WO2006121350A1

WO2006121350A1 - Entomopathogenic fungi and uses thereof

Info

Publication number: WO2006121350A1
Application number: PCT/NZ2006/000102
Authority: WO
Inventors: Stephen Reynold Ford; Qaiser Mustafa
Original assignee: Millennium Microbes Limited
Priority date: 2005-05-10
Filing date: 2006-05-10
Publication date: 2006-11-16
Also published as: NZ539935A

Abstract

The present invention provides strains of entomopathogenic fungi, compositions comprising said entomopathogenic fungi, and the use of such entomopathogenic fungi and compositions as biological control agents. Methods for the biological control of phytopathogenic insects using entomopathogenic fungi selected from strains of Lecanicillium spp., Paecilomy ces fumosoroseus, and Beauveria bassiana and compositions comprising said fungi are also provided.

Description

ENTOMOPATHOGENIC FUNGI AND USES THEREOF FIELD OF THE INVENTION

This invention relates to entomopathogenic fungi, compositions comprising said entomopathogenic fungi, and the use of such entomopathogenic fungi and compositions as biological control agents. Methods for the biological control of phytopathogenic insects, including aphids, thrips, white fly, mealy bug, and the like using entomopathogenic fungi, particularly Lecanicillium spp., Paecilomyces fumosoroseus, and Beauveria bassiana and compositions comprising said fungi are also provided. BACKGROUND OF THE INVENTION

Plant disease caused by pathogens such as insects are a significant economic cost to plant based agriculture and industries. Losses may arise through spoilage of produce both pre and post harvest, loss of plants themselves, or through reduction in growth and production abilities. Traditionally, control of plant pathogens has been pursued through the application of chemical insecticides. The use of chemicals is subject to a number of disadvantages. The pathogens can and have developed tolerance to chemicals to over time, producing resistant populations. Indeed, resistance to pesticides is the greatest challenge to the viability of the horticultural industry. The problem is particularly illustrated with reference to a number of economically important phytopathogenic insects. Populations of western flower thrips worldwide are reported to be resistant to most groups of pesticides including the following examples; acephate, abamectin, chlorpyrifos, endosulfan, methomyl, methiocarb, omethoate, pyrazophos and tau-fluvalinate. Populations of onion thrips in New Zealand have developed resistance to deltamethrin, and local populations have been reported to be resistance to diazinon and dichlorvos. Onion thrips in the United States have been reported to be resistant to many pesticides (Grossman, 1994). Greenhouse whitefly has reportedly developed resistance to organochlorine, organophosphate, carbamate and pyrethroid insecticides (e.g. Georghiou 1981, Anis & Brennan 1982, Elhag & Horn 1983, Wardlow 1985 and Hommes 1986). Resistance has also been reported in newer insecticides, buprofezin and teflubenzuron (Gorman et al. 2000). Chemical residues may also pose environmental hazards, and raise health concerns. The revival of interest in biological control such as microbial insecticides over the last 20 years has come directly from public pressure in response to concerns about chemical toxicities. Biological control presents an alternative means of controlling plant pathogens which is potentially more effective and specific than current methods, as well as reducing dependence on chemicals. Such biological control methods are perceived as a "natural" alternative to insecticides with the advantage of greater public acceptance, reduced environmental contamination, and increased sustainability. Mechanisms of biological control are diverse. One mechanism which has been demonstrated to be effective is the use of antagonistic microorganisms such as bacteria to control phytopathogenic insects. For example, the large scale production of Bacillus thuringiensis enabled the use of this bacterio-insecticide to control painted apple moth in Auckland, New Zealand. The production of bacterio-insecticides is now common world wide.

There is however little information on the successful application of entomopathogenic fungi and their industrial production is still relatively unsophisticated. Applications of entomopathogenic fungi using Lecanicillium muscarium (previously known as Verticillium lecanii), Beauveria bassiana and Metarhizium anisopliae have been developed in the US, Europe, Africa and Russia. It will therefore be appreciated that Lecanicillium and Beauveria species are clearly considered as prime candidates of effective biological control agents (BCAs), and have been the subject of significant research. However, to date none of the candidates have proved ideal, presumably through a failure to quickly establish and/or survive the environmental variability existing in the field. Indeed, existing candidates do not appear to have met with significant grower acceptance, and may be perceived to be uneconomic.

This is compounded by the frequent unavailability of entomopathogenic fungi developed in one country to the horticulturalists of another country, for example due to regulatory constraints. For example, entomopathogenic fungi developed in Europe or the United States are generally not available to the New Zealand horticultural sector. Furthermore, many non-indigenous fungi may be unlikely to be suitable for, or able to survive or flourish in, local conditions. Surprisingly, the Applicants have now identified and isolated from the New Zealand environment four Lecanicillium strains, one Paecilomyces strain, and two Beauveria strains not mentioned in any of the earlier reports as effective BCAs. The Applicants have determined that these species are highly effective in controlling phytopathogenic insects, including but not limited to thrips, aphids and white fly, and in successfully surviving and establishing in the field. The Applicants believe these to be the first indigenous New Zealand entomopathogenic strains of these species to be isolated.

It is therefore an object of the present invention to provide a strain of Lecanicillium, Paecilomyces, or Beauveria useful in the biological control of phytopathogenic insects, or at least to provide the public with a useful choice. SUMMARY OF THE INVENTION SUMMARY OF THE INVENTION

Accordingly, in one aspect the present invention provides a biologically pure culture of Lecanicillium muscariwn fungus strain K4V1 on deposit at Australian Government Analytical Laboratories (AGAL) under Accession No. NM05/44593, or a culture having the identifying characteristics thereof.

In a further aspect the present invention provides a biologically pure culture of Lecanicillium muscarium fungus strain K4V2 on deposit at AGAL under Accession No. NM05/44594, or a culture having the identifying characteristics thereof.

In another aspect the present invention provides a biologically pure culture of

Lecanicillium muscarium fungus strain K4V4 on deposit at the National Measurement

Institute of Australia (formerly the Australian Government Analytical Laboratories,

(AGAL)) under Accession No. NM06/00007, or a culture having the identifying characteristics thereof.

In another aspect the present invention provides a biologically pure culture of Lecanicillium longisporum fungus strain KT4L1 on deposit at the National Measurement Institute of Australia under Accession No. NM06/00009, or a culture having the identifying characteristics thereof. In still a further aspect the present invention provides a biologically pure culture of Beauveria hassiana fungus strain K4B1 on deposit at AGAL under Accession No. NM05/44595, or a culture having the identifying characteristics thereof. - A-

In yet a further aspect the present invention provides a biologically pure culture of Beauveria bassiana fungus strain K4B2 on deposit at the National Measurement Institute of Australiaunder Accession No. NM06/00010, or a culture having the identifying characteristics thereof. In another aspect the present invention provides a biologically pure culture of

Paecilomyces fumosoroseus fungus strain K4P1 on deposit at the National Measurement Institute of Australiaunder Accession No. NM06/00008, or a culture having the identifying characteristics thereof.

In a further aspect the present invention provides spores obtainable from a fungus of the invention. Spores obtained from the fungi of the invention are also specifically contemplated.

In another aspect, the present invention provides the use of at least one fungi as defined above together with at least one diluent, adjuvant, carrier and/or excipient in the preparation of a composition. In another aspect, the present invention provides the use of spores from at least one fungi as defined above together with at least one diluent, adjuvant, carrier and/or excipient in the preparation of a composition.

Preferably, said at least one fungi is in a reproductively viable form and amount. In a further aspect the invention provides a composition comprising spores obtainable from a least one fungi of the invention together with at least one diluent, adjuvant, carrier and/or excipient.

In a further aspect the present invention provides a composition which comprises at least one fungi as defined above together with at least one diluent, adjuvant, carrier and/or excipient.

Preferably, said at least one fungi is in a reproductively viable form and amount.

Preferably, said composition is a biological control composition, more preferably said biological control composition is an entomopathogenic composition. As used herein, a biological control composition is a composition comprising or including at least one biological control agent that is an antagonist of one or more phytopathogens. Such control agents include, but are not limited to, agents that act as repellents, agents that render the environment unfavourable for the pathogen, and agents that incapacitate, render infertile, and/or kill the pathogen.

As used herein, an entomopathogenic composition is a composition which comprises or includes at least one agent that is an antagonist of one or more phytopatho genie insect. Such an agent is herein considered to have entomopathogenic efficacy.

Preferably, said entomopathogenic efficacy is the ability to parasitise and incapacitate, render infertile, impede the growth of, or kill one or more phytopathogenic insects within 14 days of contact with said insect, more preferably within 7 days, more preferably still the ability to kill one or more phytopathogenic insects within 7 days.

The term "biological control agent" (BCA) as used herein refers to a biological agent which acts as an antagonist of one or more phytopathogenic insects, or is able to control one or more phytopathogenic insects. Antagonism may take a number of forms. In one form, the biological control agent may simply act as a repellent. In another form, the biological control agent may render the environment unfavourable for the phytopathogenic insect. In a further, preferred form, the biological control agent may parasitise, incapacitate, render infertile, impeded the growth of, and/or kill the phytopathogenic insect. Accordingly, the antagonistic mechanisms include but are not limited to antibiosis, parasitism, infertility, and toxicity. Therefore, agents which act as antagonists of one or more phytopathogenic insects can be said to have entomopathogenic efficacy.

The term "control" or "controlling" as used herein generally comprehends preventing or reducing phytopathogenic insect infection or inhibiting the rate and extent of such infection, or reducing the phytopathogenic insect population in or on a plant or its surroundings, wherein such prevention or reduction in the infection(s) or population(s) is statistically significant with respect to untreated infection(s) or population(s). Curative treatment is also contemplated. Preferably, such control is achieved by increased mortality amongst the insect population caused by the biological control agent. Preferably, said biological control composition comprises at least one agriculturally acceptable diluent, adjuvant, carrier and/or excipient. In a further aspect the present invention provides a composition which comprises at least one fungi as defined above together with at least one diluent, adjuvant, carrier and/or excipient.

Preferably, said at least one diluent, adjuvant, carrier and/or excipient is an agriculturally acceptable diluent, adjuvant, carrier and/or excipients, more preferably is selected from the group consisting of a filler stimulant, an anti-caking agent, a wetting agent, an emulsifϊer, and an antioxidant, more preferably said composition comprises at least one of each of a filler stimulant, an anti-caking agent, a wetting agent, an emulsifier, and an antioxidant.

Preferably, said filler stimulant is a carbohydrate source, such as a disaccharide including, for example, sucrose, fructose, glucose, or dextrose, said anti-caking agent is selected from talc, silicon dioxide, calcium silicate, or kaelin clay, said wetting agent is skimmed milk powder, said emulsifier is a soy-based emulsifier such as lecithin or a vegetable-based emulsifier such as monodiglyceride, and said antioxidant is sodium glutamate or citric acid.

Preferably, said composition is a biological control composition, more preferably an entomopathogenic composition. More preferably, said biological control composition is a stable composition capable of supporting reproductive viability of said fungi for a period greater than about two weeks, preferably greater than about one month, about two months, about three months, about four months, about five months, more preferably greater than about six months. In certain embodiments, the composition comprises a single strain of said fungi. Suitably, the composition comprises a strain selected from the group consisting of strain AGAL No. NM05/44593, strain AGAL No. NM05/44594, strain AGAL No. NM05/44595, strain NM06/00007, strain NM06/00008, strain NM06/00009, strain NM06/00010, and a strain having the identifying characteristics of any one of said strains.

Alternatively, the composition comprises multiple strains of said fungi, but preferably includes three strains or less. Suitably, the composition comprises any two or more strains selected from the group consisting of strain AGAL No. NM05/44593, strain AGAL No. NM05/44594, strain AGAL No. NM05/44595, strain NM06/00007, strain NM06/00008, strain NM06/00009, strain NM06/00010, and a strain having the identifying characteristics of any one of said strains.

