB borer resistance,” which is associated 1918) if a significant proportion of resistance were the development of a with at least seven genes (Onukogu et commercial maize hybrids did not have rating scale (Guthrie et al. 1960) and a al. 1978). Generally, maize plants in some degree of resistance to ECB. manual infesting apparatus, the early development (up to 25-30 cm tall bazooka (Mihm 1983a, 1983b). For this for inbreds and 40-45 cm tall for The ECB was introduced to the USA symposium, we provide further hybrids) are naturally resistant to prior to 1917, when it was described as explanation of maize plant resistance European corn borer. A chemical, 2-4 a pest of maize (Vinal 1917). Studies of by using information of Barry and dihydroxy-7-methoxy-1,4-benzoxaxine- plant resistance to ECB began in the Darrah (1991): 3-one, commonly known as DIMBOA, USA as early as 1928 (Huber et al. 1928; EUROPEAN CORN BORER RESISTANCE: EVALUATION OF COMMERCIAL MAIZE HYBRIDS AND TRANSGENIC MAIZE CULTIVARS 267 which is in relatively high However, Barry et al. (1995) have plants may have been “escapes” or concentrations in young plants, can be released three inbreds, Mo45, Mo46, “partial escapes;” i.e., something may the primary factor responsible for and Mo47 which have resistance to have happened to the manually resistance to whorl leaf feeding. The both generations of ECB. Commercial infested insects other than the effects of whorl and flowering stages of plant maize seed producers have been any resistance factors in the hybrids. development normally coincide with the improving their hybrids by using spring emergence of adults and information from public and private The data for 1986, 1987, and 1989 show oviposition for first-generation adults, research to improve ECB resistance. In a higher degree of resistance to whorl- respectively; thus, the reasoning for the order to determine whether leaf feeding in commercial maize terms “first-generation European corn commercial maize hybrids were hybrids than for sheath and sheath- borer resistance.” After borer adults resistant to ECB, a four-year study was collar feeding. Means over years show emerge in the spring, approximately 45 organized to evaluate 100 maize 10% of the hybrids rating susceptible to d are required for the moths from the hybrids each year for four years (a total whorl-leaf feeding and 25% for sheath first generation to emerge as adults and of 400 different maize hybrids were and sheath-collar feeding. An by this time, maize plants are at the evaluated) (Barry et al. 1986, 1987, explanation for this is that these are anthesis or flowering stage of 1989). Because of drought in 1988, only two distinct traits with different genes development. This stage is favorable for whorl-leaf feeding data were taken and governing the expression of resistance. the establishment of second-generation these were not publicly reported. A In Corn Belt germplasm, genes for European corn borer. The plant provides summary of the results of these resistance are present, and a technique a favorable oviposition site, and pollen evaluations are presented in Table 1 as is available to easily screen and rate grains are in abundance in the leaf axils adapted from Barry and Darrah (1991). whorl-leaf feeding resistance for a large number of genotypes. This is in for early larval feeding as the larvae migrate from the hatching site to the Hybrids that have been classified as contrast to the few genes identified as feeding site behind the leaf sheath. susceptible in all years have a very contributing to sheath and sheath- small possibility of being misclassified collar feeding resistance and the The economic significance of European for most environments, but hybrids difficult, less precise techniques corn borer has been reduced by the classified as resistant or intermediate available for evaluating this damage. identification and development of maize could possibly be more susceptible hybrids with genes for resistance to this than indicated. This is because some The results of these evaluations, however, have shown that about insect. Sources of germplasm for whorl leaf feeding resistance have been identified within corn belt breeding material (Guthrie and Dicke 1972). Germplasm sources for sheath and sheath collar feeding resistance have been identified (Pesho et al. 1965; Guthrie et al. 1971; Onukogu et al. 1978; Russell and Guthrie 1979, 1982; Table 1. Distribution of commercial maize hybrids according to the level of resistance to whorl-leaf feeding and sheath and sheath-collar feeding (tunneling) by ECB. Adapted from Barry and Darrah (1991). Only whorl-leaf feeding ratings were obtained in 1988 because the plants were under extreme drought conditions, and those data are not included. Barry et al. 1983, 1985; Barry and Zuber 1984; Klenke et al. 1986a, b, c, 1987). Because they were not readily available ECB classification of hybrids tested Percent Percent Percent Year Resistant Intermediate Susceptible laborious than for whorl leaf feeding resistance, the development of hybrids to whorl-leaf feeding and about 75% have some resistance to sheath and sheath-collar feeding. Although not statistically comparable, similar whorl-leaf feeding data collected from 226 hybrids in Ohio in 1967 and 1968 (Barry 1969) indicate some resistance in 80% of the hybrids. For approximately two-thirds of 1986 1987 1989 Mean and susceptible hybrids could be 1986 1987 1989 Mean Sheath and sheath collar feeding 20 49 31 44 50 6 17 45 38 27 48 25 resistant to sheath and sheath collar feeding has lagged behind. production have some resistance Whorl-leaf feeding rating 25 67 8 41 58 1 26 54 20 31 60 10 in Corn Belt germplasm, and the identification process was much more 90% of the hybrids currently in the hybrids evaluated in Missouri, however, resistance levels could be further enhanced improved with the introduction of additional genes for resistance. 268 B.D. BARRY AND L.L. DARRAH It has been estimated that the annual • Pioneer Brand 3378 of the plant. The concentrations of the ECB damage to maize translates into a • Pioneer Brand 3471 δ−endotoxins in leaves, sheath, and loss of several millions of dollars. If • Taylor-Evans 7055 sheath-collar sites, where young ECB host-plant resistance selection were not • Triumph 1990 larvae begin to feed, are effective in controlling both first and second a part of commercial maize breeding programs, the loss or increased cost of Several of these varieties may share the generations of this insect. The larvae production would be much greater and same parentage as other popular usually feed no more than enough to might be sufficient to reduce maize varieties in Illinois. These hybrids may make a feeding scar (not even a hole) production in some geographical areas. not be the highest yielding varieties, so on the maize leaf or sheath. Most ECB you will have to weigh the importance larvae die within the day after After the ECB resistance for the various of borer resistance against the attempting to feed and if any do not, maize hybrids was determined (Barry importance of high yields in the they usually die shortly thereafter. and Darrah 1991), the question arose of absence of borers. If you are interested Results of field evaluations of would or how could the information in more information about resistance of transgenic Bt cultivars clearly could be used by producers. As it hybrids to borers, discuss this demonstrate the effectiveness of Bt happened, two Illinois extension information with your seed dealer. It is plants as a tool for control of ECB entomologists, Drs. M. Gray and K. important to note that the results of the (Table 2). Steffy, picked up on this and the evaluations in Missouri revealed that following has been taken from their about 90% of the hybrids currently As with any management tool, use of maize entomology recommendations: produced by the seed industry have Bt transgenic cultivars should be some resistance to whorl leaf feeding considered as part of the arsenal for We have gleaned the article (Barry and and about 75% have some resistance to controlling ECB. A significant concern Darrah 1991) and have listed the corn sheath and collar feeding. is the development of resistance, over time, of pests to the insecticidal hybrids that expressed the highest levels of resistance to both first- and Our strategies and method(s) of control properties of Bt transgenic cultivars. second-generation corn borers in their for ECB are a continuous, on-going Strategies are being developed in trials. However, because tolerant program of development in which we theory and practice to prevent or delay hybrids were not identified, some corn anticipate breeding for resistance with development of resistance in pests. hybrids that tolerate corn borer damage naturally-occurring genes to play a Included are maintaining a population and produce yields at near-normal major part. We have a new tool from of ECB with a susceptible Bt transgenic levels may not be listed. The hybrids biotechnology, however, which we can cultivar (refugia), introducing more are listed alphabetically; the order of use in pest management. It is called Bt than one Bt transgenic source of the list suggests no preferences: (Bacillus thuringiensis) transgenic maize. resistance into the maize genome, and/ or adding another effective non-Bt • Agrigene 7720 Transgenic maize plants are developed origin insecticidal protein to the • Burrus 94 by bombardment of callus tissue with genome. • Cargill 7877 microprojectiles carrying Bt. By the • CFS 7615 genetic process of transformation, Bacillus thuringiensis is a naturally • Crow’s 688 insecticidal crystal proteins (δ− occurring organism which is not • DeKalb Genetics 711 endotoxins) are then able to be harmful to higher animals. It has been • Funk’s G-4635 produced in maize plants. Some of the registered as an insecticide (e.g., Bio- • Garst 8315 transgenic maize lines and hybrids bit, Dipel) since 1961, and is considered • Great Lakes GL-685 developed from these efforts have one of the least hazardous insecticides • McCurdy 7477 proven to be very effective in ever developed. • Northrup King PX9581 controlling ECB. The insecticidal • Pioneer Brand 3181 properties of these lines and hybrids The U. S. Environmental Protection • Pioneer Brand 3184 are maintained throughout the growth Agency rules for the complete evaluation and use of these transgenic EUROPEAN CORN BORER RESISTANCE: EVALUATION OF COMMERCIAL MAIZE HYBRIDS AND TRANSGENIC MAIZE CULTIVARS 269 Table 2. Effectiveness of Bt transgenic maize plants for control of ECB at Marshall, MO, 1994. Data are means from an evaluation done by D. Huckla and D. Barry (personal communication 1994). Maize type Non-Bt Bt † ‡ § ¶ # Insecticide † treatment Manual Leaf-feeding ‡ § infestation rating # No. of entry holes/plant No. of Tunnel length larvae/plant (cm/plant) Harvestable ¶ ears Yield (t/ha) None Pyrethroid weekly to post-anthesis Pyrethroid weekly from V6 to V15 None Dipel (SApp.) Pyrethroid (SApp.) Pyrethroid weekly from V6 to post-anthesis ECB 1 & 2 ECB 1 2.8 ab 2.6 b 1.7 a 0.3 cd 0.3 bc 0.0 d 8.4 b 1.3 ef 80.3 b-e 80.2 cde 8.36 cd 8.71 bcd ECB 2 1.1 c 0.9 b 0.4 ab 5.6 c 81.7 b-e 9.41 ab None ECB 1 & 2 ECB 1 & 2 None 1.1 c 2.6 b 2.8 a 1.1 c 1.6 a 1.0 b 0.5 c 0.0 d 0.5 a 0.1 cd 0.1 cd 0.0 d 10.9 a 5.1 cd 3.0 de 0.3 f 77.0 e 79.2 a 80.2 cde 82.5 a-d 7.85 d 8.38 cd 8.59 cd 9.97 a None Pyrethroid weekly to post-anthesis Pyrethroid weekly from V6 to V15 None Pyrethroid weekly from V6 to post-anthesis ECB 1 & 2 ECB 1 1.0 c 1.0 c 0.0 d 0.0 d 0.0 d 0.0 d 0.0 f 0.0 f 85.2 ab 87.0 a 8.95 bc 9.93 a ECB 2 1.0 c 0.0 d 0.0 d 0.3 f 85.0 abc 9.94 a None None 1.0 c 1.0 c 0.1 d 0.0 d 0.0 d 0.0 d 0.3 f 0.0 f 83.8 a-d 84.8 abc 9.17 abc 10.08 a Pyrethroid used was Pounce 3.2 EC. SApp. indicates a standard application done once 5d after manual infestation. ECB 1 and 2 refer to first- and second-generation of ECB. Guthrie et al. (1960) 1-9 rating scale (1 = no damage, 9 = severe damage). Mean number of harvestable ears in 18.3 m of row length. Means in a column with the same letter are not significantly different at the 0.05 probability level. plants have not been completely formulated. As for conventional insecticides and resistant maize hybrids, these evaluations should be a part of public research programs. Re fe re nce s Barry, B.D. 1969. European corn borer and corn leaf aphid resistance in corn borer and corn leaf aphid resistance in corn hybrids. Ohio Agricultural Research and Development Center Research Circular 174, Wooster. Barry, D., and L.L. Darrah. 1991. Effects of research on commercial hybrid maize resistance to European corn borer (Lepidoptera: Pyralidae). J. Econ. Entomol. 84: 1053-59. Barry, D., A.Q. Antonio, and L.L. Darrah. 1986. 1986 evaluation of commercial corn hybrids for resistance to European corn borer in Missouri. Missouri Coop. Ext. Service publication EC953. Barry, D., A.Q. Antonio, and L.L. Darrah. 1987. 1987 evaluation of commercial corn hybrids for resistance to European corn borer in Missouri. Missouri Agric. Exp. Stn. Barry, D., A.Q. Antonio, and L.L. Darrah. 1989. 1989 evaluation of commercial corn hybrids for resistance to European corn borer in Missouri. Special Report 411. University of Missouri, Columbia, MO 65211. Barry, D., A.Q. Antonio, and L.L. Darrah. 1995. Registration of Mo45, Mo46, and Mo47 germplasm lines with resistance to European corn borer (PI583350, PI583351, and PI583352). Crop Sci. In press (July-Aug, 1995). Barry, D., and M.S. Zuber. 1984. Registration of MoECB2(S1)C5 maize germplasm. Crop Sci. 24: 213. Barry, D., A.Q. Antonio, and L.L. Darrah. 1983. Selection for resistance to the second generation of the European corn borer (Lepidoptera: Pyralidae) in maize. J. Econ. Entomol. 76: 392-394. Barry, D., M.S. Zuber, and L.L. Darrah. 1985. Registration of Mo-2ECB-2 maize germplasm. Crop Sci. 25: 715-716. Beck, S.D., J.H. Lilly, and J.F. Stauffer. 1949. Nutrition of the European corn borer, Pyrausta nubilalis (Hbn.) I. Development of a satisfactory purified diet for larval growth. Ann. Entomol. Soc. Amer. 42: 483-496. Bottger, G.T. 1942. Development of synthetic food media for use in nutrition studies of the European corn borer. J. Agr. Res. 65: 493-500. Guthrie, W.D., and F.F. Dicke. 1972. Resistance of inbred lines of dent corn to leaf-feeding by first-brood European corn borers. Iowa State J. Sci. 46: 339357. Guthrie, W.D., F.F. Dicke, and C.R. Neiswander. 1960. Leaf and sheath feeding resistance to the European corn borer in eight inbred lines of dent corn. Ohio Agricultural Experiment Station Bulletin 860, Wooster. Guthrie, W.D., E.S. Raun, F.F. Dicke, G.R. Pesho, and S.W. Carter. 1965. Laboratory production of European corn borer egg masses. Iowa State J. Sci. 4: 65-83. Guthrie, W.D., W.A. Russell, and C.W. Jennings. 1971. Resistance of maize to second-brood European corn borers. pp.. In J.I. Sutherland, and R.J. Falasca (ed.), Proceedings of the Annual Corn Sorghum Industry Research Conference, vol. 26. December 14-16, 1971, 165-179. Chicago, IL.: American Seed Trade Association, Washington, D.C. 270 B.D. BARRY AND L.L. DARRAH Klenke, J.R., W.A. Russell, and W.D. Guthrie. 1986a. Recurrent selection for resistance to European corn borer on a corn synthetic and correlated effects on agronomic traits. Crop Sci. 26: 864-868. Klenke, J.R., W.A. Russell, and W.D. Guthrie. 1986b. Grain yield reduction caused by second-generation European corn borer in ‘BS9’ corn synthetic. Crop Sci. 26: 859-853. Klenke, J.R., W.A. Russell, and W.D. Guthrie. 1986c. Distributions for European corn borer (Lepidoptera: Pyralidae) ratings of S1 lines from ‘BS9’ corn synthetic. J. Econ. Entomol. 79: 1076-1081. Klenke, J.R., C.A. Martinson, and W.L. Pedersen. 1987. Disease resistance in five cycles of ‘BS9’ corn synthetic selected for resistance to two generations of European corn borer. Phytopathology 77: 735-739. Mihm, J.A. 1983a. Efficient mass rearing and infestation techniques to screen for host plant resistance to maize stem borers, Diatraea spp. Centro International de Mejoramiento de Maiz y Trigo. El Batán, Mexico. Technical Bulletin. Mihm, J.A. 1983b. Efficient mass-rearing and infestation techniques to screen for host plant resistance to fall armyworm, Spodoptera frugiperda. Centro International de Mejoramiento de Maiz y Trigo CIMMYT. El Batán, Mexico. Technical Bulletin. Onukogu, F.A., W.D. Guthrie, W.A. Russell, G.L. Reed, and J.C. Robbins. 1978. Location of genes that condition resistance in maize to sheath-collar feeding by second-generation European corn borers. J. Econ. Entomol. 71: 1-4. Patch, L.H. 1929. Some factors determining corn borer damage. J. Econ. Entomol. 22: 174-183. Patch, L.H. 1937. Resistance of a singlecross hybrid strain for field corn to European corn borer. J. Econ. Entomol. 30: 271-278. Patch, L.H. 1947. Manual infestation of dent corn to study resistance to European corn borer. J. Econ. Entomol. 40: 667-671. Patch, L.H., G.T. Bottger, and B.A. App. 1938. Comparative resistance to European corn borer of two hybrid strains of field corn at Toledo, Ohio. J. Econ. Entomol. 31: 337-340. Patch, L.H., G.W. Still, B.A. App, and C.A. Crooks. 1941. Comparative injury by the European corn borer to openpollinated and hybrid field corn. J. Agric. Res. 63: 355-368. Pesho, G.R., F.F. Dicke, and W.A. Russell. 