AU2023237039A1

AU2023237039A1 - Entomopathogenic Fungal Isolates

Info

Publication number: AU2023237039A1
Application number: AU2023237039A
Authority: AU
Inventors: Francesco Sciacca
Original assignee: Ecoganic Pty Ltd
Current assignee: Ecoganic Pty Ltd
Priority date: 2022-10-28
Filing date: 2023-09-26
Publication date: 2024-05-16

Abstract

(Figure 1) The present invention relates to entomopathogenic fungal isolates, compositions comprising entomopathogenic fungal isolates, and methods and uses thereof. More particularly, the present invention relates to compositions comprising Metarhizium spp. fungal isolates and methods of controlling banana rust thrips infestations of banana plants. 1/1 Contacting a banana plant or a banana plant propagation material with an isolated strain of Metarhizium spp. or a spore thereof Figure 1

Description

1/1

Contacting a banana plant or a banana plant propagation material with an isolated strain of Metarhizium spp. or a spore thereof

Figure 1

ENTOMOPATHOGENIC FUNGAL ISOLATES TECHNICAL FIELD

[0001] The present invention relates to entomopathogenic fungal isolates, compositions comprising entomopathogenic fungal isolates, and methods and uses thereof. More particularly, the present invention relates to compositions comprising Metarhizium spp. fungal isolates and methods of controlling banana rust thrips infestations of banana plants.

BACKGROUND

[0002] Banana rust disease is a common disease in the banana industry and may be caused by Chaetanaphothrips signipennis (rust thrips) or other thrips infestations. The disease is caused by adult thrips and larvae feeding on sap from the fruit (fingers) and pseudostem of the banana plant. Damage occurs in locations of the plant where the pest congregates to feed, such as behind the bases of the leaf sheath on the pseudostem and where the fruit contacts the tree or another fruit.

[0003] The visible symptoms of a rust thrips infestation initially appear as a water soaked smoky area which develops into a rusty-red to dark brown-black discolouration. In a severe infestation, a V-shaped rust coloured marking on the pseudostem of the plant may be observed and the peel of the fruit may split or scar. Although damage to the fruit is largely superficial and often restricted to the peel, the rusty coloured fruit is less appealing to customers who may reject or devalue the fruit, causing large economic losses to farmers.

[0004] Banana rust thrips infestations are common and persistent and are easily spread by movement of infested plant material.

[0005] Known solutions include chemical spraying of the banana plant bunches and pseudostem to target the adult thrips and larvae, and application of granules and dusts to the soil to target the thrips pupal stage. However, chemical pesticides are often non-selective and may kill beneficial insects impairing ecosystem health. Additionally, run-off from farms may contaminate water sources and cause health problems in species using those water sources.

[0006] There is also growing evidence that rust thrips are developing resistance to chemical pesticides which has necessitated more intensive spraying periods with higher pesticide concentrations, or the use of a combination of various pesticides with differing mechanisms of action.

[0007] While pesticide treatments are widely utilised solutions to rust thrips infestations, these solutions may not always present a viable option. In particular, these solutions are inappropriate on farms that have adopted organic production practices.

[0008] Thus it would be advantageous if it were possible to provide an effective pest control method that was also suitable for use in organic production practices.

[0009] It will be clearly understood that, if a prior art publication is referred to herein, this reference does not constitute an admission that the publication forms part of the common general knowledge in the art in Australia or in any other country.

SUMMARY OF INVENTION

[0010] Embodiments of the present invention are directed to entomopathogenic fungal isolates, compositions comprising entomopathogenic fungal isolates, and methods and uses thereof, which may at least partially overcome at least one of the abovementioned disadvantages or provide the consumer with a useful or commercial choice.

[0011] With the foregoing in view, the present invention in a first aspect, resides broadly in a method of controlling one or more invertebrate pests using an isolated strain of Metarhizium guizhouense or a spore thereof.