Preferably, said composition is a biological control composition that comprises, in a reproductively viable form and amount, at least one strain selected from Lecanicillium muscarium strain K4V1 (Australian Government Analytical Laboratories Accession No. NM05/44593) or a strain having the identifying characteristics thereof; Lecanicillium muscarium strain K4V2 (Australian Government Analytical Laboratories Accession No. NM05/44594) or a strain having the identifying characteristics thereof; Lecanicillium muscarium strain K4V4 (National Measurement Institute of Australia Accession No. NM06/00007) or a strain having the identifying characteristics thereof; Beauveria bassiana strain K4B1 (Australian Government Analytical Laboratories Accession No. NM05/44595) or a strain having the identifying characteristics thereof; Beauveria bassiana strain K4B2 (National Measurement Institute of Australia Accession No. NM06/00010) or a strain having the identifying characteristics thereof; Lecanicillium longisporum strain KT4L1 (National Measurement Institute of Australia Accession No. NM06/00009) or a strain having the identifying characteristics thereof; and Paecilomyces fumosoroseus strain K4P1 (National Measurement Institute of Australia Accession No. NM06/00008) or a strain having the identifying characteristics thereof, together with at least one agriculturally acceptable diluent, adjuvant, carrier and/or excipient.

In still a further aspect, the invention provides a method of producing a composition comprising one or more entomopathogenic fungi as described herein, said method comprising combining a reproductively viable form of said entomopathogenic fungi of the invention with at least one agriculturally acceptable diluent, carrier or excipient.

Preferably, said fungi is selected from the group consisting of Lecanicillium muscarium strain K4V1 (Australian Government Analytical Laboratories Accession No. NM05/44593) or a strain having the identifying characteristics thereof; Lecanicillium muscarium strain K4V2 (Australian Government Analytical Laboratories Accession No. NM05/44594) or a strain having the identifying characteristics thereof; Lecanicillium muscarium strain K4V4 (National Measurement Institute of Australia Accession No. NM06/00007) or a strain having the identifying characteristics thereof; Beauveria bassiana strain K4B1 (Australian Government Analytical Laboratories Accession No. NM05/44595) or a strain having the identifying characteristics thereof; Beauveria bassiana strain K4B2 (National Measurement Institute of Australia Accession No. NM06/00010) or a strain having the identifying characteristics thereof; Lecanicillium longisporum strain KT4L1 (National Measurement Institute of Australia Accession No. NM06/00009) or a strain having the identifying characteristics thereof; and Paecilomyces fumosoroseus strain K4P1 (National Measurement Institute of Australia Accession No. NM06/00008) or a strain having the identifying characteristics thereof.

Preferably, said at least one diluent, carrier or excipient is an agriculturally acceptable diluent, carrier or excipients, preferably said at least one diluent, adjuvant, carrier and/or excipient is selected from the group consisting of a filler stimulant, an anti-caking agent, a wetting agent, an emulsifier, and an antioxidant, more preferably said composition comprises at least one of each of a filler stimulant, an anti-caking agent, a wetting agent, an emulsifier, and an antioxidant. The invention further provides the use of a composition of the invention for the control one or more phytopathogenic insects.

Preferably, said one or more phytopathogenic insects is selected from the group consisting of Thrips (Thysanopterά), Aphids, Psyllids, Scale or Whitefly (Hemipterά), caterpillars of Moths and Butterflies (Lepidopterά), and mites including Varroa mite. In a further aspect the present invention provides a method for controlling one or more phytopathogenic insects, the method comprising applying to a plant or its surroundings a reproductively viable form and amount of at least one entomopathogenic fungi as described herein.

In one embodiment, fungal spores of the at least one entomopathogenic fungi may be applied directly to the plant or its surroundings. Preferably, said spores are admixed with water and applied as described herein.

Preferably, said fungi is selected from the group consisting of Lecanicillium muscarium strain K4V1 (Australian Government Analytical Laboratories Accession No. NM05/44593) or a strain having the identifying characteristics thereof; Lecanicillium muscarium strain K4V2 (Australian Government Analytical Laboratories Accession No. NM05/44594) or a strain having the identifying characteristics thereof; Lecanicillium muscarium strain K4V4 (National Measurement Institute of Australia Accession No. NM06/00007) or a strain having the identifying characteristics thereof; Beauveria bassiana strain K4B1 (Australian Government Analytical Laboratories Accession No. NM05/44595) or a strain having the identifying characteristics thereof; Beauveria bassiana strain K4B2 (National Measurement Institute of Australia Accession No. NM06/00010) or a strain having the identifying characteristics thereof; LecanicilUum longisporum strain K.T4L1 (National Measurement Institute of Australia Accession No. NM06/00009) or a strain having the identifying characteristics thereof; and Paecϊlomyces fumosoroseus strain K4P1 (National Measurement Institute of Australia Accession No. NM06/00008) or a strain having the identifying characteristics thereof.

In a further aspect the present invention provides a method for controlling one or more phytopathogenic insects, the method comprising applying to a plant or its surroundings a composition of the present invention.

Preferably, the composition is admixed with water to a final concentration of about 0.5gm/L to about 3gm/L prior to application, and more preferably to a final concentration of about lgm/L. Preferably, a desiccation protection agent, more preferably Fortune Plus™, is admixed to a final concentration of about lml/L prior to application.

Preferably, said composition comprises at least 10⁷ spores per millilitre at application, more preferably, said application is by spraying.

Preferably, a composition comprising LecanicilUum muscarium strain K4V1 (AGAL Accession No. NM05/44593) or a culture having the identifying characteristics thereof and/or LecanicilUum muscarium strain K4V2 (AGAL Accession No.

NM05/44594) or a culture having the identifying characteristics thereof and/or

LecanicilUum muscarium strain K4V4 (Accession No. NM06/00007) or a culture having the identifying characteristics thereof is applied at a rate of from about 1 x 10¹⁰ to about 1 x 10¹⁵ spores per hectare, preferably from about 1 x 10¹² to about 1 x 10¹⁴ spores per hectare, more preferably from about 5 x 10¹² to about 1 x 10¹⁴ spores per hectare, more preferably about 1-3 x 10 spores per hectare.

Preferably, a composition comprising LecanicilUum longisporum strain K.T4L1 (Accession No. NM06/00009) or a culture having the identifying characteristics thereof is applied at a rate of not less than about 2kg/ha in 5001tr water/ha or 1 to 3 gm/ltr in 1000 to 20001trs/ha indoor.

Preferably, a composition comprising Paecilomyces fumosoroseus strain K4P1 (Accession No. NM06/00008) or a strain having the identifying characteristics thereof is applied at a rate of not less than about 2kg/ha in 5001tr water/ha or 1 to 3 gm/ltr in 1000 to 20001trs/ha indoor.

Preferably, a composition comprising Beauveria bassiana strain K4B1 (AGAL Accession No. NM05/44595) or a culture having the identifying characteristics thereof and/or Beauveria bassiana strain K4B2 (Accession No. NM06/00010) or a culture having the identifying characteristics thereof is applied at a rate of from about 1 x 10¹⁰ to about 1 x 10¹⁵ spores per hectare, preferably from about 1 x 10¹² to about 1 x 10¹⁴ spores per hectare, more preferably from about 5 x 10¹² to about 1 x 10¹⁴ spores per hectare, more preferably about 1-3 x 10¹³ spores per hectare. Conveniently, such a rate of application can be achieved by formulating said composition at about 10⁷ spores per millilitre or more, and applying said composition at a rate of about lkg per hectare. As discussed herein, such an application rate can be conveniently achieved by dissolution of the composition in a larger volume of agriculturally acceptable solvent, for example, water. To those skilled in the art to which the invention relates, many changes in construction and differing embodiments and applications of the invention will suggest themselves without departing from the scope of the invention as defined in the appended claims. The disclosures and the descriptions herein are purely illustrative and are not intended to be in any sense limiting. DETAILED DESCRIPTION OF THE FIGURES

Figure 1 presents a graph showing the average number of whitefly scale per plant as described in Example 4 herein.

Figure 2 presents a graph depicting the number of adult and juvenile thrips observed on plants treated with a Lecanicillium muscarium BCA forumulation, a Beauveria Bassiana BCA formulation, or chemical insecticides, as described in Example 5 herein.

Figure 3 presents a graph showing the number of aphids observed on lettuces treated with a Lecanicillium muscarium BCA formulation in comparison to lettuces treated with chemical insecticides, as described in Example 7 herein.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is in part directed to strains of Lecanicillium spp., Beauveria bassiana, and Paecilomyces fumosoroseus having efficacy against phytopathogenic insects, and the use of such fungi in controlling said phytopathogenic insects.

The present invention recognises that the horticultural sectors of many countries, including for example that of New Zealand, is faced with the problem of increasing insecticide resistance amongst phytopathogenic insect pests. This is compounded under some regulatory regimes by a reduction in the availability of new chemical insecticides due to regulatory barriers.

The use of entomopathogenic fungi as biological control agents presents a solution to this problem. Effective biological control agents can be selected according their ability to incapacitate or kill a target phytopathogenic insect or insect population. Under conducive conditions, phytopathogenic insects such as aphids, thrips and whitefly may infect plants and their surroundings including soil, leaf litter, adjacent plants, supports, and the like. Entomopathogenic fungi may be applied so as to incapacitate and/or kill the phytopathogenic insect, thereby preventing or limiting the disease-causing capability of the pathogen. The effectiveness of these entomopathogenic fungi in the field is in turn dependent on their ability to survive varying climatic conditions, such as interrupted wet periods and desiccation.

The importation of entomopathogenic fungi is frequently problematic, costly, and impractical if not impossible under certain regulatory regimes. For example, entomopathogenic fungi available outside New Zealand are not available to New Zealand horticulturalists because of regulatory and legislative preclusions. The present invention therefore recognises there are distinct advantages to identifying and cultivating strains that are able to flourish under a wide variety of environmental conditions. Isolates of said fungi may conveniently be obtained from the environment, including, for example, from plants, their surroundings, and from pathogens of said plants. In certain embodiments, isolates of said fungi may be obtained from the target insect, or from the plant species (or surroundings) to which the biological control agent comprising said fungi or a composition comprising said fungi will subsequently be applied.

Methods to determine growth of said fungi under different conditions, including different temperatures and on different media or other substrates, are well known in the art. Examples of methods to determine the ability of fungi to grow at various temperatures are described herein, as are methods to determine whether a given isolate is dead or dormant at a given temperature.

Similarly, methods to establish whether an isolate is able to grow on a given artificial medium are exemplified herein. The use of such methods recognises that an isolate must be capable of being grown in sufficient quantity for it to be suitable for use as a biological control agent. Methods of growing sufficient amounts of fungi of the invention are discussed further herein.

Strains of Lecanicillium, Paecilomyces, and Beauveria effective against phytopatho genie insects, and therefore to be suitable for use in accordance with the invention, are identified as those which are effective at reducing the population of the target insect species by a statistically significant amount with respect to the control treatment against which the strains are compared. Such strains can be considered as having entomopathogenic efficacy. As described herein, the reduction in the population of the target insect may be by various antagonistic mechanisms. For example, the fungi may parasitise, incapacitate, render infertile, and/or preferably kill the phytopathogenic insect. The fungi may also reduce the population of the target insect by rendering the environment, for example the plant to which the fungi is applied or its surroundings, unfavourable for the phytopathogenic insect. In this embodiment, the fungi may be considered to be acting as a repellent, and reducing the effective population of the target insect in the vicinity of the plant or its surroundings.

Preferably, suitable strains exhibit at least 50% entomopathogenic efficacy expressed as a percentage reduction of the population of the relevant insect species compared to the control treatment. By way of illustration, the methodology described herein was employed to identify Lecanicillium, Paecilomyces, and Beauveria isolates effective against a variety of target insects, whereas procedures analogous to those described herein can be employed in relation to other fungi and insect species.

Although entomopathogenic efficacy is a principal requisite for an isolate to be considered suitable for use as a biological control agent, the fungal isolate must have additional characteristics to be suitable for use as a biological control agent. For example, the fungi must be able to be stored in a viable form for a reasonable period, ultimately so as to allow it to be applied to the target plant or its surroundings in a form and concentration that is effective as a biological control agent. The fungi must also be able to achieve infection threshold when applied to a plant or its surroundings for it to be suitable for use as a biological control agent. As used herein, infection threshold refers to the concentration of fungi required for the fungi to become established on the target plant or its surroundings so as to then have entomopathogenic efficacy. As will be appreciated, in order to achieve infection threshold, some isolates of fungi may require application at such a high rate as to be impractical or unviable. Furthermore, some fungal isolates may not be able to achieve infection threshold irrespective of the concentration or rate at which they are applied. Suitable entomopathogenic fungi are able to achieve infection threshold when applied at a rate of not less that 10¹⁰ spores per hectare, or applied at a concentration not less than 10⁷ spores per millilitre of composition when said composition is applied at a rate of about lkg/1000L/hectare.

