Entomopathogenic fungi

Abstract

The present invention relates to new fungal strains and compositions comprising these strains. The present invention also relates to the use of these fungal strains for controlling Coleopterans or Dipterans.

Description

[0001] ENTOMOPATHOGENIC FUNGI

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to new fungal strains and compositions comprising these strains. The present invention also relates to the use of these fungal strains for controlling Coleopterans or Dipterans.

[0004] BACKGROUND OF THE INVENTION

[0005] Nuts are high value crops in both Australian and international markets. Australia produces almonds, macadamias, walnuts, pecans and pistachios for both domestic and international markets. In 2022, Australia was the second biggest producer of almonds (8%) behind the United States (78%).

[0006] Carpophilus beetles can cause significant levels of damage to nut produce through direct adult and larval feeding and through vectoring bacterial and fungal diseases. Carpophilus truncatus is a major insect pest in Australian almonds and is reported to account for up to 10% loss of kernels. C. truncatus has recently been found in almond and pistachio orchards in California, although the effects of the beetles has not yet been quantified in the US.

[0007] Effective control of Carpophilus beetles in orchards utilises integrated pest management (IPM) systems where complimentary management practices such as orchard hygiene, “attract-and-kill” strategies and pesticide use, work in combination to lower Carpophilus populations and reduce damage to nut produce.

[0008] C. truncatus beetles live inside mummy nuts (nuts that remain on the tree after harvest) and in new nuts and they feed on the almond kernels. Chemical insecticide sprays are often ineffective with these beetles because it is difficult for the chemical spray to reach inside of the nuts where the beetles are feeding. Use of broad spectrum pesticides can also result in the loss of beneficial insects, such as bees, required to pollinate in the orchard or result in secondary pest infestations that would normally be controlled by invertebrate predators.

[0009] Small hive beetles (Aelhina tumida also cause significant problems in the bee keeping and honey producing industries. Infestation of hives with small hive beetle reduces the quality of honey or makes it unsuitable for consumption by humans or by bees. It can also cause significant damage to a hive resulting in the queen stopping egg production and sometimes even causes the bees to vacate the hive altogether.

[0010] The limitations and environmental concerns associated with chemical insecticides may be, at least in part, alleviated by adopting entomopathogenic fungi as a pest control agent, particularly when combined with other controls such as one or more of attractants, co-attractants, traps baiting stations and orchard hygiene.

[0011] There is a need for further means for controlling Coleopteran or Dipteran pests, particularly for use in integrated pest management systems designed to minimise pest resistance.

[0012] SUMMARY OF THE INVENTION

[0013] The present inventors have identified new strains of entomopathogenic fungi that are effective against Coleopterans such as C. truncatus, as well as effective against Dipterans such as Bactrocera tryoni.

[0014] In an aspect, the present invention provides an isolated strain of Metarhizium anisopliae TatMal deposited with the National Measurement Institute under accession number V24 / 010070 on 3 June 2024, or an active variant thereof.

[0015] In another aspect, the present invention provides an isolated strain of Beauveria bassiana ABBb2 deposited with the National Measurement Institute under accession number V24 / 010069 on 3 June 2024 or an active variant thereof.

[0016] In an embodiment, a strain of the invention is in the form of a spore, microsclerotia or blastospore. In an embodiment, a strain of the invention is in the form of a spore.

[0017] In another aspect, the present invention provides a composition comprising a strain of the invention.

[0018] In an embodiment, the composition comprises at least one diluent, carrier and / or excipient.

[0019] In an embodiment, the composition comprises spores of the strain in a dry powder.

[0020] In an embodiment, the composition comprises the strain in oil.

[0021] In an embodiment, the composition comprises spores of the strain in the form of a dry powder suspended in an oil.

[0022] In an embodiment, the composition comprises the strain suspended in an oil-in- water emulsion.

[0023] In an embodiment, the composition comprises an attractant. In an embodiment, the attractant is a pheromone. In an embodiment, the composition comprises a pheromone and / or a co-attractant.

[0024] In another aspect, the present invention provides a method for controlling a Coleopteran, and / or reducing damage caused by a Coleopteran, the method comprising delivering to the Coleopteran, or contacting the Coleopteran with, a strain of the invention.

[0025] In an embodiment, the strain is Metarhizium anisopliae TatMal. In an embodiment, the strain is Beauveria bassiana ABBb2.

[0026] In an embodiment, the method comprises applying a composition of the invention to an area which comprises, or may comprise, a Coleopteran.

[0027] In an embodiment, the area comprises an agricultural crop.

[0028] In an embodiment, the agricultural crop comprises a plant that produces nuts, grain or fruit.

[0029] In an embodiment, the plant produces a nut.

[0030] In an embodiment, the nut is an almond, pistachio, walnut, cashew nut, kemiri nut, macadamia nut, or Brazil nut.

[0031] In an embodiment, the area comprises hives, such as bee hives.

[0032] In an embodiment, the composition used in a method of the invention is a dry powder.

[0033] In an embodiment, the Coleopteran is a plant pest.

[0034] In an embodiment, the Coleopteran is a Nitidulidae.

[0035] In an embodiment, the Nitidulidae is Carpophilus spp. such as C. Iriincalus. C. davidsoni, C. hemiplerus. or C. humeralis.

[0036] In an embodiment, the Nitidulidae is Carpophilus truncatus.

[0037] In an embodiment, the Nitidulidae is an Aethina species such as Aethina tumida (small hive beetle).

[0038] In another aspect, the present invention provides a method for controlling a Dipteran, and / or reducing damage caused by a Dipteran, the method comprising delivering to the Dipteran, or contacting the Dipteran with, a strain of the invention.

[0039] In an embodiment, the strain is Metarhizium anisopliae TatMal.

[0040] In an embodiment, the strain is Beauveria bassiana ABBb2.

[0041] In an embodiment, the method comprises applying a composition of the invention to an area which comprises, or may comprise, a Dipteran.

[0042] In an embodiment, the area comprises a fruit crop or tree.

[0043] In an embodiment, the Dipteran is a plant pest.

[0044] In an embodiment, the Dipteran is a Tephritidae.

[0045] In an embodiment, the Tephritidae is a Bactrocera sp.

[0046] In an embodiment, the Bactrocera sp. is B. tryoni.

[0047] In an embodiment, the Dipteran is a Sciaridae.

[0048] Further provided is the use of a strain of the invention to control a Coleopteran and / or reduce damage, such as agricultural crop damage or hive damage, by a Coleopteran. Also provided is the use of a strain of the invention to control a Dipteran and / or reduce damage, such as agricultural crop damage or hive damage, by a Dipteran.

[0049] In another aspect, the present invention provides a device comprising a strain of the invention.

[0050] In an embodiment, the device is for attracting Coleopterans.

[0051] In an embodiment, the device is for attracting Dipterans.

[0052] In an embodiment, the device is for attracting and killing Coleopterans

[0053] In an embodiment, the device is for attracting and killing Dipterans

[0054] In an embodiment, the device provides for regulated release of an attractant.

[0055] Any embodiment herein shall be taken to apply mutatis mutandis to any other embodiment unless specifically stated otherwise.

[0056] The present invention is not to be limited in scope by the specific embodiments described herein, which are intended for the purpose of exemplification only. Functionally-equivalent products, compositions and methods are clearly within the scope of the invention, as described herein.

[0057] Throughout this specification, unless specifically stated otherwise or the context requires otherwise, reference to a single step, composition of matter, group of steps or group of compositions of matter shall be taken to encompass one and a plurality (i.e. one or more) of those steps, compositions of matter, groups of steps or group of compositions of matter.

[0058] The invention is hereinafter described by way of the following non-limiting Examples and with reference to the accompanying figures.