1965. Resistance of inbred lines of corn (Zea mays L.) to the second brood of the European corn borer (Ostrinia nubilalis Hübner). Iowa State J. Sci. 40: 85-98. Russell, W.A., and W.D. Guthrie. 1979. Registration of B85 and B86 germplasm lines of maize. Crop Sci. 19: 565. Russell, W.A., and W.D. Guthrie. 1982. Registration of BS9(CB)C4 maize germplasm. Crop Sci. 22: 694. Scott, G.E., A.R. Hallauer, and F.F. Dicke. 1964. Types of gene action conditioning resistance to European corn borer leaf feeding. Crop Sci. 4: 603-606. Scott, G.E., F.F. Dicke, and G.R. Pesho. 1966. Location of genes conditioning resistance in corn to leaf feeding of the European corn borer. Crop Sci. 6: 444446. Showers, W.B., J.F. Witkowski, C.E. Mason, F.L. Poston, S.M. Welch, A.J. Keaster, W.D. Guthrie, and H.C. Chang. 1989. European corn borer: Development and management. North Central Regional Publication 327. Iowa State University, Ames, IA. Use of CIM M YT’s M ultiple Borer Resistance Population for Developing Asian Corn Borer Resistance and Inbreds in China K. He, D. Zhou, and Y. Song, Institute of Plant Protection, CAAS, Beijing 100094, P.R. China Abst r a c t After a brief background introduction on the importance of maize, Asian corn borer (ACB), Ostrinia furnacalis (Guenée), and breeding and improving for host plant resistance (HPR) to ACB in China, we report on efforts to develop ACB resistant inbred lines for use in hybrids with CIMMYT’s multiple borer resistance (MBR) populations. In 1986, ACB resistant inbred development with CIMMYT’s MBR population was initiated. Several resistant inbreds, such as MC37, MC61, MC74, HM31 and HM67, with potential for use in hybrid crosses, were developed by selfing and selecting highly resistant types within each selfed generation after artificial infestation with ACB at whorl stage. On the basis of this work — together with additional support from CIMMYT in the form of highly resistant maize populations, financial contributions, vigorous efforts to promote cooperation between entomologists and breeders, and advanced training for young scientists — we began a new project to develop ACB-resistant inbreds using MBR-590 and the CIMMYT multiple insect resistance tropical population, MIRT-390. Finally , we describe a successful adaptation of “bazooka” technique in China. Int roduct ion ; ;; ; ; ;;; ;; ; ;; ;;; ;;; ;; ; ;;;;;; ;;; about 80% of the area in China’s Corn inexpensive, have a high and stable Belt remains untreated for economic controlling effect, be simple and easy to Asian corn borer, (ACB), Ostrinia reasons and for lack of labor. Based on apply, and not pollute. With these furnacalis (Guenée), is closely related to over 30 years experience in ACB criteria in mind, host plant resistance the European corn borer, O. nubilalis research, Prof. Zhou concluded that (HPR) in maize is considered the best (Hübner), and is the most destructive components of an integrated and most basic way to minimize losses insect pest of maize in China. From management strategy for ACB must be from ACB. north to south, it has one to seven generations a year (Fig. 1). Throughout the vast territory of the country, however, for a particular crop of maize only one or two generation(s) occur. Generally, one generation attacks at the whorl stage and the other at the pollenshedding stage. In a normal year, the annual loss caused by ACB is 10% in spring maize and 20-30% in summer maize, where no controls are used. Many effective control methods, such as chemical treatment with extended residue granular insecticides, biological control with Trichogramma, and cultural practices, have been developed. Still, Generation 1 1-2 2 3 4 5 6 7 ;; ; ;; ;;;;; ;;;;; ;;;;; ;;;;; ;;;; ;;;; ;;; ;;;; ;;; ;;; ;;; ; ;;; ; ;;; ;; ; Figure 1. Approximate distribution of generation zones of the ACB in China. 272 K. HE, D. ZHOU, AND Y. SONG Techniques for mass rearing ACB and subtropical materials as sources of lines were selfed within these rows. The evaluating resistance to ACB in maize, for use in hybrids and of genes for process carried out in year 3 was two essential elements for efficient disease and insect resistance. In this repeated for three generations. These screening and improving of HPR, have paper, we describe the use of CIMMYT inbreds were then selfed and been developed successively in China multiple borer resistance populations individually crossed onto local lines for (Zhou et al. 1980; Zhou 1982; Zhou and in resistance screening and the yield trials under artificial infestation Chen 1989). As of 1982, more than 1,048 development of ACB-resistant inbred with ACB at the whorl stage. From the inbred lines and 485 varieties and lines. results, the potential single crosses and promising lines were predicted for synthetics were evaluated by the All China Corn Borer Research Group M aterials and M ethods experimental hybrids. At all times in line selection, detailed notes were taken (ACCBRG). Although most of those lines, especially the elite ones, were In 1986, 114 families of CIMMYT’s on agronomic as well as resistance found to be susceptible, a few resistant Multiple Borer Resistant Population traits, as any new hybrid will have to ones exist. Ji404 was an outstanding (MBR) were planted in Beijing. The be competitive against the released example. Later, certain promising lines evaluations of resistance to ACB were ones. The procedure we followed is with high resistance derived from Ji404 done by artificial infestation at whorl outlined in Table 1. x elite line crosses were developed by stage. Using these materials as an using a method called second cycle exotic source of resistant germplasm, selection. From this process, a single- efforts to develop ACB resistant inbred 2. Developing Inbreds from MBR X Local Lines cross hybrid, ZHIDAN NO.1, which lines were initiated using the following When MBR populations were could be used for efficient control of two procedures. evaluated and self-pollinated in 1986, ACB at the whorl stage (Zhou et al. 1987), was released. Though the area some of their resistant families were 1. Developing inbreds from the MBR population also individually crossed as male planted to this hybrid was limited due to its substandard yield and the Self-fertilization has been used lines, such as Zi330, Ji63, E28, 122 etc., susceptibility to viral disease of the primarily for inbreeding under which combined the two groups of the female parent, it still showed that the artificial infestation with ACB at the genetic bases (Table 2). use of resistant hybrids is actually the whorl stage. In order to provide a best, most practical, most economical, broad base that permitted effective In case the MBR populations and their and most effective means to minimize selection concurrently with inbreeding progenies would not be well adapted losses from ACB in China. Zhou et al. under diverse environmental under all the diverse environmental (1987) concluded that the availability of conditions, the selection was conducted conditions, these crosses would serve sufficient resistant germplasm and the within-family in year 1. The S1 seeds as the genetic base to permit further application of modern and effective were bulked within-family to create the selection and modification of the breeding techniques are the two most respective S1 families. The following desired traits. In the following season, important factors in a successful season, year 2, S1 families were planted the crosses were planted and infested program to develop ACB-resistant and infested again. Rows that appeared with ACB at the whorl stage. Selections hybrids. to be the most resistant were selected and self-pollination were made within parents onto several locally adapted on the basis of ratings of leaf feeding rows. The resulting seeds were bulked It is well known that heterosis is damage. Within these rows the better and planted out next season, usually observed for crosses where the plants were self-pollinated and respectively. Additional selection and parent inbred lines are genetically progressed to S2. The resulting ears re-evaluation was carried out within diverse. Unlike correlation and visual from selfed plants were planted out the S1s, which were then selfed to S2. selection, the genetic diversity of ear-to-row in year 3. Selections and re- The following season, year 3, these S2s inbred lines used in crosses is generally evaluations were made not only for were planted out ear-to-row, and those recognized to be important. It is ACB resistance, but also for other major which appeared to be the most assumed that, to have a reasonable diseases resistance, earlier maturity, resistant were selected on the basis of chance of success, one should make short plant stature and tolerance to ratings of leaf feeding damage under selections from exotic tropical and lodging among rows. Better plants ACB infestation. Better plants were USE OF CIMMYT’S MULTIPLE BORER RESISTANCE POPULATION FOR DEVELOPING ASIAN CORN BORER RESISTANCE AND INBREDS IN CHINA Table 1. Procedure for developing ACB resistant lines from MBR population in Beijing. Timescale Processes 273 selfed to S3 within the rows. Then, the process of year 3 was repeated in the following seasons. Other processes and notes were taken as in procedure 1. Year 1 Plant MBR population Artificially infest plants with ACB Evaluate for resistance Self-pollinate most resistant plants Bulk S1 seed within-family Year 2 Plant S1 families Infest and evaluate Select most resistant S1 families Self-pollinate better plants in selected rows Year 3 Plant ears from selfed plants ear-to-row Infest and evaluate Select for ACB resistance, major diseases resistance, earlier maturity, short plant stature, tolerance to lodging and good plant aspect Self-pollinate better plants in selected rows Following Years Repeat the procedure described in year 3 Self to inbred Cross onto local adapted lines Evaluate crosses for ACB resistance, yield performance and other agronomic traits Select the potential crosses and promising lines Re sult s A histogram showing the numbers of families classified as resistant, intermediate, and susceptible is presented in Figure 2. Ratings were done with a 1 to 9 scale, where 1 was the most resistant and 9 the most susceptible. The resistant class included families rated from 1 to 3, intermediate from 4 to 6, and susceptible from 7 to 9. Most (85% or more) of the 114 MBR families tested were rated as resistant, and thus were comparable to the resistant check (122) which was one of a few best materials locally available for ACB resistance and showed no Table 2. Procedure for developing ACB resistant lines from MBR x local lines in Beijing. Timescale Processes significant level of insect damage. One family rated intermediate, and 15 families(13.2%) susceptible. This indicates that MBR is an excellent source material of ACB-resistance. Year 1 Form crosses between local lines and some resistant MBR families Year 2 Plant the crosses Infest and evaluate Self-pollinate better plants in selected rows Bulk S1 seeds, respectively Year 3 Plant the S1’s Infest and evaluate Self-pollinate better plants within rows Several highly resistant inbreds have been developed with the two procedures used by our program. Ratings of leaf feeding damage sustained by these inbreds and a local Year 4 Following Years Repeat the procedure described in year 3 Self to inbred Cross onto local lines Evaluate crosses for ACB resistance, yield performance and other agronomic traits Select the potential crosses and promising lines ;;; ;;; ;;; ;;; ;;; ;;; ;; ;;; ;;; ;; 100 Number of families Plant S2’s ear-to-row Infest and evaluate Select for ACB resistance, major diseases resistance, earlier maturity, short plant stature, tolerance to lodging and good plant aspect Self-pollinate better plants in selected rows 80 60 40 20 0 Resistant Intermediate Susceptible (1-3) (4-6) (7-9) Figure 2. Asian corn borer damage ratings of 114 families of CIMMYT’s MBR population planted in Beijing. 274 K. HE, D. ZHOU, AND Y. SONG susceptible check, Zi330, in 1992 are husk cover when grown under the in certain aspects since Dr. Mihm’s visit given in Table 3. The inbreds MC37, temperate environment in Beijing. It to Beijing in 1992. His viewpoint and MC61 and MC74 were derived from was recognized that considerable outstanding work on HPR to borers MBR, whereas the inbreds HM31, potential existed for screening and made a very deep impression on HM67 and HM15 were derived from developing highly adapted temperate Chinese breeders and resulted in a local lines x MBR. ACB resistant lines from MBR. The two vigorous push towards cooperation procedures used were effective in between entomologists and breeders Table 4 shows the yield and ACB developing ACB resistant inbreds. from major institution, although this resistance performance of some However, certain deficiencies remain process is just at the initial stage. potential crosses developed by our and still need to be improved for program under artificial infestation Chinese conditions. For instance, In 1993, fortunately, the senior author with ACB at the whorl stage. They continuous self-fertilization seems to be got a precious opportunity to attend were not only resistant to ACB, but also too drastic, thus the MBR population the two training courses held at demonstrated their good yield traits were lost too quickly. A milder CIMMYT, i.e., Maize Breeding for potential, and promise in probable form of inbreeding that still permits Insect Resistance and The Maize hybrid use. effective selection should be used. In Breeding Course. From these he addition, yield and topcross testing obtained a lot of knowledge in the field Discussion should be done at an earlier stage. of maize breeding and breeding for Although the MBR population was of mentioned above and our situation, tropical and subtropical adaptation and Important contributions from CIMMYT to HPR study in China is considered to contain tremendous Mihm (1985) stated that an support in giving highly resistant genetic diversity, compared with local interdisciplinary team having at least maize populations and a financial temperate materials, all 114 families an entomologist and a breeder is contribution, a new project for introduced were able to mature in spite desirable to carry out the HPR developing ACB resistant inbreds with of their relatively late maturity, high program. In China, however, most CIMMYT’s populations MBR- plant, big tassel and long and thick breeders pay no attention to HPR. They 590(temperate) and MIRT-390(tropics always consider that it is easy to and subtropics) has been actively control ACB by using insecticides, but undertaken. One seasons results, breeding and improvement of HPR to histograms showing the contributions insects is very difficult. So, until 1992 of numbers of families classified as the research on HPR had been done highly resistant (HR), resistant(R), mainly by the entomologists, who moderately resistant(MR), usually lack maize breeding skills. The susceptible(S), and highly susceptible situation, however, has been changed (HS), are presented in Figures 3 and 4. insect resistance. On the basis of work Table 3. Ratings of leaf feeding damage of inbreds developed under artificial infestation with ACB at the whorl stage. Inbred Rating MC37 MC61 MC74 HM31 HM67 HM15 Zi330 1 1 2 1 1 2 9 together with CIMMYT’s further ;; ; ;; ; ;; ; ;; ; ;; ;; ;; ;; ; ;; ; ;; ;; ;; ; ;; ;; ;; ;; ;;;;; ;; ;; ;;; 140 90 120 100 Table 4. Yield performance of the potential hybrid crosses developed Hybrid Cross MC37 X YUANFU30 MC61 X HM31 MC74 X 525 SANTUAN4 X MC61 Zi330 X HM67 HM15 X YELLOW EARLY 4 Rating Yield (g/plt.) 2 1 3 3 3 2 146.3 132.3 117.6 136.6 121.0 127.8 60 80 60 30 40 20 0 0 HS R MR S HS (1-1.9) (2-3.9) (4-5.9) (6-7.9) (8-9) Figure 3. Asian corn borer damage ratings of CIMMYT’s MBR-590 planted in Beijing. HS R MR S HS (1-1.9) (2-3.9) (4-5.9) (6-7.9) (8-9) Figure 4. Asian corn borer damage ratings of CIMMYT’s MIRT-390 planted in Beijing. USE OF CIMMYT’S MULTIPLE BORER RESISTANCE POPULATION FOR DEVELOPING ASIAN CORN BORER RESISTANCE AND INBREDS IN CHINA HR class included families rated from 1 In our laboratory, waxy paper(27 x 44 to 1.9, R from 2 to 3.9, MR from 4 to 5.9, cm) sheets are placed on top of S from 6 to 7.9, and HS from 8 to 9. It oviposition cages for oviposition. The indicated that MBR-590 and MBR-390 sheets containing egg masses are are excellent source materials of ACB removed and replaced with new ones resistance. We hope that new inbreds every morning. The egg-mass sheets with resistance to ACB, and other are then kept at 28ºC and >75% RH for major maize diseases, and with good about 2 days. When the egg masses yield performance can thus be become nearly ready to hatch, egg- developed and used in hybrid mass sheets are dehumidified in a low production by our new program. humidity room for 30 to 60 min. and then cut into 3 strips (about 9 cm wide) Modification and adaptation of the bazooka method for efficient field infestation of ACB along the long axis. After that, the same Until 1993, artificial infestation with collecting egg masses, mixing the ACB in China had always been done by hatched larvae with corn cob grits, and placing two egg masses or glass tubes infesting in the field. procedures described by Mihm(1989) are followed for removing and containing 30 to 40 newly hatched larvae into maize plant whorls. These In the other procedure, the egg-mass- techniques can be used effectively for sheets are slit into four equal-sized infestation, but they are very inefficient smaller pieces, and kept in total because of the many laborious steps, darkness at 28ºC. When egg masses such as cutting egg masses, placing egg reach the black-head stage, they are masses ready to hatch into glass tubes, incubated at 15ºC until larvae hatch. At and the slowness of field application. this relatively low temperature the egg Although the bazooka method for masses can develop continually, but larval infestation has been used to the newly hatched larvae are not active. infest many species of lepidopterous For mixing the hatched larvae with insect pests (Mihm 1987), it had not corn cob grits, the larvae are been possible to adapt it to our transferred to the mixing bottle by situation, due to the fact that egg snapping the back of the sheets with masses could not be removed from fingers. With such a procedure, the egg-mass sheets quickly. Hence, process of removing egg masses from procedures were developed to the sheets can be omitted. overcome this problem. 