[0012] Advantageously, controlling one or more invertebrate pests using an isolated strain of Metarhizium guizhouense or a spore thereof may reduce, or preclude the need to use chemical pesticides. In instances where the method of controlling one or more invertebrate pests using an isolated strain of Metarhizium guizhouense or a spore thereof precludes the need to use chemical pesticides, farmers may retain their organic certifications, thereby enabling farmers to offer their crops for sale at a higher value. Furthermore, controlling one or more invertebrate pests using an isolated strain of Metarhizium guizhouense or a spore thereof may prove beneficial to the environment when compared with existing pest treatments, as the isolated strain of Metarhizium guizhouense or a spore thereof is more selective than traditional chemical pesticides. Furthermore, Metarhizium guizhouense or a spore thereof is not harmful to humans which may reduce downstream processing and contamination concerns.

[0013] Preferably, the isolated strain of Metarhizium guizhouense or a spore thereof includes all the identifying characteristics of the strain as deposited with the United States Department of Agriculture Agricultural Research Service Collection of Entomopathogenic Fungal cultures under accession numbers ARSEF:4303; ARSEF: 7502; and ARSEF: 7412. The skilled addressee will appreciate that the isolated strain of Metarhizium guizhouense or a spore thereof may comprise identifying characteristics different from the aforementioned strains.

[0014] For convenience, the term Metarhizium spp. will be used to refer collectively to one or more of Metarhizium guizhouense and Metarhizium lepidiotae.

[0015] As used herein, the term "entomopathogenic" means that the fungal pesticide is pathogenic to at least one target insect. As used herein, "entomopathogenic fungus" is a fungus that is capable of attacking, infecting, killing, disabling, causing disease, and/or causing injury to an insect, and is thus able to be used in the control insect infestation by adversely affecting the viability or growth of the target insect.

[0016] The entomopathogenic effects may occur by antagonism of one or more phytopathogens. Antagonism of the phytopathogen may result in varied response by the phytopathogen. For example, the antagonism may simply repel the phytopathogen. Preferably, the antagonism may render the plant environment unfavourable to the phytopathogen. In some embodiments, the antagonism may impede growth, render infertile, incapacitate, or kill the phytopathogen. Preferably, the antagonism prevents the phytopathogen from feeding.

[0017] Preferably, the entomopathogenic effects exhibit approximately 5% entomopathogenic efficacy, at least about 10%, at least about 15%, at least about %, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, more preferably at least about 50% entomopathogenic efficacy expressed as a percentage reduction of the phytopathogen population.

[0018] As used herein, the term "spore" has its normal meaning which is well known and understood by those skilled in the art. In particular, the term spore refers to a microorganism in its dormant, protected state.

[0019] As used herein, the term "conidium" or "conidia" refer to a type of spore produced asexually.

[0020] As used herein, the term "pest" refers to any animal of the scientific classification (class) Insecta, including order Thysanoptera (e.g. thrips).

[0021] As used herein, the terms "life stage" or "life stages" are intended to refer to any of the developmental stages (e.g., eggs, larvae, nymphs, instars, pupae, adults, etc.) of any animal of the scientific classification (class) Insecta, including order Thysanoptera (e.g. thrips).

[0022] As used herein, the term "surfactant" refers to a molecule that belongs to a class of molecules having a hydrophilic group (or groups) and a hydrophobic group (or groups) that exhibit surface activity when the relative amounts of hydrophilic and hydrophobic parts are appropriate.

[0023] The strain or spore of Metarhizium spp. may be obtained from any suitable source. For instance, the strain or spore of Metarhizium spp. may be obtained from its natural source, substrate, habitat, or environment and grown in culture. The strain or spore of Metarhizium spp. may be obtained from an infected insect host and grown in culture. The strain or spore of Metarhizium spp. may be obtained from a suitable reference library and grown in culture. It will be understood that the isolated strain of Metarhizium spp. may be obtained from any suitable source and separated from one or more constituents with which it was associated. The isolated strain of Metarhizium spp. may be purified to obtain a pure culture of fungi.

[0024] In some embodiments, the strain of Metarhizium spp. or a spore thereof, may be provided in a composition.

[0025] In other embodiments, the composition may comprise two or more strains of Metarhizium spp. or a spore thereof.