[0059] BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS

[0060] Figure 1. The average percent mortality demonstrating the pathogenicity of entomopathogenic fungal species isolates (x axis) applied to vermiculite (lx 106conidia g'1) against fifth-instar larvae of C. truncatus beetle. The control was treated with sterile 0.05% Tween-80. Error bars represent the mean percent mortality (± standard error). Refer to Table 2 for the species of each of the tested strains.

[0061] Figure 2. Average percentage mortality of Qfly B. tryoni when exposed to different isolates of Metarhizium spp. versus a control (no-inoculum). Error bars represent standard error of the mean. DETAILED DESCRIPTION OF THE INVENTION

[0062] General Techniques and Definitions

[0063] Unless specifically defined otherwise, all technical and scientific terms used herein shall be taken to have the same meaning as commonly understood by one of ordinary skill in the art (e.g., in insect pest management, biological control of insects using entomopathogenic fungi, formulations comprising entomopathogenic fungi etc).

[0064] The term “and / or”, e.g., “X and / or Y” shall be understood to mean either “X and Y” or “X or Y” and shall be taken to provide explicit support for both meanings or for either meaning.

[0065] As used herein, the term about, unless stated to the contrary, refers to + / - 10%, or more preferably + / - 5%, more preferably + / - 1%, of the designated value.

[0066] Throughout this specification the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

[0067] Entomopathogenic Fungi

[0068] Entomopathogenic fungi usually attach to the external body surface of insects in the form of microscopic spores (usually asexual, mitosporic spores also called conidia). Under permissive conditions of temperature and moisture, these spores germinate, grow as hyphae and colonize the insect cuticle; eventually they bore through it and reach the body cavity. The fungal cells proliferate in the host body cavity, usually as walled hyphae or in the form of wall-less protoplasts (depending on the fungus involved). After some time the insect is usually killed (sometimes by fungal toxins) and new propagules (spores) are formed in / on the insect if environmental conditions are again permissive.

[0069] In an aspect, the present invention provides an isolated strain of Metarhizium anisopliae TatMal deposited with the National Measurement Institute under accession number V24 / 010070 on 3 June 2024, or an active variant thereof.

[0070] In another aspect, the present invention provides an isolated strain of Beauveria bassiana ABBb2 deposited with the National Measurement Institute under accession number V24 / 010069 on 3 June 2024 or an active variant thereof.

[0071] The term “isolated”, as used herein, is intended to mean that a strain of the disclosure has been isolated from its natural source, substrate, habitat or environment and transferred to a culture media e.g., using methods known in the field mycology. For example, an “isolated” strain of the invention may encompass fungus which has been grown or produced in culture. An “isolated” strain of the invention also encompasses fungus which have been purified.

[0072] The strain of the present disclosure may be provided in the form of mature fungus, fungal spores, microsclerotia or blastospores. In an embodiment, the strain is provided in the form of spores, microsclerotia or blastospores. In an embodiment, the spores, microsclerotia or blastospores are reproductively viable. In one example, the spores, microsclerotia or blastospores are provided in the form of a dry powder. In an embodiment, the strain is provided in the form of spores.

[0073] The term "spore" is intended to encompass any unicellular or multicellular, reproductive or distributional cells developing into a number of different phases of the complex life cycles of the strain.

[0074] The term “microsclerotia” as used herein refers to a desiccation-tolerant survival structure that is composed of compact, melanic, solid hyphae aggregated into particles with a size range of 200-600 pm and are derived from the genus Metarhizium .

[0075] The term “blastospore” is intended to encompass spores produced by budding and that can give rise to another spore or hypha.

[0076] As used herein, the term “reproductively viable spores, blastospores or microsclerotia” includes reproductive units of fungus, such as spores, blastospores and microsclerotia which, under favorable conditions, will develop into adult fungal organisms.

[0077] Spores, blastospores and microsclerotia of a strain of the present disclosure may be produced by methods known to those skilled in the art. For example, spores, blastospores and microsclerotia of a strain of the present disclosure may be produced using conventional solid substrate and liquid fermentation technologies well known in the art. The spores may be produced in bulk for field application using nutrient film, submerged culture, and grain (such as rice) substrate growing techniques. In this way, the fungi can be grown in sufficient amounts to allow use as agent in compositions and methods of the present disclosure.

[0078] One example for producing spores suitable for the present disclosure is as follows. Spores may be produced by isolating the conidia from infected Coleopterans (such as Carpophilus truncatus) or Dipterans (such as Bactrocera tryoni . and culturing it on a petri dish plate of dextrose agar. The agar may then be used to inoculate a yeast extract / dextrose broth and which is then incubated on an orbital shaker for 5-6 days at 25°C. The yeast extract / dextrose broth may then be used to inoculate a solid substrate of long grained partially boiled rice that had been sterilized in a 10-1 autoclave self-aerating culture bag. The rice-conidia mixture may then be removed by sieving to give a clean conidial powder which is dried and formulated in oil containing emulsifiers, or formulated as a dry powder talc, or pelleted using, for example, the sodium alginate method.

[0079] In one embodiment, a strain of the invention is an active variant of Metarhizium anisopliae TatMal deposited with the National Measurement Institute under accession number V24 / 010070 on 3 June 2024.

[0080] In one embodiment, a strain of the invention is an active variant of Beauveria bassiana ABBb2 deposited with the National Measurement Institute under accession number V24 / 010069 on 3 June 2024.

[0081] As used herein, the term “active variant” refers to the fungi being able to kill a Coleopteran such as C. truncatus and / or a Dipteran such as B. tryoni. In an embodiment, the active variant is able to kill at least about 90%, or at about least about 95%, of Coleopterans, such as C. truncatus, and / or a Dipteran such as B. tryoni, when tested in a suitable assay such as described in the Examples. Such variants, which may be identified using appropriate screening techniques, are a part of the present invention.

[0082] In an embodiment, the active variant is obtainable by breeding between a first strain and a second strain, wherein at least one strain is Metarhizium anisopliae TatMal or Beauveria bassiana ABBb2.

[0083] In an embodiment, the active variant is a mutant. In the present context, the term "mutant" or "mutant strain" refers to a strain derived, or a strain which can be derived, from a strain of the invention (or the parental strain) by means of e.g. genetic engineering, mutagenesis, radiation and / or chemical treatment. In an embodiment, the mutant comprises a transgene expressing a polynucleotide and / or protein of interest.

[0084] Compositions

[0085] The strain of the present disclosure may be formulated into a composition suitable for application to a plant or an area e.g., to protect the plant or a part thereof or a hive from Coleopteran pests such as Coleopteran plant or hive pests, or to protect a fruit free from Dipteran pest such as B. tryoni. Accordingly, the present disclosure provides a composition comprising an isolated strain of the invention.

[0086] In one example, the composition is formulated for application to a plant, a plant part, soil, and / or an area which comprises, or may comprises a Coleopteran and / or a Dipteran. When formulated, the composition typically comprises an effective amount of the strain of the disclosure and an agriculturally acceptable diluent, carrier and / or excipient. Such agriculturally acceptable diluents, carriers and excipients would be known to a person of skill in the art. In another example, the composition may be formulated to apply to soil under and around a hive such as a honey bee hive.

[0087] In an example, the fungus of the disclosure is present in the composition in the form of spores, blastospores or microsclerotia. In an example, the fungus of the disclosure is present in the composition in the form of spores, microsclerotia or blastospores, especially spores.

[0088] Liquid compositions of the disclosure may also include water-soluble concentrates, emulsifiable concentrates, emulsions or suspensions.

[0089] A composition of the present disclosure may be achieved by suspending spores of the fungus in an oil, optionally together with an emulsifier. Before application, the oil and optional emulsifier can be mixed with an appropriate quantity of an aqueous medium to form an emulsion before spraying onto an area such as a field or orchard, or the soil under and / or around a hive.

[0090] In one example, the composition may comprise dry powder spores. The dry powder spores may be provided alone or together with the oil component. Where the dry powder spores, microsclerotia or blastospores are provided with the oil component, the spores, microsclerotia or blastospores and oil will be packaged separately e.g., in the form of a kit.