275 Re fe re nce s Mihm, J.A. 1985. Breeding for Host plant resistance to maize stem borers. Insect Sci. Applic. 6(3): 369-377. Mihm, J.A. 1989. Evaluating maize for resistance to tropical stem borers, armyworms, and earworms. In Toward Insect Resistant Maize for the Third World: Proceedings of the International Symposium on Methodologies for Developing Host Plant Resistance to Maize Insects, 109-121. Mexico, D.F.: CIMMYT. Zhou, D., Y. Wang, B. Liu and Zh. Ju. 1980. Studies on the mass rearing of corn borer I. Development of a satisfactory artificial diet for larval growth. Acta Phytophyl. Sinica 7(2): 113-122. Zhou, D. 1982. A brief description on the resistance study of corn borer in the People’s Republic of China. Annual Plant Resistance to Insect. Newsletter 8: 69-70. Zhou, D., and C. Chen. 1989. Studies of a bioassay technique for resistance evaluation of maize to the Asian corn borer,Ostrinia furnacalis (Guenée). CIMMYT. 1987. In Toward Resistant Maize for the Third World: Proceedings of the International Symposium on Methodologies for Developing Host Plant Resistance to Maize Insects, 295. Mexico, D.F.: CIMMYT. Zhou, D., Zh. Ju, R. Wei, C. Chen, Y. Gao, L. Wen, K. He, X. Li, and Ch. Liu. 1987. Utilization of corn borer resistance in maize and introduction of a resistant single cross Zhidan No. 1. Plant Protection. 13(5): 16-18. Corn Borers Affecting M aize in Egypt M. Soliman, Agricultural Research Center, Giza, Egypt Abst r a c t In Egypt, maize plants are severely attacked by different species of Lepidopteran pests, the most important being the corn borers: the pink borer or greater sugar cane borer, Sesamia cretica Led (Noctuidae); the purple-lined borer or lesser sugar cane borer, Chilo agamemnon Bles. (Crambidae), which are the principal borers of sugar cane and rice in Egypt; and the European corn borer, Ostrinia nubilalis Hbn. (Pyraustidae). These borers are also considered the principal cause for the secondary infection of fungal and bacterial diseases. Sesamia cretica is considered the most serious of the borers. This species attacks maize plants shortly after emergence, devours the whorl leaves and may kill the growing point, causing dead hearts. It is also capable of damaging older plants and excavating tunnels into the stem, ears and/or cobs. This pest lays its eggs during March, so it causes complete death of small maize plants in April and May, leading to drastic yield losses. Chemical insecticides are commonly used to control S. cretica, but given the negative environmental side effects, associated with chemical control, development of maize cultivars with resistance to S. cretica offers a better alternative. The Egyptian national maize breeding program is concentrating its efforts to develop and release new white and yellow maize hybrids with high yielding ability, plus resistance to the major diseases such as late wilt, common smut, downy mildew and leaf blight, as well as resistance to insect pests. Int roduct ion effects of this chemical control on the agroecosystem include the destruction Evaluation and Development of New Hybrids Maize is considered one of the most of natural enemies of pests, outbreaks important cereal crops in Egypt. The of mite populations and environmental total cultivated area is about 0.84 pollution. To avoid or at least minimize New hybrids developed through the million ha for early (May-June) and late such side-effects, growing maize breeding program are evaluated and (July-August) plantings. The total cultivars resistant to S. cretica is highly advanced in two stages, before release national production of maize is about recommended (Simeada 1985). The for commercial production. The first 5.3 million tons. About 2.0 million tons Egyptian national maize breeding stage consists of four different on- of maize are imported annually as the program is concentrating its efforts to station evaluation trials: total consumption has reached 7.0 develop and release new white and • A Trials. Top crosses are evaluated million tons. The national yield average yellow maize hybrids with high in 2-3 locations to estimate general was 6.5 tons/ha in 1993, but this value yielding ability, plus resistance to the combining ability (GCA) and is still below the expected yield major diseases such as late wilt, specific combining ability (SCA), potential (Abou El-Saad 1994). Our common smut, downy mildew and leaf using the best local hybrids as target is to reach an average yield of 8.5 blight, as well as resistance to insect checks. The promising hybrids are tons/ha. This is a realistic possibility, pests. A considerable number of new advanced to the AH Trials. because there is an increased tendency white and yellow inbred lines have • AH Trials. For evaluating single, for farmers to use high yielding, been isolated and developed using three-way and double crosses disease and pest resistant single and different breeding techniques. Several derived from the A Trials in three three-way cross hybrids. genetic sources for higher yielding locations. The promising hybrids are ability, better plant type as well as Control of S. cretica in maize fields is resistance to diseases and pests have commonly done by the application of been obtained. chemical insecticides, either as sprays or granules, directly to the whorl. Side advanced to B Trials. • B Trials. Promising hybrids from the national maize program, as well as from local and foreign seed CORN BORERS AFFECTING MAIZE IN EGYPT 277 companies, are tested at five Studies of the seasonal distribution of the aforementioned “safe period”. research stations. Superior hybrids borers affecting maize, done about 20 Hence, the Egyptian national maize are advanced to C Trials. years ago, revealed that fields planted breeding program has decided to before the beginning of May are attempt to develop, and use, maize Trials are tested in C Trials. These severely infested by Sesamia (Fig. 1). with host plant resistance. trials are conducted in a disease They lay their eggs beneath the sheath nursery at five research stations to of first or second leaves on maize Any program that is to be successful in evaluate hybrids for their resistance plants 20 days after planting. After developing maize varieties resistant to to the major diseases, late wilt, hatching the larvae feed on furled insect pests and with good agronomic common smut, downy mildew and leaves causing leaf damage and dead characteristics has to have seven basic leaf blight. Promising hybrids are hearts. Maize planted after the components (J.A. Mihm pers. comm.), advanced to the verification trials in beginning of July is subject to high these are: the farmers fields. infestation with Ostrinia, which attacks • Maize germplasm, including some • C Trials. Hybrids advanced from B maize 45 days after planting. Hence with genes for resistance. • A supply of insects (a colony). The second stage of the development maize growers in Egypt are process involves verification trials (D encouraged to plant their maize during Depending on the requirements and Trials), where superior hybrids derived the period from the beginning of May desires of the program, these may be from C Trials are evaluated in the to mid-June, in order to escape severe reared on natural hosts, or on farmers fields in trials conducted in at infestation by the two borers. This least 10 governorates in the Delta and recommendation decreases the need for • Capability to artificially infest. Upper Egypt regions. intensive use of insecticides, so • Capability to rapidly assess damage, Progress Tow ards Host Plant Resistance in the Egyptian M aize Program artificial diets in the laboratory. minimizing environmental pollution. or lack of it, after infestation. This Specific biological and ecological usually means developing a rating studies revealed that the main reason scale that identifies the category and for this phenomenon was the level of resistance (antibioses type), environmental conditions occurring into high, intermediate, low or susceptible. during summer in Egypt. The hot and cretica Led., is the most important of the dry conditions were found to be borers which affect maize in Egypt. Yet, unsuitable for the adults to mate and despite the agricultural importance of lay fertile eggs. However, it was noted this pest, very few studies exist in the that a small proportion of the borer consisting of entomologists, published literature on the relative population became adapted to the breeders and pathologists. susceptibility of maize plants to Sesamia summer conditions. This proportion is cretica Led. A review of the limited expected to increase gradually and the program. This is basically the available knowledge on Sesamia threaten maize fields planted during dedication, money and trained indicated that several investigators had evaluated maize varieties commonly cultivated in Egypt, with respect to susceptibility to infestation by S. cretica. Unfortunately, most of these investigations were carried out on obsolete cultivars under natural infestations. Results obtained under these conditions do not usually reflect the real level of susceptibility or resistance in the cultivars screened. Number of egg masses/100 plants The greater sugarcane borer, Sesamia • Knowledge of the inheritance/ heritability of the resistance. • An interdisciplinary team, • The resources to execute all steps of people. 50 40 Sesam Chilo Ostr Host plant resistance is based on the presence of genes for resistance. Hence, the first stage in our program has been 30 to screen local materials for resistance. 20 If these are found to be susceptible, then the second step is to screen exotic 10 materials. We already have most of the most advanced and best materials, with 0 J F M A M J J A S O N D Months Corn borers seasonal distribution Figure 1. Corn borer seasonal distribution in Egypt. known resistance to borers. Once genes for Sesamia resistance are identified they can be utilized. 278 M. SOLIMAN There is no way to identify genes for grain yield without causing a resistance in maize plants without significant effect on the infestation having insects on the plants at the level, ranged between 144 and 216 kg/ proper stage. No program anywhere in ha. Phosphorous and potassium the world has developed resistant applications did not affect the varieties by selecting “undamaged” infestation level of this borer. For plants that were naturally infested (J.A. Sesamia, it was found that planting with Mihm pers. comm.). In order to select 5-6 kernels/hill and removing the plants with resistance genes, one has to infested plants at thinning before the see the amount and type of damage first irrigation resulted in the removal that occurs when insects are feeding on of about 80-85% of the insect the plant. In order to achieve these population (Awadallah, et al. 1980). goals we have just established a maize Other studies revealed that early maize borer rearing laboratory. can be intercropped in onion fields just before the last irrigation of onion. In Investigations into other non-chemical this case, the onion’s odor repels the control methods, such as the effect of Sesamia moth and consequently the different plant densities, as well as, infestation level with borer is greatly using different rates and combinations decreased, (Awadallah, et al. in press). of nitrogen (N), phosphorus (P) and potassium (K) fertilizers on the We hope to start up our breeding infestation level of Ostrinia, have also program for host plant resistance been carried out (Awadallah, et al. including artificial infestation for about 1980). The results indicated that the 500 families (local and exotic) during levels of N fertilizer, which increase the 1995 season. Re fe re nce s Abou El-Saad, S.F. 1994. The role of maize program in seed industry. International and Regional Maize Workshop, Cairo, Egypt, April, 4-5. Awadallah, W.H., A.A. Galal, R.M. Abdallah, and N.S. Selim. 1980. Evaluations of some local and exotic materials for the resistance in maize to the European corn borer Ostrinia nubilalis (Hubner) and studying the effect of nitrogen fertilizer and number of plants per unit area on the infestation level of the same pest. Proc. 1st. Conf. Pl. Prot. Res. Ins. Vol. 1: 187196. Awadallah, W.H., A.F. Lutfallah, M.R. Sherif, and M.H. Hanna Alla (in press). Intensification into onion fields to avoid the economic infestation with the corn stem borer Sesamia cretica Led. in Egypt Agric. Res. Rev. Vol. (in press). Simeada, A.M. 1985. Relative susceptibility of certain maize germplasm to infestation with the greater sugar cane borer, Sesamia cretica Led. (Lepitoptera:Noctuidae). M.Sc. Thesis, Fac. of Agric. Cairo Univ., Egypt. Search for M ultiple Resistance in M aize to StemBorers Under Natural Infestation in M idaltitude Intermediate M aturity Areas in Kenya M. Gethi, RRC - EMBU, P.O. Box 27, EMBU, Kenya Abst r a c t The search for multiple borer resistance in maize, mainly against Chilo partellus (Swinhoe) and Busseola fusca (Fuller), requires routine screening of a large number of germplasm sources. In the present investigation, the search for multiple borer resistance involved evaluation of inbreds, (local and exotic) synthetics, open pollinated materials and hybrids. The parameters that were used for evaluation were based on infestation level (larval and pupal density) and damage levels (foliar damage, stalk tunneling, borer exit/ entry holes). Preliminary results indicated significant (P=0.05) differences between cultivars/lines in the parameters that were used for evaluation. There was a positive and significant (P=0.05) relationship between foliar damage and tunnel length. As evaluations were done under natural infestation, results on yields as a measure of resistance were not considered. From the data presented, it can be concluded that some parameters, like foliar damage and tunnel length, may be used as possible selection characters in resistance breeding. However, controlled uniform artificial infestation is required to obtain consistent results. Int roduct ion typical control method is insecticides pollinated cultivars. However, (Warui and Kuria 1983). This is usually successful breeding for multiple borer Stem borers in maize are considered to not an economic proposition and is resistance (MBR) depends mainly on be the most important pests of all often an ineffective approach in developing suitable procedures for graminaceous crops in the world subsistence farming systems. This is screening and on identifying the (Jepson 1954; Hill 1975). These borers because the current recommendations physical traits responsible. The constitute one of the major constraints are only moderately effective, mainly objectives of this study were to: to efficient maize production in the due to the timing of application. • Identify sources of resistance to stem borers. developing world, where maize is • considered as one of the most Host plant resistance (HPR) has been important subsistence crops (Scheltes shown to offer the most effective, resistance screening in breeding 1978). economical, stable and ecologically programs. Develop procedures to be used in sound approach to reducing damage Studies in Kenya have showed that the (Ampofo 1986). HPR is an innate stem borers C. partellus, C. quality that renders the plant orichalcociliellus, B. fusca, and Sesamia unsuitable as food or shelter for insect This work was carried out at the calamistis were the most important pests. Regional Research Center, Embu, M aterials and M ethods during the 1992-94 cropping seasons. borers of maize and sorghum (Seshu Reddy 1983). C. partellus comprises 90% Since most of the cultivars developed Twenty-three maize cultivars and of all the borer species infesting maize by Kenya’s maize improvement inbred lines, obtained from the local in Kenya, causing yield losses of about program are susceptible to stem borers, breeding nursery and from the 18% to 40%. Several stem borer control