WO2018141020A1

WO2018141020A1 - Biofumigant compositions and methods thereof

Info

Publication number: WO2018141020A1
Application number: PCT/AU2018/050069
Authority: WO
Inventors: Christian KRILL; Ross MANN; Desmond AUER; Simone Jane Rochfort; German Carlos Spangenberg
Original assignee: Agriculture Victoria Services Pty Ltd
Current assignee: Agriculture Victoria Services Pty Ltd
Priority date: 2017-02-02
Filing date: 2018-02-02
Publication date: 2018-08-09
Anticipated expiration: 2019-08-02
Also published as: AU2018216628A1; CA3051899A1; BR112019016106A2; AR110925A1; EP3576532A1

Abstract

The present invention relates to biofumigant and biocidal compositions, particularly biofumigant and biocidal compositions including isoprene or an analogue thereof. The present invention also relates to uses and methods of use of said compositions, including in biofumigation or bioprotection, more particularly for pest control in products such as stored grain.

Description

BIOFUMIGANT COMPOSITIONS AND METHODS THEREOF

Field of the Invention The present invention relates to biofumigant and biocidal compositions, particularly biofumigant and biocidal compositions including isoprene and analogues thereof. The present invention also relates to uses and methods of use of said compositions, more particularly for pest control in products such as stored grain. Background of the Invention

Microbes represent an invaluable source of genes and compounds that have the potential to be utilised in a range of industrial sectors. Scientific literature gives numerous accounts of microbes being the primary source of antibiotics, immunosuppressants, anticancer agents, cholesterol-lowering drugs and agricultural chemicals, in addition to their use in environmental decontamination and in the production of food and cosmetics. A relatively unexplored group of microbes known as endophytes, which reside in the tissues of living plants, offer a particularly diverse source of novel compounds and genes that may provide important benefits to society, and in particular, agriculture.

Endophytes often form mutualistic relationships with their hosts, with the endophyte conferring increased fitness to the host, often through the production of defence compounds. At the same time, the host plant offers the benefits of a protected environment and nutriment to the endophyte. Bioprotectant endophytes that have been developed and commercialised include Neotyphodium species that produce insecticidal alkaloids, including peramine (a pyrrolopyrazine) and the lolines (pyrrolizidines). These compounds can accumulate to high levels in planta where they act as potent feeding deterrents against a range of insect pests. The insecticidal compounds, destruxins, have also been well characterised as secondary metabolites of fungi. Another antimicrobial compound of fungi is the peptaibols, produced by Trichoderma virens, Quercus suber, Trichoderma citrinoviridae, that show antifungal activity against a range of plant pathogens, including Biscogniauxia mediterranea and Apiognomonia quercine.

Recent discoveries highlight the diversity of applications of endophytes, such as in the energy (e.g. biofuels) sector, and the agricultural sector where fungal species have been identified that produce volatile biocidal metabolites which show application as fumigants. For instance, the fungus Muscodor albus from Cinnamomum zeylanicum in Honduras produces a suite of volatile antimicrobial compounds that are effective against soil borne pathogens, and this has enabled development of a commercial preparation which has been evaluated as a biological alternative (e.g. mycofumigant) to soil fumigation. Furthermore, the discovery of the endophytic fungus Ascocoryne sarcoides, which produces a variety of hydrocarbons commonly found in diesel, petrol and biodiesel, offers mankind a potential alternative to fossil fuels.

Stored grain disinfestation has utilised phosphine as the primary fumigant for many years due to its broad spectrum insecticidal activity, high volatility, negligible environmental impact (no residues) and cost-effectiveness (Warrick, 2011 ; Collins, 2015). However, there is increasing incidence of phosphine resistance in pests of stored grain in Australia, Asia and Brazil, with four of the five major grain insect pests in Australia exhibiting some level of resistance (Lesser Grain Borer - Rhyzopertha dominica; Sawtooth Grain Beetle - Oryzaephilus surinamensis; Rust Red Flour Beetle - Tribolium castaneum; Flat Grain Beetle - Cryptolestes ferrugineus) (Collins et al., 2002; Jagadeesan et al., 2012; Nayak et al., 2012). Resistance has developed, in part, due to a lack of available alternatives to phosphine, coupled with its repeated use on the same parcel of grain (Collins, 2015). Increased phosphine resistance poses an escalated risk of quarantine incursions that threaten the sustainability of the grains industry (Collins, 2015). Furthermore, the compromised disinfestation practices that arise from phosphine resistance have the potential to impact on market access for Australian grains (Collins, 2015).

There is an increasing need to identify alternative soil and quarantine fumigants, as a number of chemicals have environmental or resistance issues. For instance, the widely used fumigant methyl bromide has been identified as a significant ozone depleter, contributing to the degradation of the Antarctic ozone hole, which has led to increased UV exposure and higher incidence of skin cancer in the southern hemisphere. Furthermore, the multi-purpose fumigant phosphine has shown increased incidence of resistance in insect populations following fumigation, which has biosecurity implications and market access issues globally (e.g. stored grain).

It is an object of the present invention to overcome, or at least alleviate, one or more of the difficulties or deficiencies associated with the prior art. Summary of the Invention

In one aspect, the invention provides a biofumigant or biocidal composition including isoprene or an analogue thereof.