[0091] In an embodiment, the composition comprises an abrasive substance. Examples of abrasive substances include, but are not limited to, calcite, emery, pumice powder, diatomaceous earth, or a mixture of two or more thereof.

[0092] In an embodiment, the abrasive substance is diatomaceous earth. As described in WO 2019 / 165413, diatomaceous earth comprises fossilized remains of a type of hard- shelled protest known as diatoms. Microscopically, the particles are very sharp and can stick to an insect, becoming lodged between its exoskeletal joints. As the insect moves, its body receives cuts and lacerations from the sharp particles. Furthermore, the abrasive particles can scratch away at the insects waxy exoskeletal layer, which then allows internal moisture to escape from the insect’s body. The moisturized surface of the insect and the “bleeding” liquids from the circulatory system due to the cuts allow germination of fungal spores, thus increasing the propagation of the fungi and hastening the death of the insect. In an embodiment, the abrasive substance is in the form of a powder having a particle size of about 1 micrometer to about 1 millimeter, preferably from about 10 to about 200 micrometers.

[0093] In another example, the fungus may be provided in the form of spores, microsclerotia or blastospores suspended in an oil-in-water emulsion. Suitable oils for use in a composition of the present disclosure include low molecular weight oil formulations, but other suitable petroleum oils are white oils, DC Tron oil (nC 21 and nC 24 oils), Canopy oil (nC27 oil), Biopest oil (nC 24 oils), dormant oil, or summer oil as known in the horticultural industry. Other suitable oils include horticultural oil, olive oil, soy oil, com oil, sunflower oil, canola oil, linseed oil, castor oil, fish oil, tung oil, sesame oil and / or MCT oils. Most of these oils are nCi9-nC27 but other hydrocarbons having acceptable safety profile may be used. There are number of such products in the market which are suitable for use with the present disclosure. These are Sunspray oil, tea tree oil, Sunspray Ultra fine manufactured by the Sun Refining and Marketing Company.

[0094] In another example, the spores, microsclerotia or blastospores may be provided in the form of an insecticidal soap. For example, the spores may be provided in a composition containing salts of fatty acids, such as potassium salts of fatty acids. An example is Natrasoap®.

[0095] It will be appreciated that the petroleum spray oil and / or polysaccharide may be used in conjunction with suitable agronomically acceptable diluents, carriers and / or excipients as described herein, as well as with other additives common in the art such as emulsifiers, wetting agents, humectants, surfactants, stabilizers, stickers, spreaders, penetrants or the like.

[0096] The compositions of the disclosure may also include so-called 'stressing' additives to improve spore, microsclerotia or blastospore vigor, germination and survivability such as potassium chloride, glycerol, sodium chloride and glucose.

[0097] Additives for use in compositions of the disclosure may also include compositions which assist in maintaining viability of the strain 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 (which may be gelled by adding a salt such as a calcium salt) and conidia on the inside.

[0098] Examples of surfactants, spreaders and stickers include Fortune®, carola (capsicums), C-Daxoil®, Codacide oil®, Kanolacide 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) and Triton X (available from Sigma Aldrich), 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 strain to control Coleopteran and / or Dipteran pests, and / or protect plants or parts thereof such as nuts or fruits, from Coleopteran and / or Dipteran pests. For compositions intended for application to plants, other natural or synthetic, organic or inorganic materials may be included in the composition to facilitate application of the composition to an area. Such a carrier is thus generally inert and it should be agriculturally acceptable, in particular on the plant treated. The carrier can be solid (clays, natural or synthetic silicates, silica, resins, waxes, solid fertilizers, etc.) or liquid (water, alcohols, in particular butanol, etc.).

[0099] In another embodiment, the composition of the disclosure is provided as a controlled release formulation that is capable of slowly releasing the strain to its intended target i.e., plant, plant part, soil, Coleopteran pest, Dipteran pest or a habitat of Coleopteran and / or Dipteran pest. As used herein, a controlled release formulation comprises a strain of the present disclosure in a controlled release vehicle. Such controlled release vehicles suitable for use in a composition of the present disclosure are known in the art.

[0100] In an embodiment, the composition is a stable composition capable of supporting reproductive viability of the strain for a period greater than about two weeks, greater than about one month, about two months, about three months, about four months, about five months, or greater than about six months. To be suitable for use in controlling Coleopterans and / or Dipterans, and / or protecting plants or plant parts from Coleopteran and / or Dipteran pests e.g., in a method of the disclosure, the composition is preferably able to support reproductive viability of the fungi e.g., such as in the form of spores, for a period greater than about six months. Accordingly, the composition will be formulated to support reproductive viability of the fungi.

[0101] In an embodiment, the composition may comprise a biopolymer that protects the strain or spores of the strain from stress such as UV irradiation and desiccation. Suitable biopolymers include cellulose derivatives such as hydroxyethylcellulose and carboxymethylcellulose, chitosan, alginate and starch. The biopolymer may be used to encapsulate the fungi enhancing conidial stability, survival under field conditions, adherence and sporulation on the surface of the target Coleopteran or Dipteran, and enhance product shelf life.

[0102] In an embodiment, spores will be present in the composition (when formulated as a liquid) at a concentration of at least about 10 spores / mL, at a concentration of at least about IxlO2spores / mL, at a concentration of at least about IxlO3spores / mL, at a concentration of at least about IxlO4spores / mL, at a concentration of at least about IxlO5spores / mL, at a concentration of at least about IxlO6spores / mL, or at a concentration of at least about IxlO7spores / mL. In an embodiment, the composition comprises spores at a concentration of at least about IxlO7spores / mL. For example, a typical composition of the present disclosure may comprise 25- 50g dry spore powder formulated in oil, with effective amounts of the formulation dissolved in 30-100 litres of water per hectare of plants to control pests. These amounts may also apply to applications to soil underneath and / or around hives such as bee hives.

[0103] In one example, the method(s) may comprise applying about 1 to about 500g spore powder per hectare of plants. Equally, the method may comprises applying about 2 to about 200g, about 5 to about 100g, about 5 to about 80g, about 10 to about 75g, about 10 to about 50, about 20 about 50g, or about 25 to about 50g spore powder per hectare of plants. These amounts may also apply to applications to soil underneath and / or around hives such as bee hives.

[0104] The frequency of application of the strain i.e., frequency of treatment, will vary depending on, for instance, environmental conditions including rainfall, humidity and temperature, and the population size or prevalence of the Coleopterans or Dipterans. Typically, the treatment may involve at least two sprays at about 14 to about 28 days intervals. In an embodiment, the treatment may involve at least four sprays at about 14 to about 28 days intervals. Repeated applications at the same or different times in a crop cycle is also contemplated.

[0105] In one example, the composition of the disclosure may be formulated to comprise, or be packaged with, a further substance having insecticidal or pesticidal activity. Suitable substances having insecticidal or pesticidal activity will vary depending on the plant, part thereof or area to which the composition is to be applied. For example, insecticides which may be included in the composition include synthetic insecticides and natural insecticides. Suitable insecticides include endosulfan, dichlorvos, dicofol, chlorpyrifos, dimethoate, disulfoton, omethoate, parathion, phorate, profenofos, sulprofos, thiometon, naled, parathion, malathion, S-benzyl diisopropyl phosphorothiolate (IBP), aldicarb, carbaryl, beta-cyfluthrin, deltamethrin, esfenvalerate, fenvalerate, fluvalinate, lamda-cyhalothrin, chlorfluazuron, piperonyl butoxide, (S)- methoprene or other insect growth regulators, petroleum spray oils and the like. Pesticides include other biological pesticides.

[0106] The present disclosure may allow for reduced label rates of synthetic chemical treatments and / or biological treatments against relevant Coleopterans or Dipterans. In one embodiment, the present disclosure allows for half or one-third label rates.