it was necessary to look for ways of International Maize and Wheat methods have been utilized, but the incorporating HPR into the currently Improvement Center (CIMMYT), recommended hybrids and open 280 M. GETHI Mexico, were evaluated for resistance and damage levels for all the Re sult s parameters that were tested when to stem borers. Two local commercial hybrids (H511 and H512) were Most of the cultivars/lines that were compared to susceptible check inbred included, together with two open screened under natural conditions for A (Table 2). Foliar rating and tunnel pollinated cultivars (KCB and DLC 1). MBR showed significant (P=0.05) length were significantly (P=0.05) During the experimental period, inbred differences in their response to damage higher in all those lines that showed A was used as a susceptible check. and infestation levels. CIMMYT- higher means for all other parameters derived materials that were initially used. Similarly, these inbreds also had Each cultivar/line was planted in the reported as borer resistant and the local significantly (P=0.05) higher larval/ field in triple row plots at a spacing of composites showed lower levels of pupal densities than those showing 90 x 30 cm between and within rows, infestation and damage (Table 1). lower means. There were indications respectively, in a randomized complete However, most of the inbreds derived that lines extracted from H511, Embu block design with three replications. from H511, E11 and E12 had 11 and 12 have a higher degree of borer This design was used to give all plants significantly (P=0.05) higher infestation susceptibility. This was the same in an equal opportunity of being selected by the ovipositing adult moths. The parameters that were tested as possible sources of resistance or susceptibility were: • Foliar damage rating. This was done on 10 plants selected at random using a scale of 1 to 9, where 1 was regarded as no damage and 9 meant severe foliar damage (Guthrie et al. 1960). • Stalk tunneling. Measurements were taken at harvesting from plants selected at random. The length of the tunnel above and below the ear was expressed as a percentage of Table 1. Levels of damage and infestation by the stem borer in different maize cultivars/lines. Foliar damage rating % tunnel length Exit holes per plant No. of larvae and pupae per plant Inbred A H512 E 11 PR 86 MBR DLC1 KCB PR 8523 SCB H511 PR 86 CHICO 2.15a 1.0b 0.95b 0.23b 0.68b 0.50b 0.73b 0.77b 0.71b 2.20a 1.34a 2.73ab 1.67ab 1.95b 2.00ab 1.39ab 1.83ab 1.33b 1.33 1.03 1.87 1.07 1.29 1.14 0.82 1.05 0.99 0.9b 1.80a 1.61a 1.09b 1.37ab 1.05b 1.02b 1.13b 1.21ab LSD 1.41 1.29 0.63 0.61 Cultivar Table 2. Levels of damage and infestation by stem borers in different maize lines under natural infestation. plant height. • Number of larvae and pupae of each species. This was determined at 3week intervals from another random set of 10 plants per plot. • Entry/exit holes. Below and above the ear from the plants that were used in (3) above. The holes were detected by the presence of frass deposits. An analysis of variance was carried out for the various parameters used (damage and infestation levels), and multiple regression analysis was also done to test the relationship between these parameters. Exit holes Cultivar E11 Syn1 E11 L.18 KCB DLC 1 E12 L139 E12 L163 H511 L225 H511 L8 Popu X1 MUVC9014SR E11 L133 H511 Syn1 E12 Syn1 Popu X2 E12 L3 E12 L210 Inbred A H511 L196 H511 Comm LSD CV Tunnels Foliar damage Above ear Below ear Chilo spp. Busseola Above spp. ear Below ear 1.08 1.04 1.02 1.01 1.09 1.16 1.13 1.20 1.08 1.07 1.13 1.09 1.00 1.02 1.06 1.06 1.13 1.09 1.14 0.93 0.71 0.72 0.72 0.72 0.77 0.71 0.73 0.72 0.89 0.79 0.78 0.74 0.73 0.79 0.72 0.71 0.72 0.75 0.98 0.72 0.73 0.73 0.87 0.76 0.73 0.97 0.78 0.77 0.77 0.87 0.81 0.77 0.94 0.86 0.95 0.82 0.95 0.73 0.71 0.71 0.71 0.71 0.71 0.73 0.82 0.71 0.78 0.74 0.73 0.71 0.71 0.73 0.71 0.71 0.73 0.71 0.76 0.71 0.72 0.72 0.77 0.73 0.71 0.73 0.73 0.71 0.71 0.71 0.74 0.71 0.75 0.76 0.72 0.72 0.84 1.03 0.71 0.71 0.77 0.71 0.82 0.71 0.76 0.77 0.94 0.78 0.73 0.78 0.72 0.81 0.77 0.71 0.72 0.77 0.99 0.71 0.78 0.76 0.91 0.82 0.82 1.02 0.92 0.77 0.87 1.00 0.87 0.82 1.30 1.29 1.02 0.84 0.88 0.04 12.50 0.05 19.33 0.08 28.71 0.03 9.36 0.03 9.78 0.07 26.01 0.14 41.84 SEARCH FOR MULTIPLE RESISTANCE IN MAIZE TO STEM-BORERS UNDER NATURAL INFESTATION varietal cross MUVC 9014 SR and Discussion 281 Similarly, synthetics that may be adapted to a wide range of double crosses that had the same parentage as H511. The most Locally grown open pollinated maize environments showed high levels of distinguishable parameters were the cultivars (composite) are more resistant susceptibility, as they were from the level of foliar damage, number of to stem borers than the hybrids and same parentage as the inbreds. larvae and pupae, and stalk tunneling. inbred lines. Omolo (1983) had earlier However, some of the lines and crosses attributed this to their early maturing screened had lower values and hence In multiple regressions to determine nature, resulting in avoidance of may have good combining ability for the correlation between parameters, all second generation borers. This was also specific characters. This is due to the were positively correlated, with the true for MBR materials from CIMMYT, fact that sources of resistance are correlation coefficient being highly which were early-to-medium in diverse and have a different significant (P=0.01) r =0.496. For maturity. It is also evident that most of combination of resistance factors. example, there was a positive the inbreds derived from H511, E11 relationship (r=0.35) between foliar and E12 or their progenies have no From this study it is clear that foliar damage, tunnel length below the ear, resistance to borer damage. These lines, damage and stalk tunneling are good and the larval/pupal density (Figs. 1 although of medium maturity, were indicators of resistance or and 2). Regression analysis also clearly attacked by second generation borers, susceptibility. Conversely, there are indicated that tunnel length increases as evidenced by data on the mean characters which, though singly of little considerably as rating increases. number of exit holes and mean tunnel importance, may contribute to reduce length above the ear. yields significantly when occurring in combination. For example when borer Rate 1.6 exit holes are coupled with stalk breakage due to weakened stems, there 1.5 is a high reduction in yield due to 1.4 reduced plant stand. 1.3 1.2 Thus, resistance sources are diverse, 1.1 varying by maturity, morphology, and genetic traits, as reported by Sharma 1.0 (1993). These sources can be adapted 0.6 0.8 1.0 1.2 1.4 Tunnel length 1.6 1.8 Figure 1. Relationship between foliar damage and tunnel length in maize under natural infestation with stem borers. per se or used in maize improvement in different regions. This means that a breeding program focusing on different ecozones is advantageous. Those materials that are known to possess moderate levels of stem borer Tunnel length 1.8 resistance could be used in breeding programs to generate better hybrids 1.6 which are heterotically superior, 1.4 removing those morphological and 1.2 genetical characters contributing to 1.0 susceptibility. However, these results need to be supported by challenging 0.8 the materials with artificial infestation 0.6 in the field. 0.7 0.8 0.9 chilo 1.0 1.1 Figure 2. Relationship between tunnel length and borer numbers in maize under natural infestation with stem borers. 282 M. GETHI Re fe re nce s Ampofo, J.K.O. 1986. Maize stalk borer (Lepidoptera: Pyralidae) damage and plant resistance. Environs. Entomol. 15: 24-1129. Anon. 1990. Agroecological zoning to maize research priorities in Kenya. In Proceedings of the Review of the National Maize Research Program, Kakamega, Kenya. Guthrie, W.D., Dickie, F.F., and Neiswander, C.R. 1960. Leaf and sheath feeding resistance to the European corn borer in eight inbred lines of dent lines of dent corn. Ohio Agric. Exp. Stn. Res. Bull. 860. Hill, D.S. 1975. Agricultural insect pest of the tropics and their control. Cambridge University Press Cambridge. Jepson, W.F. 1954. Critical review of the World Literature of the Lepidopterous stalk borers of the tropical graminaceous crops. London Common W. Inst. Ent. Omolo, E.O. 1983. Screening of local and exotic maize lines for stem-borer resistance with special reference to Chilo partellus. Insect Sci. Application 4 (1/2): 105-108. Scheltes, P. 1978. Ecological and physiological aspect of aestivation diapause in the larvae of two pyralid stalk-borers of maize in Kenya. Landbourwhoge School, Wageningen, The Netherlands. Seshu Reddy, K.V. 1983. Studies on the stem borer. Complex of sorghum in Kenya. Insect. Sci. Applic. 4: 3-10. Sharma, H.C. 1993. Host plant resistance in insects in sorghum and its role in integrated pest management. Crop Protection 12(1): 11-34. Warui, C.M., and Kuria J.N. 1983. Population incidence and the control of maize stalk borers Chilo partellus (Swinhoe) C. orichalcociliellus strand and Sesamia calamistis Namps in coastal Province, Kenya. Insect. Sci. Application 4: 11-18. Developing Rootw orm, Diabrotica virgifera zeae Krysan and Smith, Resistant M aize in M éxico J.F. Pérez Domínguez, J.B. Maya Lozano, INIFAP, Ocotlán, Jalisco, México. and J.A. Mihm, French Agric. Res. Inc., Lamberton, MN, USA. Abst r a c t The Mexican corn rootworm, (CRW) Diabrotica virgifera zeae, is one of the most important insect pests of maize in the Mexican “Corn Belt” - the Bajio region of central Mexico. Field evaluations are presented for resistance characteristics of S1, S2 and S3 maize lines derived from CIMMYT Population 593, selected for resistance to corn rootworms. The techniques used included: the use of a susceptible hybrid check planted at regular, repeated intervals throughout the screening nurseries; comparison of phenological development of maize in paired plots, with and without chemical protection against rootworms; degree and amount of root lodging; visual estimates of root pruning by CRW larvae; secondary root development; firmness of root anchoring, as measured by force required for vertical root pulling; and percentage of plants surviving CRW damage. Results are presented for two years of evaluation and selection for resistance. Lines selected in the 1993 summer screening nursery were planted for increase and improvement in a winter nursery. Of 16 materials selected for advancement, 8 were outstanding for rootworm resistance characteristics. In the 1994 summer nursery, advanced S3 lines were screened at two locations, where 25 and 15 lines were selected, respectively. Considering the resources and techniques available for screening, the resistance mechanisms we are seeking are antibiosis and tolerance. In the coming winter nursery we are planning to make test crosses using selected resistant lines crossed onto a susceptible population tester, as well as to advance lines to another cycle of inbreeding. Int roduct ion D. virgifera zeae is one of the principal species. In the case of maize, research root pests in 20 Mexican states, while D. has been done on fall armyworm Maize is grown in practically all longicornis has been reported in 6 states (Salazar 1991; Loera 1990), stem borers farming areas of Mexico, with the (Krysan and Smith 1987). and leafhoppers. As for resistance to rootworms and specifically to Diabrotica greatest production in the states of México, Jalisco, Sinaloa, Tamaulipas, Few studies in Mexico have focused on no research has been reported, other Puebla, Michoacan, Veracruz and host plant resistance to insect pests, and than studies conducted in Jalisco by Sonora. A considerable range of insect those have been conducted under the Pérez and Maya (1991). pests can cause maize production auspices of the National Institute for losses, but in the central part of the Research on Agriculture, Livestock and Consequently, we present our current country, root pests are among the most Forestry (INIFAP), the International research on corn rootworm (CRW) important. Among the species which Maize and Wheat Improvement Center resistant maize germplasm. Our inflict root damage are: rootworms, (CIMMYT), and several universities. objective is to identify sources of CRW resistance in maize and subsequently Diabrotica virgifera zeae and Diabrotica longicornis; white grubs Phyllophaga spp., Studies by Mexican scientists have incorporate desirable resistance traits Anomala spp. and Cyclocephala spp.; looked at natural insect populations into advanced maize lines with high wireworms Agriotes spp.; cutworms and have concentrated on maize, yield potential and good adaptation. Agrotis spp. and Colaspis spp. Of these, wheat, cotton, soybeans and other 284 J.F. PÉREZ DOMÍNGUEZ, J.B. MAYA LOZANO, AND J.A. MIHM M aterials and M ethods treated rows were compared with those 1994 in untreated rows. The S4 seeds resulting from selfing and crossing in Tlaltizapán were planted at All tests were conducted under natural infestation, since facilities for mass- When the crop reached the hard dough two locations in Jalisco: Sabino, rearing CRW larvae were not available. stage, plants were tested for firmness of municipality of Tototlán, on June 24, Sites with high egg and larval root anchoring, measured by the force and Jocotepec on July 6. One hundred infestations were selected. required for vertical root pulling. At twenty lines were evaluated in flowering, selected lines were selfed Jocotepec and 237 were evaluated in 1993 and pollinated and some crosses were Tototlán. Trial design and A screening nursery was established done among the same materials. The management, as well as testing (June to December) in Zapotitan, in the resulting lines were advanced to S3 in techniques, were similar to those used municipality of Jocotepec, Jalisco, in a CIMMYT’s winter nursery at in the 1993 experiment. During both field with a history of very high CRW Tlaltizapán, Morelos, under rootworm years, sampling was done in the test infestations. We screened 194 maize S1 free conditions. All materials were plots to gauge the size of Diabrotica lines from CIMMYT’s population 593, selfed and some crosses were carried larval populations. selected for rootworm resistance. out at this location. Planting was done on June 23 in a plot having two 2.5 m rows, with two seeds from each line sown every 20 cm. Table 1. Maize lines screened for corn rootworm resistance in Jocotepec, Jalisco, México 1993. Insecticide was applied to one row, while the other received no chemical treatment. The treated row received Root damage1 Pedigree live plants at the 8-10 leaf stage and Guat 166 x CO 289 Guat 189 ƒƒ3 200-6 x Guat 189 200-6 x Guat 189 200-6 x Guat 189 Guat 633 x CO 289 Agscal 6 x CO 272 Agscal 6 x CO 272 2 B68 Ht x Guat 165 Guat 166 x B68 Guat 189 x B68 200-1 x Guat 189 200-1 x Guat 189 200-1 x Guat 633 200-7 x Maíz San Andrés 200-7 x Maíz San Andrés 200-7 x Maíz San Andrés 200-7 x Guat 189 200-6 x Guat 189 Agscal 6 x CO 289 Agscal 6 x CO 289 Guat 166 x CO 272 Guat 189 ƒƒ1 Guat 189 ƒƒ2 Guat 189 ƒƒ5 B68 Ht2 x Guat 633 2 B68 Ht x Guat 166 Guat 166 x B68 Guat 166 x B68 Guat 166 x B68 Guat 189 x B68 again at the milk stage for comparative Check: H 355 two insecticide applications: a dose equivalent to 15 kg/ha was applied at planting and again with the second fertilization. In all cases the insecticide treatment consisted of granulated 5% isozofos mixed with fertilizer. One out of every four test plots included a susceptible hybrid (H-355) as a replicated check. The hybrid was planted in the same manner as the test lines. A few squash plants (Cucurbita pepo) were sown, at the beginning of the cycle, in each plot to stimulate the development of rootworm populations for the following cycle. Experimental plots received adequate protection against weeds and leaf insect pests, and tillage operations were carried out periodically. Test materials were evaluated twice, once for comparative growth, root lodging and number of growth, root damage, secondary root development and general appearance of the crop. For each variable, plants in 2 3 With Without 2 4 3 4 4 5 5 3 3 3 1 3 4 3 3 3 4 4 3 3 3 3 4 4 4 3 4 3 4 4 4 4 5 2 4 4 5 5 3 5 4 4 3 4 3 3 3 4 4 4 3 4 4 4 3 3 3 5 3 4 4 4 4.0 0.2 5.4 0.2 No. plants With 9 8 10 8 9 6 4 1 5 8 10 3 6 2 12 7 7 8 5 8 5 5 8 11 7 4 8 7 6 6 11 8.5 2.2 Without 7 10 5 8 7 6 5 8 9 6 9 10 9 7 6 9 10 10 12 6 3 7 3 6 10 2 6 7 6 3 10 8.1 2.2 Root lodging With Without 0 0 0 0 0 3 3 0 1 1 1 0 0 0 2 2 0 3 4 1 1 1 0 0 0 0 4 1 3 2 2 3 1 0 2 0 3 1 1 1 0 1 0 0 1 0 0 0 0 0 2 0 1 1 2 0 0 1 1 0 0 2 0.8 1.31 Column headings refer to results from rows with and without pesticide treatments. 1 Root damage evaluated on a 1-6 scale (Hills and Peters 1971). 2 Average of 47 check plots. 3 Standard deviation of check. 