[0026] The composition comprising the strain of Metarhizium spp. may include any suitable form from the life cycle of the species. For instance, the strain may take the form of spores (such as, e.g., conidia, blastospores), vegetative cells (such as, e.g., hyphae, conidiogenous cells) or a mass of hyphae (such as, e.g., thallus, mycelium). In some such embodiments, the composition may include one or more vegetative cells. In other such embodiments, the composition may include a mass of hyphae. In yet other such embodiments, the composition may include one or more spores. Generally, the composition includes one or more conidia.

[0027] The strain of Metarhizium spp. or a spore thereof may preferably be provided with a growth sustaining treatment before application.

[0028] In an embodiment of the invention, the strain of Metarhizium spp. or a spore thereof may be provided with a source of carbon to sustain growth. The skilled addressee will appreciate that provision of carbon may facilitate and sustain the growth of the strain of Metarhizium spp. or a spore thereof. Similarly, the strain of Metarhizium spp. or a spore thereof may be provided with a source of nitrogen to facilitate and sustain the growth of the strain of Metarhizium spp. or a spore thereof.

[0029] Typically, the composition includes a strain or a spore of one or more entomopathogenic fungi from a group consisting of: Metarhizium guizhouense or Metarhizium lepidiotae.

[0030] The skilled addressee will appreciate that the strain or a spore of one or more entomopathogenic fungi may be selected for its ability to survive in atmospheric conditions without significant growth sustaining treatment.

[0031] Preferably, the one or more entomopathogenic fungi have all of the identifying characteristics of the aforementioned deposited strains.

[0032] The composition including a strain of Metarhizium spp. or a spore thereof may be of any suitable type. Suitably, the composition may provide a stable environment for the strain of Metarhizium spp. or a spore thereof such that the spores keep their viability over time. However, it will be understood that the type of composition may vary depending on numerous factors, such as, e.g., the number of plants to be treated, the type of plant to be treated, the location of the infestation on the plant, the pest species being treated, the life stage of the pest species being treated and the equipment available to treat the plant. For example, the type of composition may be a liquid composition (such as a concentrate, an emulsion, a suspension), a dry composition (such as a powder, granules, microcapsules, baits), or any suitable combination thereof. The composition may be dispersed in a liquid for application to the plant or may be applied to the plant without dispersion in a liquid.

[0033] The composition including a strain of Metarhizium spp. or a spore thereof may be prepared according to procedures and compositions which are conventional in the relevant art, such as, e.g., agricultural chemical art.

[0034] For example, in some embodiments, the composition may include the strain of Metarhizium spp. or a spore thereof and at least one diluent, carrier and/or excipient.

[0035] Any suitable diluent, carrier and/or excipient may be used. However, it will be understood that the type of diluent, carrier and excipient used may vary depending on a number of factors, such as what the plant to be treated can tolerate and the most effective delivery method of a strain of Metarhizium spp. or a spore thereof to the plant to be treated. Such agriculturally acceptable diluents, carriers and excipients would be known to a person of skill in the art.

[0036] The composition comprising a strain of Metarhizium spp. or a spore thereof, may additionally include one or more additives, such as, polymers, sticking agents or adherents, adjuvants, emulsifying agents, surfactants, foams, humectants, or wetting agents, antioxidants, colorants, UV protectants, thickeners, fillers, anti-freeze agents, solvents, nutritive additives, fertilizers, chemical pesticides and biopesticides (insecticides, fungicides and/or herbicides).

[0037] The skilled addressee will appreciate that the composition may be formulated in any suitable manner. For example, the composition may be formulated as a concentrate, solution, spray, aerosol, immersion bath, dip, emulsion, wettable powder, soluble powder, suspension concentrate, dust, granule, paste, gel, or other.