[0107] The other treatment can be applied as a mixture or simultaneously or sequentially. For example, for a sequential treatment with a particular treatment regime of the present disclosure, if the regime requires treatment every about 14 days or about 21 days, then a reduced label rate of another treatment can be applied every alternate fortnight to suppress Coleopteran or Dipteran numbers if earlier application of the composition of the present disclosure has not brought numbers below the economic threshold. Alternatively, the present disclosure can be applied as a mixture with reduced label rate of another treatment to utilise the synergistic effect of the combination to reduce Coleopteran or Dipteran numbers below the recommended threshold.

[0108] Thus, the present disclosure is suitable for combining with other treatments such as reduced rate of synthetic or biological insecticides to prevent build up populations of Coleopterans or Dipterans above economic threshold levels or build up resistance by Coleopterans or Dipterans.

[0109] Attractants

[0110] A composition of the invention may comprise one or more compounds that attract (an attractant) the Coleopteran or Dipterans, or may be used with one or more compositions that attract the Coleopteran or Dipteran. Such compounds are well known in the art.

[0111] In an embodiment, the attractant is a pheromone. In an embodiment, the pheromone is oil soluble and non-polar.

[0112] Examples of pheromones suitable for the invention include, but are not limited to, (2E,4E,6E)-5-ethyl-3-methyl-2,4,6-nonatriene, (3E,5E,7E)-6-ethyl-4-methyl-3 ,5,7- decatriene, (2E,4E,6E,8E)-3,5,7-Trimethyl-2,4,6,8-decatetraene, (2E,4E,6E,8E)-7-

[0113] Ethyl-3,5-dimethyl-2,4,6,8-decatetraene, (2E,4E,6E,8E)-3,5,7-trimethyl-2,4,6,8- undecatetraene, (2E,4E,6E,8E)-7-ethyl-3,5-dimethyl-2,4,6,8-undecatetraene, or a mixture of two or more thereof.

[0114] In an embodiment, the pheromone is (2E,4E,6E,8E)-7-Ethyl-3,5-dimethyl- 2,4,6, 8-decatetraene, (2E,4E,6E,8E)-3,5,7-trimethyl-2,4,6,8-undecatetraene, or a combination thereof.

[0115] In an embodiment, the pheromone is (2E,4E,6E,8E)-3,5,7-trimethyl-2,4,6,8- undecatetraene.

[0116] The pheromone composition may include one or more Ce-Ci6 aldehydes. In an embodiment, the one or more Ce-Ci6 aldehydes are a saturated aldehyde. In one embodiment, the one or more Ce-Ci6 aldehydes are a straight chain aldehyde. In yet another embodiment, the one or more Ce-Ci6 aldehydes are a straight chain, saturated aldehyde. In an embodiment, the one or more Ce-Ci6 aldehydes are selected from hexadecanal, pentadecanal, tetradecanal, tridecanal, dodecanal, undecanal, decanal, nonanal, octanal, heptanal and hexanal. In an embodiment, the Ce-Ci6 aldehyde is tetradecanal, hexanal or nonanal. In an embodiment, the Ce-Ci6 aldehyde is tetradecanal. In an embodiment, where the Coleopteran is a Carpophilus sp. such as Carpophilus Iruncalus. a composition of the invention comprises (2E,4E,6E,8E)-3,5,7- trimethyl-2,4,6,8-undecatetraene and tetradecanal.

[0117] In some embodiments, the ratio of 3,5,7-trimethyl-2,4,6,8-undecatetraene and Ce- Ci6 aldehyde, especially (E,E,E,E)-3,5,7-trimethyl-2,4,6,8-undecatetraene and tetradecanal, is one that results in emission of a 2:1 to 1 :5 ratio of 3,5, 7-trimethyl-2, 4,6,8- undecatetraene and Ce-Ci6 aldehyde, especially a 2: 1 to 1 :2 ratio and more especially about a 1 : 1 ratio of 3,5,7-trimethyl-2,4,6,8-undecatetraene and Ce-Ci6 aldehyde. In some embodiments, the ratio of the 3,5,7-trimethyl-2,4,6,8-undecatetraene and Ce-Ci6 aldehyde in the composition is 2:1 to 1 :5, especially 1 : 1 to 1 :4, more especially 1 :2 to 1 :4 or 1 :3 to 1 :3.5 and most especially about 1 :3.3. Compositions comprising one of these ratios may result in emission of a 1 : 1 ratio of 3,5,7-trimethyl-2,4,6,8- undecatetraene and C6-C16 aldehyde.

[0118] In an embodiment, the attractant for a Dipteran is Cue Lure (4-(4- acetoxyphenyl)butan-2-one), anisylacetone (4-(4-methoxyphenyl)butan-2-one), a- copaene, methyl eugenol, raspberry ketone, zingerone, methyl eugenol derivatives or analogues such as E-3,4-dimethoxycinnamyl alcohol, E-3,4-dimethoxycinnamyl acetate and fluorinated derivatives, i-butyl-2-methyl-4-chlorocyclohexane carboxylate (Trimedlure), a-ionol (Latilure), 3-oxo-7,8-dihydro-a-ionone or raspberry ketone formate. Other attractants for Dipterans include those that can be obtained from ripening or ripe fruit. One such attractant is described in WO 2014 / 053016. The lure described in WO 2014 / 053016 comprises at least two lower alkyl esters. An exemplary composition includes methyl acetate, ethyl acetate, ethyl propionate and ethanol in a ratio of 0.1 to 1 .5 : 2 : 0.1 to 1 .5 : 0.5 to 2.5, especially in a ratio of 1 : 2 : 1 : 2. Another exemplary composition includes ethyl butanoate, ethyl acetate, methyl butanoate, ethyl propionate and isobutyl acetate in a ratio of 0.5 to 1 .5: 1 : 0.5 to 1 .5 : 0.5 to 1 .5 : 0.5 to 1 .5, especially in a ratio of 1 : 1 : 1 : 1 : 1 (referred to herein as 5-Mix Lure). A further example of an attractant for use on Dipterans is described in WO 2022 / 109662.

[0119] In an embodiment, a composition of the invention, or a composition useful in combination with a composition of the invention (for example in a device described herein), comprises a co-attractant. As the skilled person would be aware, a co-attractant can enhance the effects of a pheromone. Examples of co-attractants include, but are not limited to, Ci-Ce alcohols, C1-C4 aldehydes, indoles and C1-C12 esters. In an embodiment, the co-attractant is ethanol, isopentyl alcohol, acetaldehyde, isobutanol, 2- methylbutanol, ethyl acetate, isopentyl acetate, isobutyl acetate, 2-phenylethyl acetate, (E)-4,8-dimethyl-l,3,7-nonatriene (DMNT), methyl benzoate, (Z)-3-hexenyl acetate, or a mixture of two or more thereof.

[0120] In an embodiment, the co-attractant is ethanol and isopentyl alcohol. In an embodiment, the co-attractant is aqueous ethanol and isopentyl alcohol. In an embodiment, the Coleopteran Carpophilus sp., such as Carpophilus Iruncalus. and the co-attractant is ethanol and isopentyl alcohol. In an embodiment, the Coleopteran Carpophilus sp., such as Carpophilus truncatus, and the co-attractant is aqueous ethanol and isopentyl alcohol.

[0121] In some embodiments, aqueous ethanol is about 30% to about 60% ethanol in water, about 40% to about 55% ethanol in water, about 40% to about 50% ethanol in water, for example, about 45% ethanol in water.