2.2 2.2 DEVELOPING ROOTWORM, DIABROTICA VIRGIFERA ZEAE KRYSAN AND SMITH, RESISTANT MAIZE IN MÉXICO 285 averaged 4 larvae per plant, compared to the normally high variation in with an average of 5 larvae per plant in natural rootworm populations. Testing Rootworm damage was found in all Jocotepec during the period of heaviest techniques were those proposed by materials planted in the three trials infestation. Campbell (1989) and Branson and Results and Discussion Sutter (1989). conducted to date. In 1993, 16 lines were selected, along with 15 others, Inclusion of a susceptible hybrid as a that showed good traits for potential replicated check allowed us to study Selected materials have advanced in resistance (Table 1). Selection was the variation of pest populations the breeding process and are being based on the results of all tested distributed throughout the study area. crossed with CIMMYT’s Population variables. Lines selected in 1994 will be Root damage assessments have shown 390 MIRT to find potential sources of included in future tests. The level of that some selected materials have large, multiple insect resistance. However, corn rootworm infestation was, on vigorous root systems with lots of since the data obtained so far do not average, 8.5 larvae per plant in the secondary roots, while others have root indicate a definitive source of heaviest infestations, which allowed systems that are not very large but resistance, these results should be satisfactory evaluation. develop abundant secondary roots after considered preliminary. being damaged by rootworms. During In the 1994 cycle, 25 lines were selected the two years of trials, selected in Tototlán and 15 in Jocotepec (Tables materials typically responded to 2 and 3) . Even when root damage was rootworm damage by rapidly forming severe, as it was in some cases due to an abundance of new secondary roots. Re fe re nce s the intensity of the attack, resistance traits were observed. Most of these Throughout the study, all variables and selected materials had been crossed testing techniques utilized were given with the S2 lines from Tlaltizapán. Corn equal weight to ensure more reliable rootworm incidence in Tototlán results, since evaluations were subject Branson, T.F., and G.R. Sutter. 1989. Evaluating and breeding for maize resistance to the rootworm complex. In Toward Insect Resistant Maize for the Third World: Proceedings of the International Symposium on Methodologies for Developing Host Plant Resistance to Maize Insects, 130-139. México, D.F.: CIMMYT. Table 2. Maize lines screened for corn rootworm resistance in Tototlán, Jalisco, México 1993. Root damage Pedigree 200-7 x Maíz San Andrés 200-7 x Maíz San Andrés 200-7 x Maíz San Andrés 200-6 x Guat 189 200-6 x Guat 189 200-6 x Guat 189 200-6 x Guat 189 Guat-166 x B68 Guat-166 x B68 (200-1xGuat 189) x (68-3-1) (200-3 x Guat-189) x (20 x 244-1) (200-7 x Maíz San Andrés) x (68-3-1) (200-7 x Maíz San Andrés) x (70-1-1) (200-7 x Maíz San Andrés) x (51-2-1) (200-7 x Maíz San Andrés) x (125-2-2) (200-6 x Guat 189) x (408-3-1) Check: H 355 3 4 1 No. plants Root lodging Plant height With Without With Without With Without With Without 3 3 4 3 4 5 4 3 3 4 3 3 3 4 4 3 4 3 4 5 4 5 4 4 4 4 4 5 2 4 3 3 6 7 2 14 5 7 15 6 12 4 12 4 7 5 6 8 11 6 2 10 5 5 11 4 3 4 11 4 6 4 4 8 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 2 0 1 0 0 0 0 0 0 0 0 0 0 0 0 1 0 1.95 1.90 1.65 2.00 2.00 2.00 2.05 1.95 2.35 2.50 2.15 2.25 2.65 2.40 2.10 2.25 1.60 1.85 1.60 1.95 1.85 2.05 1.80 1.75 2.60 2.35 2.25 2.20 2.45 2.35 1.90 1.70 3.6 0.23 4.8 0.26 16.2 2.66 13.9 2.7 0.2 0.94 0.3 0.83 2.21 0.25 2.18 0.26 Column headings refer to results from rows with and without pesticide treatments. Plant height is in meters. 1 Root damage evaluated on a 1-6 scale (Hills and Peters 1971). 2 These were selected at the two 1994 test sites. 3 Average of 57 check plots. 4 Standard deviation of check. 286 J.F. PÉREZ DOMÍNGUEZ, J.B. MAYA LOZANO, AND J.A. MIHM Campbell, J.E. 1989. Corn rootworm rearing methodologies. In Toward Insect Resistant Maize for the Third World: Proceedings of the International Symposium on Methodologies for Developing Host Plant Resistance to Maize Insects, 60-66. México, D.F.: CIMMYT. Hills, T.M., and D.C. Peters. 1971. A method of evaluating post planting insecticide treatments for control of western corn rootworm. J. Econ. Entom. 64: 764-765. Krysan, J.L., and R.F. Smith. 1987. Systematics of the virgifera species group of Diabrotica (Chrysomelidae: Galerucinae). Entomography 5: 375-484. Loera, G.,J. 1990. Resistencia de maices a gusano cogollero Spodoptera frugiperda Smith (Lepidoptera: Noctuidae). In Memorias XXV Congreso Nacional de Entomología. Sociedad Mexicana de Entomología, 442. Oaxaca, Oaxaca, México. Pérez, D.,J.F., and J.B. Maya L. 1991. Evaluación de poblaciones de maíz para resistencia al complejo de plagas de la raíz en el centro de Jalisco. In Memorias III Simposio sobre plantas resistentes a insectos, 13-26. Veracruz, México.: Sociedad Mexicana de Entomología. Salazar, P.,A. 1991. Estudio sobre resistencia de plantas a insectos en el estado de Morelos. In Memorias III Simposio sobre plantas resistentes a insectos, 5-12. Veracruz, Veracruz, México.: Sociedad Mexicana de Entomología. Table 3. Maize lines screened for corn rootworm resistance in Jocotepec, Jalisco, México 1994. 1 Root damage 200-3 x Guat 189 200-7 x Guat 633 200-7 x Maíz San Andrés 200-7 x Maíz San Andrés 200-6 x Guat 189 200-6 x Guat 189 200-6 x Guat 189 200-6 x Guat 189 200-6 x Guat 189 Agscal 6 x Co 272 (200-7x Guat 633) x (232-3-1) (200-7x Guat 633) x (406-2-1) (200-7x Guat 633) x (64-1-1) (200-7x Guat 633) x (232-3-1) (200-7x Guat 633) x (406-2-1) (200-7x Maíz San Andrés) x (45-1-1) (200-7x Maíz San Andrés) x (232-3-1) (200-7x Maíz San Andrés) x (20x 217)-1 (200-7x Maíz San Andrés) x (70-1-1) (200-7x Maíz San Andrés) x (70-2-1) (200-7x Maíz San Andrés) x (51-2-1) (200-7x Maíz San Andrés) x (45-1-1) (200-7x Maíz San Andrés) x (70-1-1) (Guat 189 x 1368) x (232-3-1) Check: H 355 2 3 No. plants Root lodging Plant height With Without With Without With Without With Without 3 5 4 4 4 3 4 2 4 4 4 3 5 5 3 5 4 5 4 4 5 4 4 4 5 4 5 4 4 4 4 3 4 6 4 3 4 4 4 5 3 3 4 4 4 4 4 4 15 4 6 4 6 12 12 13 8 9 2 12 5 13 18 12 12 14 12 12 15 8 9 12 14 6 10 5 9 11 11 13 4 8 3 10 5 12 16 8 13 12 12 11 13 7 13 6 8 0 5 0 0 9 4 0 1 0 0 1 0 5 3 8 0 9 4 0 9 0 2 7 11 0 8 0 1 6 3 0 0 1 0 2 3 2 3 3 3 2 7 0 0 1 1 0 2.10 2.10 2.00 1.85 2.35 2.00 2.10 1.80 1.85 1.60 2.35 2.30 2.65 2.35 2.65 2.65 2.50 2.65 2.45 2.60 2.90 2.70 2.90 2.45 2.40 1.90 1.70 1.85 2.35 1.95 2.05 2.00 1.55 1.45 1.90 2.35 2.60 2.05 2.85 2.65 2.10 2.50 2.30 2.50 2.75 2.50 2.55 2.50 2.91 4.01 3.0 2.37 2.79 0.24 2.80 0.25 3.8 0.3 4.9 0.28 13.09 2.31 11.0 2.48 Column headings refer to results from rows with and without pesticide treatments. 1 Root damage evaluated on a 1-6 scale (Hills and Peters 1971). 2 Average of 47 check plots. 3 Standard deviation of check. Selection M ethodology for Resistance to Dalbulus maidis and Fine Stripe Virus Disease in M aize in Peru P.H. Injante Silva, and J. Lescano Muñoz, National Institute of Agricultural Research (INIA), Cajamarca, Peru Abst r a c t This paper describes the methods used in the INIA Maize Research Program to obtain and maintain mass colonies of Dalbulus maidis, and in the near future to improve resistance to Maize Fine Stripe Virus. The following steps were followed to achieve these objectives: 1) collection, identification and mass rearing of D. maidis; 2) greenhouse cultivation of a population of high-altitude maize (Peruvian Complexes), and subsequent inoculation with the virus; 3) transplanting into the field; 4) ELISA serological testing; 5) selfing of families showing tolerance and/or resistance to the virus; and 6) new potential sources of resistance in the Peruvian populations were identified through this approach. Int roduct ion tolerant and/or resistant to the virus or M e t hodology its vector is an efficient method of Maize is one of the principal sources of controlling the disease. The release of food in Peru, grown on some 400,000 resistant varieties is the best option Mass rearing of the fine stripe vector in greenhouses ha. However, yields are low (1.2 t/ha), which researchers can provide to Collection and multiplication of the due mainly to inadequate technology, farmers. Developing a workable vector - Formulating a mass-rearing diseases and pests. Maize fine stripe method of mass-rearing the vector in technique required the collection of the virus is one of the most serious captivity permitted us to make this vector in valleys which experienced the diseases, transmitted by the leafhopper alternative a reality. greatest incidence of fine stripe virus in D. maidis, which is common to the recent years. Using Inter-Andean valleys of Peru (Fig. 1a) suction tubes and insect (Sarmiento et al. 1992). The INIA Maize collection jars, the vector Research Program (MRP) recognizes was captured from maize that the use of materials which are plants showing virus symptoms (Fig. 1b). The collected insects were taken to the entomology laboratory at the National University of Cajamarca, where an average of 150 adult insects were identified and sexed. They were then taken to the MRP rearing laboratory and 288 P.H. INJANTE SILVA, AND J. LESCANO MUÑOZ placed in wooden rearing boxes (1 x 0.5 varieties and soil similar to that Greenhouse planting of materials - The x 0.5 m) lined with anti-aphid mesh described above. These larger boxes MRP began planting 254 families of screen (Fig. 2), thus providing adequate were maintained at 24-26ºC, and a Population IV canchero tardío in conditions for their development relative humidity of 70%. The insects greenhouses, sowing ten seeds per (Dabrowski 1989). remained there for 40 days, the family in plastic bags containing 1 kg of duration of the biological cycle of the soil (Fig. 4). Each family was placed in Maize plants of the susceptible variety insect. Asymptomatic plants were closed wooden boxes (1.2 x 0.5 x 0.35 Blanco Urubamba were placed in the removed from the cages in order to m) with Saran screen mesh and glass. boxes. The plants were sown in plastics obtain a high percentage of diseased Planting was staggered over time to pots containing a soil mixture of 2:1:1 plants and infected insects, and permit placement of insects in each box earth:sand:moss. The first virus material which was biologically pure. (30 insects per family). symptoms were observed 10 days after This method guaranteed a population feeding by the Dalbulus, and were of approximately 20,000 insect vectors When the plants reached an average confirmed through ELISA testing. in each cycle, in cages of (3.0 x 1.2 x height of 10 cm, they were infested 1.2 m) (Fig. 3). with the insect vector for a period of 6 The infected vectors were subsequently days, adequate time to ensure transferred to larger wooden cages (3.0 transmission of the virus (Fig. 6) Once x 1.2 x 1.2 m) lined with heavy plastic and glass windows, containing maize SELECTION METHODOLOGY FOR RESISTANCE TO DALBULUS MAIDIS AND FINE STRIPE VIRUS DISEASE IN MAIZE IN PERU 289 this was completed, the maize plants ELISA serological test from each family were ready to be Asymptomatic materials, at the pre- transplanted to the field (Fig. 7). At this flowering phase, were subjected to The following results were achieved point, it was important to apply a serological tests at the National under the experimental conditions: systematic insecticide to the inoculated University of Cajamarca. The best • material to eliminate the insects and plants from the best families showing causing maize streak virus is D. propagate the virus, before replicating tolerance and/or resistance were self- maidis, common in the inter-Andean the plants in the experimental station pollinated and planted in the next cycle valleys of Peru. fields. of selection. Field stage Once the fields were in optimal condition for plant development, the families were transferred to the field in plastic strips; the plastic was removed and the plants were carefully placed in the bottom of the furrow with a distance of 0.25 m between each plant (Figs. 8 and 9). Results and Discussion Confirmation that the insect vector 290 • • • P.H. INJANTE SILVA, AND J. LESCANO MUÑOZ Symptoms develop 2 weeks after follows: (1992-93 entries, listed as infestation, with young plants being family (row) number - plant number the most affected. (within each row)) 1, 2, 3, 4-2, 5, 7, 8, To Dr. John Mihm and to Luís Narro, ELISA proved to be the most 9, 9-1, 10, 11, 12, 13, 14, 15, 16, 17, 19, CIMMYT Maize Program, for their effective serological test for 23-1, 28-3, 30, 32, 33, 37, 40-1, 42, 43, constant support. To the staff of the detection of maize fine stripe virus. 47, 50, 55, 63-6, 68, 73-2, 74, 75, 76, Regional Maize Program, Peru, for Of the total number of experimental 78, 94, 98, 99-1, 99-2, 102-2, 104, 104- their invaluable help in carrying out samples collected from 1, 107, 116-1, 116-2, 116-3, 122, 122-1, the present work, and to Ing. Gonzales asymptomatic plants, 84% were 124, 125-2, 125-4, 127, 182-1, 128-5, Muñoz for his insightful suggestions. positive. 130, 136-2, 140, 145, 148, 148-4, 148-5, A high percentage of serologically 149, 150, 150-4, 165, 166-1, 173-1, positive plants developed normally 173-3, 173-6, 173-7, 182, 190, 195-1, and produced ears. The susceptible 206, 227, 228, 243, and 253. families were heavily affected; most • • In the second selection cycle, the ten failed to achieve normal growth and most susceptible families were did not produce ears. identified in the greenhouse, as Eighty ears were harvested and follows: 9-1, 23-1, 28-3, 48, 104, 145, identified from virus-infected and 149, 190, 206, and 227. These were non-infected plants, one from each confirmed by comparison with plant and up to four from a family. materials planted in the field, which failed to produce any ears. Conclusions • 27 families were selected which are currently being screened in the field: The experiment resulted in the 1, 2, 3, 4, 7, 8, 9, 10, 11, 12, 13, 14, 15, following conclusions: 16, 17, 19, 27, 30. 32, 33, 43, 47, 55, 65, • 68, 74, and 76. In the first cycle, 80 ears were selected from 56 families as being the most tolerant to the virus, as Acknow le dgm e nt s Re fe re nce s Dabrowski, Z.T. 1989. Procedures and techniques for rearing Cicadulina leafhoppers. In Toward Insect Resistant Maize for the Third World: Proceedings of the International Symposium on Methodologies for Developing Host Plant Resistance to Maize Insects, 84-93. México, D.F.: CIMMYT. Sarmiento, J.; G. Sanchez; y J. Herrera. 1992. Plagas de los Cultivos de Caña de Azúcar, Maíz y Arroz. Universidad Nacional Agraria La Molina, Lima, Perú. M ass Rearing of Helicoverpa zeae in Peru P.H. Injante, National Institute of Agricultural Research (INIA), Cajamarca, Peru Abst r a c t The Maize Research Program (MRP), INIA-Peru, has successfully raised and maintained colonies of corn earworms, Helicoverpa zeae (Boddie). With technical assistance from the International Maize and Wheat Improvement Center (CIMMYT), our laboratory currently can produce large quantities of this species to infest high-altitude maize varieties, facilitating the selection and breeding process for corn earworm (CEW) resistance. Int roduct ion M e t hodology Discussion The cosmopolitan species Helicoverpa The rearing and efficient field • zeae attacks more than 68 species of infestation techniques used are similar host plants (Vela and Quispe 1988), to CIMMYT’s. These techniques were belonging to 26 different families (Paliz initiated last year. Previous efforts best materials will be re-planted and and Mendoza 1985). The insect species failed due to inadequate diets for selected. is distributed throughout all maize- Helicoverpa, and consequently a growing regions, although it has a contamination of the samples. Once a with bazookas in the screening of higher incidence in the inter-Andean reliable source of insects was obtained, high-altitude maize populations. valleys of Peru where the largest artificial infestations with CEW were cultivation areas are found of the more carried out on materials introduced susceptible sweet and waxy kernel from Mexico, which showed resistance maize varieties. there. These first infestations were done manually using camel-hair The most recent advances for brushes, a method which has been used combating CEW rely on integrated for more than 40 years (Blanchard et al. control measures, including the use of 1942), but which is extremely time- and resistant varieties, to maintain insect labor-intensive. The innovation of populations below economic threshold using manual “bazookas” will simplify levels. To confront this challenge, the future infestations in Peruvian high- MRP began mass-rearing of CEW altitude maize populations. under CIMMYT guidance, adapting the latest techniques and selection methods Re sult s for maize breeding. • Materials provided by CIMMYT (197 families) were infested, with encouraging results obtained in 32 of the families. • Large populations of Helicoverpa can be raised using the meridic diet. Thirty-two materials showed superior resistance (Table 1). • • In the current selection cycle, the We will initiate CEW infestation Re fe re nce s Blanchard, R.A., A.F. Satterwait, and R.O. Snelling. 1942. Manual infestation of corn strains as a method of determining differential earworm damage. J. Econ. Entomol. 35: 508-511. Paliz, V. and J. Mendoza. 1985. Plagas del Maíz en el Litorial Ecuatoriano. Vela, A. and L. Quispe. 1988. Plagas de Papa y Maíz. Universidad Nacional de Cajamarca. 