The isoprene component in the composition of the present invention may be produced from an isoprene-producing fungus; for example, by culturing a fungus and recovering isoprene produced by the fungus from fungal cells, from the culture medium, or from air space associated with the culture medium or fungus.

Alternatively, the isoprene may be synthesised or otherwise obtained, and compositions thereof where desirable may be manufactured by admixture. For example, one or more organic compounds that are substantially identical with isoprene produced by a fungus, or are analogues thereof, may be provided, and may be mixed with other components to form a composition. The one or more organic compounds may be synthesised by suitable chemical reactions.

In the context of this invention, a "biofumigant or biocidal composition" is a composition capable of reducing, suppressing or protecting a product (such as stored grain) against the activity of pests (such as insects) or micro-organisms, including fungi and bacteria.

In the context of the invention, the term 'bioactivity' or "bioactive" refers to the component including a biocidal acitivity. A biocidal activity includes insecticidal and microbial activity. Reference to microbial activity includes fungicidal and bactericidal activity.

In the context of the invention, the term "bioprotection" refers to use of a composition to reduce, suppress or protect a product (such as stored grain) against the activity of pests (such as insects) or micro-organisms, including fungi and bacteria. The biocidal or biofumigant composition according to this aspect of the invention may further include one or more excipients, such as binders, carriers, propellants, azeotropes, surfactants, etc., depending on the desired application. These materials and methods of preparation thereof would be familiar to a skilled worker in the art. By an 'analogue' is meant a compound similar to isoprene, differing in respect of one or more structural components. The term encompasses both 'substantially identical' compounds and derivatives, along with other similar compounds. By a 'derivative' is meant an organic compound obtained from, or regarded as derived from, another compound. Examples of derivatives include compounds where the degree of saturation of one or more bonds has been changed (e.g., a single bond has been changed to a double or triple bond) or wherein one or more atoms are replaced with a different atom or functional group. Examples of different atoms and functional groups may include, but are not limited to, hydrogen, halogen, oxygen, nitrogen, sulphur, hydroxy, alkoxy, alkyl, alkenyl, alkynyl, amine, amide, ketone and aldehyde. By 'substantially identical' is meant for example, a stereoisomer, regioisomer, skeletal isomer, positional isomer, functional group isomer, structural isomer, conformational isomer, tautomer, or other isomer, isotopic variant, derivative or salt thereof.

In a preferred embodiment, the biofumigant or biocidal composition of the present invention includes isoprene or an analogue thereof and a further bioactive component.

In a more preferred embodiment, the further bioactive component may be selected from alcohols, ketones, aldehydes and monoterpenoids. The alcohol may be selected from the group consisting of: 3-Methyl-1-butanol, Isoamyl alcohol, 3-Methyl-2-butanone, p-Cresol (s), 2-Methyl-3-buten-2-ol, n-Butyl alcohol, or 2-Methyl-1-butanol. The monoterpenoid may be selected from the group consisting of: (+)-trans-p-Menth-2-ene, a-Terpinene, Sabinene,(+)- α Pinene, (R)-Carvone R+Limonene, 3-Carene, a-Phellandrene, (-)-Linalool, Terpinolene, 1 ,4-Cineole,Terpinen-4-ol,lsoborneol (s), n-Butyl, alcohol, Camphene (s), Menthofuran,(-)- Menthol(s), Eucalyptol. The ester may be selected from the group consisting of: Ethyl 2- methylbutyrate, Methyl isobutyrate, Ethyl Isobutyrate, or Isobutyl acetate. The aldehyde may be selected from the group consisting of: trans-2-Hexenal, or Acetaldehyde. The ketone may 3-Penten-2-one.

Preferably, the bioactive component is selected from the group consisting of: 2-methyl-1- butanol, 3-methyl-2-butanone, n-butyl alcohol, acetaldehyde, linalool, sabinene and eucalyptol.

It has surprisingly been found that the combination of isoprene or an analogue thereof and one or more of the above bioactive components may generate a synergistic biocidal effect. The further bioactive component may be one or a combination of any two or more of the above. For example, preferred combinations include: isoprene or an analogue thereof and 2-methyl-1-butanol; isoprene or an analogue thereof and 3-methyl-2-butanone; isoprene or an analogue thereof and n-butyl alcohol; isoprene or an analogue thereof and acetaldehyde; isoprene or an analogue thereof and linalool; isoprene or an analogue thereof and sabinene; isoprene or an analogue thereof and eucalyptol. In a particularly preferred embodiment, the biocidal composition may include isoprene, or an analogue thereof and acetaldehyde, either alone, or in combination with 2-methyl-1-butanol. In a preferred embodiment, the composition includes a further bioactive component, as hereinbefore described. In this case, the isoprene or analogue thereof may be administered at a sub-lethal dose.

Preferably, at least one of the bioactive components is derived from an endophyte. The endophyte may be for example Nodulisporium sp. Dandenong Ranges isolate 1 (DR1).

In a further aspect, the present invention provides use of a composition including isoprene or an analogue thereof in pest control such as biofumigation or bioprotection of a product.