[0122] In an embodiment, the co-attractant includes one or more compounds selected from: acetaldehyde at a concentration of between about 5 pl and about 170 pl / 100 ml of composition, or between about 50 pl and about 70 pl / 100 ml, ethyl acetate at a concentration of between about 75 pl and about 125 pl / 100 ml of composition, or between about 100 pl and about 110 pl / 100 ml, isobutanol at a concentration of between about 0.1 pl and about 50 pl / 100 ml of composition, or between about 15 pl and about 40 pl / 100 ml,

[0123] 2-methyl-butanol at a concentration of between about 0.1 pl and about 40 pl / 100 ml of composition, or between about 15 pl and about 30 pl / 100 ml, isopentyl alcohol at a concentration of between about 0.01 pl and about 1750 pl / 100 ml of composition, or between about 400 pl and about 1500 pl / 100 ml, isobutyl acetate at a concentration of between about 0.05 pl and about 5 pl / 100 ml of composition, or between about 0.1 pl and about 20 pl / 100 ml, isopentyl acetate at a concentration of between about 0.01 pl and about 150 pl / 100 ml of composition, or between about 0.1 pl and about 20 pl / 100 ml; or

[0124] 2-phenethyl acetate at a concentration of between about 15 pl and about 30 pl / 100 ml of composition, or between about 5 pl and about 50 pl / 100 ml.

[0125] Control of Coleopterans or Dipterans

[0126] The strains, and compositions comprising the strain, of the invention can be used for controlling a Coleopteran and / or reducing damage by a Coleopteran, such as damage to an agricultural crop. In one aspect there is provided a method for controlling a Coleopteran, and / or reducing damage caused by a Coleopteran, the method comprising delivering to the Coleopteran, or contacting the Coleopteran with, a strain of the invention.

[0127] The strains, and compositions comprising the strain, of the invention can also be used for controlling a Dipteran and / or reducing damage by a Dipteran, such as damage to an agricultural crop. In another aspect there is provided a method for controlling a Dipteran, and / or reducing damage caused by a Dipteran, the method comprising delivering to the Dipteran, or contacting the Dipteran with, a strain of the invention.

[0128] The terms "controlling" or "control" as used herein are meant to include any pesticidal (killing) or pestistatic (inhibiting, maiming or generally interfering) activities of the strain against a Coleopteran or Dipteran at any stage in its life cycle. Thus, these terms not only include killing, but also include such activities as the production of behavioural abnormalities (e.g., tremor, incoordination, hyperactivity, anorexia) which interfere with such activities such as but not limited to eating, molting, hatching, mobility, or plant or part thereof attachment. Thus, in one embodiment, “controlling” may be understood to mean that the fungus kills or interferes with the pest, and includes reducing the number of the pests on a plant, animal or hive compared to the number of pests on the plant, animal or hive prior to application of the strain.

[0129] As used herein, the term "reducing damage” or similar refers to a method of the invention providing a benefit, such as an economic, health (such as plant or bee health) and / or production benefit.

[0130] As used herein, the term "reducing agricultural crop damage” or similar refers to a method of the invention providing an increased yield, such of nuts, fruit or grain. The improved crop yield may be determined relative to the yield of a crop of the same plant variety grown under similar conditions which has not received treatment with the strain of the disclosure. The reduction in crop damage may be due to reduced Coleopteran or Dipteran activity within the crop or may be due to reduced disease load within the crop that is spread by the Coleopteran or Dipteran.

[0131] The term "plant" as used herein encompasses not only whole plants, but extends to plant parts including nuts, fruit and grain. Plants that may benefit from the present disclosure cover a broad range of agricultural and horticultural crops, which are described in further detail herein.

[0132] In an embodiment, the plant produces nuts. Such plants, and nuts thereof, may be in an orchard. Examples of such plants include, but are not limited to, those which produce almonds, pistachios, walnuts, cashew nuts, kemiri nuts, macadamia nuts or Brazil nuts. In an embodiment, the plant produces almonds, pistachios or walnuts. In an embodiment, the plant produces almonds. The nut may be a mummy nut. The nuts may be on the plant, on the ground, or stored in stockpiles ready for export or commercial sale. In the embodiment, the nut is an almond, pistachio, walnut, cashew nut, kemiri nut, macadamia nut or Brazil nut. In an embodiment, the nut is an almond, pistachio or walnut. In an embodiment, the nut is an almond.

[0133] In an embodiment, the plant produces fruit. Such plants, and fruit thereof, may be in an orchard.

[0134] In an embodiment, the plant produces stone fruit. Examples of such plants, but are not limited to, those which produce peaches, apricots, nectarines, plums, or cherries.

[0135] In an embodiment, the plant produces pome fruit. Examples of such plants, but are not limited to, those which produce apples or pears.

[0136] In an embodiment, the plant produces citrus fruit. Examples of such plants, but are not limited to, those which produce lemons, orange or limes.

[0137] In an embodiment, the plant produces persimmons.

[0138] In an embodiment, the plant produces figs.

[0139] In an embodiment, the fruit is, but not limited to, a peach, an apricot, a nectarine, a plum, a cherry, an apple, a pear, a lemon, orange, a lime, a persimmon, or a fig.

[0140] In an embodiment, the plant produces grain. Such plants, and grain thereof, may be in a cereal crop. Examples of such plants include, but are not limited to, wheat, corn, rice, oats, or barley.

[0141] In an embodiment, the grain is, but not limited to, wheat grain, corn grain, rice grain, oat grain, or barley grain.

[0142] With specific relation to Dipterans, in an embodiment the plant is a fruit tree or a vegetable. Examples of fruit and vegetable crops that are susceptible to Dipteran, such as fruit fly, infestation include, but are not limited to, abiu, acerola, apple, achachairu, apricot, avocado, babaco, banana, black sapote, blackberry, boysenberry, blueberry, brazil cherry, breadfruit, caimito, cape gooseberry, capsicum, carambola, cashew apple, casimiroa, cherimoya, cherry, chilli, choko, citrus, cocoa berry, coffee berry, cumquat, custard apple, date (dried or fresh), dragonfruit, durian, eggplant, feijoa, fig, goji berry, granadilla, grape, grumichama, guava, hog plum, jaboticaba, jackfruit, jew plum, jujube, kiwifruit, loganberry, longan, loquat, lychee, mandarin, mango, nectarine, papaya, passionfruit, peach, pear, persimmon, plum, pomegranate, prune, quince, loquat, santol, tamarillo, tomato, and wax jambu.

[0143] The strain and methods of the present disclosure are useful against Coleopterans or Dipterans during one or more stages of the life-cycle e.g., egg, larva, pupae, and / or adult. In this respect, contacting the strain of the present disclosure with a Coleopteran or Dipteran will preferably prevent reproduction, development and / or survival of the Coleopteran or Dipteran. For example, the strain or methods may prevent or reduce eggs of a Coleopteran or Dipteran from hatching into larva. Alternatively, or in addition, the strain or methods may prevent or reduce the development of larva of a Coleopteran or a Dipteran into pupae form. Alternatively, or in addition, the strain or methods may prevent or reduce the emergence of adult forms of a Coleopteran or a Dipteran from pupae. In an embodiment, contacting of the strain of the present disclosure with the Coleopteran or Dipteran (at any one or more life stages) results in mortality of the Coleopteran or Dipteran.

[0144] In an embodiment, the Coleopteran or Dipteran is a plant pest. In an embodiment, the Coleopteran or Dipteran plant pest reduces the yield of a plant, such as of a plant described herein.

[0145] In an embodiment, the Coleopteran is a bee hive pest, such as a honey bee hive pest. In an embodiment, the Coleopteran pest may reduce the viability of the hive, reduce the productivity of the hive, cause honey fermentation, cause the queen to stop laying eggs and in some instances may result in the bee colony leaving the hive.

[0146] In an embodiment, the Coleopteran is a member of one of the following Families; Nitidulidae, Carabidae, Cerambycidae, Chrysomelidae, Curculionidae or Scarabaeidae.

[0147] In an embodiment, the Coleopteran is a Nitidulidae.