Methods to determine infection threshold are well known in the art, and examples of such methods are presented herein. In certain embodiments, infection threshold can be determined directly, for example by analysing one or more samples obtained from a target plant, its surroundings, and/or a pathogen of said plant, and determining the presence or amount of fungus on or in said sample. In other embodiments, infection threshold can be determined indirectly, for example by observing a reduction in the population of one or more phytopathogenic insects. Combinations of such methods are also envisaged.

Entomopathogenic fungi of the invention include strains of Lecanicillium spp., Beauveria bassiana, and Paecilomyces fumosoroseus. Such fungi are described in more detail below.

Lecanicillium muscarium is an entomopathogenic fungi with a broad host range including homopteran insects and other arthropod groups. L. muscarium is considered a species complex, which includes isolates of varied morphological and biochemical characteristics. Typically, L. muscarium can be isolated from insect cadavers, such as aphids, thrips, whitefly, and mealy bugs, and may also be isolated from soil. Isolates have the following identifying characteristics:

Mycelium: Colonies on potato dextrose agar (PDA), malt extract agar (MEA) or oatmeal agar (OA) are white, creamy, thin, cottony, with reverse colourless to pale or deep yellow. Conidiophores: Phialides are formed singly or directly from mycelium or in whorls of 3 or 4 erect conidiophores much like vegetative mycelium. Phialides delicate, of variable size depending on both strain and the age of the culture. Size from 8.5-16x 0.8- 1.2μm to 30-4Ox 2-2.2μm. Conidia: Produced singly and aggregating on heads at the tips of the phialides in a mucilaginous matrix. Ellipsoidal to cylindrical with rounded ends varying in size with the strain from 2.3-3.4x 1-1.3μm to 7.2-1Ox 2.1-2.6μm. Blastospores are produced in submerged culture. Hydrophilic.

K4V1 - 60% Conidia 1.0x1.0 micron on whitefly scale, 30% Conidia 2.0x1.0 micron on thrip juveniles (nymphs), 10% Conidia 2.5x1.3 micron on thrip pupae. Underside of mycelium thallus sparsely creased, Mycelium thallus removes from the agar very easily.

K4V2 - 50% Conidia 2.0x1.5 micron, 30% Conidia 2.0x1.0 micron, 20% Conidia 1.0x1.0 micron, pathogenic to Whitefly adults, while Blastospores pathogenic to aphids. Underside of mycelium thallus frequently creased, Mycelium thallus difficult to remove from agar surface.

K4V4 — 50% Conidia 1.0x0.5 micron, pathogenic to whitefly scale and adults, very aggressive at low humidity 65-75%, high temp 28-32°. Generally v.l > 75%. 50% Condidia 0.5x0.5 micron. Underside of mycelium thallus sparsely creased, Mycelium thallus diffuses custard yellow to light orange pigment in media.

One strain of Lecanicillium muscarium meeting the above requirements was isolated from whitefly in a greenhouse tomato crop in Pukekohe, New Zealand. The insect was placed onto MEA and PDA and incubated at 24°C at 93% relative humidity which is considered optimum conditions for sporulation. It demonstrates the growth characteristics typical of the species when grown on MEA. Details of the isolation and selection process employed to obtain this isolate, strain K4V1, are set out in the Examples. This L. muscarium isolate has been deposited in the Australian Government Analytical Laboratories, (AGAL), 1 Suakin Street, Pymble, New South Wales, Australia on 16 March 2005 according to the Budapest Treaty for the purposes of patent procedure. The isolate has been accorded the deposit number NM05/44593. The deposit certificate and viability statement are presented herein. Accordingly, in one aspect the present invention provides a biologically pure culture of Z. muscaήum AGAL No.NM05/44593. Similarly provided are Lecanicillium having the identifying characteristics of AGAL NO. NM05/44593.

A second strain of Z. muscariwn meeting the above requirements was isolated from whitefly in a cucumber greenhouse in Ruakaka, New Zealand. The insect was placed onto MEA and PDA and incubated at 24°C at 93% as above. It demonstrates the growth characteristics typical of the species when grown on MEA. Details of the isolation and selection process employed to obtain this isolate, strain K4V2, are set out in the Examples. This Z. muscariwn isolate has been deposited in the Australian Government Analytical Laboratories, (AGAL), 1 Suakin Street, Pymble, New South

Wales, Australia on 16 March 2005 according to the Budapest Treaty for the purposes of patent procedure. The isolate has been accorded the deposit number NM05/44594.

The deposit certificate and viability statement are presented herein.

Accordingly, in one aspect the present invention provides a biologically pure culture of Z. muscarium AGAL No. NM05/44594. Similarly provided are Lecanicillium having the identifying characteristics of AGAL NO. NM05/44594.

A third strain of Z. muscarium meeting the above requirements was isolated from isolated from an outdoor organic tamarillo crop. The insect was placed onto MEA and PDA and incubated at 24°C at 93% as above. It demonstrates the growth characteristics typical of the species when grown on MEA. Details of the isolation and selection process employed to obtain this isolate, strain K4V4, are as set out in the Examples. This Z. muscarium isolate has been deposited in the National Measurement Institute of Australia (formerly the Australian Government Analytical Laboratories, (AGAL)), 1 Suakin Street, Pymble, New South Wales, Australia on 3 March 2006 according to the Budapest Treaty for the purposes of patent procedure. The isolate has been accorded the deposit number NM06/00007. The deposit certificate and viability statement are presented herein.

Accordingly, in one aspect the present invention provides a biologically pure culture of Z. muscarium AGAL No. NM06/00007. Similarly provided are Lecanicillium having the identifying characteristics of AGAL NO. NM06/00007.

AGAL No. NM05/44593, AGAL No. NM05/44594, AGAL No. NM06/00007 and other suitable isolates of Z. muscarium are particularly effective biological control agents, being capable of surviving interrupted wet periods, desiccation, and colonising, incapacitating and killing phytopathogenic insects such as, but not limited to, aphids, whitefly, mealy bugs, Varroa mite, and thrips., in the field. The degree of killing of whitefiy, Varroa mite, and thrips, and aphids using a blastospore or condial composition by these isolates of L. muscarium is generally as good as the commonly used 5 insecticides employed by growers. However, resistance to such insectides by insects, for example thrips, whitefly and aphids, has become the greatest threat to the horticultural industry.

For example, overseas populations of Western flower thrips are resistant to most groups of pesticides. The following pesticides gave inadequate control in the 24

10 hour bioassay: acephate, abamectin, chlorpyrifos, endosulfan, methomyl, methiocarb, omethoate, pyrazophos, and tau-fluvalinate.

In New Zealand, resistance to deltamethrin is present in Onion thrips in the North and South Islands, but resistance to diazinon and dichlorvos has only been found near Auckland (Martin et al. in prep). Onion thrips have been reported to be resistant to

15 many pesticides in the USA, but still susceptible to synthetic pyrethroids (Grossman 1994).

Resistance to chlorpyrifos in Kelly's citrus thrips has been reported from South Australia (Purvis 2002).

There have been reports of insecticide resistance to greenhouse whitefiy, but

20 only the most recent, to buprofezin, has been confirmed (Workman & Martin 1995). Overseas, greenhouse whitefly has developed resistance to organochlorine, organophosphate, carbamate and pyrethroid insecticides (e.g. Georghiou 1981, Anis & Brennan 1982, Elhag & Horn 1983, Wardlow 1985, and Hommes 1986). Resistance has also been found in newer insecticides, buprofezin and tefmbenzuron (Gorman et al.

25 2000).

It is therefore apparent that many plant pathogenic insects have developed resistance to a number of insecticides; in these and other instances, L. muscarium isolates selected in accordance with the invention provide an effective alternative for insect control. This potent activity in the control of plant disease coupled with the

30 absence of any observations of plant pathogenicity induced by L. muscarium demonstrate that isolates of these species have desirable attributes for use as a biological control agent. Lecanicillium longisporum is an entomopathogenic fungi that is particularly pathogenic to aphids. The isolate KT4L1 has the following identifying characteristics:

100% Condidia 6.0x2.1 micron, Mycelium thallus is offwhite to yellow growing very roughly which could be described as lumpy in consistency. Mycelium thallus diffuses light red brown colour into agar.

One strain of Lecanicillium longisporum meeting the above requirements was isolated from aphids in Barley grass Banker plants in Franklin, Auckland, New Zealand. The insect was placed onto MEA and PDA and incubated at 24⁰C at 93% as above. It demonstrates the growth characteristics typical of the species when grown on MEA. Details of the isolation and selection process employed to obtain this isolate, strain KT4L1, are as set out in the Examples. This L. longisporum isolate has been deposited in the National Measurement Institute of Australia (formerly the Australian Government Analytical Laboratories, (AGAL)), 1 Suakin Street, Pymble, New South Wales, Australia on 3 March 2006 according to the Budapest Treaty for the purposes of patent procedure. The isolate has been accorded the deposit number NM06/00009. The deposit certificate and viability statement are presented herein.

Accordingly, in one aspect the present invention provides a biologically pure culture of L. longisporum AGAL No. NM06/00009. Similarly provided are Lecanicillium having the identifying characteristics of AGAL NO. NM06/00009. AGAL No. NM06/00009 and other suitable isolates of L. longisporum are particularly effective biological control agents, being capable of surviving interrupted wet periods, desiccation, and colonising, incapacitating and killing phytopathogenic insects such as aphids, in the field. The degree of killing of aphids using a blastospore or condial composition by these isolates of L. longisporum is generally as good as the commonly used insecticides employed by growers.

As discussed above, many plant pathogenic insects have developed resistance to a number of insecticides; in these and other instances, L. longisporum isolates selected in accordance with the invention provide an effective alternative for insect control. This potent activity in the control of plant disease coupled with the absence of any observations of plant pathogenicity induced by L. longisporum demonstrate that isolates of these species have desirable attributes for use as a biological control agent.

Beauveria bassiana is a soil born fungi that attacks both immature and adult insects including, for example, grasshoppers, aphids, thrips, moths, and several other species. Typically, B. bassiana can be isolated from insect cadavers, such as aphids, borers, and thrips, and may also be isolated from soil.

The isolate K4B1 shows a preference for thrips adults, while K4B2 exhibits a preference for caterpillars, including soybean looper caterpillar and white butterfly and army worm caterpillar. These isolates are also pathogenic to thrip juveniles, adults, and pupae, aphids and whitefly. These isolates have the following identifying characteristics:

Mycelium: Grows readily on MEA. Colonies are generally white at the edge becoming cream to pale yellow. Very occasionally reddish. Underside of mycelium thallus infuses a red blush pigment into agar.

Conidiophores: Abundant, rising from hyphae. l-2μm wide bearing groups of clustered conidiogenous cells 3-6x 3-5μm which may branch to give rise to further conidiogenous cells, globular to flask shape with well developed stalk up to 20μm long by 1 μm wide, geniculate with denticles up to lμm wide. Conidia: Clear globose conidia that are 2-3x 2-2.5μm. Blastospores are formed in submerged culture. Hydrophobic. Dusty, granular appearance in aggregation on agar. K4B2 forms yellow dusty aggregations, while K4B1 is cream.

One strain of B. bassiana meeting the above requirements was isolated from a borer larva within a pine forest in Bombay, New Zealand. It demonstrates the growth characteristics typical of the species when grown on MEA. Details of the isolation and selection process employed to obtain this isolate, strain K4B1, are set out in the Examples. This B. bassiana isolate has been deposited in the Australian Government Analytical Laboratories, (AGAL), 1 Suakin Street, Pymble, New South Wales, Australia on 16 March 2005 according to the Budapest Treaty for the purposes of patent procedure. The isolate has been accorded the deposit number NM05/44595.

Accordingly, in one aspect the present invention provides a biologically pure culture of B. bassiana AGAL No. NM05/44595. Similarly provided are Beauveria having the identifying characteristics of AGAL NO. NM05/44595.

A second strain of B. bassiana meeting the above requirements was isolated from a Lepidoptera caterpillar on a sunflower in the Aka Aka flats, New Zealand. The insect was placed onto MEA and PDA and incubated at 24°C at 93% as above. It demonstrates the growth characteristics typical of the species when grown on MEA. Details of the isolation and selection process employed to obtain this isolate, strain K4B2, are as set out in the Examples. This B. bassiana isolate has been deposited in the National Measurement Institute of Australia (formerly the Australian Government Analytical Laboratories, (AGAL)), 1 Suakin Street, Pymble, New South Wales, Australia on 3 March 2006 according to the Budapest Treaty for the purposes of patent procedure. The isolate has been accorded the deposit number NM06/00010. The deposit certificate and viability statement are presented herein.