For example, isoprene may be used as a fumigant. Isoprene may be used to fumigate various products or commodities, including but not limited to, stored grain, soil, timber, buildings, fresh produce and import/export goods. In particular, isoprene may be used for quarantine and pre-shipment (QPS), structural or soil fumigation.

Compositions, e.g. fumigants containing isoprene or an analogue thereof, may be applied by any suitable method. Suitable methods for applying compositions such as fumigants would be familiar to a person skilled in the art. For example, compositions containing isoprene may be applied by application directly to the fumigation area and/or product to be treated, e.g. fumigated. This may include application by spraying, gassing, clouding, wetting, injecting, sublimating and dusting. For example, fumigants containing isoprene may be applied by direct injection into a fumigation area. Application may be with or without a carrier gas such as C0₂ and air, and with or without heating. Application may also be by moisture activation of a pelleted form, with or without a binding agent such as metal binding agents of aluminium, zinc and calcium.

Isoprene or an analogue thereof may be effective against pests and diseases including but not limited to insects such as grain borers and beetles, including grain borers and beetles selected from the group consisting of Lesser Grain Borer (Rhyzopertha dominica), Sawtooth Grain Beetle (Oryzaephilus suinamensis), Rust Red Flour Beetle (Tribolium castaneum) and Flat Grain Beetle (Crryptolestes ferrugineus). Isoprene or an analogue thereof may have benefits selected from the group consisting of being safer, less damaging to the environment, less susceptible to resistance and faster acting than commonly used fumigants such as methyl bromide and phosphine.

For example, insect mortality may be evident after approximately 1 hour to approximately 10 days of fumigation, more preferably after approximately 3 to approximately 7 days of fumigation. With currently used fumigants, such as phosphine, insect mortality may not be evident until up to approximately 20 days of fumigation.

In another aspect, the present invention provides a method for inhibiting an insect or a micro-organism including exposing the insect or micro-organism to isoprene or an analogue thereof. In a preferred embodiment, the isoprene or analogue thereof may be present in a composition for use as a fumigant or a biocidal composition as hereinbefore described.

In a preferred embodiment, the insect is a pest of stored grain, including but not limited to Tribolium castaneum, Rhyzopertha dominica, Cryptolestes ferrugineus and Oryzaephilus suinamensis. In a further preferred embodiment, the micro-organism is a fungus selected from one or more of the genus Fusarium, Botrytis, Alternaria or Rhizoctonia, such as species Fusarium verticillioides, Botrytis cinerea, Alternaria alternata and Rhizoctonia cerealis, and a bacteria of the genus Pseudomonas such as species Pseudomonas syringae. As used herein, the terms 'an insect' and 'a micro-organism' is taken to include a population thereof.

Inhibition of an insect or micro-organism may be by way of a decrease in a normal activity. For an insect, this may include, for example, prevention or reduction of insect proliferation, growth or breeding. In preferred embodiments, the biocidal composition causes insect mortality, for example, by fumigation as hereinbefore described. The amount to which the insect is exposed may be from about 20 to about 200, preferably about 50 to about 200, more preferably about 100 to about 200, microlitres of isoprene or an analogue thereof per litre of environment (e.g. container, vessel, silo etc.). For a micro-organism, inhibition may include prevention or reduction of proliferation or growth. For example, a reduction in growth may be evident after approximately 1 hour to approximately 10 days of exposure, e.g. fumigation, more preferably after approximately 1 day to approximately 4 days of exposure.

The present invention will now be more fully described with reference to the accompanying Examples and drawings. It should be understood, however, that the description following is illustrative only and should not be taken in any way as a restriction on the generality of the invention described above.

Brief Description of the Drawings/Figures

In the Figures: Figure 1. A. Conidiophore ex-culture (Nodulisporium sp., DR1); B. Conidia ex-culture (Nodulisporium sp., DR1); C - Growth on PDA (Nodulisporium sp., DR1).

Figure 2. A MP phenogram (1 of 8631) based on 5.8 S / ITS rRNA gene sequences from 55 isolates of Nodulisporium and Hypoxylon species. Highlighted area (grey) shows Victorian Nodulisporium isolate DR1. The phenogram was obtained using the Close- Neighbour-Interchange algorithm of MEGA4.1 (deletion of gaps and missing data). Numbers on the nodes represent frequency (in percent) with which a cluster appears in 1000 bootstrap tests. Scale bar equals 5 changes per 100 bases. Figure 3. Image of the split plate bioassay that evaluates the insecticidal activity of volatile compounds produced by Nodulisporium sp. (DR1) and their structural analogues, against T. castaneum.

Detailed Description of the Embodiments

Example 1 - Fungal Isolate

Pieces of leaf and stem of Lomatia fraserii were collected during surveys in the Dandenong Ranges. Sections of leaf and stem were surface sterilised (70 % Ethanol for 30 sees, flame sterilisation) prior to the excision of internal tissues, which were then plated onto potato dextrose agar (PDA) (39g/L) (Amyl Media, Dandenong, Australia) amended with achromycin (50 ppm). Endophytic fungi growing from the plant tissue were removed by excising a hyphal tip from each colony, and plated onto PDA. Each hyphal tip constituted one endophytic fungal isolate. Isolates then underwent a preliminary screen for bioactivity by challenging them against Rhizoctonia solani on PDA. One isolate inhibited the growth of R. solani and was selected for further analysis. A pure culture of the isolate (i.e. hyphal plugs) was placed in SDW and stored at room temperature and at 4°C, and in 15 % glycerol at -70°C.