[0038] Preferably, the composition comprising a strain of Metarhizium spp. or a spore thereof may additionally include an adjuvant. Any suitable adjuvant may be used. Such agriculturally acceptable adjuvants would be known to a person of skill in the art. It is envisaged that, in use, the adjuvant may enhance pesticide activity by allowing a water-based spray solution to penetrate the waxy surface of the plant. Accordingly, the adjuvant may include properties or characteristics of surfactants, anti foaming agents, buffering agents, crop oils, spreader stickers and other compatibility agents. In preferred embodiments , the composition may include a surfactant, most preferably Tween@ 80 (polysorbate 80; Tween is a registered trade mark of Croda Americas LLC).

[0039] In some embodiments , the composition may have an approximate cell density of at least 10 cells/mL, typically at a concentration of at least 1x102 cells/mL, preferably at a concentration of at least 1x10 3 cells/mL, and more preferably at a concentration of at least x104 cells/mL.

[0040] In some embodiments, the composition when formulated as a liquid may have an approximate cell density of 4000 cells/mL, preferably at least about 4000 Metarhizium spp. cells per millilitre of composition, wherein the Metarhizium spp. cells comprise at least one spore.

[0041] Preferably, the method of controlling one or more invertebrate pests using an isolated strain of Metarhizium guizhouense or a spore thereof is carried out on a banana plant or banana plant propagation material.

[0042] While the embodiments described herein are in reference to banana plant or banana plant propagation material, the skilled addressee will appreciate that the teachings disclosed may be applied to any number of plant or plant propagation materials. In particular, the teachings may be applied to citrus fruits, or native Australian plants which have been documented to host Thysanoptera.

[0043] The banana plant or banana plant propagation material may be of any suitable form. For example, the banana plant may be a new plant (i.e., sucker), a mature plant in a vegetative stage, a mature plant in a flowering stage or a mature fruiting plant. Most preferably, the banana plant includes a banana spike, bunch, hand, and/or individual fruit. The banana plant propagation material may include suckers (such as maidenhead suckers, water suckers or sword suckers), bits comprising a section of banana rhizome containing an eye or bud (developing sucker) or tissue cultured plants.

[0044] In some embodiments, the banana plant propagation material may be a sucker. In other embodiments, the banana plant may be a mature banana plant.

[0045] The banana plant or banana plant propagation material may be any suitable variety. The banana plant or banana plant propagation material may be derived from Musa acuminata, Musa balbisiana or Musa x paradisiaca varieties. The banana plant may be Cavendish and Cavendish cultivars (such as Valery, Williams (Mons Mari), Grande Naine), Pome bananas (such as Australian Lady Finger), Lady Finger bananas (such as Sucrier, Lady's Finger, Sugar banana), Silk bananas (such as Apple Banana, Figue pomme) and plantains.

[0046] In some embodiments, the banana plant may be a triploid AAA cultivar.

[0047] In preferred embodiments, the banana plant may be a Cavendish variety.

[0048] In some embodiments, the method of controlling one or more invertebrate pests may include contacting the plant material with Metarhizium spp.

[0049] As used herein, the term "control" or the phrase "controlling one or more invertebrate pests" may refer to preventing a thrips infestation, treating a banana plant or banana plant propagation material for a thrips infestation, treating the soil at the base of a banana plant of banana plant propagation material for a thrips infestation, reducing the population of an already infested area, preventing a thrips infestation from feeding, preventing a thrips infestation from becoming more severe, or preventing the spread of a thrips infestation within or between banana plants or banana plant propagation materials.

[0050] In some embodiments, there is provided a method to protect a banana plant or banana plant propagation material from one or more invertebrate pests. In such embodiments, it will be understood that the Metarhizium spp. may be applied to a portion of the banana plant or banana plant propagation material and the Metarhizium spp. may be further distributed on the banana plant or banana plant propagation material, or to nearby banana plants or banana plant propagation materials during movement of the one or more invertebrate pests.

[0051] In further embodiments, there is provided a method to treat a banana plant or banana plant propagation material for an infestation of one or more invertebrate pests.

[0052] The one or more invertebrate pests to be controlled may be an insect, preferably a sap-feeding insect, more preferably from the order Thysanoptera, such as family Thripidae. In preferred embodiments, the pest species to be controlled may be Chaetanaphothrips signipennis or banana rust thrips.