Accordingly, in one aspect the present invention provides a biologically pure culture of B. bassiana AGAL No. NM06/00010. Similarly provided are Beauveria having the identifying characteristics of AGAL NO. NM06/00010. AGAL No. NM05/44595, AGAL No. NM06/00010 and other suitable isolates of B. bassiana are particularly effective biological control agents, being capable of surviving interrupted wet periods, desiccation, and colonising, incapacitating and killing phytopathogenic insects such as, but not limited to, aphids, caterpillars, whitefly, moths, Varroa mite and thrips in the field. The degree of killing of whitefly, thrips and aphids by these isolates of B. bassiana is generally as good as the commonly used insecticides as described above. Resistance to these insecticides has developed; in these and other instances, B. bassiana isolates selected in accordance with the invention provide an effective alternative for insect control. This potent activity in the control of plant disease coupled with the absence of any observations of plant pathogenicity induced by B. bassiana demonstrate that isolates of these species have desirable attributes for use as a biological control agent.

Paecilomyces fumosoroseus is an entomopathogenic fungi found in infected and dead insects, and in some soils. P. fumosoroseus typically infects whiteflies, thrips, aphids, and caterpillars. K4P1 has the following identifying characteristics:

Growth on insect: Produces simple mononematous conidiophores or distinct but loose synnemata. The synnemata are erect, up to 3 cm long and maybe branched, appearing dusty with conidia.

Growth on agar: On malt agar (MA) and PDA, growth is moderately rapid at room temperature (25°C) 4-8 cm in 14 days, with a basal felt with regular or irregular raised floccose overgrowth, or maybe thinner, dusty and granular, and producing definite coremia which are powdery when first isolated. White at first, remaining so or changing to shades of pink which may become tinged grey with age. Vegetative hyphae: Smooth walled, hyaline, 1-5 - 3.5 μm diameter. Conidial sti'uctures: Tend to be complex consisting of erect conidiophores arising from the basal felt or from aerial hyphae.

Conidiophores: Produced singly or together to form synnemata, up to 100 μm long x 1.5-2 (3) μm diameter. Smooth walled, hyaline, bearing verticils of branches, in turn bearing whorls of 3-6 phialides, occasional phialides produced at the same level as the branches and in the same verticil. Sometimes the verticillate pattern is broken and single branches are produced irregularly on the conidiophore. Phialides: 5-7 x 2.5 (3) μm, with a swollen base which tapers to a long thin neck about 0.5 μm diam.

Conidia: Cylindrical to fusiform with rounded ends, smooth, hyaline, borne in chains, 2-4 x 1-2 μm, occasionally up to 5 μm long.

On insects the conidiogenous apparatus tends to be more compacted with the branches and phialides inflated, slightly shorter and more rounded, 3.5-6 x 1-2.5. Conidia as in culture.

The K4P1 strain of Paecilomyces fumosoroseus meeting the above requirements was isolated from Diamond Back Moth caterpillar present on cabbage in Runciman, New Zealand. The insect was placed onto MEA and PDA and incubated at 24⁰C at 93% as above. It demonstrates the growth characteristics typical of the species when grown on MEA. Details of the isolation and selection process employed to obtain this isolate, strain K4P1, are as set out in the Examples. This P. fumosoroseus isolate has been deposited in the National Measurement Institute of Australia (formerly the Australian Government Analytical Laboratories, (AGAL)), 1 Suakin Street, Pymble, New South Wales, Australia on 3 March 2006 according to the Budapest Treaty for the purposes of patent procedure. The isolate has been .accorded the deposit number NM06/00008. The deposit certificate and viability statement are presented herein.

Accordingly, in one aspect the present invention provides a biologically pure culture of P. fumosoroseus AGAL No. NM06/00008. Similarly provided are Paecilomyces having the identifying characteristics of AGAL NO. NM06/00008. AGAL No. NM06/00008 and other suitable isolates of P. fumosoroseus are particularly effective biological control agents, being capable of surviving interrupted wet periods, desiccation, and colonising, incapacitating and killing phytopathogenic insects such as, but not limited to, whitefly, Varroa mite, and Lepidoptera caterpillar in the field. The degree of killing of whitefly, Varroa mite, and thrips, and aphids using a blastospore or condial composition by these isolates of P. fumosoroseus is generally as good as the commonly used insecticides employed by growers.

As discussed above, many plant pathogenic insects have developed resistance to a number of insecticides; in these and other instances, P. fumosoroseus isolates selected in accordance with the invention provide an effective alternative for insect control. This potent activity in the control of plant disease coupled with the absence of any observations of plant pathogenicity induced by P. fumosoroseus demonstrate that isolates of these species have desirable attributes for use as a biological control agent. In a further aspect the present invention provides a composition which comprises at least one fungi as defined above together with at least one diluent, adjuvant, carrier and/or excipient.

The composition may include multiple strains of entomopathogenic fungi, and in certain embodiments, multiple strains may be utilised to target a number of phytopathogenic species, or a number of different developmental stages of a single phytopathogen, or indeed a combination of same. For example, the pupal form of a phytopathogenic insect may be targeted with one fungal strain, while the adult form of the phytopathogenic insect may be targeted with another fungal strain, wherein both strains are included in a composition of the invention. In other embodiments, three strains or less will be preferred, and frequently a single strain will be preferred.

Suitably, the composition comprises fungi is selected from the group consisting of Lecanicillium muscarium strain K4V1 (Australian Government Analytical Laboratories Accession No. NM05/44593) or a strain having the identifying characteristics thereof; Lecanicillium muscarium strain K4V2 (Australian Government Analytical Laboratories Accession No. NM05/44594) or a strain having the identifying characteristics thereof; Lecanicillium muscarium strain K4V4 (National Measurement Institute of Australia Accession No. NM06/00007) or a strain having the identifying characteristics thereof; Beauveria bassiana strain K4B1 (Australian Government Analytical Laboratories Accession No. NM05/44595) or a strain having the identifying characteristics thereof; Beauveria bassiana strain K4B2 (National Measurement Institute of Australia Accession No. NM06/00010) or a strain having the identifying characteristics thereof; Lecanicillium longisporum strain KT4L1 (National Measurement Institute of Australia Accession No. NM06/00009) or a strain having the identifying characteristics thereof; and Paecilomyces fwnosoroseus strain K4P1 (National Measurement Institute of Australia Accession No. NM06/00008) or a strain having the identifying characteristics thereof.

Examples of compositions comprising entomopathogenic fungi are well known in the art, and include those described in, for example, WO95/10597 (published as PCT/US94/11542) to Mycotech Corporation, WO2003/043417 (published as PCT/US2002/037218) to The United States of America as represented by The Secretary of Agriculture, US Patent No. 4,530,834 to McCabe et al., and US Patent Application No. 10/657,982 (published as US 2004/0047841) to Wright et al., each incorporated by reference herein in its entirety.

To be suitable for application to a plant or its surroundings, said at least one diluent, adjuvant, carrier and/or excipient is an agriculturally acceptable diluent, adjuvant, carrier and/or excipient, more preferably is selected from the group consisting of a filler stimulant, an anti-caking agent, a wetting agent, an emulsifier, and an antioxidant, more preferably said composition comprises at least one of each of a filler stimulant, an anti-caking agent, a wetting agent, an emulsifier, and an antioxidant. Preferably, said filler stimulant is a carbohydrate source, such as a disaccharide including, for example, sucrose, fructose, glucose, or dextrose, said anti-caking agent is selected from talc, silicon dioxide, calcium silicate, or kaelin clay, said wetting agent is skimmed milk powder, said emulsifier is a soy-based emulsifier such as lecithin or a vegetable-based emulsifier such as monodiglyceride, and said antioxidant is sodium glutamate or citric acid. However, other examples well known in the art may be substituted, provided the ability of the composition to support fungal viability is maintained. Preferably, said composition is a biological control composition. The concentration of the entomopathogenic fungi of the invention present in the composition that is required to be effective as biological control agents may vary depending on the end use, physiological condition of the plant; type (including insect species), concentration and degree of pathogen infection; temperature, season, humidity, stage in the growing season and the age of plant; number and type of conventional insecticides or other treatments (including fungicides) being applied; and plant treatments (such as deleafing and pruning) may all be taken into account in formulating the composition. For use as a biological control agent, the entomopathogenic fungi of the invention present in the composition must be in a reproductively viable form. The term reproductively viable as used herein includes mycelial and spore forms of the fungi. For example, for most purposes, fungal strains are desirably incorporated into the composition in the form of spores (conidia or blastospores). Spores are obtainable from all the fungal strains of the invention, and may be produced using known art techniques. Spores obtained from the fungal strains of the invention form a further aspect of the invention. The concentration of the fungal spores in the composition will depend on the utility to which the composition is to be put. An exemplary concentration range is from about 1 x 10⁶ to 1 x 10¹² spores per ml, preferably from about 1 x 10⁷ to 2 x 10¹⁰, and more preferably 1 x 10⁷ to 1 x 10⁸ spores per ml.

In theory one infective unit should be sufficient to infect a host but in actual situations a minimum number of units are required to initiate an infection. The concepts of Lethal dose regularly used with chemical pesticides are inappropriate for microbial pesticides. Concepts of Infective Dose or Infective Concentration are more precise or applicable. ID or IC refer to the actual number of infective units needed to initiate infection or the number of infective units exposed to the host to cause death. Therefore, the number of infective units applied in the field or greenhouse against a host will affect the degree of control. It is important to apply the desired concentration of the pathogen, property placed and at the right time, to obtain good control of the pest, this is known as the Infection Threshold.

It will be apparent that the concentration of fungal spores in a composition formulated for application may be less than that in a composition formulated for, for example, storage. The Applicants have determined that with the entomopathogenic fungi of the present invention, infection threshold occurs at about 10⁷ spores per ml of sprayable solution, when applied at a rate of about lkg per hectare. Accordingly, in one example, a composition formulated for application will preferably have a concentration of at least about 10⁷ spores per ml. In another example, a composition formulated for storage (for example, a composition such as a wettable powder capable of formulation into a composition suitable for application) will preferably have a concentration of about 10¹⁰ spores per gram. It will be apparent that the spore concentration of a composition formulated for storage and subsequent formulation into a composition suitable for application must be adequate to allow said composition for application to also be sufficiently concentrated so as to be able to be applied to reach infection threshold.

Preferably, the composition is a stable composition capable of supporting reproductive viability of said fungi for a period greater than about two weeks, preferably greater than about one month, about two months, about three months, about four months, about five months, more preferably greater than about six months. To be suitable for use as a biological control composition, the composition preferably is able to support reproductive viability of the fungi for a period greater than about six months.

Using conventional solid substrate and liquid fermentation technologies well known in the art, the entomopathogenic fungi of the invention can be grown in sufficient amounts to allow use as biological control agents. For example, spores from selected strains can be produced in bulk for field application using nutrient film, submerged culture, and rice substrate growing techniques. Growth is generally effected under aerobic conditions at any temperature satisfactory for growth of the organism. For example, for L. muscariiim, a temperature range of from 8 to 3O⁰C, preferably 15 to 25°C, and most preferably 24°C, is preferred. The pH of the growth medium is slightly acid to neutral, that is, about 5.0 to 7.0, and most usually 5.5. Incubation time is sufficient for the isolate to reach a stationary growth phase, about 14 days when incubated at 24⁰C, and will occur under artificial photoperiod. For B. bassiana, a temperature range of from 10 to 32°C, preferably 25 to 3O⁰C, and most preferably 23°C, is preferred. The pH of the growth medium is slightly acid to neutral, that is, about 5.0 to 7.0, and most preferably 5.5. Incubation time is sufficient for the isolate to reach a stationary growth phase, about 21 days when incubated at 23⁰C, and will occur in normal photoperiod. The spores may be harvested by methods well known in the art, for example, by conventional filtering or sedimentary methodologies (eg. centrifugation) or harvested dry using a cyclone system. Spores can be used immediately or stored, chilled at 0° to 6°C, preferably 2°C, for as long as they remain reproductively viable. It is however generally preferred that when not incorporated into a composition of the invention, use occurs within two weeks of harvesting.