A representative sample of Nodulisporium sp. DR1 isolate has been deposited at The National Measurement Institute on 3 May 201 1 with accession number V1 1/011039 as disclosed in WO2012/159161 (PCT/AU2012/000574) entitled "Fungi and products thereof" in the name of Agriculture Victoria Services Pty Ltd (incorporated herein by reference).

Example 2 - Morphology

Isolates were removed from storage and placed onto PDA and allowed to grow at 25°C (in the dark) until the formation of conidiophores. Sections of hyphae containing conidiophores were mounted in lactic acid and examined under light microscopy (in vitro description). Nodulisporium state of Hypoxylon

Description in vitro

Colonies on PDA initially white, becoming pale yellow to grey yellow. Conidiophores branching loosely, pale brown, paler towards the apex, verruculose, 2.5-3 μηι wide. Conidiogenous cells usually produced singly, pale brown, verruculose, 12-20 x 2.5-3 um. Conidia bourne from minutely visible denticles, pale brown, more or less smooth, ellipsoidal, 6-8 x 3-4 μm (Figure 1). By evaluating the microscopic features of the isolates growing in culture (in vitro stage) we confirmed that they were characteristic of an undescribed species of Nodulisporium. Example 3 - Genotyping

Genomic DNA was extracted from cultures of the Nodulisporium sp. DR1 isolate grown in potato dextrose broth (PDB) using a DNeasy Plant Mini Kit (Qiagen). A section of the ribosomal RNA loci (5.8S / ITS) was amplified with primers ITS4 and ITS5 (White et al. , 1990). PCR amplification was performed in 25 μL_ reaction volumes containing 1.0 U of Platinum Taq DNA Polymerase (Invitrogen), x 1 PCR buffer, 0.2 mM of each dNTP, 1 .5 mM MgCI₂, 0.5 μΜ of each primer, and 15 - 25 μg DNA. The reaction was performed in a thermocycler (Gradient Palm-Cycler, Corbett Research) with cycling conditions consisting of denaturation at 94°C (3 min), followed by 35 cycles at 94°C (30 s), 50°C (30 s), and 72°C (2 min), with a final extension step at 72°C (3 min) to complete the reaction. The PCR product was separated by electrophoresis at 100 V for 45 min in a 1 .5% (w/v) agarose gel (containing ethidium bromide, 0.1 ppm) in 0.5 X TBE running buffer and visualised under UV light. The amplification product was purified using a PCR Purification Kit (Qiagen), and sequenced using the BigDye Terminator Cycle v 3.1 sequencing kit (Applied Biosystems) on the ABI 3730x1 Capillary Sequencer (Applied Biosystems), according to manufacturers' instructions.

The rDNA-ITS sequence of Nodulisporium sp. DR1 was compared to reference sequences from Nodulisporium (or related teleomorphs, i.e. Hypoxylon and Daldinia) accessions from around world (closest matches from GenBank). A total of 55 Nodulisporium-re\ated sequences were aligned with MUSCLE. Aligned sequences were adjusted with ClustalW / Alignment Explorer in MEGA 4.1 . Based on these sequences phylogenetic relationships were inferred using distance and maximum parsimony (MP) analyses. For distance analysis, phenograms were obtained using the neighbour-joining (NJ) algorithm, applying the Kimura-2- parameter model, as implemented in MEGA4.1 . For MP analysis, phenograms were obtained using the Close-Neighbour-Interchange algorithm (search level 3), as implemented in MEGA4.1. To find the global optimum phenogram 10 random sequences were added. Measurements calculated for MP included tree length, consistency index, retention index and rescaled consistency index (TL, CI , Rl, RCI). In both analyses, alignment gaps and missing data were eliminated from the dataset (Complete deletion option) and the confidence of branching was assessed by computing 1000 bootstrap replications. Of the 55 Nodulisporium-re\atedi isolates the size of the rRNA (5.8S / ITS) gene sequence ranged from 436 - 664 base pairs, of which 371 were included in the final data set for analysis. In the NJ analysis the optimal phenogram had a sum of branch length of 0.525. The MP analysis yielded 8631 most parsimonious phenograms (TL = 211 , Cl= 0.654 Rl = 0.916, RCI = 0.569, for the parsimony informative sites). NJ and MP analyses yielded phenograms with similar topology and bootstrap values. Therefore, only the MP phenogram is presented (1 of 8631 , Figure 2).

Isolates tended to cluster according to the teleomorph of Nodulisporium species, Hypoxylon and Daldinia. Nodulisporium sp. DR1 clustered with Hypoxylon species, with an 80 % bootstrap support. This group formed a cluster with other Nodulisporium and Hypoxylon isolates, with a bootstrap support of 14 % (Clade 1). This cluster was alongside another group of Hypoxylon isolates with a bootstrap support of 41 % (Clade 2). A large group of Daldinia isolates formed the next related cluster with a 37 % bootstrap support (Clade 3).

