BR112019016106A2 - biofumigant compositions and methods for using them - Google Patents

Abstract

the present invention relates to biofumigant and biocidal compositions, in particular to biofumigant and biocidal compositions that include isoprene or an analog thereof. the present invention also relates to uses and methods of use of said compositions, including in biofumigation or bioprotection, more specifically for the control of pests in products such as stored grains.

Description

BIOFUMIGANT COMPOSITIONS AND METHODS FOR USING THEM

FIELD OF THE INVENTION [001] The present invention relates to biofumigant and biocidal compositions, in particular to biofumigant and biocidal compositions that include isoprene and analogues thereof. The present invention also relates to uses and methods of use of said compositions, more specifically for the control of pests in products such as stored grains.

BACKGROUND OF THE INVENTION [002] Microbes represent an invaluable source of genes and compounds with potential for use in a variety of industrial sectors. The scientific literature brings several reports of microbes as the main source for 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 untapped group of microbes known as endophytes, which reside in the tissues of living plants, offers a particularly diverse source for new compounds and genes capable of providing important benefits to society and, in particular, to agriculture.

[003] Endophytes often form mutualistic relationships with their hosts, with the endophyte providing greater physical fitness to the host, usually through the production of defense compounds. At the same time, the host vegetable offers the benefits of a protected environment and nutrition to the endophyte. The bioprotective endophytes developed and marketed include Neotyphodium species that produce insecticidal alkaloids, including peramine (a pyrrolopyrazine) and lolins (pyrrolizidines). These compounds can accumulate at high levels in vegetables where they act as potent food deterrents against a variety of insect pests. The compounds

Petition 870190074672, of 8/2/2019, p. 10/68

2/41 insecticides, destruxins, are also well characterized as secondary fungi metabolites. Other fungal antimicrobial compounds are peptaiboles, produced by Trichoderma virens, Quercus suber, Trichoderma citrinoviridae, which exhibit antifungal activity against a variety of plant pathogens, including Mediterranean Biscogniauxia and Quercine Apiognomy.

[004] Recent discoveries highlight the diversity of endophyte applications, such as in the energy sector (for example, biofuels) and in the agricultural sector, where species of fungus have been identified that produce biocidal metabolites that exhibit application as fumigants. For example, the fungus Muscodor albus from Cinnamomum zeylanicum in Honduras produces a set of volatile antimicrobial compounds that are effective against pathogens found in the soil, which allowed the development of a commercial preparation that was evaluated as a biological alternative (for example, mycofumigant) against soil fumigation. In addition, the discovery of the endophytic fungus Ascocoryne sarcoides, which produces a variety of hydrocarbons commonly found in diesel, gasoline and biodiesel, offers humanity a potential alternative to fossil fuels.

[005] Disinfestation of stored grains has been using phosphine as the main fumigant for many years thanks to its broad spectrum insecticidal activity, high volatility, negligible environmental impact (leaves no residue) and cost-effective (Warrick, 2011; Collins, 2015) . However, there are more and more incidents of phosphine resistance among grain pests stored in Australia, Asia and Brazil, with four of the top five pest insects in Australia exhibiting some level of resistance (cereal beetle - Rhyzopertha dominica ; Suriname beetle - Oryzaephilus surinamensis; rice beetle and other grains Tribolium castaneum; beetle - Cryptolestes ferrugineus) (Collins et al., 2002; Jagadeesan et al., 2012; Nayak et al., 2012). Resistance developed, in

Petition 870190074672, of 8/2/2019, p. 11/68

3/41 part, due to the lack of alternatives available to phosphine, combined with its repeated use in the same batch of grains (Collins, 2015). The greater resistance to phosphine imposes an increasing risk of quarantined insulation, which threatens the sustainability of the grain industry (Collins, 2015). In addition, disinfestation practices compromised due to phosphine resistance have the potential to impact access to the Australian grain market (Collins, 2015).

[006] There is a growing need to identify alternative soil and quarantine fumigants, since a series of chemicals imposes environmental problems or resistance problems. For example, the widely used fumigant methyl bromide has been identified as a significant ozone depletion, thus contributing to the degradation of the antarctic hole in the ozone layer, which has led to increased exposure to UV rays and a higher incidence of skin cancer in the South hemisphere. In addition, the phosphine multipurpose fumigant has shown a higher incidence of resistance in insect populations after fumigation, which brings complications for biosafety and problems of access to the market at a global level (for example, stored grains).

[007] One of the objectives of the present invention is to overcome, or at least alleviate, one or more difficulties or deficiencies associated with the prior art.