The composition of the invention may also include agriculturally acceptable carriers, diluents or excipients. The compositions may also comprise a broad range of additives such as surfactants, wetters, humectants, stickers, spreaders, stablisers and penetrants used to enhance the active ingredients and so-called 'stressing' additives to improve spore vigor, germination and survivability such as potassium chloride, glycerol, sodium chloride and glucose. Additives may also include compositions which assist in maintaining microorganism viability in long term storage, for example unrefined corn oil and so called invert emulsions containing a mixture of oils and waxes on the outside and water, sodium alginate and conidia on the inside.

Examples of surfactants, spreaders and stickers include Fortune®, carola (capsicums), C-Daxoil®, Codacide oil®, D-C. Tate®, Supamet Oil, Bond® Penetrant, Citowett®, Fortune Plus™, Fortune Plus Lite, Fruimec, and Fruimec lite. Where selected for inclusion, common agricultural surfactants, such as Tween

(available from Rohm & Haas) are desirably included in the composition according to known protocols. It is important that any additives used are present in amounts that do not interfere with the effectiveness of the biological control agents.

Examples of suitable compositions including carriers, preservations, surfactants and wetting agents, spreaders, and nutrients are provided in US 5780023, incorporated herein in its entirety by reference.

The Applicants have also determined that many commonly used fungicides do not adversely affect the entomopathogenic fungi of the invention. The compositions of the invention may therefore also include such fungicides. Alternatively, the compositions may be used separately but in conjunction with such fungicides in control programmes.

The invention also provides a method of producing a composition comprising one or more entomopathogenic fungi of the invention, said method comprising obtaining a reproductively viable form of said entomopathogenic fungi, and combining said reproductively viable form of said entomopathogenic fungi with at least one agriculturally acceptable diluent, carrier or excipient.

The compositions may be prepared in a number of forms. One preparation comprises a powdered form of a composition of the invention which may be dusted on to a plant or its surroundings. In a further form, the composition is mixed with a diluent such as water to form a spray, foam, gel or dip and applied appropriately using known protocols. In the presently preferred embodiment, a L. muscarium composition formulated as described above is mixed with water at about lgm/L, or about 1 to 3 kg/ha in no less than IOOOL water per ha. Conveniently, when the composition is to be administered by spraying, this may be done directly in the pressurised sprayer with which the composition is to be administered. Preferably, Fortune Plus™ is added to the composition as a UV and desiccation protection agent at about lml/L. In the presently preferred embodiment, a B. bassiana composition formulated as described above is mixed with water using a pressurised sprayer at about lgm/L, or about 1 to 3 kg/ha in no less than IOOOL water per ha. Preferably, Fortune Plus™ is added to the composition as a UV and desiccation protection agent at about lml/L. Compositions comprising L. longisponim, or P. fumosoroseus can be applied in a similar manner.

Compositions formulated for other methods of application such as injection, rubbing or brushing, may also be used, as indeed may any known art method. Indirect applications of the composition to the plant surroundings or environment such as soil, water, or as seed coatings are potentially possible.

As discussed above, the concentration at which the compositions comprising entomopathogenic fungi of the invention are to be applied so as to be effective biological control agents may vary depending on the end use, physiological condition of the plant; type (including insect species), concentration and degree of pathogen infection; temperature, season, humidity, stage in the growing season and the age of plant; number and type of conventional insecticides or other treatments (including fungicides) being applied; and plant treatments (such as leaf plucking and pruning). For example, in certain applications, a composition comprising B. bassiana may be applied, at a rate of from about 1 x 10¹⁰ to about 1 x 10¹⁵ spores per hectare, preferably from about 1 x 10 to about 1 x 10¹⁴ spores per hectare, more preferably from about 5 x 10¹² to about 1 x 10¹⁴ spores per hectare, more preferably about 1-3 x 10¹³ spores per hectare. In another example, in certain applications, a composition comprising L. muscarϊum may be applied, at a rate of from about 1 x 10¹⁰ to about 1 x 10¹⁵ spores per hectare, preferably from about 1 x 10¹² to about 1 x 10¹⁴ spores per hectare, more preferably from about 5 x 10¹² to about 1 x 10¹⁴ spores per hectare, more preferably about 1-3 x 10¹³ spores per hectare. In a further aspect the present invention provides a method for controlling one or more phytopatho genie insects, the method comprising applying to a plant or its surroundings a reproductively viable form and amount of at least one entomopathogenic fungi as described herein. Preferably, said fungi is selected from the group consisting of Lecanicillium muscarium strain K4V1 (Australian Government Analytical Laboratories Accession No. NM05/44593) or a strain having the identifying characteristics thereof; Lecanicillium muscarium strain K4V2 (Australian Government Analytical Laboratories Accession No. NM05/44594) or a strain having the identifying characteristics thereof; Lecanicillium muscarium strain K4V4 (National Measurement Institute of Australia Accession No. NM06/00007) or a strain having the identifying characteristics thereof; Beauveria bassiana strain K4B1 (Australian Government Analytical Laboratories Accession No. NM05/44595) or a strain having the identifying characteristics thereof; Beauveria bassiana strain K4B2 (National Measurement Institute of Australia Accession No. NM06/00010) or a strain having the identifying characteristics thereof; Lecanicillium longisporum strain KT4L1 (National Measurement Institute of Australia Accession No. NM06/00009) or a strain having the identifying characteristics thereof; and Paecilomyces fumosoroseus strain K4P1 (National Measurement Institute of Australia Accession No. NM06/00008) or a strain having the identifying characteristics thereof.

As described above, the term "controlling" as used herein generally comprehends preventing or reducing phytopathogenic insect infection or inhibiting the rate and extent of such infection.

Again, while multiple strains of the entomopathogenic fungi of the invention with activity against one or more phytopathogenic insect species may be employed in the control process, usually three strains or less, and more commonly a single strain is used in the process. In a presently preferred embodiment for the treatment of, for example, apbids, whitefly, Varroa mite, or thrips, the single strain L. muscarium AGAL No. NM05/44593 is employed. In an alternate preferred embodiment for the treatment of, for example, aphids, whitefly, Varroa mite, or thrips, the single strain L. muscarium AGAL No. NM05/44594 is employed. In yet a futher preferred embodiment for the treatment of, for example, aphids, thrips, Varroa mite, or whitefly, the single strain B. Beauveria AGAL No. NM05/44595 is employed.

Repeated applications at the same or different times in a crop cycle are also contemplated. The entomopathogenic fungi of the invention may be applied either earlier or later in the season. This may be over flowering or during fruiting. The entomopathogenic fungi of the invention may also be applied immediately prior to harvest, or after harvest to rapidly colonise necrotic or senescing leaves, fruit, stems, machine harvested stalks and the like to prevent insect colonisation. The entomopathogenic fungi of the invention may also be applied to dormant plants in winter to slow insect growth on dormant tissues.

Application may be at a time before or after bud burst and before and after harvest. However, treatment preferably occurs between flowering and harvest. To increase efficacy, multiple applications (for example, 2 to 6 applications over the stages of flowering through fruiting) of the entomopathogenic fungi of the invention or a composition of the invention is preferred.

Reapplication of the entomopathogenic fungi of the invention or composition should also be considered after rain. Using insect infectivity prediction models or infection analysis data, application of the BCA can also be timed to account for insect infection risk periods.

In the presently preferred embodiments, the entomopathogenic fungi of the invention or a composition comprising same is applied in a solution, for example as described above, using a pressurised sprayer. The plant parts should be lightly sprayed until just before run off. Applications may be made to any part of the plant and/or its surroundings, for example to the whole plant canopy, to the area in the canopy where the flowers and developing fruit are concentrated, or to the plant stem and/or soil, water or growth media adjacent to or surrounding the roots, tubers or the like. Preferably the entomopathogenic fungi-comprising composition is stable. As used herein, the term "stable" refers to a composition capable of supporting reproductive viability of said fungi for several weeks, preferably about one, about two, about three, about four, preferably about five, more preferably about six months, or longer. Preferably, the composition is stable without a requirement for storage under special conditions, such as, for example, refrigeration or freezing.

The applied compositions control phytopathogenic insects. Phytopatho genie insects are responsible for many of the pre- and post-harvest diseases which attack plant parts and reduce growth rate, flowering, fruiting, production and may cause death of afflicted plants. As used herein, phytopathogenic insects include insects which are themselves plant pathogens, and insects which may act as a vector for other plant pathogens, for example, phytopathogenic fungi and bacteria. It will be appreciated that by controlling host insects which act as vectors for other phytopathogens, the incidence and/or severity of plant disease can be minimised. Examples of the major phytopathogenic insects afflicting a number of important horticultural crops grown in New Zealand are presented in Table 1 below. Table 1. Major Insect Pests

Control of whitefly, thrips, aphids, and caterpillars in the crops outlined above using the compositions and method of the present invention is particularly contemplated. Control of Varroa mite using L. muscarium, B. bassiana, or Paecilomyces fumosoroseus and compositions of the present invention comprising same are also particularly contemplated. The term "plant" as used herein encompasses not only whole plants, but extends to plant parts, cuttings as well as plant products including roots, leaves, flowers, seeds, stems, callus tissue, nuts and fruit. Plants that may benefit from the application of the present invention cover a broad range of agricultural and horticultural crops. The compositions of the present invention are also especially suitable for application in organic production systems.

The process of the invention has particular application to plants and plant products, either pre- or post-harvest. For example, the composition of the invention may be applied to stored products of the type listed above including fruits, vegetables, cut flowers and seeds. Suitable application techniques encompass those identified above, particularly spraying. The composition can potentially be used to treat or pretreat soils or seeds, as opposed to direct application to a plant. The composition may find use in plant processing materials such as protective coatings, boxes and wrappers.

The term "surroundings" when used in reference to a plant subject to the fungi, methods and compositions of the present invention includes soil, water, Pleaf litter, and/or growth media adjacent to or around the plant or the roots, tubers or the like thereof, adjacent plants, supports, water to be administered to the plant, and coatings including seed coatings. It further includes storage, packaging or processing materials such as protective coatings, boxes and wrappers. Also encompassed by the present invention are plants, plant products, soils and seeds treated directly with an active strain of the entomopathogenic fungi of the invention or a composition of the invention.

In a further aspect, the present invention extends to the use of entomopathogenic fungi of the invention in a composition of the invention. The invention consists in the foregoing and also envisages constructions of which the following gives examples only and in no way limit the scope thereof.

EXAMPLE 1 - IDENTIFICATION AND ISOLATION OF Lecanicillium muscarium STRAIN K4V1 This strain was originally isolated during a screen of thousands of insects for entomopathogenic fungi from a whitefly in a greenhouse tomato crop at Pukekohe, New Zealand. Fungi was isolated from the insect sample using standard procedures, including growth at 24 C at 93% relative humidity to maximise sporulation. Individual colonies were then sub-cultured onto MEA to yield pure strains for screening for entomopathogenic efficacy. Lecanicillium characteristics

The isolate was identified as Lecanicillium muscarium using taxonomic references well known in the art. Morphological characteristics Colonies on agar. Colonies on PDA, MA or OA are white, creamy thin cottony with reverse colourless to pale or deep yellow.

Conidiophores: Phialides are formed singly or directly from mycelium or in whorls of 3 or 4 erect conidiophores much like vegetative mycelium. Phialides delicate, of variable size depending on both strain and the age of the culture. Size from 8.5-16x 0.8-1.2μm to 30-4Ox 2-2.2μm.

Conidial Morphology: Produced singly and aggregating on heads at the tips of the phialides in a mucilaginous matrix. Ellipsoidal to cylindrical with rounded ends varying in size with 60% Conidia 1.0x1.0 micron on whitefly scale, 30% Conidia 2.0x1.0 micron on thrip juveniles (nymphs), 10% Conidia 2.5x1.3 micron on thrip pupae. Blastospores are produced in submerged culture. Hydrophilic. Underside of mycelium thallus sparsely creased, Mycelium thallus removes from the agar very easily.

All media including MEA, OA, PDA and SA were prepared according to methods well known in the art

EXAMPLE 2 - IDENTIFICATION AND ISOLATION OF Lecanicillium muscarium STRAIN K4V2

This strain was originally isolated during a screen of thousands of insects for entomopathogenic fungi from a whitefly from a cucumber greenhouse at Ruakaka, New Zealand. Fungi was isolated from the insect sample using standard procedures, including growth at 24 C at 93% relative humidity to maximise sporulation. Individual colonies were then sub-cultured onto MEA to yield pure strains for screening for entomopathogenic efficacy. Lecanicillium characteristics

The isolate was identified as Lecanicillium muscarium using taxonomic references well known in the art. Morphological characteristics Colonies on agar. Colonies on PDA ,MA or OA are white, creamy thin cottony with reverse colourless to pale or deep yellow.