Example 4 - Insecticidal bioactivity of Nodulisporium sp. (DR1 ) and other endophytic fungi

In vitro bioassays were established to test the bioactivity of Nodulisporium sp. (DR1) and other endophytic fungi (Endophyte A - U) against a range of insect pests of stored grain, Tribolium castaneum (red flour beetle), Rhyzopertha dominica (lesser grain borer) and Cryptolestes ferrugineus (rusty grain beetle). The bioassays were conducted in 90 mm split Petri plates. The split plates consisted of an impermeable barrier through the centre of the plate, which completely separated the plate into two halves, with only volatile compounds capable of passing over the septum (i.e. no direct contact between the endophyte and their liquid exudates with the test insect). The isolates were inoculated on to Petri plates containing PDA by placing a 6 mm agar plug containing actively growing mycelia, 13 mm from the edge of the plate (i.e. on one half of the plate). Isolates were allowed to grow at 25°C (in the dark) for 6 days. Subsequently, the insect pests were inoculated on to the other half of the plate by placing 3 insects onto their respective feed (T. castaneum - wheat flour and yeast, R. dominica - whole wheat seed, C. ferrungineus - rolled oats). Plates were sealed with LDPE plastic film (approximately 0.01 mm thick) and covered in aluminium foil (i.e. in the dark). After 3, 7 and 10 days the mortality of insects was determined by assessing insect movement as an indicator of mortality. The mortality was calculated by comparing the number of dead insects to the total number in the plate, and expressed as percentage mortality. The Spectrum of Activity was calculated for each isolate by summing the number of insect species that were completely controlled (i.e. 100 % mortality). The Biocidal Activity Ranking was calculated for each isolate by averaging the % mortality across all insects and all time points. Data were analysed using ANOVA as performed in GenStat, version 14. The experiment was fully randomised with 4 replicates for each endophyte. Nodulisporium sp. DR1 showed broad spectrum biocidal activity against the three insect pests, completely controlling C. ferrugineus and R. dominica (100% mortality), and severely effecting the survival of T. castaneum (89% mortality) (Table 1). The biocidal activity of Nodulisporium sp. (DR1) was rapid, with biocidal activity against the insect pests observed within 3 days, and complete control within 7 - 10 days. Nodulisporium sp. (DR1) had the highest Biocidal Activity Ranking (1) of all endophytes tested, as it had the highest average mortality.

Example 5 - Volatolome of Nodulisporium sp. (DR1)

Gases were analysed in the head space above cultures of Nodulisporium sp. (DR1). The isolate was cultured under microaerophilic conditions, which consisted of growing the fungus on PDA and Yeast Malt Extract (YME) slopes in 20 ml headspace vials, with an agarair ratio of 1 :2.5. Vials were sealed with a screw cap lid with PTFE septum, and grown for 10 days at room temperature.

A head space solid phase microextraction (SPME) was performed to capture volatiles produced by the endophytes. A StableFlex fibre (Supelco) coated with either (i) 75 μηι CAR/PDMS or (ii) 30-50 μηι DVB/CAR/PDMS was used to absorb volatiles from the head space of vials. Automated sampling was performed by a Gerstel Multi-Purpose Sampler using the proprietary Maestro software. The fibre was conditioned at 250°C for 60 mins prior to commencement of activities and for 30 minutes between each sample. For each sample the fibre was inserted into the vial and incubated at room temperature for 7 minutes to absorb volatiles, after which the fibre was inserted into a splitless injection port of an Agilent 7890 GC System where the contents was thermally desorbed (250°C for 6 mins) onto a capillary column (Agilent DB-624, 30 m x 250 μm id., 1.4 μηι film thickness or Agilent DB-5MS, 30 m x 250 μηι id., 0.25 μηι film thickness). The column oven was programmed as follows: 35°C (3 min), 3°C/min to 200°C, then 25°C/min to 250°C (2 min). The carrier gas was helium with a constant flow rate of 1 mL/min. The GC was interfaced with an Agilent 7000 GC/MS triple quadruple mass selective detector (mass spectrometer, MS) operating in electron impact ionization mode at 70 eV. The temperature of the transfer line was held at 280°C during the chromatographic run. The source temperature was 280°C. Acquisitions were carried out over a mass range of mz 29 - 330, with a scan time of 200 ms.

Initial identification of the volatiles produced by Nodulisporium sp. (DR1) was made through library comparison using standard chemical databases. Secondary confirmatory identification was made by comparing mass spectral data of authentic standards with data of the fungal volatiles. All chemical names in this report follow the nomenclature of the standard chemical databases. In all cases, uninoculated control vials were also analysed and the compounds found therein were subtracted from those appearing in the vials supporting fungal growth. Tentative identification of the fungal volatiles was based on observed mass spectral data as compared to those in these chemical databases and those of authentic standards (where possible). The GC-MS analysis (0 to 65 minutes) identified 54 volatile metabolites produced by Nodulisponum sp. (DR1) when grown for 10 days on PDA and YME at room temperature (Table 2). These compounds represented a range of structural classes including monoterpenoids (predominating), alcohols, esters, aldehydes and sesquiterpenoids. The volatolome of Nodulisponum sp. (DR1 ) was more complex when grown on PDA than YME, with 54 compounds identified on PDA, compared to 49 on YME. Similarly, a further seven compounds were produced in higher concentration on PDA than on YME.