292 P.H. INJANTE Table 1. CIMMYT experimental maize varieties tested at the the La Victoria experiment station, National University of Cajamarca, Peru (1993-94). Entry 2 11 12 14 16 18 19 20 21 23 24 28 30 37 77 85 89 90 95 99 106 116 131 132 133 134 135 148 176 182 186 194 Parentage Pedigree Ba-92 ( 2501 X 2501 ) F4 ( 20 X 83 ) 4 283-4 F18 ( 60 X 127 ) - 3 297-3 F18 ( 60 X 127 ) - 5 297-5 F23 ( 9 5X 74 ) - 1 302-1 F27 ( 175 X 163 ) - 1 305-1 F267( 175 X 163 ) -3 305-3 F27 ( 175 X 163 ) - 4 305-4 F27 ( 175 X 163 ) - 5 305-5 F27 ( 175 X 163 ) - 6 305-6 (2501 X 2517 ) F31 (21X102)-2 310-2 ( 2501 X 2517 ) F31 ( 21 X 102 )-4 310-4 F40 ( 49 X 201 )-1 319-1 F43 ( 54 X 100 )-3 322-3 F49 ( 121 X 145 )-3 328-3 ( 144 X 109 )-1-2-1- # 41 # F10 46X28 ( 2503 X 2503 ) F3 ( 100X1 )-4 225-4 Linea S1 230-1 F26 ( 38 x 73 )-1 248-1 F30 ( 57 X 105 )-4 252-4 F46( 228 X 87 )-4 268-4 F16 ( 27 X 25 )-1-1 201 F2 4X3 F3 10X5 F4 11X23 F6 33X27 F11 93X87 F35 170X163 ( 119 X 129 )-5-2-3- # 77 # ( 2504 X 2504 ) F1 cruzas 4X32 F27 69X80 F43 103X63 Ear length (cm) 10.00 8.00 9.00 8.00 10.00 11.00 9.00 12.00 10.00 9.00 11.00 7.00 4.50 8.00 9.50 9.00 8.00 9.00 8.00 11.00 10.00 5.00 12.00 10.00 12.00 11.00 9.80 9.30 11.10 9.10 8.70 11.50 Damage (cm from tip) 4.00 5.00 5.00 2.00 3.00 4.00 4.00 5.00 5.00 3.00 3.00 3.00 3.00 4.00 3.00 3.00 4.50 5.00 4.00 4.00 2.50 3.00 5.00 5.00 6.00 4.00 4.80 4.50 3.90 4.00 4.10 5.90 Selection I I I R I I I I I I I I I I I I I I I I R I I I I I I I I I I I R = Resistant plants. I = Plants of intermediate resistance. Of the 197 families of CIMMYT maize, 32 showed superior resistance to Helicoverpa zea; the same families showed resistance in trials at CIMMYT, Mexico, 1992-93. (Blanchard et al. 1942). Progress of Host Plant Resistance Research to the Asiatic Corn Borer in the Philippines E.C. Fernandez, and D.M. Legacion, Institute of Plant Breeding, Laguna 4031, Philippines Abst r a c t The Asiatic Corn Borer (ACB), Ostrinia furnacalis (Guenee), remains the most serious insect pest of maize in the Philippines and parts of Tropical Asia. Advances in ACB resistance work have been obtained through an increase in information and materials, that have served as bases for future activities. Several hybrid varieties with resistance or tolerance to ACB were developed and released from 1992 to 1993. Possible genetic differentiation was identified in the local populations of ACB. Collaborative work with CIMMYT-ARMP was started in 1990 on the development of Asian Multiple Borer Resistant populations of maize. Int roduct ion During the symposium held in March 1987 at CIMMYT, Mexico, with the Information and M aterials Generated The Asiatic Corn Borer (ACB), Ostrinia theme “Towards Insect Resistant Maize furnacalis (Guenee), remains the most for the Third World”, Lit et al. (1989) According to Salazar and Legacion serious insect pest of maize in the presented the status of research (1991), past studies indicate that there is Philippines and some parts of the activities on host plant resistance to still genetic variation to be exploited in Tropical Asia. In commercial ACB in the Philippines. This breeding for resistance to ACB damage. production, the use of chemicals to presentation covered the following So, what is needed is a greater control this pest is recommended. areas: understanding of the mechanisms of However, this is seldom practiced by • • • Biology of the ACB. resistance, coupled with more effective Techniques for ACB mass rearing. selection procedures. small-scale farmers due to the high cost of the pesticides and also because of their increasing awareness of the hazardous effect of these chemicals to human life, non-target organisms and the environment. In recent years, • • • Infestation and evaluation procedures. The accomplishments achieved in the Sources of resistance. work for ACB resistance during the Breeding methodologies. early 1990s, as summarized by Salazar Mechanisms of resistance. and Legacion (1991), are in the form of information and genetic materials that farmers are learning to appreciate and use crop varieties with built-in At present, breeding for resistance to serve as a foundation for future resistance to insect pests. In the corn borer remains a high priority in the research activity: Philippines, the establishment of the over-all maize breeding program of the Institute of Plant Breeding (IPB) in 1975 IPB. This paper presents the progress of • • helped advance the growing awareness ACB resistance work in the Philippines corn borer damage are not resistant of host plant resistance, as an approach since the last symposium. Most of the to post-tasseling damage. to pest population regulation and work was done at the Institute of Plant management (Lit et al. 1987). Breeding in collaboration with other of resistance to pre-tasseling corn Units/Institutions. borer damage. • Information obtained Materials resistant to pre-tasseling Antigua Grupo I is a reliable source 294 • • • E.C. FERNANDEZ AND D.M. LEGACION Heavy fertilization favors corn borer of Dr. Legacion to assess the limitations to the results obtained and damage. performance of identified resistant further studies are needed. If there is ACB is more severe during the wet materials against the three populations indeed local differentiation, the season, especially in late plantings. of corn borer: Laguna, VISCA and USM question is raised as to which DIMBOA was positively correlated in Mindanao. Furthermore, it was ecological variable(s) is responsible for to pre-tasseling corn borer damage, aimed at determining whether local the population differentiation? but not to post-tasseling borer population differences existed. damage. • • Varie t ie s De ve lope d Plants with erect leaves tend to Preliminary results from exhibit less borer egg mass electrophoretic studies of population The progress and success of any deposition. structure and population breeding program is measured in terms In a pre-tasseling corn borer differentiation, within the Philippine of the final output - a variety. To fully resistant (CBR) composite corn borer species (Mendoza et al. appreciate the status and progress of population, significant additive 1992), showed that Laguna and USM host plant resistance activity to ACB in genetic variance was found populations had 5 alleles while VISCA the Philippines, the list of corn varieties suggesting progress from recurrent had 6. Allele y was only observed in developed by IPB and approved by the selection. the VISCA population. Laguna and Philippine Seed Board from 1990 to USM were more variable than VISCA, 1993 are presented in Table 2. Genetic materials available due to higher heterozygosity values. • A CBR composite population, made Significant heterogeneity was observed IPB Var 5 a varietal hybrid between IPB up of 14 populations previously among the populations. However, Var 1 x Suwan 2 was released in 1990. found to be resistant to pre-tasseling when specific comparisons were made This was the first commercial varietal borer damage. the Laguna population was hybrid released by the public sector in Inbred lines which have undergone significantly different from VISCA and the Philippines. Another varietal a general combining ability (GCA) USM population, but the latter two hybrid, IPB Var 4 (IPB Var 2 x Antigua test, extracted from superior families were not different (Table 1). The results GPo1) followed in 1991. No indication, of CBR. suggest local genetic differentiation however, was reported regarding their Crosses of CBR populations with an among the different populations of the performance against pests, particularly elite breeding population. borer. Of the three, the Laguna the corn borer O. furnacalis. In 1992, a population seemed to be the most yellow corn hybrid named IPB 913 was differentiated. However, the developed with a moderately resistant investigators believed that there were reaction to ACB and earworm. Three • • Biological and Biochemical Studies on ACB Populations A study on the biological and Table 2. Corn varieties developed at IPB and approved by the Philippine Seed Board from 1990 to 1993. biochemical aspects of ACB populations was initiated by the group Table 1. Homogeneity tests among the gene frequencies between the three local populations of the corn borer, Ostrinia furnacalis (Guenee). a Population Los Baños /location Laguna VISCA Los Baños Laguna VISCA USM Mindanao a - 13.34 - ** USM Mindanao * Variety name Year released Type Yield (t/ha) Reaction to pests IPB Var 5 1990 (IPB Var 1 x Suwan 2) Improved Macapuno 1991 (fresh) IPB Var 4 1991 (IPB Var 2 x Antigua GPo 1) IPB 913 1992 Yellow Hybrid - - Glutinous White 6.26 - Yellow Hybrid 4.89 - Yellow Hybrid 6.58 PSB Cn 1993 93-49 (DLU Sweet) IPB Var 7 1993 IPB 919 1993 IPB 921 1993 IPB 929 1993 Glutimous White O.P Yellow O.P Yellow Hybrid Yellow Hybrid Yellow Hybrid 6.10 moderately resistant to ACB and earworm susceptible to ACB 5.57 6.35 6.89 7.01 some resistance to DM tolerant to ACB, resistant to DM tolerant to ACB, resistant to DM tolerant to ACB, resistant to DM 5.226 2.671ns - From the report of Mendoza et al. (1992). PROGRESS OF HOST PLANT RESISTANCE RESEARCH TO THE ASIATIC CORN BORER IN THE PHILIPPINES 295 more commercial hybrids: IPB 919, IPB borers most prevalent in Asia and selections included Mbita 86 MBR Chilo 921 and IPB 929 were developed in Southeast Asia (Granados 1994). The (Yellow), MBR 86 Across borers, Across 1993. Although these hybrids were not Entomology Laboratory of the IPB, 8432, CBR-1, MBR 86 Stars and purposely developed for corn borer University of the Philippines, Los Diamonds and Pop. 24 bulk (Table 3). resistance, all turned out to have high Baños was identified as one of the three According to Granados (1994), MBR- level of tolerance to ACB. It is worth original collaborators. This was due to SCB Res. EV (yellow), Population 24 mentioning also, as shown Table 2, that the fact that O. furnacalis is being and MBR 86 Stars and Diamonds were yield level increased as new hybrids successfully reared at IPB (Rangdang also found to be resistant to Chilo were developed. However, all these 1971; Hirai and Legacion 1985) for partellus in India. These three materials, hybrid varieties bearing resistance or artificial infestation of test materials. however, are very susceptible to downy mildew. tolerance to ACB were for industrial purposes. We are yet to see a variety In 1990, 25 cultivars were screened for with resistance or tolerance to ACB that ACB resistance. These included 11 Collaborative work in 1991-92 was is utilized as “green corn”. DMR materials, 6 borer tolerant concentrated on the evaluation of the varieties from CIMMYT’s MBR derived EVs and inbred lines that population, 5 borer tolerant varieties CIMMYT’s resident entomology Collaborative Work from the Philippines, 5 EV’s from program had generated from the MBR The CIMMYT-Asian Regional Maize CIMMYT’s population 28, 30, 32, and (Population 590) and MIRT (Population Program (ARMP), initiated in 1990, is a 36, and bulks of populations 24 and 26. 390). A number of materials were regional project for the development of The results showed that MBR-SCB Res. identified as intermediate in their maize populations resistant to downy EV (yellow) had the lowest leaf feeding tolerance to O. furnacalis (Table 4). mildew and tolerant to the species of damage (1.8). The other resistant At present, the focus of the collaborative work with CIMMYT- Table 3. Reaction of 25 corn materials artificially infested with larvae of Ostrinia furnacalis at IPB, Summer 1990. Entry No. Description 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 a Multiple Corn Borer Tolerant, Downy feeding Leaf % a damage of check CBR-1 Pop. 26 Bulk Philippines 06 Philippines 17 MBR 86 Stars and Diamonds MBR-SCB Res. EV (Yellow) MBR 86 Across borers Mbita 86 MBR Chilo (Yellow) EY-DMR Pool C3 HS bulk LY-DMR Pool C3 HS bulk Across 8336 Poza Rica 8336 Phil. DMR Comp. 1 Pop. 28 DMR C3 HS bulk Suwan 85 28 Pop. 24 Bulk Pop. 31 DMR C4 HS bulk Improved Tiniguib Mbita 86 MBR Chilo (White) MBR-SCB Res. EV (White) EW-DMR Pool C3 HS LW-DMR Pool C3 HS Tiniguib Synthetic Across 8432 Poza Rica 8530 Phil. Super Sweet (suscept. Check) 2.6 3.4 3.5 3.0 2.6 1.8 2.4 2.3 3.4 3.8 3.3 3.0 3.2 2.8 2.9 4.7 2.7 2.9 3.7 3.1 3.6 2.5 4.1 5.2 50.0 65.3 67.3 57.6 50.0 34.6 46.1 44.2 65.2 73.0 63.4 57.6 57.1 53.8 55.7 90.3 51.9 55.7 71.1 59.6 69.2 48.0 78.0 100.0 Mean 3.1 59.3 Scale Rating: 1-9. ARMP is on the development of Asian Mildew Resistant (AMBT-DMR) Early Table 4. Reaction of the materials from Pop. 590 (MBR) and Pop. 390 (MIRT) artificially infested with larvae of Ostrinia furnacalis (Guenee). IPB, Los Baños, 1991-92 trial. Entry No. Description Leaf feeding damage a Population 590 (MBR) 1 Across 86590 (IR) 2 Across 86590-2 (ECB) 3 Poza Rica 86590 (SCB) 4 Mbita 86590 (Chilo) 5 Tlaltizapan 85590 6 CML 135/CML 139 7 CML 135/CML 67 8 Ki3/CML 131 9 MBR HT Local check (Susceptible) 3.5 3.8 3.5 4.1 4.1 3.9 3.8 5.2 4.2 4.8 Population 390 (MIRT) 1 Across 90390-W (IR) 2 Across 90390-Y (IR) 3 SCB-GCA 4 FAW-GCA 5 Ki3/CML 139 6 CML 69/Ki3 Local Check (Susceptible) 3.6 3.6 3.6 3.8 3.8 4.0 4.8 a Scale Rating: 1-9. 296 E.C. E.C.FF ERNANDEZ ERNANDEZ , AND ANDD.M. D.M.LLEGACION EGACION White, Early Yellow, and Late Yellow towards which the resistant varieties populations. The IPB, Los Baños, was developed to date may behave designated the primary location for the differently. Re fe re nce s Hirai, Y., and D.M. Legacion. 1985. Improvement of the mass rearing techniques for the ACB, Ostrinia furnacalis (Guenee) in the Philippines. JARQ 19(3): 224-233. Granados, G. 1994. Status of cornborer research conducted in collaboration with India, Philippines and Taiwan. Progress Report 1990-1993. Lit,M.C., C.B. Adalla, and M.M. Lantin. 1987. Host Plant resistance to the Asiatic Corn Borer, Ostrinia furnacalis, in the Philippines. In Towards Insect Resistant Maize for the Third World: Proceeding of the International Symposium on Methodologies for Developing Host Plant Resistance to Maize Insects, 277-280. Mexico D.F.: CIMMYT. Mendoza, E.M.T., C. Demayo, and D.M. Legacion. 1992. Electrophoretic studies of population structure and population differentiation within the Philippine cornborer species, Ostrinia furnacalis. Unpublished Study report (Study 4) of the project Biological and Biochemical Studies of ACB Populations in the Philippines. Rangdang, Y. 1971. Artificial media and rearing techniques for the corn stem borer, Ostrinia salentialis (Snel.). In Proc. Seven Inter Asian Corn Improvement Workshop, 116-123. Los Baños, Philippines. Salazar, A.M., and D.M. Legacion. 1991. Breeding for Resistance to Corn Borer, Phase II. Unpublished Research Proposal for Local funding. development of Early White-DMRBorer Resistant populations. Table 5 Several years ago Lit et al. (1987) show the material composition of the mentioned that, while efforts on field three populations being developed. screening are modestly supported, funds for basic research have been very In addition to the above, further limited. The situation remains the same evaluation of the IPB selected ACB today or even worse. Despite this resistant populations are continuing. limitation, we recognize the need for a The materials currently being advanced continuing effort to develop new to develop better resistant lines, that varieties with a better and higher level may be of value to the breeders of resistance to ACB. Likewise, there is particularly for the hybrid program, are a need to continue the work on shown in Table 6. Materials from the determining the extent of population breeding group are also being field differentiation of the Philippine ACB. evaluated for ACB resistance. Further work must also be put in place to establish how the developed Looking to the Future resistant varieties, and other resistant materials, will respond to these Despite the gains we have attained in differentiated ACB populations if their the last few years through the release of existence is confirmed. varieties with built-in resistance or tolerance to ACB, there is no Ac know le dgm e nt s complacency in our efforts to effectively manage this pest. There are Thanks to Dr. A.M. Salazar, Asst. indications that the insect has Professor, IPB, UP Los Baños for the differentiated into several populations, comments and suggestions on this paper. Table 5. Composition of the three populations being developed for Asian Multiple Borer Tolerant-Downy Mildew Resistant (AMBT-DMR). Population Description Material source description 1 AMBT-DMR Early White AMBT-DMR Early Yellow a) Pop 100 EW-DMR S2 Bulk b) EEW-DMR Pool FS a) Pop 31 DMR S2 Bulk b) Viemyt 49 (Y) S2 Bulk c) Pop 145 EY-DMR Pool S2 Bulk d) EY TAK-FA HS e) EEY DMR Pool FS a) Pop 345 LY-DMR S2 Bulk b) Pop 28 DMR C6 S2 Bulk 2 3 AMBT-DMR Late Yellow LY TAK-FA HS Across 90390 W (IR) Across 86590 (IR) FAW - GCA Table 6. IPB selected populations continually evaluated for ACB a resistance. Population 1. XV3 2. Antigua Grupo I 3. IPB Var 1 4. S3 (9PG-238) 5. S4 (YOF-62 6. MIRT I 7. MIRT II 8. Other germplasm a Number of lines Generation 132 120 84 52 8 21 15 36 S6 S5 S6 S5 S6 S4 S4 S2 Materials available as of June 1994. Tw o Experimental M aize Varieties Selected for Resistance to Fall Armyworm and Sugarcane Borer in Tabasco, M exico Obdulia L. Segura-León, Graduate School of Agriculture, Tabasco Campus, Tabasco, Mexico