Conidiophores: Phialides are formed singly or directly from mycelium or in whorls of 3 or 4 erect conidiophores much like vegetative mycelium. Phialides delicate, of variable size depending on both strain and the age of the culture. Size from 8.5-16x 0.8-1.2μm to 30-4Ox 2-2.2μm. Conidial Morphology: Produced singly and aggregating on heads at the tips of the phialides in a mucilaginous matrix. Ellipsoidal to cylindrical with rounded ends varying in size with 50% Conidia 2.0x1.5 micron, 30% Conidia 2.0x1.0 micron, 20% Conidia 1.0x1.0 micron. Pathogenic to Whitefly adults, while Blastospores pathogenic to aphids. Underside of mycelium thallus frequently creased, Mycelium thallus difficult to remove from agar surface. Blastospores are produced in submerged culture. Hydrophilic.

EXAMPLE 3 - IDENTIFICATION AND ISOLATION OF Beauveria bassiana

STRAIN K4B1

This was originally isolated during a screen of thousands of insects for entomopathogenic fungi from a borer larvae in a pine forest at Bombay, New Zealand.

Fungi was isolated from the insect sample using standard procedures, including growth at 24 C at 93% relative humidity to maximise sporulation. Individual colonies were then sub-cultured onto MEA to yield pure strains for screening for entomopathogenic efficacy.

Beauverium characteristics

The isolate was identified as Beauverium bassiana using taxonomic references well known in the art.

Morphological characteristics

The isolate K4B1 exhibits a preference for caterpillars - soybean looper caterpillar and white butterfly and army worm caterpillar. This isolate is also pathogenic to thrip juveniles, adults, and pupae, aphids and whitefly. This isolate has the following identifying characteristics:

Conidiophores: Abundant, rising from hyphae. l-2μm wide bearing groups of clustered conidiogenous cells 3-6x 3-5μm which may branch to give rise to further conidiogenous cells, globular to flask shape with well developed stalk up to 20μm long by 1 μm wide, geniculate with denticles up to 1 μm wide.

Conidia: Clear globose conidia that are 2-3x 2-2.5μm. Blastospores are formed in submerged culture. Hydrophobic. Dusty, granular appearance in aggregation on agar.

EXAMPLE 4 - COMPARISON OF WHITEFLY CONTROL USING Lecanicilliiim muscarium AND CHEMICAL INSECTICIDES

Introduction This example describes field trials conducted to assess the efficacy of Lecanicillium muscarium strain K4V2 as a biological control agent of whitefly, and comparing same to established chemical treatment procedures. The trial was conducted in two 1680 m² Venlo style Faber glasshouses, complete with coal fired boilers and Chemtest environment and irrigation controllers. The glasshouses were constructed one year apart and were in all cases, apart from drainage of runoff, identical. A chemical pesticide regime was conducted in Glasshouse 1, and a trial of the BCA of the present invention was conducted in Glasshouse 2. Methods Glasshouse 1

This glasshouse was planted with the De Ruiter variety Antarctica, one month earlier than Glasshouse 2 (due in part to hormone damage from neighbouring properties). As is normal practice after establishment, plants were allowed to reach knee high before Vydate (240gm/L oxamyl) was applied via the irrigation at lOOml/lOOOm². As the crop progressed, the dose of Vydate was increased to 200ml/100m² and finally 300ml/1000m when the crop had reached shoulder height. Vydate with an LD50 of 37mg/kg must be withdrawn within 4 weeks of harvest so as to not exceed the maximum recommended level (MRL) for oxamyl in the fruit. Normal regimes of Lannate (200gm/L methomyl) and Thiodan (350gm/L endosulfan) mixture, Chess (250gm/kg pymetrozine) and Attack (25gm/L permethrin plus 475gm/L pirimiphos methyl) were then followed. Glasshouse 2

This glasshouse was planted with the De Ruiter variety Toronto. This variety is a much harder variety to manage than the Antartica variety planted in Glasshouse 1. As is normal practice, Whitefly control was initially performed with Vydate and followed the same regime as described above for Glasshouse 1. On withdrawal of the Vydate, Lecanicillium muscarium was applied. Lecanicillium muscarium was introduced into IL of 0.1%Triton x 100, and built up to a spore concentration of 1010/ml using a haemocytometer. The spore solution was chilled to 2°C and then transported immediately to Glasshouse 2. This spore solution was then added to the IOOL spray tank to achieve a spore count of 10⁷AnI to achieve infection threshold. Fortune Plus™, a food grade vegetable oil, was then added as a humectant at the rate of lOOml/lOOL. Spores were applied in falling temperatures and high humidity so that the greenhouse would exceed 80% rH during the night. This process was repeated 4 times in December and then repeated 4 times in February. Results The difference in crop health and the numbers of Whitefly scale on the lower leaves of each crop was clearly evident. Plants to which the BCA had been applied were in excellent condition with noticeably less whitefly scale observed than those of Glasshouse No. 1 which were treated with chemical insecticides.

Infection rate was determined quantitatively by counting the number of whitefly scale on a representative number of plants in each glasshouse, so as to determine a whitefly scale average per plant. As can be seen in Figure 1 and Table 2 below, the average number of Whitefly scale on plants treated with the BCA formulation were dramatically lower (approximately two orders of magnitude lower) than that on plants treated with chemical insecticides.

Table 2. Average number of Whitefly scale per plant

Discussion

As shown above the Lecanicillim muscarium BCA provided excellent control of whitefly coupled with a simple application regimen.

The Lecanicillium muscarium BCA accordingly presents a very good option for growers to control Whitefly infestation. This trial further demonstrates that many of the chemical pesticides being used as normal practice for Tomato growing have lost efficacy as Whitefly have become resistant.

EXAMPLE 5 - CONTROL OF THRIPS USING SELECTED STRAINS

Lecanicillium muscarium AND Beauveria bassiana Introduction

The growers of this trial traditionally lose control of their Thrip populations by December of every growing season. Spray trials were conducted within two 1.4ha greenhouse blocks within the 7ha Glasshouse to assess the efficacy of two BCA formulations in lowering and controlling the population of thrips. Methods

The grower's 7ha Venlo style Faber glasshouse was divided by plastic walls into 5 1.4ha greenhouse blocks. Two greenhouses were set aside for the entomopathogenic trial, these being Greenhouse I₅ (the most western end) and Greenhouse 5, (the most eastern end). Greenhouse 1 was selected for Lecanicillium muscarium strain K4V1 and

Greenhouse 5 was selected for Beauveria bassiana strain K4B1. These greenhouses were assessed as having the highest population of thrips. The remaining three greenhouses were control greenhouses in which normal chemical insecticide regimes were conducted. On average each 1.4ha block has a water rate of 3000L per application.

Spraying was conducted in the evening to disrupt as little as possible the work activities within the greenhouse.

Greenhouse 1 Lecanicillium muscarium was introduced into 3L of 0.1%Triton x 100, and built up to a spore solution of 10¹⁰/ml using a haemocytometer. The spore solution was chilled to 2⁰C and then transported immediately to the Glasshouse. This spore solution was then added to the 3000L spray tank to achieve a spore count of 10⁷/ml to achieve infection threshold. Fortune Plus™, a food grade vegetable oil, was then added as a humectant at the rate of 1 OOml/100L. Greenhouse 5

Beauveria bassiana was introduced into 3L of 0.1%Triton x 100, and built up to a spore solution of 10¹⁰/ml using a Haemocytometer. The spore solution was chilled to 2°C and then transported immediately to the Glasshouse. This spore solution was then added to the 3000L spray tank to achieve a spore count of 10⁷/ml to achieve infection threshold. Fortune Plus™, a food grade vegetable oil, was then added as a humectant at the rate of lOOml/lOOL.

Control Greenhouses 2, 3 and 4

The control houses were sprayed with a common mixture of insecticides, being Match, (50gm/L lufenuron) at lOOml/lOOL, and Success, (120gm/L Spinosad) at 40ml/100L, on a weekly basis. Assessment Procedures Greenhouse 1, Greenhouse 3 and Greenhouse 5 had an area selected that was exactly Im². Each area has 4 mature fruiting plants which had been split to grow two stems so there was a total of 8 stems per area. One flower was selected from the head of one plant for each week. Each week one flower from each area was sprayed with p- anisaldehyde at lml/L to immobilize the thrips and the flower was then harvested and placed into a plastic vial for analysis.

Each flower was then retreated with p-anisaldehyde to immobilize the thrips and the thrips were then counted and adults and juveniles were identified. Results As can be seen in Figure 2 and Table 3 below, both the Lecanicillim and

Beauveria BCA formulations were highly effective against thrip infestation. Notably, after 28 days of treatment with Lecanicillium BCA, no juvenile thrips were observed. Similarly after 35 days of treatment with Beauveria BCA, no juvenile thrips were observed.

In contrast, treatment with chemical insecticides did not reduce thrip infestation, and indeed the number of adult thrips and juvenile tlirips steadily increased during the trial.

Table 3. BCA efficacy against Thrips

Discussion

The above results clearly indicated that the growers had developed a population of thrips that were resistant to the insecticides Match and Success. The application of both the Beauveria hassiana BCA formulation and the Lecanicillium muscarium BCA formulation successfully controlled the resident population of thrips. The short term increase in adult thrips observed in week two of the experiment is consistent with other trials, and is believed to result from adult thrips flying in through the vents of the Greenhouses.

The amount of eggs and egg laying observed in the head of the plants also decreased significantly. This is believed to suggest that not only are the BCAs effective in controlling existing populations of insects, but are effective to prevent or reduce the emergence of new populations. At the completion of the trial the growers implemented the spraying of the whole 7ha glasshouse with both Beauveria bassiana and Lecanicillium muscarium BCAs.

EXAMPLE 6 - CONTROL OF LETTUCE APHID USING A SELECTED

STRAIN OF Lecanicillium muscarium Introduction

Trials were peformed at the greenhouse of a grower of organic lettuce to assess and achieve control of Lettuce Aphid without the introduction of chemical pesticides. Methods

The lettuces spend 5 weeks on the benches in the greenhouse, coming from the nursery very small and growing to a saleable size. The greenhouse, a 1000m plastic house, was divided into 4 benches of NFT gullies. Lecanicillium muscarium BCA was applied to one full bench. The remaining 3 benches within the greenhouse were designated the control, to which the insecticides currently being used were applied.

The current insecticides being applied are Pyganic (13gm/L Pyrethrins) at lOOml/lOOL, alternated weekly withNeemazal (lOgm/L azadiractin) at 300ml/100L. The normal water rate is IOOL per bench. Trial BCA

Lecanicillium muscarium blastospores were harvested from broth and built up to a spore solution of 1010/ml using a haemocytometer. The spore solution was chilled to 2⁰C and then transported immediately to the greenhouse. This spore solution was then added to the IOOL spray tank to achieve a spore count of 10⁷/ml to achieve infection threshold. Fortune Plus™, a food grade vegetable oil, was then added as a humectant at the rate of 100ml/ IOOL. The application of the spore solution was done in falling temperatures and rising humidity. This procedure was repeated on a weekly basis for 5 weeks. Assessment Procedures

One lettuce was harvested from one of the control blocks and the Trial block at random. These lettuce samples were bagged to prevent the escape of any aphids and taken for analysis. The two samples were refrigerated for 2hrs to slow down the aphids to facilitate counting. In the latter stages, the lettuces were destructively analyzed as they had begun to form hearts. The results reflect only the numbers of healthy aphids present, not the number of infected cadavers. Results

As can be seen in Figure 3 and Table 4 below, the Lecanicillium BCA was highly effective in the control of aphid infestation of lettuce. After a single application of BCA, aphid numbers were approximately half those seen on control plants. After 28 days of Lecanicillim BCA application, no live aphids were observed. In comparison, control lettuces were heavily infested with aphids. Table 4: Control of Lettuce Aphids

Discussion

As shown above the Lecanicillium BCA was highly effective in control of aphids on lettuce plants.

In comparison, the control insecticides demonstrated little if any efficacy in reducing populations of the Lettuce aphid. Indeed, at the completion of the trial the Lecanicillium row was harvested, while the control rows were dumped.