[0148] In an embodiment, the Coleopteran is a member of one of the following Genera, Carpophilus spp., Acanthoscelides spp, Aethina spp., Agriotes spp., Anthonomus spp., Aphidius spp., Apion spp., Apogonia spp., Araecerus spp., Aulacophora spp., Bruchus spp., Cerosterna spp., Cerotoma spp., Ceutorhynchus spp., Chaetocnema spp., Colaspis spp., Ctenicera spp., Curculio spp., Cyclocephala spp., Diabrotica spp., Dinoderus spp., Gnathocerus spp., Hemicoelus spp., Heterobostruchus spp., Hyper a spp., Ips spp., Lyctus spp., Megascelis spp., Meligethes spp., Mezium spp., Niptus spp., Otiorhynchus spp., Pantomorus spp., Phyllophaga spp., Phyllotreta spp., Ptinus spp., Rhizotrogus spp., Rhynchites spp., Rhynchophorus spp., Scolytus spp., Sphenophorus spp., Sitophilus spp., Tenebrio spp., or Tribolium spp..

[0149] In an embodiment, the Coleopteran is a Carpophilus spp..

[0150] In another embodiment, the Coleopteran is an Aethina spp.

[0151] In an embodiment, the Coleopteran is Acanthoscelides obtectus (common bean weevil), Aethina lumida. Agrilus planipennis (emerald ash borer), Ahasverus advena, Alphitobius diaperinus, Anoplophora glabripennis (Asian longhomed beetle), Anthonomus grandis (boll weevil), Anthrenus verbasci, Anthrenus falvipes, Ataenius spretulus (Black Turgrass Ataenius), Atomaria linearis (pygmy mangold beetle), Attagenus unicolor, Bothynoderes punctiventris (beet root weevil), Bruchus pisorum (pea weevil), Callosobruchus maculatus (southern cow pea weevil), Carpophilus hemipterus (dried fruit beetle), Carpophilus davidsoni, Carpophilus humeralis, Carpophilus truncatus, Cassida vittata, Cathartus quadricollis, Cerotoma trifurcate (bean leaf beetle), Ceutorhynchus assimilis (cabbage seedpod weevil), Ceutorhynchus napi (cabbage curculio), Conoderus scalaris, Conoderus stigmosus, Conotrachelus nenuphar (plum curculio), Cotinus nitidis (Green June beetle), Crioceris asparagi (asparagus beetle), Cryptolestes ferrugineus (rusty grain beetle), Cryptolestes pusillus (flat grain beetle), Cryptolestes turcicus (Turkish grain beetle), Cylindr ocpturus adspersus (sunflower stem weevil), Deporaus marginatus (mango leaf-cutting weevil), Dermestes lardarius (larder beetle), Dermestes maculates (hide beetle), Diabrotica virgifera, Epilachna varivestis (Mexican bean beetle), Euvrilletta peltata, Faustinus cubae, Hylobius pales (pales weevil), Hylotrupes bajulus, Hypera postica (alfalfa weevil), Hypothenemus hampei (coffee berry beetle), Lasioderma serricorne (cigarette beetle), Leptinotarsa decemlineata (Colorado potato beetle), Liogenys futscus, Limonius canus, Liogenys fuscus, Liogenys suturalis, Lissorhoptrus oryzophilus (rice water weevil), Lophocateres pusillus, Lyctus planicollis, Maecolaspis joliveti, Melanotus communis, Meligethes aeneus (blossom beetle), Melolontha melolontha (common European cockchafer), Necrobia rufipes, Oberea brevis, Oberea linearis, Oryctes rhinoceros (date palm beetle), Oryzaephilus mercator (merchant grain beetle), Oryzaephilus surinamensis (sawtoothed grain beetle), Oulema melanopus (cereal leaf beetle), Oulema oryzae, Phyllophaga cuyabana, Polycaon stoutti, Popillia japonica (Japanese beetle), Prostephanus truncatus (larger grain borer), Rhizopertha dominica (lesser grain borer), Sitona lineatus (pea leaf weevil), Sitophilus granaries (granary weevil), Sitophilus oryzae (rice weevil), Sitophilus zeamais, Stegobium paniceum (drugstore beetle), Tenebroides mauritanicus, Tribolium castaneum (red flour beetle), Tribolium confusum (confused flour beetle), Trogoderma granarium, Trogoderma variabile (warehouse beetle), Xestobium rufovillosum and Zabrus tenebioides.

[0152] In an embodiment, the Coleopteran is Carpophilus truncatus.

[0153] In an embodiment, the Coleopteran is Carpophilus davidsoni. In an embodiment, the Coleopteran is Carpophilus hemipterus. In an embodiment, the Coleopteran is Carpophilus humeralis. In an embodiment, the Coleopteran C Aelhina tumida.

[0154] In an embodiment, the Dipteran is a Tephritidae.

[0155] In an embodiment, the Tephritidae is a Dacinae. In an embodiment, the Dipteran is a Bactrocera sp., Dacus sp., Ceratitis sp., Zeugodacus sp., Anastrepha sp. or Rhagoletis sp.. In an embodiment, the Dipteran is a Bactrocera sp..

[0156] In an embodiment, the Dipteran is Bactrocera tryoni (Queensland fruit fly), Bactrocera curvipennis. Bactrocera facialis, Bactrocera frauenfeldi, Bactrocera jarvi si, Bactrocera kraussi, Bactrocera kirki, Bactrocera melanotus, Bactrocera neohumeralis, Bactrocera passijlorae, Bactrocera psidii, Bactrocera tan, Bactrocera Irilineola, Bactrocera trivialis, Dacus demmerezi, Dacus frontalis, Dacus solomonensis, Ceratitis capitata (Mediterranean fruit fly), Ceratitis brachychaeta, Ceratitis caetrata, Ceratitis catoirii, Ceratitis cornuta, Ceratitis malgassa, Ceratitis manjakatompo, Ceratitis pinax o Anastrepha iudens.

[0157] In an embodiment the Dipteran is Bactrocera tryoni.

[0158] In an embodiment, the Dipteran is a Sciaridae.

[0159] In an embodiment, the Sciaridae is a Orfelia sp. or Bradysia sp.

[0160] In an embodiment, the strain of the invention or a composition comprising a strain of the invention is delivered to an area which comprises or may comprise a Coleopteran or a Dipteran by application to soil or ground in which the plant is growing or will be sown or the soil or ground upon which a hive is situated. For example, the strain may be applied as a powder, a liquid spray or in pellet form, to the soil before or after planting or to an orchard floor at the start of the fruiting season and if necessary, applied at subsequent time(s) during the fruiting season and harvest. The strain may be applied as a powder, liquid or spray or in pellet form under a hive when a hive is being moved or when an infestation of a Coleopteran such as small hive beetle, or a Dipteran, is first identified and / or to the soil or ground around the hive or hives.

[0161] In an embodiment, the strain or composition of the invention may be included in an integrated pest management system (IPM). For example, the strain or composition may be used in one or more baiting stations located within a crop, such as in an orchard or in a field or an area around a hive or hives. In this embodiment, the strain or composition of the invention may be included in a device that further comprises one or more attractants, such as a pheromone and / or co-attractant as described above. The one or more attractants attracting the Coleopteran or Dipteran to the baiting station where it comes into contact with the strain or composition comprising the strain of the invention. The Coleopteran or Dipteran may then leave the baiting station and infect other Coleoptera or Diptera with which it comes into contact. Devices

[0162] The invention further provides a device comprising a strain of the invention, typically in the form of a composition of the invention. In an embodiment, the device is for delivering the strain to a Coleopteran or a Dipteran, or to an area which comprises, or may comprise a Coleopteran or a Dipteran. In an embodiment, the device is for storing the strain or for providing regulated release of the strain.

[0163] The device may be any device that is able to bring the strain into contact with the Coleopteran or Dipteran while providing an environment that protects the strain from adverse environmental conditions such as UV light and desiccation. Examples of suitable devices are bait stations that have an entry / exit that allow the Coleoptera or Dipteran to enter and exit the station. The strain or composition comprising the strain may be located within the bait station diffusely throughout the station or located in one or more particular areas of the bait station. For example, the strain or composition comprising the strain may be dispersed around the inner surface of the bait station contacting the Coleopteran or Dipteran as it moves around the bait station. Alternatively, the strain or composition comprising the strain may be located at the entrance and / or exit of the bait station such that as the Coleopteran or Dipteran enters or exits the station, it brushes against a reservoir or composition comprising the strain.