Int roduct ion A heavy infestation of FAW was in mid-July 1994 following the station’s detected in subsistence maize crops in recommended agronomic practices. Stalk borers and fall armyworms farmers’ fields during the second half Seeds were treated with Furadan- (FAW), Spodoptera frugiperda (J.E. of May, 1994, in the municipality of thiram prior to planting to avoid Smith), are the principal causes of Cardenas, Tabasco, Mexico, when the damage by soil pests and according to maize crop damage, resulting in serious plants were at the 4-6 leaf stage. the practices carried out at CIMMYT. grain production problems. One option Chemical controls were not used in for reducing losses is the use of resistant these plots. As a result, an evaluation Planting was carried out using a varieties (Wiseman and Davis 1979). was carried out of the damage to, and divided-plot design. Eight furrows yields of, two varieties selected by were sown with genotypes of the four The selection of genotypes with CIMMYT for resistance to FAW and varieties indicated in Table 1 (large resistance to FAW began in 1956 in stalk borers, and a comparison with plot); due to a lack of seed, only six Brazil with amargo-type varieties, from two varieties commonly grown in the furrows were planted with the hybrids. which maize germplasm was identified region plus two hybrids — one Furrows were 2.5 m long with ten with resistance to this pest (Wiseman identified by CIMMYT as susceptible plants per row, and the plot was and Davis 1979). The International and another as resistant — as checks on divided in half. One half was treated Maize and Wheat Improvement Center the infestation levels in the region. with Methyl Parathion dust (3%) at the (CIMMYT) has worked since 1986 to Finally, a comparison was done of the 8-10 leaf stage (small plots), and the develop maize germplasm with host damage caused by FAW and SCB other was left untreated. The harvested plant resistance (HPR) to multiple between plants with and without plot corresponded to the two central species of Lepidoptera identified as insecticide applications. rows of each experimental unit. Four replications were done for each tropical maize pests. The CIMMYT M aterials and M ethods treatment. Population 390 Multiple Insect This research was conducted at the The test variables consisted of: FAW Resistance Tropical (MIRT) selected Colegio de Postgraduados’ Tabasco foliar damage, first and second under artificial infestation in Mexico. Campus experiment station in generations of borers, number of Subsequently, CIMMYT developed the Cárdenas, Tabasco. Planting took place damaged stalks, internodes damaged in materials demonstrate acceptable agronomic traits, beginning with experimental varieties Across 90390 (W) and Across 90390 (Y), which show resistance to Diatraea grandiosella, D. Table 1. Genotypes, genetic composition, and origin of materials screened for damage by FAW and SCB in Cardenas, Tabasco, Mexico. saccharalis (Sugarcane borer, SCB) and Genotype Genetic composition Origin Across 90390 IRW Across 90390 IRY VS-536 Mejen Ki3 x CML131 CML135 x CML67 Variety resistant to FAW and SCB Variety resistant to FAW and SCB Variety Variety Susceptible hybrid Resistant hybrid CIMMYT CIMMYT Local commercial Local criollo CIMMYT CIMMYT FAW (Mihm et al. 1991). However, the plants’ resistance levels may vary if they are moved to a different environment (Wiseman and Davis 1979). 298 OBDULIA L. SEGURA-LEÓN the first and second generation, and ear scores of 2.1 and 3.2 with and without internodes were at the ear or the base and grain yield adjusted to 11% insecticide, respectively. The least of the ear, a location and phenological humidity. The first two screenings were affected was Across 90390 IRY, with stage considered susceptible to the done 7 days after applying the damage scores of 1.5 under both second generation of borers (Guthrie insecticide and the last prior to treatments. and Barry 1989; Chippendale 1978). of 0-9 for FAW, and a damage scale of In the first borer generation, damage With regard to the level of borer 1-9 for SCB, where 1 is resistant and 9 is was quite low (1.0), an observation damage in plants with and without susceptible (Mihm 1989). For the data which was confirmed at harvest when protection (small plot), variance on damage by FAW and borers only the an average of 0.22 damaged internodes analysis of the factors damaged stalks average values were obtained, while for were recorded (Table 2). In the second (DS), internodes damaged in the first the number of damaged stalks and generation, damage scores ranged from and second generation and in total, and internodes data were analyzed under a 1.0 to 3.0 in all varieties and correlated maize and grain yields indicates no divided-plot design and means were to the number of damaged internodes, evidence of differences between plants compared using a Tukey test. For the which averaged 0.558 (Table 2). In this with and without insecticide for any of large plot (genotypes), an F-test was case, one of the most affected the tested variables (Table 3). This may done using the mean squared of error genotypes was the susceptible check be related to the low level of damage of Gen*rep, to detect differences among K13 x CML131, with damage scores of (2-3) detected during plant them. Data for the number of damaged 2.0 and 1.3 with and without pesticides, development. internodes was transformed before respectively — but much below the analysis due to the presence of zeros in expected score of 7-9, suggesting either The genotype response study indicated the data (Steel and Torrie 1988). The that the borer population was low that significant differences existed for means presented in the tables are not during this stage of plant development, damaged stalks, total number of transformed. or that the effect of the Furadan was damaged internodes, internodes still persisting. damaged by second-generation borers, flowering, using a foliar damage scale and ear and grain yield (Table 3). Results and Discussion Table 2 indicates that the variety VS- However, for the variable of damaged Table 2 presents the average scores for 536 showed more foliar damage than internodes, significant differences were FAW and borer damage prior to the susceptible check, and that the also noted in the interaction of varieties flowering. The low level of damage by least-affected genotypes were the and repetitions, hence the means and F FAW (1.5 and 1.8) and borers (1.0) seen resistant check and the variety Across tests for genotypes were not significant in the susceptible check Ki3 x CLM131 90390 IRY. The foliar damage caused (Tables 3 and 4). This is reflected as indicates a low level of infestation, by borers was greater during well in the high coefficients of since the expected damage ratings flowering, as it was observed at harvest variation. would be 7-9 and 7-10, respectively. that the majority of the damaged This response may be related to the seed treatment used prior to planting, since the product used is a systemic Table 2. Average scores of maize foliar damage by FAW and SCB, in Cárdenas, Tabasco. insecticide, but it normally persists only 10-12 days under tropical conditions. (Plots near the experiment and planted Genotype in the same period with VS-536, but Across 90390 IRW Across 90390 IRY VS-536 (local) Mejen (local) Ki3 x CML131 (susceptible) CML135 x CML 67 (resistant) without the seed treatment, showed natural FAW infestation levels of 18.5%+/- 3 of plants at the 6-8 leaf stage, corresponding to 16 days after planting.) The variety most affected by FAW was Mejen, with average damage Armyworm damage1 Borer damage2 Treatment 1 Treatment 2 Treatment 1 Treatment 2 2.1 1.5 2.7 3.2 1.5 1.3 1.8 1.5 2.0 2.1 1.8 1.7 1.6 1.6 2.2 1.7 2.0 1.0 1.1 1.3 2.0 1.7 1.3 1.1 Note: Treatment 1 = no insecticide; treatment 2 = protected with 3% methyl parathion. 1 Seven days after chemical protection. 2 Pre-flowering. TWO EXPERIMENTAL MAIZE VARIETIES SELECTED FOR RESISTANCE TO FALL ARMYWORM AND SUGARCANE BORER IN TABASCO, MEXICO 299 • In every case, internode damage in It is possible to explain the variability in uncontrollable environmental the damage response by irregularity in conditions with the result that usually the first generation was less than 0.4, the distribution of the natural insect they are neither uniform nor predictable with the susceptible check Ki3 x populations, except that it over time, space, nor infestation level. CML131 showing the greatest damage (0.343) and Mejen the least approximates a negative binomial affected (0.152). distribution belonging to a contagious Nevertheless, the tendencies in the distribution family (Rojas 1970). Given results show evidence of genotype the high response variability and low response, even though they are not affected genotype was the resistant level of uniformity in natural statistically different for the above- check (0.419), followed by Across infestations, Ortega et al. (1984) and mentioned reasons. Table 5 shows the 90390 IRY (0.424), with Mejen Davis and Williams (1989) recorded average values of the test variables. The limited efficiency for selection of following findings can be observed: resistant genotypes. Mihm (1989) • The variety Across 90390 IRY • In the second generation, the least showing the most damage (0.824). • This response is similar to that shown for total internode damage. considers this variability a limitation on showed the least stalk damage (at the selection of insect-resistant 3.375), and Mejen showed the most As for yields, the varieties with high ear genotypes, in that the natural insect (at 5.875). and grain weights were the two hybrids (CML135 x CML67 with 901.44 g per populations are subject to harvested plot, and Ki3 x CML 131 with Table 3. Summary of F-values calculated for variance analysis of stalk damage, damaged internodes (first and second generations, and total), and ear and grain yield, for maize affected by FAW and SCB. Damaged Damaged internodes GL stalks 1st gen. 2nd gen. F-value Replications (Repl.) 3 Genotypes (Gen.) 5 Repl. x Gen. 15 Treatments (Trtmt.) 1 Gen. x Trtmt. 5 Variance coefficient X 2.08NS 3.81* 2.59NS 0.00NS 0.41NS 40.63 4.479 24.37** 4.74NS 2.02NS 7.57** 1.90NS 5.65** 0.21NS 1.84NS 1.16NS 0.93NS 32.24 36.66 0.220 0.558 868.82 g). Of the varieties, VS-536 had the highest ear yield (744.79 g) followed by Across 90390 IRY (735.95 g); Mejen was the lowest yielding, with an Yield Ears Total Grain 2.68 2.68NS 0.83NS 4.82** 11.84** 9.65** 4.15** 1.02NS 1.10NS 0.81 2.41NS 0.00NS 0.91 1.83NS 1.13NS 39.00 17.81 21.047 1.050756.332 549.02 average of 582.88 g. However, in terms of grain yield, Across 90390 IRY and the two hybrids all exceeded the local varieties. Conclusion Based on the results obtained in the Table 4. F-values calculated to prove the varieties hypothesis for the variables: stalk damage, internode damage (first and second generations, and total), and ear and grain yield, for maize affected by FAW and SCB. F-value Replications Genotypes GL 3 5 Damaged Damaged internodes stalks 1st gen. 2nd gen. 0.80NS 0.25NS 12.85** 1.06NS 0.84NS 1.34NS present research, it is suggested that the scarcity of FAW and first generation borers in the experimental plot may be attributed to the lack of uniformity in natural infestations, and/or to the Total Yield Ears Grain preventive seed treatment applied 0.65NS 1.16NS 2.57NS 11.56** 0.75NS 8.75** before planting. Therefore, in future research the seed treatment needs to be eliminated, to determine whether the Table 5. Average values of the following variables: stalk damage, internode damage (first and second generations, and total), and ear and grain yield, for maize affected by FAW and SCB. Genotypes Across 90390 IRW Across 90390 IRY VS 536 Mejen Ki3 x CML131 CML135 x CML67 Damaged Damaged internodes stalks 1st gen. 2nd gen. Total 4.250A 3.375A 5.000A 5.875A 4.562A 3.813A 0.189A 0.170A 0.207A 0.152A 0.343A 0.256A 0.462A 0.424A 0.766A 0.824A 0.459A 0.419A 1.000A 0.965A 1.129A 1.143A 1.059A 1.010A Yield Ears 703.40CD 735.95BCD 744.79ABC 582.88D 868.82AB 901.44A plant response was due to antixenosis or the interference caused by the systemic insecticide. The low infestation levels detected prevented a clear Grain 524.90AC 636.16BC 467.74BC 431.93C 652.99A 580.42AB demonstration of the antibiotic resistance of the materials in terms of the test variables. Future trials should be artificially infested, assuring results simulating what occurs when natural epidemics do exist in the region. 300 OBDULIA L. SEGURA-LEÓN One hypothesis which emerges is that Ac know le dgm e nt s differences in the number of damaged internodes in the varieties might be Thanks to Dr. John A. Mihm, CIMMYT related to stalk hardness. This variable entomologist, for providing me with was not evaluated in the current the materials tested in this research research, but differences were detected project; to Dr. Felipe Romero Rosales, in the course of field observations, with entomologist at the CP-Phytosanitary the selected resistant varieties harder Institute, and to Dr. David Palma than the local ones. This trait seems to López, member of the editorial suggest the resistance mechanism committee at the CP-Tabasco Campus, which the plants develop against the for review and critique of the insects. manuscript. With regard to damage and yield, it is Re fe re nce s clear that the local variety Mejen was the most affected by borers and had the lowest ear and grain yields. The selected resistant varieties suffered less damage and Across 90390 YRI also showed better yields, implying that it could compete with the commercial and criollo varieties planted in this region. Chippendale, G.M. 1979. The southwestern corn borer Diatraea gandiosella: Case history of an invading insect. Research Bulletin 1031. University of Missouri, Columbia of Agriculture. 1-52. Davis, F.M., and W.P. Williams. 1989. Methods used to screen maize for and to determine mechanism of resistance to the southwestern corn borer and fall armyworm. In Toward Insect Resistant Maize for the Third World: Proceedings of the International Symposium on Methodologies for Developing Host Plant Resistance to Maize Insects, 101-108. Mexico, D.F.: CIMMYT. Guthrie, W.D., and B.D. Barry. 1989. Methodologies used for screening and determining resistance in maize to the European Corn borer. In Toward Insect Resistant Maize for the Third World: Proceedings of the International Symposium on Methodologies for Developing Host Plant Resistance to Maize Insects, 122-129. Mexico D.F.: CIMMYT. Mihm, J.A. 1989. Evaluating maize for resistance to tropical stem borers, armyworms, and earworms. In Toward Insect Resistant Maize for the Third World: Proceedings of the International Symposium on Methodologies for Developing Host Plant Resistance to Maize Insects, 109-121. Mexico, D.F.: CIMMYT. Mihm, J.A., D.C. Jewell, and J. Deutsch. 1991. CIMMYT population 390 — Resistant to multiple species of lepidopterous maize pests. Plant resistance to insects newsletter 17: 69-71. Ortega, A., S.K. Vasal, J.A. Mihm, and C. Hershey. 1984. Mejoramiento de maíz resistente a los insectos. In F.G. Maxwell, and P. Jennings (eds.), Mejoramiento de plantas resistentes a insectos, 391-442. Mexico: Limusa. Rojas, B.A. 1970. El combate de plagas como problema de decisión estadística. Agrociencia 5: 101-107. Steel, R.G.D., and J.H. Torrie. 1988. Bioestadística: Principios y procedimientos. Mexico: McGraw-Hill. Wiseman, B.R., and F.M. Davis. 