EXAMPLE 7 - HONEY BEE RESPONSE ON EXPOSURE TO Vβiticillium lecanii AND Beauveria bassiana Introduction The European Honeybee Apis mellifera is an economically and environmentally important insect in New Zealand. This trial was conducted to establish whether the entomopathogenic fungi LecanicilHum muscarium and Beauveria Bassiana exhibit any threat to Apis mellifera. Furthermore, the efficacy of these fungi against Varroa mite {Varroa destructor), an ectoparasite of the European honeybee, was investigated. Methods

Materials

Three BCA formulations were tested: Beaugenic™, containing Beauveria bassiana K4B1 at 10¹⁰ spores per gram, Vertiblast™, containing LecanicilHum muscarium K4V2 at 10¹⁰ blastospores per gram, and Vertildl™, containing Verticillium lecanii K4V1 at 10¹⁰ spores per gram. These formulations were tested both topically and orally and at concentrations that would be considered a typical spray concentration of 10⁷ spores per ml. As recommended by the OECD guidelines, 3 concentrations of

Dimethoate were used as a toxic standard. For the topical test a control of sterile distilled water was employed while the oral test control bees were fed sugar water. All trials were replicated three times.

Bees and Cages

For each test, 1000 Apis mellifera ligustica were collected from brood combs and were anaesthetized with CO₂. After topical and oral treatments, bees were held in groups of 10, in units of 3 wooden cages measuring 68mm long, 25mm wide and 25mm deep. The top and bottom were covered in wire mesh, and cages were rested slightly elevated on wooden dowel to allow complete circulation of air. Feeder tubes holding 8ml were fitted through a drilled hole at the end of each cage. One frame held 3 cages of 30 bees which were subjected to each formulation. To maximize the pathogenicity of the formulations employed, the frames were maintained at the fungi's optimum temperature of 24⁰C and at a relative humidity of 80%. The incubators used had no light, but the bees were exposed to normal lighting during evaluation and mortality assessment.

Topical Tests Test formulations were diluted at lgm/L of distilled and sterile water to achieve a solution of 10 spores per ml. lμl of test solution was applied with a micropipette to the dorsum of the mesosoma of each anaesthetized bee. Each cage containing 10 treated bees was supplied with 6ml of 50:50 sugar water in the feeder tube. Oral Tests

Test formulations were diluted at lgm/L of prepared 50:50 sugar water to achieve a solution of 10⁷ spores per ml. 3ml was dispensed per feeder tube. The volume of fluid consumed was recorded over the duration of the experiment. Assessment and Statistical analysis

The number of dead bees per cage was assessed at 4, 24 and 48 hrs after treatment. Probit analysis was used to estimate LD₅₀ values where the data showed a dose response. Mortality due to each of the treatments was adjusted for control mortality using the correction of Abbott, (1925). The 95% confidence limits were calculated on the scale of the linear predictor and back transformed to percentages. Where there was no mortality, upper 95% confidence limits for percent mortality were calculated assuming standard binominal distribution theory. Results

Topical Test As shown in Table 5 below, at each assessment time no mortality was observed with either the BCA formulations or the controls.

LD_5O values could not be calculated. Mortality after 24 and 48hrs for Dimethoate was 100% at all 3 doses. Therefore, the LD₅₀ for the BCA formulations after 24hrs was less than 200ml/l 00L. Oral Test

An average of 435μl was consumed from each feeding tube. No mortality was observed in either the control or BCA formulation. Due to the lack of mortality in any of the three formulation tests, LD₅₀ values could not be calculated. The mortality after 24hrs for Dimethoate was 100% for all three doses. Therefore the LD₅₀ for the BCA formulations after 24hrs was less than 20OmIAOOL. Table 5. Effect of BCA on Bee Mortality

Discussion

All three BCA formulations tested topically and orally under the OECD guidelines were non toxic to honey bees at levels that foraging bees would likely come into contact with from spray residue.

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JNDUSTRIAL APPLICATION

As will be evident from the above description, the present invention provides strains of entomopathogenic fungi, together with the compositions comprising said fungi, useful for the control of phytopathogenic insects. The use of such fungi in the control of phytopathogenic insects, and methods to control phytopathogenic insects, are also provided.

Publications

Abbott,W.S. 1925: A method of computing the effectiveness of an insecticide. J. Econ.Entomol.18:265-267

Anis, A.I.M.; Brennan, P. 1982 Susceptibility of different populations of glasshouse whitefly Trialeurodes vaporariorum, Westwood to a range of chemical insecticides. Faculty of General Agriculture University College of Dublin, Research report 1980- 1981: 55. Donovan, B.J. ;EUiot, G.S. 2000: Honey bee response to high concentrations of some new spray adjuvants. KZ. Plant Prot. 53:230-234

Elhag, E.A.; Horn, DJ. 1983 Resistance of greenhouse whitefly (Homoptera: Aleyrodidae) to insecticides in selected Ohio greenhouses. Journal of Economic Entomology 76: 945-948. Georghiou, G.P. 1981 The occurrence of resistance to pesticides in arthropods, an index of cases reported through 1980. FAO of UN, Rome 1981. 172 p.

Gorman, K.; Devine, GJ.; Denholm, I. 2000 Status of pesticide resistance in UK populations of glasshouse whitefly, Trialeurodes vaporariorum, and the two-spotted spider mite, Tetranychus urticae. The BCPC Conference: Pests and diseases: 1: 459- 464

Grossman, J. 1994 Onion thrips. IPM Practitioner. 16(4): 12-13

Hommes, M. 1986 Insecticide resistance in greenhouse whitefly {Trialeurodes vaporariorum, Westw.) to synthetic pyrethroids. Mitteilungen aus der Biologischen Bundesanstalt fur Land-und Forstwirtschaft 232: 376. Purvis, S. 2002 Are KCT developing resistance to chlorpyrifos. Talking thrips in citrus October 2002 issue 2: 1

Martin, N. A., Workman, PJ. 1994 Confirmation of a pesticide-resistant strain of western flower thrips in New Zealand. Proceedings of the 47th KZ. Plant Protection conference: 144-148.

Martin, N. A. 1996. Whitefly resistance management strategy. Pp 194-203. In: Bourdot, G.W., Suckling, D.M. (eds). Pesticide Resistance: Prevention & Management., New Zealand Plant Protection Society, Lincoln, NZ.

OECD 1998: Guidelines for the Testing of Chemicals, www.oecd.org Wardlow, L.R. 1985 Pyrethroid resistance in glasshouse whitefly (Trialeurodes vaporariorum, Westw.). Mededelingen van de Faculteit Landbouwwetenschappen, Rijksuniversiteit, Gent 50 (2b): 164-165.

Those persons skilled in the art will understand that the above description is provided by way of illustration only and that the invention is not limited thereto.

All patents, publications, scientific articles, and other documents and materials referenced or mentioned herein are indicative of the levels of skill of those skilled in the art to which the invention pertains, and each such referenced document and material is hereby incorporated by reference to the same extent as if it had been incorporated by reference in its entirety individually or set forth herein in its entirety. Applicants reserve the right to physically incorporate into this specification any and all materials and information from any such patents, publications, scientific articles, web sites, electronically available information, and other referenced materials or documents. The specific methods and compositions described herein are representative of various embodiments or preferred embodiments and are exemplary only and not intended as limitations on the scope of the invention. Other objects, aspects, examples and embodiments will occur to those skilled in the art upon consideration of this specification, and are encompassed within the spirit of the invention as defined by the scope of the claims. It will be readily apparent to one skilled in the art that varying substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention. The invention illustratively described herein suitably may be practiced in the absence of any element or elements, or limitation or limitations, which is not specifically disclosed herein as essential. Thus, for example, in each instance herein, in embodiments or examples of the present invention, any of the terms "comprising", "consisting essentially of, and "consisting of may be replaced with either of the other two terms in the specification. Also, the terms "comprising", "including", containing", etc. are to be read expansively and without limitation. The methods and processes illustratively described herein suitably may be practiced in differing orders of steps, and that they are not necessarily restricted to the orders of steps indicated herein or in the claims. It is also that as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural reference unless the context clearly dictates otherwise. Thus, for example, a reference to "a host cell" includes a plurality (for example, a culture or population) of such host cells, and so forth. Under no circumstances may the patent be inteipreted to be limited to the specific examples or embodiments or methods specifically disclosed herein. Under no circumstances may the patent be interpreted to be limited by any statement made by any Examiner or any other official or employee of the Patent and Trademark Office unless such statement is specifically and without qualification or reservation expressly adopted in a responsive writing by Applicants .

The terms and expressions that have been employed are used as terms of description and not of limitation, and there is no intent in the use of such terms and expressions to exclude any equivalent of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention as claimed. Thus, it will be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the appended claims.

The invention has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein. Other embodiments are within the following claims. In addition, where features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group.

Claims

1. A biologically pure culture of Lecanicillium muscarium fungus strain K4V1 on deposit at Australian Government Analytical Laboratories under Accession No. NM05/44593, or a culture having the identifying characteristics thereof.

2. A biologically pure culture of Lecanicillium muscarium fungus strain K4V2 on deposit at Australian Government Analytical Laboratories under Accession No. NM05/44594, or a culture having the identifying characteristics thereof.

3. A biologically pure culture of Lecanicillium muscarium fungus strain K4V4 on deposit at the National Measurement Institute of Australia under Accession No. NM06/00007, or a culture having the identifying characteristics thereof.

4. A biologically pure culture of Lecanicillium longisporum fungus strain KT4L1 on deposit at the National Measurement Institute of Australia under Accession No. NM06/00009, or a culture having the identifying characteristics thereof.

5. A biologically pure culture of Beauveria bassiana fungus strain K4B1 on deposit at Australian Government Analytical Laboratories under Accession No. NM05/44595, or a culture having the identifying characteristics thereof.

6. A biologically pure culture of Beauveria bassiana fungus strain K4B2 on deposit at the National Measurement Institute of Australia under Accession No. NM06/00010, or a culture having the identifying characteristics thereof.

7. A biologically pure culture of Paecilomyces fumosoroseus fungus strain K4P1 on deposit at the National Measurement Institute of Australia under Accession No. NM06/00008, or a culture having the identifying characteristics thereof.

8. Spores obtainable from a fungus as claimed in any one of claims 1 to 7.

9. Spores as claimed in claim 8 which are conidia or blastophores.

10. The use of at least one fungus as claimed in any of claims 1 to 7 together with at least one diluent, adjuvant, carrier and/or excipient in the preparation of a composition.

11. The use of spores from at least one fungus as claimed in any one of the claims 1 to 7 together with at least one diluent, adjuvant, carrier and/or excipient in the preparation of a composition.

12. The use according to claim 10 or claim 11 wherein said composition is a biological control composition.

13. The use according to claim 12 wherein said biological control composition is an entomopathogenic composition.

14. The use according to any one of claims 10 to 13 wherein said composition comprises at least one agriculturally acceptable diluent, adjuvant, carrier and/or excipient.

15. A method of producing a composition comprising combining a reproductively viable form of one or more fungi as claimed in any one of claims 1 to 7 with at least one diluent, adjuvant, carrier and/or excipient.

16. A method according to claim 15 wherein said fungi is selected from the group consisting of Lecanicillium muscarium strain K4V1 (Australian Government Analytical Laboratories Accession No. NM05/44593) or a strain having the identifying characteristics thereof; Lecanicillium muscarium strain K4V2 (Australian Government Analytical Laboratories Accession No. NM05/44594) or a strain having the identifying characteristics thereof; Lecanicillium muscarium strain K4V4 (National Measurement Institute of Australia Accession No. NM06/00007) or a strain having the identifying characteristics thereof; Beauveria bassiana strain K4B1 (Australian Government Analytical Laboratories Accession No. NM05/44595) or a strain having the identifying characteristics thereof; Beauveria bassiana strain K4B2 (National Measurement Institute of Australia Accession No. NM06/00010) or a strain having the identifying characteristics thereof; Lecanicillium longisporum strain KT4L1 (National Measurement Institute of Australia Accession No. NM06/00009) or a strain having the identifying characteristics thereof; and Paecilomyces fumosoroseus strain K4P1 (National Measurement Institute of Australia Accession No. NM06/00008) or a strain having the identifying characteristics thereof.

17. A method according to claim 15 or claim 16 wherein said at least one diluent, adjuvant, carrier and/or excipient is an agriculturally acceptable diluent, carrier or excipient.

18. A method according to any one of claims 15 to 17 wherein said at least one diluent, adjuvant, carrier and/or excipient is selected from the group consisting of a filler stimulant, an anti-caking agent, a wetting agent, an emulsifier, and an antioxidant.