SUMMARY OF THE INVENTION [008] In one aspect, the invention proposes a biofumigant or biocidal composition that includes isoprene or an analog thereof.

[009] The isoprene component in the composition of the present invention can be produced from an isoprene-producing fungus; for example, growing a fungus and recovering the isoprene produced by it from the fungus cells, the culture medium or the air space associated with the culture medium or fungus.

Petition 870190074672, of 8/2/2019, p. 12/68

4/41 [010] Alternatively, isoprene can be synthesized or obtained in some other way, and, when desirable, it is possible to manufacture compositions of it by mixing. For example, one or more organic compounds that are substantially identical to the isoprene produced by a fungus, or that are analogous to it, can be obtained and can be mixed with other components to form a composition. The one or more organic compounds can be synthesized by suitable chemical reactions.

[011] In the context of the present invention, a biofumigant or biocidal composition is a composition capable of reducing, suppressing or protecting a product (such as stored grains) against the activity of pests (such as insects) or microorganisms, including fungi and bacteria.

[012] In the context of the invention, the terms bioactivity or bioactive refer to a component that exerts biocidal activity. Biocidal activity includes insecticidal and microbial activity. References to microbial activity include fungicidal and bactericidal activity.

[013] In the context of the invention, the term bioprotection refers to the use of a composition to reduce, suppress or protect a product (such as stored grains) against the activity of pests (such as insects) or microorganisms, including fungi and bacteria .

[014] The biocidal or biofumigant composition according to that 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 preparing them would be familiar to those skilled in the art.

[015] 'Analog' is understood to mean a compound similar to isoprene, but which differs from that with respect to one or more structural components. The term covers both 'substantially identical' compounds and derivatives, in addition to

Petition 870190074672, of 8/2/2019, p. 13/68

5/41 other similar compounds. 'Derivative' means an organic compound obtained from another compound or considered to be derived from another compound. Examples of derivatives include compounds whose degree of saturation of one or more bonds has changed (for example, a single bond has been changed to a double or triple bond) or in which one or more atoms are replaced by a different atom or functional group. Examples of different atoms and functional groups may include, but are not limited to, hydrogen, halogen, oxygen, nitrogen, sulfur, hydroxy, alkoxy, alkyl, alkenyl, alkynyl, amine, amide, ketone and aldehyde.

[016] By 'substantially identical', 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 .

[017] In a preferred embodiment, the biofumigant or biocidal composition of the present invention includes isoprene or an analog thereof and an additional bioactive component.

[018] In a more preferred embodiment, the additional bioactive component can be selected from alcohols, ketones, aldehydes and monoterpenoids. The alcohol can 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, Alcohol n-butyl or 2-Methyl-1-butanol. The monoterpenoid can be selected from the group consisting of: (+) - tra /? Sp-Ment-2-eno, α-Terpineno, Sabineno, (+) - a-Pineno, (R) -Carvona, R + Limonene, 3-Careno, α-Felandreno, (-) - Linalool, Terpinolene, 1,4-Cineol, Terpinen-4-ol, Isoborneol (s), n-Butyl, alcohol, Camphene (s), Mentofuran, (-) - Menthol (s), Eucalyptol. The ester can be selected from the group consisting of: Ethyl 2-methylbutyrate, methyl isobutyrate, ethyl isobutyrate or isobutyl acetate. The aldehyde can be selected from the group consisting of: trans-2Hexenal or Acetaldehyde. The ketone can be 3-Penten-2-one.

Petition 870190074672, of 8/2/2019, p. 14/68

6/41 [019] 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.

[020] To everyone's surprise, it was discovered that the composition of isoprene or an analogue of it and one or more of the above bioactive components can generate a synergistic biocidal effect. The additional bioactive component can be one or a combination of any two or more of the above. For example, preferred combinations include: isoprene or an analog thereof and 2-methyl-1-butanol; isoprene or an analog thereof and 3-methyl-2-butanone; isoprene or an analog thereof and n-butyl alcohol; isoprene or an analog thereof and acetaldehyde; isoprene or an analog thereof and linalool; isoprene or an analogue thereof and sabinene; isoprene or an analogue of it and eucalyptol. In a particularly preferred embodiment, the biocidal composition can include isoprene or an analog thereof and acetaldehyde, either alone or in combination with 2-methyl-1-butanol. In a preferred embodiment, the composition includes an additional bioactive component, as described above. In this case, the isoprene or its analog can be administered in a sublethal dose.

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

[022] In another aspect, the present invention proposes the use of a composition that includes isoprene or an analog thereof in the control of pests, such as in the biofumigation or bioprotection of a product.