[0053] Most preferably, control of the banana rust thrip includes preventing the banana rust thrip from feeding on, or otherwise damaging, the skin of a banana. Even more preferably, control of the banana rust thrip initially includes preventing the banana rust thrip from feeding and subsequently causing the death of the banana rust thrip.

[0054] Preferably, once the banana rust thrip is contacted with the Metarhizium spp. the banana rust thrip is precluded from further damaging the banana plant or banana plant propagation material. Most preferably, once the banana rust thrip is contacted with the Metarhizium spp. the banana rust thrip is immediately precluded from further damaging the banana plant or banana plant propagation material. Advantageously, this may reduce aesthetic damage to the banana fruit such that it may be a higher grade and subsequently sold at a higher price.

[0055] Advantageously, once the banana rust thrip is contacted with the Metarhizium spp. the banana rust thrip is not immediately killed, rather the banana rust thrip is prevented from feeding. This enables the banana rust thrip to spread the Metarhizium spp. to other banana rust thrips in the vicinity without further damaging the banana plant or banana plant propagation material. Advantageously, by preventing the banana rust thrip from feeding, the banana rust thrip may be mobilised to seek alternate foods and/or to alert other banana rust thrips of potential dangers thereby spreading the Metarhizium spp.

[0056] The banana plant or banana plant propagation material may be contacted with the composition comprising Metarhizium spp. by any suitable means. However, it will be understood that the mechanism of contacting the banana plant with a composition may vary depending on a number of factors, such as the number of plants to be treated, the type of plant to be treated, the location of the infestation on the plant, the pest species being treated, the life stage of the pest species being treated and the equipment available to treat the plant.

[0057] For example, in some embodiments an aerosol or a liquid composition may effectively contact the surface of a mature banana plant and allow penetration of the Metarhizium spp. into the banana plant bunches and pseudostem.

[0058] Most preferably, an aerosol or liquid composition is utilised to contact with the banana plant or banana plant propagation material. In such embodiments, the aerosol or liquid composition may first be placed in a storage vessel and subsequently dispensed. For example, the aerosol or liquid composition may be placed in a backpack sprayer, agricultural sprayer, or like piece of equipment for subsequent distribution on the banana plant or banana plant propagation material.

[0059] Contact with the banana plant or banana plant propagation material may include contact of the composition with a combination of soil, water, leaf litter and/or growth media proximal the banana plant or banana plant propagation material and roots.

[0060] In some embodiments, contact with the banana may occur by immersing the banana plant propagation material in the composition. It is envisaged that this method of contact may be preferred in a nursery.

[0061] In other embodiments in which the roots of a banana plant need to be treated, a granular or liquid composition may be applied to the base of a banana plant and the surrounding soil.

[0062] For example, the composition may be applied to the base of the banana plant or banana plant propagation material through the process of watering the banana plant or banana plant propagation material.

[0063] In embodiments in which the banana plant or banana plant propagation material is cultivated hydroponically, the composition may be applied directly to the hydroponic medium. In such instances, it is envisaged that the composition concentration will be adjusted to a suitable level in the hydroponic medium so as to provide adequate effects the banana plants and banana plant propagation material therein.

[0064] In some embodiments, the contact with the banana plant or banana plant propagation material may occur indirectly. For example, the composition may be contacted with storage, packaging, or processing materials including boxes, wrappers, farming, and/or harvesting equipment.

[0065] In a second aspect, the invention resides broadly in a banana plant or banana plant propagation material when treated with a strain of Metarhizium spp. or a spore thereof.

[0066] The treated banana plant or banana plant propagation material may include banana plant or banana plant propagation material wherein only a parent generation was treated with a strain of Metarhizium spp. or a spore thereof.

[0067] In a third aspect, the invention resides broadly in use of a strain of Metarhizium spp., or a spore thereof, to control one or more invertebrate pests infesting a banana plant or banana plant propagation material.

[0068] Any of the features described herein can be combined in any combination with any one or more of the other features described herein within the scope of the invention.

