Classifications

• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
  • A01N27/00—Biocides, pest repellants or attractants, or plant growth regulators containing hydrocarbons
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
  • A01N31/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic oxygen or sulfur compounds
  • A01N31/02—Acyclic compounds
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
  • A01N31/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic oxygen or sulfur compounds
  • A01N31/06—Oxygen or sulfur directly attached to a cycloaliphatic ring system
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
  • A01N31/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic oxygen or sulfur compounds
  • A01N31/08—Oxygen or sulfur directly attached to an aromatic ring system
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
  • A01N35/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having two bonds to hetero atoms with at the most one bond to halogen, e.g. aldehyde radical
  • A01N35/02—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having two bonds to hetero atoms with at the most one bond to halogen, e.g. aldehyde radical containing aliphatically bound aldehyde or keto groups, or thio analogues thereof; Derivatives thereof, e.g. acetals
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
  • A01N37/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids
  • A01N37/02—Saturated carboxylic acids or thio analogues thereof; Derivatives thereof
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
  • A01N43/00—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds
  • A01N43/02—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms
  • A01N43/04—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms with one hetero atom
  • A01N43/06—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms with one hetero atom five-membered rings
  • A01N43/12—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms with one hetero atom five-membered rings condensed with a carbocyclic ring
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
  • A01N43/00—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds
  • A01N43/90—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having two or more relevant hetero rings, condensed among themselves or with a common carbocyclic ring system

Definitions

• 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.
• Endophytes often form mutualistic relationships with their hosts, with the endophyte conferring increased fitness to the host, often through the production of defence compounds.
• 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.
• 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).
• 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.
• the isoprene may be synthesised or otherwise obtained, and compositions thereof where desirable may be manufactured by admixture.
• 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.
• 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.
• biocidal activity 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.
• 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.
• an 'analogue' is meant a compound similar to isoprene, differing in respect of one or more structural components.
• a 'derivative' is meant an organic compound obtained from, or regarded as derived from, another compound.
• 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.
• 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.
• 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.
• the biofumigant or biocidal composition of the present invention includes isoprene or an analogue thereof and a further bioactive component.
• 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,(+)- ⁇ 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.
• 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.
• 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.
• 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.
• the biocidal composition may include isoprene, or an analogue thereof and acetaldehyde, either alone, or in combination with 2-methyl-1-butanol.
• the composition includes a further bioactive component, as hereinbefore described.
• the isoprene or analogue thereof may be administered at a sub-lethal dose.
• 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).
• 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.
• 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.
• isoprene may be used for quarantine and pre-shipment (QPS), structural or soil fumigation.
• QPS quarantine and pre-shipment
• 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.
• 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.
• fumigants containing isoprene may be applied by direct injection into a fumigation area. Application may be with or without a carrier gas such as C0 2 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.
• 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.
• fumigants such as phosphine
• insect mortality may not be evident until up to approximately 20 days of fumigation.
• 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.
• the isoprene or analogue thereof may be present in a composition for use as a fumigant or a biocidal composition as hereinbefore described.
• the insect is a pest of stored grain, including but not limited to Tribolium castaneum, Rhyzopertha dominica, Cryptolestes ferrugineus and Oryzaephilus suinamensis.
• 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.
• 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.
• this may include, for example, prevention or reduction of insect proliferation, growth or breeding.
• 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.).
• inhibition may include prevention or reduction of proliferation or growth.
• 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.
• Figure 1 A. Conidiophore ex-culture (Nodulisporium sp., DR1); B. Conidia ex-culture (Nodulisporium sp., DR1); C - Growth on PDA (Nodulisporium sp., DR1).
• FIG. 1 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 renovated 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.
• 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.
• 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.
• 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).
• 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 ⁇ L_ 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 MgCI 2 , 0.5 ⁇ of each primer, and 15 - 25 ⁇ g 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 .
• 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.
• 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
• 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).
• 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.
• Nodulisporium sp. 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 was performed to capture volatiles produced by the endophytes.
• a StableFlex fibre (Supelco) coated with either (i) 75 ⁇ CAR/PDMS or (ii) 30-50 ⁇ 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.
• 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 ⁇ m id., 1.4 ⁇ film thickness or Agilent DB-5MS, 30 m x 250 ⁇ id., 0.25 ⁇ 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.
• 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.
• 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 ⁇ L , except isoprene - 10 ⁇ L) on to the other half of the Petri plate on filter paper. Plates were immediately sealed with Parafilm®, covered in aluminium foil (i.e.
• Table 3 Classification of the level of insecticidal activity of volatile compounds produced by Nodulisporium sp. (DR1 ) and their structural analogues, against T. castaneum.
• 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 ⁇ L (Table 5). The most rapid biocidal activity was observed after a 1 day exposure, with volumes ranging from 100 - 250 ⁇ L (59.2 - 100.0 % mortality). The lowest volume that exhibited 100 % mortality in the shortest period of time (1 day exposure) was 150 ⁇ L, while 100 ⁇ L 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 ⁇ L / 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 ⁇ L @) (Table 6).
• isoprene had no insecticidal effect when applied alone with the same volumes (20 and 40 ⁇ L).
• 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).
• 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 ⁇ L_) on to another third of the Petri plate on filter paper.
• 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 ⁇ L_) on to another third of the Petri plate on filter paper.
• 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.
• Table 8 Bactericidal activity of isoprene, alone and in synergy with other biocidal compounds from the volatolome of Nodulisporium sp. (DR1), against P. syringae.

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