[023] For example, isoprene can be used as a fumigant. Isopropene can be used to fumigate various products or commodities, including, but not limited to, stored grains, soil, hardwood, buildings, fresh products and import / export goods. In particular, isoprene can be used

Petition 870190074672, of 8/2/2019, p. 15/68

7/41 in quarantine and pre-shipment (QPS) fumigation, structural fumigation or soil fumigation.

[024] Compositions, for example, fumigants containing isoprene or an analog thereof, can be applied by any suitable method. Suitable methods for applying compositions as fumigants would be familiar to those skilled in the art. For example, compositions containing isoprene can be applied directly to the fumigation area and / or to the product to be treated, that is, fumigated. This may include spray application, gasification, atomization, wetting, injection, sublimation and dusting. For example, fumigants containing isoprene can be applied by direct injection into a fumigation area. The application can be with or without a carrier gas, such as CO2 and air, and with or without heating. The application can also take place by activation of moisture in a pelleted form, with or without a binding agent, such as aluminum, zinc and calcium metal binding agents.

[025] Isopropene or an analog thereof can be effective against pests and diseases, including, but not limited to, insects such as beetles and beetles, including beetles and beetles selected from the group consisting of cereal beetle (Rhyzopertha dominica), woodworm from Suriname (Oryzaephilus suinamensis), rice beetle and other grains (Tribolium castaneum) and beetle (Crryptolestes ferrugineus). Isopropene or an analogue thereof may have benefits selected from the group consisting of being safe, less harmful to the environment, less susceptible to resistance and faster acting than other fumigants commonly used such as methyl bromide and phosphine.

[026] For example, insect mortality may be evident after about 1 hour to about 10 days of fumigation, more preferably after about 3 to about 7 days of fumigation. With the fumigants currently used, such as

Petition 870190074672, of 8/2/2019, p. 16/68

8/41 phosphine, insect mortality may not be evident until about 20 days of fumigation.

[027] In another aspect, the present invention proposes a method for inhibiting an insect or microorganism that includes exposing the insect or microorganism to isoprene or an analog thereof. In a preferred embodiment, the isoprene or analog thereof may be present in a composition for use as a fumigant or biocidal composition as described further above in this document.

[028] In a preferred embodiment, the insect is a pest of stored grains, including, among others, Tribolium castaneum, Rhyzopertha dominica, Cryptolestes ferrugineus and Oryzaephilus suinamensis.

[029] In another preferred embodiment, the microorganism is a fungus selected from one or more of the genus Fusarium, Botrytis, Alternaria or Rhizoctonia, such as the species Fusarium verticillioides, Botrytis cinerea, Alternaria alternata and Rhizoctonia cerealis, and a bacterium from the genus Pseudomonas, as well as the species Pseudomonas syringae.

[030] As used in this document, the terms ‘an insect’ and ‘a microorganism’ are used to include a population of them.

[031] The inhibition of an insect or microorganism can occur through the reduction of a normal activity. In the case of an insect, this may include, for example, preventing or reducing the proliferation, growth or reproduction of the insect. In preferred embodiments, the biocidal composition causes insect mortality, for example, by fumigation, as described above. The amount to which the insect is exposed can be from about 20 to about 200, preferably from about 50 to about 200, more preferably from about 100 to about 200 microliters of isoprene or an analog thereof. liter of the environment (for example, a container, vase, silo etc.).

Petition 870190074672, of 8/2/2019, p. 17/68

9/41 [032] In the case of a microorganism, inhibition may include preventing or reducing proliferation or growth. For example, a reduction in growth may become evident after about 1 hour to about 10 days of exposure, for example, fumigation, more preferably after about 1 day to about 4 days of exposure.

[033] Hereinafter, the present invention will be more fully described with reference to the accompanying examples and drawings. It should be borne in mind, however, that the following description is merely illustrative and should not be construed in any way as a restriction on the generality of the invention described above.

BRIEF DESCRIPTION OF THE DRAWINGS / FIGURES

In the Figures:

[034] Figure 1: A. Ex-culture of conidiophores (Nodulisporium sp., DR1); B. Conidia exculture (Nodulisporium sp., DR1); C. Growth in PDA (Nodulisporium sp., DR1).

[035] Figure 2: An MP phenogram (1 of 8,631) based on gene sequences of 5.8 S / ITS rRNA from 55 isolates from Nodulisporium and Hypoxylon species. The highlighted area (gray) represents the Victorian Nodulisporium DR1 isolate. The phenogram was obtained using the Close-Neighbor-Interchange algorithm of MEGA4.1 (deletion of gaps and missing data). The numbers in the nodes represent the frequency (in percent) with which a cluster appears in 1,000 bootstrap tests. The scale bar is 5 changes per 100 bases.