[0164] In an embodiment, the device may further comprise in the same composition or as one or more separate compositions, one or more attractants. For example, the device may further comprise a pheromone attractant as described above that attracts the Coleoptera or Dipteran to the bait station to come into contact with the strain of the invention. In an embodiment, the device may further comprise a co-attractant as described above.

[0165] In an embodiment, the device provides for regulated release of the strain, or at least one component of a composition such as an attractant or co-attractant. In an embodiment, the device provides for regulated release of the strain, or at least one component of a composition such as an attractant or co-attractant, for between about 1 to about 6 weeks, or between about 1 to about 4 weeks.

[0166] In an embodiment, the device comprises a composition comprising a strain of the invention, a pheromone composition and a co-attractant composition, all as separate compositions. In another embodiment, the device comprises a composition comprising a strain of the invention and at least one pheromone component and a separate co- attractant composition. In an embodiment, the co-attractant may be a single composition or separate compositions and may be housed in one or more deposit elements as described in W02022 / 120404.

[0167] EXAMPLES

[0168] Example 1 - Materials

[0169] Insects

[0170] Adult Carpophilus truncatus beetles were collected from almond orchards in Robinvale, Victoria, Australia and maintained in culture on a diet of soybean meal, almond meal and sugar. Insects were maintained in an environment-controlled cabinet (25°C, 16:8 light: dark, 40% RH). Pre-pupating larvae were separated and held in containers of moist vermiculite or used in bioassays.

[0171] Fungal Isolates

[0172] Five new isolates of Beauveria sp. and two new isolates of Metarhizium sp. were identified and screened. The commercial product Velifer® (BASF, Vic, Australia) which contains B. bassiana isolate PPRI 5339 (PPRI: Plant Protection Research Institute) is the only biopesticide product that is currently registered in in Australia (APVMA #85696) based on B. bassiana. For comparison, both the commercial formulation (Velifer) and the isolate (PPRI 5339 - fresh spores prepared in suspension) were also analysed. Non-commercial isolates were obtained from orchard soil (Tatura SmartFarm, Agriculture Victoria) and / or from insect cadavers using the insect-bait method (Zimmerman, 1986) (Table 1).

[0173] Briefly, soil samples were collected at a depth of 10-15 cm, using a manual core sampler. Each sample consisted of 20 subsamples (approximately 120 g of soil) and was transferred into polyethylene bags, sealed, and stored at 4 °C. The sampler was sterilised (80 % ethanol) after each collection to avoid cross-contamination. Samples were sieved through a 2 mm sieve to remove stones and organic debris. Mealworm larvae (Tenehrio molitor) were coated with independent samples of soil based on the methods described by Inglis et al. (2012), using five freshly shed mealworm larvae. These insects were then incubated at 26 °C and 70 % RH. After 13 to 17 days, dead larvae were removed from the soil, surface sterilised (70 % ethanol for 30 s, then rinsed three times in sterile distilled water), and transferred to Petri dishes lined with moistened filter paper, for further incubation at 26 °C. Cadavers were monitored daily and fungal colonies were isolated from hyphae emerging from the cadavers. The fungal cultures were initially identified using morphological keys (Humber, 2012; Barnett and Hunter, 1972). Table 1: Details of entomopathogenic fungi screened against Carpophilus truncatus .

[0174] All fungal isolates were grown on oatmeal agar plates for 2-3 weeks at 25°C 14: 10 L:D. Spore suspensions were prepared for each replicate on each day of experimentation using fresh sporulating cultures, according to the method described by McKinnon (2011).

[0175] Harvested spores of each fungal isolate were suspended in sterile 0.05% Tween 80, quantified with Neubauer Improved Hemocytometer, and adjusted to 106conidia / mL. The viability of the spores in each suspension was assessed for each isolate by plating 100 pL of a 106conidia / mL spore suspension at the end of each assay, onto PDA plates and incubating at 25 °C for 18 h. Conidia were considered to have germinated when the germ tube length was greater than the spore width. The first 100 spores seen were counted, this was replicated four times across different fields of view. Only assays with a germination rate over 90% were considered valid and used in the following assessment. Molecular identification of novel fungal species

[0176] To identify novel fungi species, the translation elongation factor 1 alpha (TEF1- a) locus was extracted from whole genome sequence data for evaluation. Genomic DNA was isolated from fungal hyphae using the Qiagen DNesy Soil PowerPro Kit. Library preparation for whole genome sequencing on the Illumina NovaSeq 6000 platform was conducted using the Nextflex Rapid XP V2 DNA-seq kit, using the Perkin-Elmer 10 NT unique dual indexes, prepared at a concentration of 2.5 nM per library. Fastq files were demultiplexed and trimmed using usearch and bbtools (https: / / sourceforge.net / projects / bbmap / ). Genomes were assembled using the SPAdes assembler (Bankevich et al., 2012), and QUAST (Gurevich et al., 2013) was used to assess and compare assembly quality. BBmap was used to locate marker loci of interest, such as TEF, from assembly scaffolds and imported into Geneious Prime (Biomatters). Species identity was then tentatively assigned by querying the GenBank non-redundant nucleotide database with the TEF loci using the Basic Local Alignment Search Tool (BLASTn) and by constructing phylogenetic trees based on loci.

[0177] Larval Bioassay

[0178] Suspensions of each fungal isolate were prepared as described above to 106conidia / mL in sterile 0.05% Tween 80. 2 mL of each isolate suspension was mixed with 2 g of vermiculite in a 30 mL plastic cup with a perforated plastic lid. Ten final instar “wandering stage” larvae were added to each cup, comprising one replicate. A minimum of five replicates were completed per each bioassay, and only the most virulent isolates were tested in repeated bioassay experiments. Control treatments contained 2 mL of sterile 0.05% Tween 80. The treatments were randomly arranged in trays (28 x 24 cm) and incubated at 25 °C, 50 - 60% RH, in the dark in a controlled environment cabinet. All replicates were assessed for mortality after 10 days.

[0179] Larval mortality was indicated by lack of movement when grasped with forceps colour change (extensive brown melanisation spots and / or cream to pink) or mycelial growth. During the incubation period some larvae underwent pupation, especially for less virulent isolates which likely have a longer incubation period. In that instance, death of the pupae was indicated by the same features as the larvae.

[0180] Statistical Analysis

[0181] A probit model was used to analyse the proportion of juveniles dead between the isolate treatments, using a binomial generalised linear mixed model (GLMM) with a probit-link function, conducted with package lme4 (v. 1.1-21) (Bates et al., 2014) in R (v. 3.5.1). The fungal isolate factor was treated as a fixed effect in the model, and the bioassay experiment number was treated as a random effect, to account for any variation arising between experiments. Model assumptions were inspected by plotting residuals versus fitted values, and by testing for overdispersion based on Pearson’s chi-squared residual test. To further compare the formulation treatments, post hoc Tukey tests were performed using a generalised linear hypotheses multiple comparisons procedure (package multcomp) (Hothorn et al., 2023). Mean percent mortality data for larvae / pupae were graphed (package ggplot2) (Wickham et al., 2014) to visualise and present the treatment effects tested.