1 - Fragmentation pattern matches spectra of compound in NIST library (>75%)

2 - Fragmentation pattern matches spectra of compound in NIST library (>90%)

3 - Fragmentation pattern matches spectra of authentic standard

* - Fragmentation pattern compared against an authentic standard

+ - significant peak; ++ - major peak; +++ - dominant peak Example 6 - Insecticidal activity of compounds from the volatolome of Nodulisponum sp. (DR1 )

A total of 76 chemical standards were evaluated for their insecticidal activity against the stored grain pest, Tribolium castaneum. These chemical standards represented compounds in the volatolome of Nodulisponum sp. (DR1) or were structural analogues of these compounds. The bioassays were conducted in 90 mm split Petri plates (as per example 4) (Figure 3). The insect pest was inoculated on to one half of the Petri plate by placing 4 insects onto feed (wheat flour and yeast). A chemical standard was then aliquoted (5 μL , except isoprene - 10 μL) on to the other half of the Petri plate on filter paper. Plates were immediately sealed with Parafilm®, covered in aluminium foil (i.e. in the dark) and maintained at room temperature. The mortality of insects was monitored daily by assessing insect movement as an indicator of mortality. The mortality was calculated by comparing the number of dead insects to the total number in the plate, and expressed as percentage mortality. Data were analysed using ANOVA as performed in GenStat, version 14. The experiment was fully randomised with 5 replicates for each compound. A total of 4 compounds exhibited high insecticidal activity against T. castaneum, providing 100% mortality following 24 - 48 hrs exposure (Table 3). These compounds included isoprene (10 μL_), menthofuran, (-)-menthol and 2-methyl-3-buten-2-ol (Table 4). These four compounds were either monoterpenoids or alcohols. Furthermore, only one of these compounds was confirmed within the volatolome of Nodulisporium sp. (DR1), isoprene (10 μL_). A total of 12 compounds exhibited medium insecticidal activity (36 - 99 % mortality following 24 - 76 hrs exposure), while 20 compounds exhibited low insecticidal activity (1 - 35% % mortality following 24 - 96 hrs exposure). A total of 40 compounds exhibited no insecticidal activity.

Table 3 - Classification of the level of insecticidal activity of volatile compounds produced by Nodulisporium sp. (DR1 ) and their structural analogues, against T. castaneum.

Table 4 - Insecticidal activity of volatile compounds (76) produced by Nodulisporium sp. (DR1 ) and their structural analogues, against T. castaneum (dark grey: 100% mortality, med grey: 36-99% mortality, light grey: 1 - 35 % mortality, white: 0% mortality).

Example 7 - Insecticidal dose response of a key compound (isoprene) from the volatolome of Nodulisporium sp. (DR1) An insecticidal dose response bioassay was established to evaluate isoprene against the stored grain pest, T. castaneum. The bioassay was conducted in 1 L Schott bottles. The insect pest was inoculated into the bioassay by placing 9 - 14 insects onto feed (wheat flour and yeast). Isoprene was then aliquoted into the bioassay at volumes ranging from 20 - 250 uL, ensuring no direct contact with the insect. Bottles were immediately sealed with Parafilm® and maintained at room temperature. The mortality of insects was monitored following 1 , 6, 8 and 11 days exposure, by assessing insect movement as an indicator of mortality. The mortality was calculated by comparing the number of dead insects to the total number in the bioassay, and expressed as percentage mortality. Data were analysed using ANOVA as performed in GenStat, version 14. The experiment was fully randomised with 4 replicates.

Isoprene exhibited biocidal activity against T. castaneum with volumes ranging from 20 - 250 μL (Table 5). The most rapid biocidal activity was observed after a 1 day exposure, with volumes ranging from 100 - 250 μL (59.2 - 100.0 % mortality). The lowest volume that exhibited 100 % mortality in the shortest period of time (1 day exposure) was 150 μL, while 100 μL exhibited 100 % mortality after a 6 day exposure. Table 5 - Dose response (20 - 250 uL) of isoprene against T. castaneum

Example 8 - Insecticidal synergy of isoprene with other key compounds from the volatolome of Nodulisporium sp. (DR1)

An insecticidal synergy bioassay was established to evaluate the combinatorial effect of isoprene with other compounds (2-methyl-1-butanol; 3-methyl-2-butanone; n-butyl alcohol; eucalyptol) from the volatolome of Nodulisporium sp. (DR1) against the stored grain pest, T. castaneum. The bioassays were conducted in 500 mL Schott bottles. The insect pest was inoculated into the bioassay by placing 10 - 14 insects onto feed (wheat flour and yeast). The biocidal compounds were then aliquoted into the bioassay at sub-lethal doses (20 μL / compound), ensuring no direct contact with the insect. Bottles were immediately sealed with Parafilm® and maintained at room temperature. The mortality of insects was monitored following 5 - 24 hrs exposure, by assessing insect movement as an indicator of mortality. The mortality was calculated by comparing the number of dead insects to the total number in the bioassay, and expressed as percentage mortality. Data were analysed using ANOVA as performed in GenStat, version 14. The experiment was fully randomised with 4 replicates for each compound combination. A synergistic insecticidal effect was observed when isoprene was combined with 2-methyl- 1-butanol, 3-methyl-2-butanone, n-butyl alcohol and eucalyptol, at sub-lethal doses (20 μL @) (Table 6). In contrast, isoprene had no insecticidal effect when applied alone with the same volumes (20 and 40 μL). The synergistic insecticidal activity of isoprene with 2- methyl-1-butanol, 3-methyl-2-butanone or n-butyl alcohol was rapid, with 100 % mortality observed after 5 hours, while isoprene with eucalyptol exhibited 100 % mortality after 24 hrs. It is thought that the enhanced bioactivity of isoprene when applied at sub-lethal doses with other bioactive compounds could be attributed to different modes of action of the compounds, as they have diverse structures and are from varying chemical classes (alcohols, ketones and monoterpenoids). Table 6 - Synergistic effect (insecticidal) of isoprene and other key compounds from the volatolome of Nodulisporium sp. (DR1 ).