[0069] The reference to any prior art in this specification is not, and should not be taken as an acknowledgement or any form of suggestion that the prior art forms part of the common general knowledge.

BRIEF DESCRIPTION OF DRAWINGS

[0070] Preferred features, embodiments and variations of the invention may be discerned from the following Detailed Description which provides sufficient information for those skilled in the art to perform the invention. The Detailed Description is not to be regarded as limiting the scope of the preceding Summary of Invention in any way. The Detailed Description will make reference to a number of drawings as follows:

[0071] Figure 1 is a flow chart showing steps in a method of controlling one or more invertebrate pests.

DETAILED DESCRIPTION

[0072] Referring to Figure 1, as an initial step the method (100) of controlling one or more invertebrate pests may include contacting a banana plant or a banana plant propagation material with an isolated strain of Metarhizium spp. or a spore thereof 105).

[0073] Prior to contacting the banana plant or a banana plant propagation material, the strain of Metarhizium spp. or a spore thereof is typically provided with a growth sustaining treatment. Generally, the strain of Metarhizium spp. or a spore thereof is provided with carbon or nitrogen. Advantageously, the growth sustaining treatment improves the longevity of the strain of Metarhizium spp. or a spore thereof, such that the treated strain of Metarhizium spp. or a spore thereof may have an increased shelf life.

[0074] Generally, the strain of Metarhizium spp. or a spore thereof, is provided in a composition. The composition typically includes a strain or a spore of one or more entomopathogenic fungi from a group consisting of: Metarhizium guizhouense or Metarhizium lepidiotae.

[0075] Generally, the composition further includes a diluent, carrier and/or excipient. The type of diluent, carrier and/or excipient will be sufficient to ensure the control of one or more invertebrate pests without damaging and/or impairing the banana plant or a banana plant propagation material and the growth of the banana plant or a banana plant propagation material.

[0076] Typically, the composition is a liquid composition (such as a concentrate, an emulsion, a suspension).

[0077] Typically, the liquid composition is first placed in a storage vessel and subsequently dispensed.

[0078] It is envisaged that the liquid composition will be dispensed on the banana plant or a banana plant propagation material such that the banana plant or a banana plant propagation material is saturated by the liquid composition.

[0079] In instances where the banana plant or a banana plant propagation material are exposed to the elements, it will be understood that additional contact may be required if the composition is not provided adequate time to treat the banana plant or a banana plant propagation material due to rainfall, or other weather events. Typically, the banana plant or a banana plant propagation material contacted with the composition will remain in contact with the composition for at least a continuous 12 hour period.

[0080] Generally, the banana plant or a banana plant propagation material contacted with the composition will be contacted with the composition a plurality of times.

[0081] Contact with the banana plant or banana plant propagation material occurs by contact of the composition with a combination of soil, water, leaf litter and/or growth media proximal the banana plant or banana plant propagation material and roots.

[0082] Where contact with the banana plant or banana plant propagation material occurs by contact of the composition with a combination of soil, water, leaf litter and/or growth media proximal the banana plant or banana plant propagation material and roots, the contact occurs a plurality of times. Generally, the contact occurs by way of drip irrigation. Typically, the contact occurs four times per day for a 10-minute period each contact time.

[0083] In alternate embodiments, contact with the banana occurs by immersing the banana plant propagation material in the composition.

[0084] In embodiments in which the roots of a banana plant need to be treated, a granular or liquid composition is applied to the base of a banana plant and the surrounding soil.

[0085] In embodiments in which the banana plant or banana plant propagation material is cultivated hydroponically, the composition is applied directly to the hydroponic medium. In such instances, it is envisaged that the composition concentration will be adjusted to a suitable level in the hydroponic medium so as to provide adequate effects the banana plants and banana plant propagation material therein.

[0086] In some embodiments, the contact with the banana plant or banana plant propagation material occurs indirectly. For example, the composition is contacted with storage, packaging, or processing materials including boxes, wrappers, farming, and/or harvesting equipment.

[0087] In the present specification and claims (if any), the word 'comprising' and its derivatives including 'comprises' and 'comprise' include each of the stated integers but does not exclude the inclusion of one or more further integers.