[0182] Example 2 - Identification of highly pathogenic strains of entomopathogenic fungi

[0183] Two fungal species isolates, Metarhizium anisopliae TatMal and B. bassiana isolate ABBb2, demonstrated the greatest biocontrol potential against C. truncatus larvae with 96% and 95% mortality (Table 2; Figure 1). Statistically significant differences were indicated by the probit model for the proportion of dead juveniles quantified for all fungal isolate treatments compared to the negative control treatment, except for Beauveria australis isolate ‘Bmet’. The post-hoc multiple comparisons procedure demonstrated that while the PPRI 5339 / Velifer treatments were statistically different to the no-inoculum control at the 5% significance level, isolates’ ABBb2, ABMA10, TatMal and B54 were all statistically different (<1% significance level) when directly contrasted with isolate PPRI 5339, demonstrating the higher pathogenicity in these novel fungal isolates to Carpophilus beetle larvae compared to the commercial strain. While the data replication for isolates’ B54 and PPRI 5339 was minimal (N = 5), the results are similar to what was observed previously by Boston et al. (2020) for the same treatment, providing confidence in the validity of these results.

[0184] Table 2: Summary statistics including mean percent mortality of juvenile Carpophilus truncatus treated with entomopathogenic fungi species isolates, N designates the number of replicates, SD = standard deviation, SE = standard error and CI = confidence interval. Example 3 - Use of Metarhizium anisopliae TatMal to control Dipterans

[0185] Metarhizium anisopliae TatMal was tested for activity against larvae of the Dipteran Bactrocera tryoni (Queensland fruit fly). TatMal was found to have the highest virulence ever observed in Bactrocera tryoni when compared to other known strains of M. anisopliae, achieving 100% mortality of pupae in every replicate tested (Figure 2).

[0186] The present application claims priority from AU 2024901919 filed 24 June 2024, the entire contents of which are incorporated by reference.

[0187] It will be appreciated by persons skilled in the art that numerous variations and / or modifications may be made to the invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.

[0188] All publications discussed and / or referenced herein are incorporated herein in their entirety.

[0189] Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is solely for the purpose of providing a context for the present invention. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed before the priority date of each claim of this application.

[0190] REFERENCES

[0191] Bankevich et al. (2012) J Comput Biol, 19:455-77.

[0192] Barnett and Hunter (1972) Illustrated genera of imperfect fungi. Illustrated genera of imperfect fungi. 3rd ed. CABI.

[0193] Bates (2014) J Stat Softw, 67:48.

[0194] Boston et al. (2020) Agronomy, 10: 1207.

[0195] Gurevich et al. (2013) Bioinformatics, 29:1072-1075.

[0196] Hothorn et al. (2023) Biom J, 50:346-363.

[0197] Humber (2012) Identification of entomopathogenic fungi. In: Lacey LA, editor. Manual of techniques in invertebrate pathology. Academic Press; pp. 2012. pp: 151-87.

[0198] Inglis et al. (2012) Laboratory techniques used for entomopathogenic fungi: Hypocreales. In: Lacey LA, editor. Manual of techniques in invertebrate pathology. Academic Press; pp. 189-253, 2:18-53.

[0199] McKinnon (2011) Rhizosphere colonisation of Beauveria Vuillemin species (Ascomycota: Hypocreales) (B. bassiana and B. caledonica). Lincoln University, Christchurch, New Zealand.

[0200] Wickham et al. (2014) Ggplot2: Create elegant data visualisations using the grammar of graphics. New York, NY: Springer-Verlag, 2: 1-189.

[0201] Zimmermann (1986) J Appl Entomol, 102: 213-5.

Claims

CLAIMS1. An isolated strain of Metarhizium anisopliae TatMal deposited with the National Measurement Institute under accession number V24 / 010070 on 3 June 2024, or an active variant thereof.

2. An isolated strain of Beauveria bassiana ABBb2 deposited with the National Measurement Institute under accession number V24 / 010069 on 3 June 2024 or an active variant thereof.

3. The strain of claim 1 or claim 2 which is in the form of a spore, microsclerotia or blastospore.

4. A composition comprising a strain according to any one of claims 1 to 3.

5. The composition of claim 4 which comprises at least one diluent, carrier and / or excipient.

6. The composition of claim 4 or claim 5, wherein the composition comprises:(i) the strain in oil;(ii) spores of the strain in the form of a dry powder suspended in an oil;(iii) spores of the strain in a dry powder; or(iii) the strain suspended in an oil-in-water emulsion.

7. The composition according to any one of claims 4 to 6 which comprises an attractant.

8. The composition of claim 7, wherein the attractant is a pheromone.

9. The composition of claim 8, wherein the pheromone is (2E,4E,6E)-5-ethyl-3- methyl-2,4,6-nonatriene, (3E,5E,7E)-6-ethyl-4-methyl-3 ,5,7-decatriene, (2E,4E,6E,8E)-3,5,7-Trimethyl-2,4,6,8-decatetraene, (2E,4E,6E,8E)-7-Ethyl-3,5- dimethyl-2,4,6,8-decatetraene, (2E,4E,6E,8E)-3,5,7-trimethyl-2,4,6,8-undecatetraene,(2E,4E,6E,8E)-7-ethyl-3,5-dimethyl-2,4,6,8-undecatetraene, or a mixture of two or more thereof.

10. The composition according to any one of claims 7 to 9 which comprises a pheromone and / or a co-attractant.

11. The composition of claim 10, wherein the co-attractant is ethanol, isopentyl alcohol, acetaldehyde, isobutanol, 2-methylbutanol, ethyl acetate, isopentyl acetate, isobutyl acetate, 2-phenylethyl acetate, (E)-4,8-dimethyl-l,3,7-nonatriene (DMNT), methyl benzoate and (Z)-3 -hexenyl acetate, or a mixture of two or more thereof.

12. A method for controlling a Coleopteran, and / or reducing damage caused by a Coleopteran, the method comprising delivering to the Coleopteran, or contacting the Coleopteran with, a strain according to any one of claims 1 to 3.

13. The method of claim 12 which comprises applying a composition according to any one of claims 4 to 11 to an area which comprises, or may comprise, a Coleopteran.

14. The method of claim 13 wherein the area comprises an agricultural crop or a hive.

15. The method of claim 14, wherein the agricultural crop comprises a plant that produces nuts, grain or fruit.

16. The method of claim 15, wherein the plant produces a nut.

17. The method of claim 16, wherein the nut is an almond, pistachio, walnut, cashew nut, kemiri nut, macadamia nut, or Brazil nut.

18. The method according to any one of claims 12 to 17, wherein the Coleopteran is a plant pest.

19. The method of claim 14, wherein the hive is a honey bee hive.

20. The method according to any one of claims 13 to 19, wherein the composition is a dry powder.

21. The method according to any one of claims 12 to 20, wherein the Coleopteran is a Nitidulidae.

22. The method of claim 21, wherein the Nitidulidae is a Carpophilus spp. such as C. Iriincalus. C. davidsoni, C. hemiplerus. or C. humeralis.

23. The method of claim 21 or claim 22, wherein the Coleopteran is Carpophilus truncatus.

24. The method according to claim 21, wherein the Nitidulidae is Aethina tumida.

25. A method for controlling a Dipteran, and / or reducing damage caused by a Dipteran, the method comprising delivering to the Dipteran, or contacting the Dipteran with, a strain according to any one of claims 1 to 3.

26. The method of claim 25, wherein the strain is Metarhizium anisopliae TatMal.

27. The method of claim 25 or claim 26 which comprises applying a composition according to any one of claims 4 to 11 to an area which comprises, or may comprise, a Dipteran.

28. The method of claim 27, wherein the area comprises a fruit crop or tree.

29. The method according to any one of claims 25 to 28, wherein the Dipteran is a plant pest.

30. The method according to any one of claims 25 to 29, wherein the Dipteran is a Tephritidae.

31. The method of claim 30, wherein the Tephritidae is a Bactrocera sp. such as B. tryoni.

32. Use of a strain according to any one of claims 1 to 3 to control a Coleopteran and / or reduce damage caused by a Coleopteran.

33. Use of a strain according to any one of claims 1 to 3 to control a Dipteran and / or reduce damage caused by a Dipteran.

34. A device comprising a strain according to any one of claims 1 to 3.

35. The device of claim 34 for attracting Coleopterans or Dipterans.

36. The device of claim 35 which provides for regulated release of an attractant.

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