Example 9 - Fungicidal activity of isoprene, alone and in synergy with other key compounds from the volatolome of Nodulisporium sp. (DR1)

A fungicidal bioassay was established to evaluate the effect of isoprene against the plant pathogenic fungus, Fusarium verticillioides, when applied alone and in synergy with other compounds (2-methyl-1-butanol; 3-methyl-2-butanone; acetaldehyde; eucalyptol; linalool; sabinene) from the volatolome of Nodulisporium sp. (DR1). The pathogen was inoculated on to one third of the Petri plate by placing an agar plug of actively growing hyphae onto PDA. Isoprene was then aliquoted (10 μL_) on to another third of the Petri plate on filter paper. In the synergy treatments a second compound (2-methyl-1-butanol, 3-methyl-2- butanone, acetaldehyde, eucalyptol, linalool or sabinene) was added to the final third of the Petri plate on filter paper. Plates were immediately sealed with Parafilm® and maintained at room temperature. After 4 days the growth of the pathogen was determined by measuring the radius of colony (on two alternate planes). Measurements were compared to the control and expressed as percentage inhibition relative to the control plate (no growth = 100 %). Compound combinations that exhibited 100 % inhibition were assessed for fungistatic or fungicidal activity by placing the original plug onto a fresh PDA plate. Data were analysed using ANOVA as performed in GenStat, version 14. The experiment was fully randomised with 4 replicates for each compound combination. Isoprene had a fungicidal effect when applied alone inhibiting growth by 40.9 % (Table 7). When isoprene was combined with acetaldehyde the growth of F. verticilliodies was inhibited by 100 %, while isoprene with linalool inhibited the growth by 99.9 %. When hyphal plugs from these compound combinations were transferred to fresh PDA no growth was observed in the isoprene-acetaldehyde treatment indicating fungicidal bioactivity, while growth was observed in the isoprene-linalool treatment indicating fungistatic bioactivity. As per the insecticidal synergy experiments, it is thought that the enhanced bioactivity of isoprene when applied with other bioactive compounds could be attributed to different modes of action of the compounds, as they have diverse structures and are from varying chemical classes (alcohols, ketones, aldehydes and monoterpenoids).

Table 7 - Fungicidal activity of isoprene, alone and in synergy with other biocidal compounds from the volatolome of Nodulisporium sp. (DR1 ), against F. verticillioides.

Example 10 - Bactericidal effect of isoprene, alone and in synergy with other key compounds from the volatolome of Nodulisporium sp. (DR1)

A bactericidal bioassay was established to evaluate the effect of isoprene against the plant pathogenic bacterium Pseudomonas syringae, when applied alone or in synergy with other compounds (2-methyl-1-butanol; 3-methyl-2-butanone; acetaldehyde; eucalyptol; linalool; sabinene) from the volatolome of Nodulisporium sp. (DR1). The pathogen was inoculated on to one third of the Petri plate by streaking bacterial cells from an actively growing culture (overnight) on to nutrient agar (NA). Isoprene was then aliquoted (10 μL_) on to another third of the Petri plate on filter paper. In the synergy treatments a second compound (2- methyl-1-butanol, 3-methyl-2-butanone, acetaldehyde, eucalyptol, linalool or sabinene) was added to the final third of the Petri plate on filter paper. An untreated control was included and consisted of no compounds. Plates were immediately sealed with Parafilm® and maintained at room temperature. After 3 days the growth of the pathogen was visually assessed and scored based on the following scale: 2 - equivalent growth to the control; 1 - less growth than the control; 0 - no growth. Compound combinations that exhibited 100 % inhibition were assessed for bacteristatic or bactericidal activity by subculturing from the original streak on to a fresh NA plate. Data were analysed using ANOVA as performed in GenStat, version 14. The experiment was fully randomised with 4 replicates for each compound combination. Isoprene had no bactericidal effect when applied alone. A bactericidal effect was only observed when isoprene was combined with acetaldehyde and 2-methyl-1-butanol, however the data were not significant (Table 8). Isoprene with acetaldehyde was the only combination to completely inhibit the growth of P. syringae, while isoprene with 2-methyl-1- butanol reduced the growth. When colonies from these compound combinations were transferred to fresh NA no growth was observed in the isoprene-acetaldehyde treatment indicating bactericidal bioactivity. As per the insecticidal synergy experiments, it is thought that the enhanced bioactivity of isoprene when applied with other bioactive compounds could be attributed to different modes of action of the compounds, as they have diverse structures and are from varying chemical classes (alcohols, ketones, aldehydes and monoterpenoids).