[0088] Reference throughout this specification to 'one embodiment' or 'an embodiment' means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, the appearance of the phrases 'in one embodiment' or 'in an embodiment' in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more combinations.

[0089] In compliance with the statute, the invention has been described in language more or less specific to structural or methodical features. It is to be understood that the invention is not limited to specific features shown or described since the means herein described comprises preferred forms of putting the invention into effect. The invention is, therefore, claimed in any of its forms or modifications within the proper scope of the appended claims (if any) appropriately interpreted by those skilled in the art.

EXAMPLES

[0090] Example 1 - Culturing of Metarhizium spp. strains

Soil sampling

[0091] Soil was sampled from an ecoganic banana farm and in the adjoining hedgerow. Three soil samples were taken 10 metres apart, along two 20 metre transects (on and off-farm) to a depth of 10 cm with an auger (15 cm diameter), then mixed together in separate polyethylene bags per transect.

Soil baiting to rear fungal isolates out of soil samples

[0092] Soil-baiting experiments followed similar protocol to Zimmermann (1986), though substituting Galleria mellonella with Tenebrio molitor.

[0093] The beetle larvae were placed into containers holding soil samples at room temperature and checked weekly for signs of sporulation. After signs of sporulation, conidia were removed from the dead beetle larvae and placed onto potato dextrose agar (PDA) plates containing 0.3 g/L of chloramphenicol.

Preparation of cultures for downstream PCR

[0094] The DNA was extracted from the fungal cultures using the DNeasy Plant Mini Kit (Qiagen, Australia), and quantified using the Thermo Scientific NanoDrop 2000 UV-Vis spectrophotometer. The extracted DNA was analysed and sequenced through the nuclear gene region elongation factor 1-alpha (EF1- a), using forwards and backwards primers EF1T (5'-ATGGGTAAGGARGACAAGAC) and 1567R (5' ACHGTRCCRATACCACCSATCTT) (Rehner & Buckley, 2005).

[0095] Three distinct Metarhizium isolates were selected based off their high spore production. The chosen isolates M. guizhouense was reared from soil on-farm, and M. lepidiote (M13) was obtained from soil in the adjoining hedgerow.

Preparation of conidial suspensions for spray application in pot and laboratory trials

[0096] After determining the preferred growth media of the Metarhizium isolates, they were sub-cultured onto full strength malt extract agar (MEA) plates and left for three weeks.

[0097] Conidial suspensions were prepared by scraping 20 large plates of each isolate with a sterile spatula into separate falcon tubes, before adding 50 mL of TWEEN (0.1%) and vortexing for 30 seconds. The concentration of each stock solution was determined using a haemocytometer.

[0098] The required calculated volumes of the stock solution were then added to give a final concentration of 2 x10 6 in 500 mL of Tween (0.1%).

[0099] Example 2 - Banana Plant Pot trials

[00100] 16 Cavendish tissue cultures were placed in a banana row on an ecoganic farm for five weeks to allow infection by thrips. After five weeks, the Cavendish tissue cultures were placed into individual entomological cages (24x24x56" BioQuip Products, Inc. CA, USA) and fitted with fine nylon mesh netting to prevent thrips escaping the enclosure.

[00101] At the start of the trial, the second emerging leaf was marked to keep track of growth. The banana plant was sprayed with a composition comprising 4000 Metarhizium spp. spores/mL of 0.1% w/v Tween 80. Each emerging leaf above that marked point was included in the analysis.

[00102] The banana plants were drip irrigated four times a day for 10 minutes and fertilised each fortnight over the one month trial period.

[00103] Each banana plant was assessed for visible signs of rust scaring on the banana pseudostem over the one month trial period. Each emerging leaf was evaluated at the pseudostem as either'damage - visible signs of thrips activity' or'no damage - no thrips activity'. The percentage of total rust damage over the trial period was calculated based on the emerging leaf data.

[00104] Example 3 - Banana finger Laboratory Trials

[00105] Immature banana fingers were placed into individual polyethylene bags together with 15 thrips in each polyethylene bag. The thrips were of different life stages, although predominantly of the nymphal stage.