19. A method according to claim 18 wherein said composition comprises at least one of each of a filler stimulant, an anti-caking agent, a wetting agent, an emulsifϊer, and an antioxidant.

20. A method according to any one of claims 15 to 19 wherein the composition produced is a biological control composition.

21. A method according to claim 20 wherein the biological control composition is an entomopathogenic composition.

22. A composition which comprises at least one fungi as claimed in any one of claims 1 to 7 together with at least one diluent, adjuvant, carrier and/or excipient.

23. A composition comprising spores obtainable from at least one fungi as claimed in any one of claims 1 to 7 together with at least one diluent, adjuvant, carrier and/or excipient.

24. A composition according to claim 22 or claim 23 wherein said at least one diluent, adjuvant, carrier and/or excipient is an agriculturally acceptable diluent, adjuvant, carrier and/or excipients.

25. A composition according to any one of claims 22 to 24 wherein said at least one diluent, adjuvant, carrier and/or excipient is selected from the group consisting of a filler stimulant, an anti-caking agent, a wetting agent, an emulsifier, and an antioxidant.

26. A composition according to claim 25 wherein said composition comprises at least one of each of a filler stimulant, an anti-caking agent, a wetting agent, an emulsifier, and an antioxidant.

27. A composition according to claim 25 or 26 wherein said filler stimulant is glucose, said anti-caking agent is silicon dioxide, said wetting agent is skimmed milk powder, said emulsifier is lecithin, and said antioxidant is sodium glutamate.

28. A composition according to any one of claims 22 to 27 wherein said composition is a biological control composition.

29. A composition according to claim 28 wherein said biological control composition is an entomopathogenic composition.

30. A composition according to any one of claims 22 to 29 wherein said composition is a stable composition capable of supporting reproductive viability of said fungi for a period greater than about two weeks.

31. A composition according to any one of claims 22 to 30 wherein said composition is a stable composition capable of supporting reproductive viability of said fungi for a period greater than about one month.

32. A composition according to any one of claims 22 to 31 wherein said composition is a stable composition capable of supporting reproductive viability of said fungi for a period greater than about three months.

33. A composition according to any one of claims 22 to 32 wherein said composition is a stable composition capable of supporting reproductive viability of said fungi for a period greater than about six months.

34. A composition according to any one of claims 22 to 33 wherein said composition comprises a strain selected from the group consisting of Lecanicillium muscarium strain K4V1 (Australian Government Analytical Laboratories Accession No. NM05/44593) or a strain having the identifying characteristics thereof; Lecanicillium muscarium strain K4V2 (Australian Government Analytical Laboratories Accession No. NM05/44594) or a strain having the identifying characteristics thereof; Lecanicillium muscarium strain K4V4 (National Measurement Institute of Australia Accession No. NM06/00007) or a strain having the identifying characteristics thereof; Beauveria bassiana strain K4B1 (Australian Government Analytical Laboratories Accession No. NM05/44595) or a strain having the identifying characteristics thereof; Beauveria bassiana strain K4B2 (National Measurement Institute of Australia Accession No. NM06/00010) or a strain having the identifying characteristics thereof; Lecanicillium longisporum strain KT4L1 (National Measurement Institute of Australia Accession No. NM06/00009) or a strain having the identifying characteristics thereof; and Paecilomyces fumosoroseus strain K4P1 (National Measurement Institute of Australia Accession No. NM06/00008) or a strain having the identifying characteristics thereof.

35. A composition according to any one of claims 22 to 33 wherein said composition comprises any two or more strains selected from the group consisting of Lecanicillium muscarium strain K4V1 (Australian Government Analytical Laboratories Accession No. NM05/44593) or a strain having the identifying characteristics thereof; Lecanicillium muscarium strain K4V2 (Australian Government Analytical Laboratories Accession No. NM05/44594) or a strain having the identifying characteristics thereof; Lecanicillium muscarium strain K4V4 (National Measurement Institute of Australia Accession No. NM06/00007) or a strain having the identifying characteristics thereof; Beauveria bassiana strain K4B1 (Australian Government Analytical Laboratories Accession No. NM05/44595) or a strain having the identifying characteristics thereof; Beauveria bassiana strain K4B2 (National Measurement Institute of Australia Accession No. NM06/00010) or a strain having the identifying characteristics thereof; Lecanicillium longisporum strain KT4L1 (National Measurement Institute of Australia Accession No. NM06/00009) or a strain having the identifying characteristics thereof; and Paecilomyces fumosoroseus strain K4P1 (National Measurement Institute of Australia Accession No. NM06/00008) or a strain having the identifying characteristics thereof.

36. A composition according to claim 34 or 35 wherein the strain is, or the composition includes, Lecanicillium muscarium strain K4V1, on deposit at the Australian Government Analytical Laboratories under Accession No. NM05/44593 or a strain having the identifying characteristics thereof.

37. A composition according to claim 34 or 35 wherein the strain is, or the composition includes, Lecanicillium muscarium strain K4V2 (Australian Government Analytical Laboratories Accession No. NM05/44594) or a strain having the identifying characteristics thereof.

38. A composition according to claim 34 or 35 wherein the strain is, or the composition includes, Lecanicillium muscarium strain K4V4 (National Measurement Institute of Australia Accession No. NM06/00007) or a strain having the identifying characteristics thereof.

39. A composition according to claim 34 or 35 wherein the strain is, or the composition includes, Beauveria bassiana strain K4B1 (Australian Government Analytical Laboratories Accession No. NM05/44595) or a strain having the identifying characteristics thereof.

40. A composition according to claim 34 or 35 wherein the strain is, or the composition includes, Beauveria bassiana strain K4B2 (National Measurement Institute of Australia Accession No. NM06/00010) or a strain having the identifying characteristics thereof.

41. A composition according to claim 34 or 35 wherein the strain is, or the composition includes, Lecanicillium longisporum strain KT4L1 (National Measurement Institute of Australia Accession No. NM06/00009) or a strain having the identifying characteristics thereof.

42. A composition according to claim 34 or 35 wherein the strain is, or the composition includes, Paecilomyces fumosoroseus strain K4P1 (National Measurement Institute of Australia Accession No. NM06/00008) or a strain having the identifying characteristics thereof.

43. The use of one or more fungi as claimed in any one of claims 1 to 7 in the control one or more phytopathogenic insects.

44. The use of spores obtainable from one or more fungi as claimed in any one of claims 1 to 7 in the control one or more phytopathogenic insects.

45. The use of a composition as claimed in any one of claims 22 to 42 in the control one or more phytopathogenic insects.

46. The use according to claim any one of claims 43 to 45 wherein said one or more phytopathogenic insects is selected from the group consisting of thrips, aphids, whitefly, caterpillars, and Varroa mite.

47. A method for controlling one or more phytopathogenic insects, the method comprising applying to a plant or its surroundings a reproductively viable form and amount of at least one fungi as claimed in any one of claims 1 to 7.

48. A method as claimed in claim 47 wherein the reproductively viable form is spores.

49. A method for controlling one or more phytopathogenic insects, the method comprising applying to a plant or its surroundings a composition as claimed in any one of claims 22 to 42.

50. A method according to any one of claims 47 to claim 49 wherein said fungi is or said composition comprises a fungi is selected from the group consisting of Lecanicillium muscarium strain K4V1 (Australian Government Analytical Laboratories Accession No. NM05/44593) or a strain having the identifying characteristics thereof; Lecanicillium muscarium strain K4V2 (Australian Government Analytical Laboratories Accession No. NM05/44594) or a strain having the identifying characteristics thereof; Lecanicillium muscarium strain K4V4 (National Measurement Institute of Australia Accession No. NM06/00007) or a strain having the identifying characteristics thereof; Beauveria bassiana strain K4B1 (Australian Government Analytical Laboratories Accession No. NM05/44595) or a strain having the identifying characteristics thereof; Beauveria bassiana strain K4B2 (National Measurement Institute of Australia Accession No. NM06/00010) or a strain having the identifying characteristics thereof; Lecanicillium longisporum strain KT4L1 (National Measurement Institute of Australia Accession No. NM06/00009) or a strain having the identifying characteristics thereof; and Paecilomyces fumosoroseus strain K4P1 (National Measurement Institute of Australia Accession No. NM06/00008) or a strain having the identifying characteristics thereof.

51. A method according to any one of claims 47 to 50 wherein said fungi is or said composition comprises Lecanicillium muscarium strain K4V1 (Australian Government Analytical Laboratories Accession No. NM05/44593) or a strain having the identifying characteristics thereof.

52. A method according to any one of claims 47 to 50 wherein said fungi is or said composition comprises Lecanicillium muscarium strain K4V2 (Australian Government Analytical Laboratories Accession No. NM05/44594) or a strain having the identifying characteristics thereof.

53. A method according to any one of claims 47 to 50 wherein said fungi is or said composition comprises Lecanicillium muscarium strain K4V4 (National Measurement Institute of Australia Accession No. NM06/00007) or a strain having the identifying characteristics thereof.

54. A method according to any one of claims 47 to 50 wherein said fungi is or said composition comprises Lecanicillium lopngisporum strain KT4L1 (National Measurement Institute of Australia Accession No. NM06/00009) or a strain having the identifying characteristics thereof.

55. A method according to any one of claims 47 to 50 wherein said fungi is or said composition comprises Beauveria bassiana strain K4B1 (Australian Government Analytical Laboratories Accession No. NM05/44595) or a strain having the identifying characteristics thereof.

56. A method according to any one of claims 47 to 50 wherein said fungi is or said composition comprises Beauveria bassiana strain K4B2 (National Measurement Institute of Australia Accession No. NM06/00010) or a strain having the identifying characteristics thereof.

57. A method according to any one of claims 47 to 50 wherein said fungi is or said composition comprises Paeciliomyces fumosoroseus strain K4P1 (National Measurement Institute of Australia Accession No. NM06/00008) or a strain having the identifying characteristics thereof.

58. A method according to any one of claims 47 to 57 wherein the composition is admixed with water to a final concentration of about lgm/L to about 3gm/L prior to application, and more preferably to a final concentration of about lgm/L.

59. A method according to any one of claims 47 to 58 wherein a desiccation protection agent is admixed to a final concentration of about lml/L prior to application.

60. A method according to claim 59 wherein said desiccation protection agent is a vegetable oil.

61. A method according to any one of claims 47 to 60 wherein said composition comprises at least 10⁷ fungal spores per millilitre at application.

62. A method according to any one of claims 47 to 61 wherein said composition comprises fungal spores and said composition is applied at a rate of from about 1 x 10¹ to about 1 x 10¹⁵ fungal spores per hectare.

63. A method according to claim 62 wherein said composition is applied at a rate of from about 1 x 10¹² to about 1 x 10¹⁴ spores per hectare.

64. A method according to claim 63 wherein said composition is applied at a rate of from about 5 x 10¹² to about 1 x 10¹⁴ spores per hectare.

65. A method according to claim 64 wherein said composition is applied at a rate of about 1-3 x 10¹³ spores per hectare.

66. A method according to any one of claims 47 to 65 wherein said composition comprises Lecanicillium muscarium strain K4V1 (Australian Government Analytical Laboratories Accession No. NM05/44593) or a culture having the identifying characteristics thereof and/or Lecanicillium muscarium strain K4V2 (Australian Government Analytical Laboratories Accession No. NM05/44594) or a culture having the identifying characteristics thereof; and/or Lecanicillium muscarium strain K4V4 (National Measurement Institute of Australia Accession No. NM06/00007) or a culture having the identifying characteristics thereof.

67. A method according to any one of claims 47 to 6661 wherein said composition comprises Lecanicillium longisporum strain KT4L1 (National Measurement Institute of Australia Accession No. NM06/00009) or a culture having the identifying characteristics thereof.

68. A method according to any one of claims 47 to 66 wherein said composition comprises Paecilomyces fumosoroseus strain K4P1 (National Measurement Institute of Australia Accession No. NM06/00008) or a strain having the identifying characteristics thereof.

69. A method according to any one of claims 47 to 66 wherein said composition comprises Beauveria hassiana strain K4B1 (Australian Government Analytical Laboratories Accession No. NM05/44595) or a culture having the identifying characteristics thereof and/or Beauveria bassiana strain K4B2 (National Measurement Institute of Australia Accession No. NM06/00010) or a culture having the identifying characteristics thereof or a culture having the identifying characteristics thereof.

70. A method according to any one of claims 47 to 69 wherein said application is by spraying.