Table 8 - Bactericidal activity of isoprene, alone and in synergy with other biocidal compounds from the volatolome of Nodulisporium sp. (DR1), against P. syringae.

As used herein, except where the context requires otherwise, the term "comprise" and variations of the term, such as "comprising", "comprises" and "comprised", are not intended to exclude further additives, components, integers or steps. Reference to any prior art in the specification is not, and should not be taken as, an acknowledgment or any form of suggestion that this prior art forms part of the common general knowledge in Australia or any other jurisdiction or that this prior art could reasonably be expected to be ascertained, understood and regarded as relevant by a person skilled in the art.

Finally, it is to be understood that various alterations, modifications and/or additions may be made without departing from the spirit of the present invention as outlined herein. References

Collins, P. J., Daglish, G. J., Bengston, M., Lambkin, T. M., & Pavic, H. (2002). Genetics of resistance to phosphine in Rhyzopertha dominica (Coleoptera: Bostrichidae). Journal of Economic Entomology, 95(4), 862-869.

Collins, P. J. (2015). Strategy to manage resistance to phosphine in the Australian grain industry. Plant Biosecurity Cooperative Research Centre / National Working Group Party on Grain Protection.

Jagadeesan, R., Collins, P. J., Daglish, G. J., Ebert, P. R., & Schlipalius, D. I. (2012). Phosphine resistance in the rust red flour beetle, Tribolium castaneum (Coleoptera: Tenebrionidae): inheritance, gene interactions and fitness costs. PLos one, 7(2), e31582. Nayak, M. K., Holloway, J. C, Emery, R. N., Pavic, H., Bartlet, J., & Collins, P. J. (2013). Strong resistance to phosphine in the rusty grain beetle, Cryptolestes ferrugineus (Stephens)(Coleoptera: Laemophloeidae): its characterisation, a rapid assay for diagnosis and its distribution in Australia. Pest management science, 69(1), 48-53.

Warrick, C. (2011). Fumigating with phosphine and other fumigants in controlled atmospheres. GRDC Final Report.

Claims

THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS:

1. A biofumigant or biocidal composition including isoprene or an analogue thereof.

2. A biofumigant or biocidal composition according to claim 1 , wherein the composition includes a further bioactive component selected from alcohols, ketones, aldehydes and monoterpenoids.

3. A biofumigant or biocidal composition according to claim 1 , wherein the further bioactive component is selected from the group consisting of 2-methyl-1-butanol, 3-methyl-

2-butanone, n-butyl alcohol, acetaldehyde, linalool, sabinene and eucalyptol.

4. A biofumigant or biocidal composition according to claim 3, wherein the further bioactive component includes acetaldehyde, either alone, or in combination with, 2-methyl- 1-butanol.

5. A biofumigant or biocidal composition according to any one of claims 1 to 4, wherein at least one of the bioactive components is derived from an endophyte.

6. Use of a composition including isoprene or an analogue thereof in biofumigation or bioprotection.

7. Use according to claim 6, wherein the composition includes a further bioactive component selected from the group consisting of 2-methyl-1-butanol, 3-methyl-2-butanone, n-butyl alcohol, acetaldehyde, linalool, sabinene and eucalyptol.

8. Use according to claim 7, wherein the further bioactive component includes acetaldehyde, either alone, or in combination with 2-methyl-1-butanol.

9. A method for inhibiting an insect or a micro-organism including exposing the insect or micro-organism to a biocidal or biofumigant composition including isoprene or an analogue thereof.

10. A method according to claim 9, wherein the biofumigant or biocidal composition includes a further bioactive component selected from the group consisting of 2-methyl-1- butanol, 3-methyl-2-butanone, n-butyl alcohol, acetaldehyde, linalool, sabinene and eucalyptol.

11. A method according to claim 10, wherein the further bioactive component includes acetaldehyde, either alone, or in combination with, 2-methyl-1 -butanol.

12. A method according to any one of claims 9 to 11 , wherein the method includes inhibiting an insect which is a pest of stored grain.

13. A method according to claim 9, wherein the insect is of the species selected from one or more of Tribolium castaneum, Rhyzopertha dominica, Cryptolestes ferrugineus and Oryzaephilus suinamensis.

14. A method according to any one of claims 9 to 11 , wherein the method includes inhibiting a micro-organism, and wherein the microorganism is a fungus selected from one or more of the genus Fusarium, Botrytis, Alternaria or Rhizoctonia, or a bacterium of the genus Pseudomonas.

15. A method according to claim 14, wherein the microorganism is a fungus selected from one or more of Fusarium verticillioides, Botrytis cinerea, Alternaria alternata and

Rhizoctonia cerealis.

16. A method according to any one of claims 9 to 15, wherein the method includes inhibiting a micro-organism, and wherein the microorganism is a bacterium of the species Pseudomonas syringae.

17. Use of a composition including isoprene or an analogue thereof as a biofumigant for the disinfestation of stored grain.