[00106] A composition comprising 4000 Metarhizium spp. spores/mL of 0.1% w/v Tween 80 was sprayed into each polyethylene bag.

[00107] Each banana finger was assessed for: visible signs of rust scaring; sporulating thrips; fungal persistence; and thrips population counts over the three-week trial period.

References

Rehner, S., & Buckley, E. (2005). A Beauveria phylogeny inferred from nuclear ITS and EFI-alpha sequences: evidence for cryptic diversification and links to Cordyceps teleomorphs. (Vol. 97).

Zimmermann, G. (1986). The Galleria bait method for detection of entomopathogenic fungi in soil. Journal of Applied Entomology, 102, 213-215.

Claims

1. A method of controlling one or more invertebrate pests, the method comprising

contacting a banana plant or a banana plant propagation material with an isolated strain of Metarhizium spp. or a spore thereof.

2. The method as claimed in claim 1, wherein the strain of Metarhizium spp. is one of, or a combination of, Metarhizium guizhouense or Metarhizium lepidotae

3. The method as claimed in claim 1 wherein the one or more invertebrate pest belongs to the family Thripidae.

4. The method as claimed in claim 1, wherein the one or more invertebrate pest is a Chaetanaphothrips signipennis (rust thrip).

5. The method as claimed in claim 1, wherein the isolated strain or Metarhizium spp. or a spore thereof is provided in a composition.

6. The method as claimed in claim 5, wherein the composition comprises Metarhizium spp. or a spore thereof and at least one diluent, carrier, and/or excipient.

7. The method as claimed in claim 5, wherein the composition comprises a cell density of at least 4000 Metarhizium spp. cells per millilitre, and wherein the Metarhizium spp. cells comprise at least one spore.

8. The method as claimed in claim 1, wherein contacting the banana plant or banana plant propagation material includes spraying or otherwise coating the banana plant or banana plant propagation material.

9. The method as claimed in claim 1, wherein contacting the banana plant or banana plant propagation material includes contacting the spike, bunch, hand and/or individual fruit of the banana plant.

10. The method as claimed in claim 1, wherein contacting the banana plant includes applying the isolated strain of Metarhizium spp. or a spore thereof to the banana plant or banana plant propagation material, or soil, water, leaf litter or growth media proximal the banana plant or banana plant propagation material.

11. A banana plant or banana plant propagation material when treated with a strain of Metarhizium spp. or a spore thereof.

12. The banana plant or banana plant propagation material of claim 11, wherein the banana plant or banana plant propagation is treated by applying the strain of Metarhizium spp. or a spore thereof to the coil, water, leaf litter, or growth media proximal the banana plant or banana plant propagation material.

13. The banana plant or banana plant propagation material as claimed in claim 11, wherein the composition as claimed in any one of claims 5 to 7 is used to treat the banana plant or banana plant propagation material.

14. The banana plant or banana plant propagation material as claimed in claim 11, wherein the method as claimed in any one of claims 8 to 10 is used to treat the banana plant or banana plant propagation material.

15. Use of a strain of Metarhizium spp. or a spore thereof, to control one or more invertebrate pests infesting a banana plant or banana plant propagation material.

16. The use of Metarhizium spp. or a spore thereof according to claim 15, wherein the one or more invertebrate pest belongs to the family Thripidae.

17. The use of Metarhizium spp. or a spore thereof according to claim 16, wherein the one or more invertebrate pest is a Chaetanaphothrips signipennis (rust thrip).

18. The use of Metarhizium spp. or a spore thereof according to claim 15, wherein control of the one or more invertebrate pests includes repulsion, growth impediment, infertility, incapacitation, or death of the one or more invertebrate pests,

19. The use according to claim 15, wherein the composition as claimed in any one of claims 5 to 7 is used to treat the banana plant or banana plant propagation material.

20. The use according to claim 15, wherein the method as claimed in any one of claims 1 to 10 is used to treat the banana plant or banana plant propagation material.