1979. Plant resistance to the fall armyworm. Florida Entomologist 62(2): 123-130. Conclusion Host Plant Resistance — Alleviating Poverty and Improving Environmental Stability D.L. Winkelmann, Director General, International Maize and Wheat Improvement Center (CIMMYT), Mexico, 1985-1994 On behalf of the CIMMYT trustees, developing countries and motivating technologies that increase agricultural staff, and central management, I want development assistance agencies. productivity while protecting soil, to congratulate the participants in this Poverty is the pivotal element in this water, and forest resources, as well as symposium. Over the past week, you triad of interacting problems. Poverty crop biodiversity. Among other things, have worked through a marathon is toxic to the agricultural environment, in concert with agricultural research agenda comprising over 60 as the poor press on fragile lands and institutions worldwide: presentations on critical themes relating forest margins to subsist. Poverty also • to insect resistant maize, including increases the pace of population improved varieties of maize and mechanisms and bases of resistance, growth, which in itself aggravates wheat that yield more while using advances in conventional techniques environmental deterioration. available resources more efficiently; • and the application of new We develop and disseminate We contribute to the development of biotechnology tools, and research to Inasmuch as poverty is the fulcrum of productivity increasing, resource verify and utilize resistance. Certainly a this nexus of problems, much of their conserving management rich and varied menu about insects. solution then lies with raising the real technologies for maize- or wheat- incomes of the developing world’s based systems, as well as helping to And thanks will go to wild applause, poor. How to raise incomes? For the formulate efficient approaches to maybe a WAVE, when these poorest developing countries, research on such technologies; and speculations are reflected in new achieving higher incomes will depend varieties and hybrids that both resist largely on improved productivity in maize and wheat genetic resources, insect pests and meet the other pressing agriculture. Agricultural productivity and assist others engaged in the needs of developing country farmers. can serve as an engine of growth in same activities. Of particular relevance to CIMMYT, a poor economies, stimulating the center working for the benefit of the demand for goods and services and High yielding, insect resistant maize poor in developing countries, is that the leading to widening rounds of has enormous potential as a part of products of your work can be delivered spending. Productivity gains in productivity enhancing, resource to farmers in that utterly traditional and agriculture also lower the real price of conserving maize farming. As convenient package—the seed. food to consumers, further lubricating mentioned throughout the symposium, • We preserve, catalog, and utilize economic growth. Few poor societies insect pests cause enormous damage to The importance of helping poor have achieved increased incomes maize crops worldwide, but their farmers to improve their well-being can without having first improved effects are especially acute in the hardly be overstated. Like others productivity in agriculture. tropical environments that predominate in developing countries. involved in development, we at CIMMYT see poverty, environmental Which brings us to the role of According to Dr. Mihm’s recent decline, and rapid population growth CIMMYT. The heart of our work is estimates, the 19 leading maize as the principal dilemmas affecting collaborative research to develop producing nations of the developing world could augment their harvests by resistant varieties which not only pledge to continue to facilitate your approximately 4 million tons of grain prevent damage losses but cause actual research through the free exchange of annually — representing some US$400 declines in pest populations, lessening germplasm and knowledge. Moreover, million — if even a fourth of their the need for other control measures. as your work proceeds, know that we farmers had access to insect resistant Moreover, as specialists we know that will be open to new forms of varieties and hybrids. Because these once insecticides are removed from the collaboration that bring your talents benefits are inherent in the seed, poorer cropping system, the natural dynamics closer to our needs. farmers could obtain increased yields between populations of insect and yield stability without investing in predators and maize pests will come I would like to acknowledge the special pesticides or additional manual labor. into play, helping regulate pests in a support of UNDP and the Rockefeller As well, more prosperous farmers who more sustainable fashion. Foundation, as well as the private companies Mahyco, UpJohn, Pioneer, normally protect their crops with chemicals would obtain additional What is often not sufficiently Cargill, and Dekalb, for this savings in the form of reduced appreciated are the indirect symposium. As well, I wish to join with pesticide and labor costs. Farmers consequences of host plant resistance you in congratulating Dr. H.C. Chiang, everywhere would find seed of for the environment. By raising to whom the symposium is dedicated, genetically resistant maize easier and productivity on current maize lands, for his pioneering research in host safer to use than knowledge-intensive use of resistant seed will lessen the plant resistance and integrated pest IPM methods, such as tailoring pressure to open more marginal lands management. pesticide use to quantitative estimates and tropical forests to agriculture. This of pest and predator populations. It is a fact acquires special pertinence in view Finally, I want to bid an appreciative, case where substituting chromosomes of recent predictions that, over the respectful, and a fond farewell to John for chemicals has clear advantage. coming decade, demand for maize in Mihm, who has played a pivotal role, a developing countries will grow more crucial role, in our progress in than 4% each year. developing insect resistant tropical Along with the productivity-enhancing maize. In addition to the outstanding features of insect resistant maize come significant environmental benefits. It is So you see that your work in quality of his research during his 19 obvious that reducing pesticide use developing insect resistant maize ties years at CIMMYT, John has become will lessen health hazards for the directly into efforts to alleviate poverty well-known for his individualistic farmer and workers who apply such and to reduce threats to the fashion statements and his finely honed chemicals, for farm animals and environment. Resistant varieties will alertness during meetings and wildlife that share the ecosystem, for make maize farming more productive presentations. John is leaving CIMMYT consumers of farm products, and for and sustainable, while increasing the as of January. CIMMYT will certainly ground water. We know that it is well-being of farmers and consumers. miss his imposing presence and wishes theoretically possible to develop highly We value your collaboration. We him happiness and success in his new undertakings. To all participants, may you have a safe trip home and continue your valuable research. Participants and Contact Information Florentino Amasende León U.A.A.A.N. Buenavista,Saltillo,Coahuila Tel:17-30-22,18-06-26 Dr. Tom Archer Texas A&M University Agricultural Research and Extension Center Rt.3,Box219 Lubbock, TX 79401 USA Tel.(806)746-6101 Fax:(806)746-6528 M.C. Concepción Arenas Luna Universidad Autónoma Chapingo PreparatoriaAgrícola,AreadeBiología Carr. Fed. Mexico-Texcoco Km. 38 56230 Chapingo, Edo. de Mexico Tel:(595)422-00Ext.5289-5282 Fax:(595)5-05-54 Dr. J. Thor Arnason BiologyDept. Ottawa Carleton University 30 George Glinski Ottawa, Ontario Canada K1N 6N5 Tel: (613) 564-2338, 564-3458 Fax:(613)364-9295 Dr. Dean Barry USDA-ARS- Univ. MO 243Agric.Engr.Bldg. Columbia, MO 65211 USA Tel:(314)882-1116 Fax:(314)882-1115 Dr. David Beck CIMMYT/Mexico Lisboa 27, Col. Juarez, Deleg. Cuauhtemoc 06600 Mexico, D.F. Mexico FAX (5) 726-7559 FAX (595) 4-10-69 TEL:(595)4-21-00/(5)726-90-91 E-mail: dbeck@cimmyt.mx Dr. Dirk Benson ICISeeds P.O.Box8 Thomasville, GA 31799 USA Tel:(912)228-7333 Fax:(912)-228-7847 Dr. David Bergvinson CIMMYT/Mexico Lisboa 27, Col. Juarez, Deleg. Cuauhtemoc 06600 Mexico, D.F. Mexico FAX (5) 726-7559 FAX (595) 4-10-69 TEL:(595)4-21-00/(5)726-90-91 E-mail: dbergvinson@cimmyt.mx Natasha Bohorova CIMMYT/Mexico Lisboa 27, Col. Juarez, Deleg. Cuauhtemoc 06600 Mexico, D.F. Mexico FAX (5) 726-7559 FAX (595) 4-10-69 TEL:(595)4-21-00/(5)726-90-91 E-mail: nbohorova@cimmyt.mx Dra. Nilsa A. Bosque-Perez Department of Plant, Soil and Entomological Sciences CollegeofAgriculture UniversityofIdaho Moscow, Idaho, USA 83844-2339 Phone: (208) 885-7544 Fax:(208)885-7760 E mail: nbosque@uidaho.edu Kaine Bondari CPES P.O. Box 748 Tifton, GA 31793 USA Miss Julia Bonga University of Zimbabwe Crop Science Dept. P.O. Box MP 167 Mount Pleasant Harare, Zimbabwe Tel: (263) 4-303211 Ext. 1139 or 1469 Fax:(263)4-333407 E-mail: descole@zimbix.uz.zw Dr. Eduardo Brambila Northrup King Co. 317-330thStreet Stanton, MN 55018-4308 USA Tel:(507)663-7619 Fax:(507)645-7519 Prof. Emer. Huai C. Chiang University of Minnesota St. Paul, MN 55108 USA Tel: (612) 631-0023 (Home) E-mail: chian004@maroont.c.umn.edu Hugo H. Cueto Flores U.A.A.A.N. Buenavista,Saltillo,Coahuila Tel:17-30-22 Dr. Frank M. Davis USDA/ARS P.O. Box 5367 Mississippi State, MS 39762 USA Tel:(601)323-2230 Fax: (601) 325-8441 or 323-0915 Ing. Luis Othon Espinosa Carrillo Universidad Autónoma Chapingo 56230 Chapingo, Edo. de México México Tel:(595)4-22-00 Dr. Juan José Estruch CIBA Corp. 3054 Cornwallis Rd. Research Triangle Park Durham, NC 27709 USA Tel:(919) 541-8609 Fax:(919)541-8585 Dr. Eduardo C. Fernandez InstituteofPlantBreeding U.P. Los Baños, College 4031 Laguna,Philippines Tel: 2512, 3304, 2339 Loc. 223 Fax: 63-94-3438 Ing. Alfredo Fernández Gaytán U.A.A.A.N. Buenavista,Saltillo,Coahuila México Tel:17-03-99-17-30-22Ext.125 Dr. John E. Foster UniversityofNebraska-Lincoln Dept. of Entomology 312FPIBldg.E.C. Lincoln, NE 68583-0816 USA Tel: 402-472-8686 Fax:402-472-4687 Dr. Lee K. French FrenchAgriculturalResearch R.R. 2 Box 294 Lamberton, MN 56152 USA Tel:(507)752-7274 Fax: 507-7526132 Dr. Osvaldo Garcia Prof.Parasitologia U.A.A.N. Buenavista 25315Saltillo,Coahuila Assefa Gebre-Amlak Awassa College of Agriculture P.O.Box5 Awassa, Ethiopia Tel: (251) 6-200094, 200211 Fax:(251)6-200072 Dr. Macharia Gethi KenyaAgriculturalResearchInstitute R.R.C.-Embu P.O.Box27 Embu, Kenya Tel. 0161-20116 or 20873 Fax: 0161-30064 E-mail: icraf-embu@cgnet.com Dr. Diego González de León CIMMYT/Mexico Lisboa 27, Col. Juarez, Deleg. Cuauhtemoc 06600 Mexico, D.F. Mexico FAX (5) 726-7559 FAX (595) 4-10-69 TEL:(595)4-21-00/(5)726-90-91 E-mail: dgdeleon@cimmyt.mx Dra. Patricia Guevara Fefer U.N.A.M. Fac. Ciencias CiudadUniversitaria Mexico,D.F. Mexico Tel.(5)622-49-05 Fax:(5)622-48-28 Carlos Harjes CornellUniversity 307BradfieldHall Ithaca,N.Y.14850 USA Tel:(607)255-3104 Fax:(607)225-6683 E-mail: ch20@cornell.edu Ing. José Luis Herrera Ayala U.A.A.A.N. Buenavista,Saltillo,Coahuila Mexico Tel:17-03-99-17-30-22Ext.215 Dra. Edith Hesse CIMMYT/Mexico Lisboa 27, Col. Juarez, Deleg. Cuauhtemoc 06600 Mexico, D.F. Mexico FAX (5) 726-7559 FAX (595) 4-10-69 TEL:(595)4-21-00/(5)726-90-91 E-mail: ehesse@cimmyt.mx Dr. David Hoisington CIMMYT/Mexico Lisboa 27, Col. Juarez, Deleg. Cuauhtemoc 06600 Mexico, D.F. Mexico FAX (5) 726-7559 FAX (595) 4-10-69 TEL:(595)4-21-00/(5)726-90-91 E-mail: dhoisington@cimmyt.mx Ing. Pedro Injante Silva PIM-INIA Cajamarca, Peru Dr. Daniel Jeffers CIMMYT/Mexico Lisboa 27, Col. Juarez, Deleg. Cuauhtemoc 06600 Mexico, D.F. Mexico FAX (5) 726-7559 FAX (595) 4-10-69 TEL:(595)4-21-00/(5)726-90-91 E-mail: djeffers@cimmyt.mx Dr. David Jewell CIMMYT Maize Research Station P.O. Box MP 163 Mount Pleasant, Harare, Zimbabwe E-mail: d.jewell@cgnet.com Tel: 263-4-301807 Fax:263-4-301327 E-mail: d.jewell@cgnet.com or cimmyt-zimbabwe@cgnet.com Home: Fax/Phone: 263 (4) 885090 Nora Cecilia Jimenez Mass CORPOICA Regional 2 C.I. Turipana AA 602 Monteria, Colombia, S.A. Tel:(947)860211 Fax:(947)860219 Dr. Alice Kamau EgertonUniversity Nakuru, Kenya Dr. He Kanglai CAAS Beijing The People’s Republic of China Dr. Mireille Kairallah CIMMYT/Mexico Lisboa 27, Col. Juarez, Deleg. Cuauhtemoc 06600 Mexico, D.F. Mexico FAX (5) 726-7559 FAX (595) 4-10-69 TEL:(595)4-21-00/(5)726-90-91 E-mail: mkhairallah@cimmyt.mx Dr. Z.R. Khan ICIPE P.O.Box 30772 Nairobi,Kenya Tel: 254-35-43281 Fax: 254-35-43779 Dr. S.Tej Kumar Maize Research Station (A.P.AgriculturalUniversity) Amberpet, Hyderabad 500029 A.P.India Off.Tel:(+040)868498 Res.Tel:(+040)638316 Dr. Harish Kumar 5J/56;N.I.T. Faridabad - 121001 Haryana,India Tel. 91-129-221-342 Dr. Krishen Kumar Marwaha IndianAgriculturalResearchInstituteNew Delhi Divisionof EntomologyIARI New Delhi, India 110012 Tel: 5781482 Dr. Karim M. Maredia Michigan State University 416 Plant & Soil Science Bldg. P.O. Box 6301 East Lansing, MI 48824 USA Tel: 517-353-5262 Fax: 517-432-1982 E-mail: kmaredia@msu.edu Ing. Jose Blas Maya Lozano Investigador, Porgrama Maiz INIFAP,CIPACJal. Campo Auxiliar Ameca Apdo. Postal No. 10 46600Ocotlan,Jalisco Dr. John A. Mihm Entomologist FrenchAgriculturalResearch,Inc. R.R.2,Box294 Lamberton MN 56152 Tel:(507)752-7274 Fax:(507)752-6132 E-mail: jmihm@prairie.lakes.com Dr. Dan Moellenbeck PioneerHi-BredInternational 7301 NW 62nd P.O.Box85 Johnston, IA 50131-0085 USA Tel.(515)270-4083 Fax:(515)253-2221 E-mail: moellenbeckd@phibred.com Noe Monroy Arcos U.A.A.A.N. Buenavista,Saltillo,Coahuila Mexico Tel:12-30-22 Dr. Alfonso Monteiro CargillInvestigacionesS.deR.L. Av. Cuauhtemoc No. 421 45050 Zapopan, Jalisco Mexico Fax:(379)80431 Dr. Kiarie Njoroge KenyaAgriculturalResearchInstitute P.O. Box 57811 Nairobi,Kenya Tel.444144 Dr. Rafael Obando S. INTA CentroNacionaldeInvest.Agrícolas Km. 14 C. Norte 2 Km. al Sur Apdo. 2648 Managua, Nicaragua Tel: 31512, 31334 y 31340 Fax: 31738 Victor Hugo Ochoa Antuna Univ. Autónoma Agraria “ Antonio Narro” Buenavista,Saltillo,Coahuila México Tel:12-30-22 Dr. James L. Overman DEKALB Genetics Inc. P.O. Box 504 Union City, TN 38261 USA Tel:(901)885-7421 Fax:(901)885-7422 Dr Daniel F. Palmer DEKALB Genetics Corporation 2135 W. Lincoln Ave. Olivia, MN 56277 USA Tel:(612)523-2222 Fax:(612)523-2224 Ing. Juan Francisco Perez Dominguez Investigador, Programa de Entomologia Campo Experimental Ocotlan Apdo. Postal 79 47800Ocotlan,Jalisco Benito Reséndiz Garcia Universidad Autónoma Chapingo Km. 37.5 Carretera México-Texcoco 56230 Chapingo, Edo. de Mexico Tel.4-22-00Ext.5003 Fax:4-06-92 Dr. Luis A. Rodriguez del Bosque Director,Div.Agricola INIFAP Apdo. Postal # 172 Rio Bravo, Tamaulipas Biol. Armando Rodríguez Garcia Universidad Autónoma Agraria Antonio Narro (UAAAN) Buenavista,Saltillo,Coahuila Tel:17-30-22Ext.310 Dr. Felipe Romero Rosales ColegiodePostgraduadosI.F. Montecillo, Edo. de Mexico Mexico Tel:(595)53007 Fax:(595)5-30-07 Dr. S.S. Sekhon PunjabAgriculturalUniversity Dept.PlantBreeding Ludhiana,India Tel:401960Ext.224 Dr. Margaret E. Smith CornellUniversity 252 Emerson Hall Ithaca,N.Y.14853 USA Tel:(607)255-1654 Fax:(607)255-6683 E-mail: mes25@cornell.edu Prof. C. Michael Smith KansasStateUniversity Dept. of Entomology WatersHall, Manhattan, KS 66506-4004 USA Tel.(913)532-4700 Fax:(913)532-6232 E-mail: cmsmith@ksuvm.ksu.edu Dr. Maurice E. Snook USDA-ARS Athens, GA USA Tel: 706-546-3597 Fax: 706-546-3454 Mohamed Soliman Maize Research Station FieldCropsResearchInstitute AgriculturalResearchCenter 9 Gamaa St., Giza, Egypt Tel.(202)5731580 Fax:(202)624-668 Dr. Jeanne Romero-Severson Linkage Genetics PO Box 157 Mazomanie WI 53560 Tel:(608)767-3237 Fax:(608)795-4563 E-mail: romeros@mailbag.com CORPORATE 2411 South 1070 West, Suite B Salt Lake City, Utah 84119 Tel:(801)975-1188 Fax:(801)975-1244 E-mail: lingene@xmission.com Prof. Jennifer Ann Thomson University of Cape Town Dept. of Microbiology, UCT Private Bag, Rondesbosch 7700 South Africa Tel.(21)650-3269/70 Fax:(21)650-4023 E-mail:jat@molbiol.uct.ac.za Dr. Jonathan Sagers Northrup King Co. 317330thSt. Stanton, MN 55018-4308 USA Tel.(507)663-7629 Fax:(507)645-7519 Home Phone: 612-460-6176 Ing. Roger Urbina Algabas DirectorGeneral Programa de Maiz Instituto Nicaraguense de Tecnologia Agropecuaria EdificioMariaCastill,Modulo416 Apdo. Postal 1247 Managua, Nicaragua Phone: +505(2) 490 583 Fax: +505(2) 490 583 M.C. Ma. del Carmen Sánchez Gálvez Universidad Autónoma Chapingo 56230 Chapingo, Edo. de México Mexico Tel:(595)4-22-00 M.C. Obdulia Segura Leon CEICADES-C.P. PerifericoCarlosA.Molinas/n Km. 3 Carr. Cardenas-Huimanguillo Apdo. Postal No. 24 86500 H. Cardenas, Tabasco FAX:(937)2-22-97 Dr. J. Antonio Serratos CIMMYT/Mexico Lisboa 27, Col. Juarez, Deleg. Cuauhtemoc 06600 Mexico, D.F. Mexico FAX (5) 726-7559 FAX (595) 4-10-69 TEL:(595)4-21-00/(5)726-90-91 E-mail: aserratos@cimmyt.mx Dr. Chin Tien Tseng TiananDistricAgric.Imp.Sta. PO Box 30 Pot-Tzu Chia-Yi, Taiwan 61314 Dr. J.B.J. van Rensburg GrainCropsInstitute Private Bag X1251 Potchefstroom 2520 Rep.S.Africa Tel: 27-148-2977211 Fax: 27-148-2947146 E-mail:JBJ@igg2.agric.za Dr. Paul Afonso Viana EMBRAPA/CNPMS C.P. 151-Rod. MG 424-Km 65 35701-970, Sete Lagoas, MinasGerais,Brasil Tel:(031)923-5644 Fax:(031)923-9252 Telex: 31.2099EBPA E-mail: cpms@ntiaa.embrapa.ansp.br Dr. Richard Wedderburn CIMMYT/Mexico Lisboa 27, Col. Juarez, Deleg. Cuauhtemoc 06600 Mexico, D.F. Mexico FAX (5) 726-7559 FAX (595) 4-10-69 TEL:(595)4-21-00/(5)726-90-91 E-mail: rwedderburn@cimmyt.mx Dr. Claude Welcker INRA U.R.P.V.,AmeliorationdesPlantes B.P.515 97165 Pointe-a-Pitre Cedex Guadeloupe, F. West Indies Tel:(590)25-59-15 Fax:0590.94.11.72 Telex: INRAAG 919867 GL E-mail:welcker@antilles.inra.fr G. Febvay INSA, UA INRA 203, LaboratoiredeBiologieappliquee,F-69621, Villeurbannecedex,France. Tel: 33-(0)472438356 Fax: 33-(0)472 438 534 febvay@jouy.inra.fr D. Clavel and I. Guinet CIRAD-CA Programme Mais 2477 Av Val de Montferrand BP5035, F-34032, Montpellier cedex1, France. Dr. Martha Willcox CIMMYT/Mexico Lisboa 27, Col. Juarez, Deleg. Cuauhtemoc 06600 Mexico, D.F. Mexico FAX (5) 726-7559 FAX (595) 4-10-69 TEL:(595)4-21-00/(5)726-90-91 E-mail: mwillcox@cimmyt.mx Dr. W. Paul Williams USDA/ARS Corn Host Plant Research Laboratory Box 9555 Mississippi State, MS 39762 USA Tel:(601)325-2735 Fax:(601)325-8441 E-mail: wpw1@ra.msstate.edu Dr. Richard L. Wilson USDA-ARS Plant Introduction Station G-204 Agronomy Hall IowaStateUniversity Ames, IA 50011 USA Tel:(515)294-8583 Fax:(515)294-4880 E-mail:rlwilson@iastate.edu Dr. Billy R. Wiseman USDA-ARS Insect Biology and Population Management Research Laboratory CoastalPlainExperimentStation P.O. Box 748 Tifton, GA 31793 USA Tel:(912)387-2340 Fax:(912)387-2321

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