[036] Figure 3: Image of the bioassay in split plates that assesses the insecticidal activity of volatile compounds produced by Nodulisporium sp. (DR1) and its structural analogues against T. castaneum.

DETAILED DESCRIPTION OF THE MODALITIES

Petition 870190074672, of 8/2/2019, p. 18/68

10/41

Example 1 - Fungal Isolate [037] Parts of the leaf and stem of Lomatia fraserii were collected during research in the Dandenong Cordillera. Sections of the leaf and stem were sterilized (70% ethanol for 30 seconds, flame sterilized) before extirpation of the internal tissues, which were then plated on potato dextrose agar (PDA) (39 g / L) (Amyl Media , Dandenong, Australia) spliced with acromycin (50 ppm). The endophytic fungi that developed from the plant tissue were removed by extirpating a hyphal tip from each colony and plated on PDA. Each hyphal tip constituted an endophytic fungal isolate. Then, the isolates were subjected to preliminary screening for bioactivity by challenging them against Rhizoctonia solani in PDA. One of the isolates inhibited the development of R. solani and was selected for further analysis. A pure culture of the isolate (ie, hyphal plugs) was placed in SDW and stored at room temperature and at 4 ^o C and in 15% glycerol at -70 ° C.

[038] A representative sample of the Nodulisporium sp. was deposited with the National Measurement Institute on May 3, 2011 under access number V11 / 011039, as disclosed in WO2012 / 159161 (PCT / AU2012 / 000574) entitled “Fungi and products thereof 'on behalf of Agriculture Victoria Services Pty Ltd. (which is incorporated herein by reference).

Example 2 - Morphology [039] Isolates were removed from storage, placed in PDA and allowed to grow at 25 ° C (in the dark) until the formation of conidiophores. Himalayan sections containing conidiophores were mounted in lactic acid and examined under light microscopy (in vitro description).

Hypoxylon Nodulisporium State

In vitro description

Petition 870190074672, of 8/2/2019, p. 19/68

11/41 [040] PDA colonies initially white, turning from pale yellow to grayish yellow. Conidiophores branching freely, pale brown, paler towards the apex, verruculose, 2.5 to 3 pm wide. Conidiogenic cells typically produced individually, pale brown, verruculose, 12 to 20 x 2.5 to 3 pm. Conidia transported from minimally visible, pale brown, more or less smooth, ellipsoid denticles, 6 to 8 x 3 at 4 pm (Figure 1).

[041] When evaluating the microscopic attributes of the isolates growing in culture (in vitro stage), it was confirmed that they were characteristic of a species of Nodulisporium not described.

Example 3 - Genotipaqem [042] Genomic DNA was extracted from cultures of Nodulisporium sp. grown in potato dextrose broth (PDB) using a DNeasy Vegetable Mini Kit (Qiagen). A section of the ribosomal RNA locus (5.8S / ITS) was amplified with ITS4 and ITS5 primers (White etal., 1990). PCR amplification was performed in 25 pL 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 of MgCb, 0, 5 pM of each primer and 15 to 25 pg of DNA. The reaction was carried out in a thermocycler (Gradient Palm-Cycler, Corbett Research) with cycling conditions composed by 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 viewed in UV light. The amplification product was purified using a PCR Purification Kit (Qiagen) and sequenced using the BigDye Terminator Cycle v 3.1 (Applied Biosystems) sequencing kit on

Petition 870190074672, of 8/2/2019, p. 20/68

12/41

ABI 3730x1 Capillary Sequencer (Applied Biosystems), according to the manufacturer's instructions.

[043] The DR1 rDNA-ITS sequence of Nodulisporium sp. it was compared to reference sequences of accessions of Nodulisporium (or related teleomorphs, that is, Hypoxylon and Daldinia) from around the world (closest matches to GenBank). A total of 55 Nodulisporium-related strings were aligned using MUSCLE. The aligned sequences were adjusted with ClustalW / Alignment Explorer in MEGA 4.1. Based on these sequences, phylogenetic relationships were deduced using analysis of distance and maximum parsimony (MP). In the distance analysis, phenograms were obtained using the neighbor-joining (NJ) algorithm, applying the Kimura-2-parameter model, as implemented in MEGA4.1. In the MP analysis, phenograms were obtained using the Close-Neighbor-Interchange algorithm (search level 3), as implemented in MEGA4.1. To find the ideal global phenogram, 10 random sequences were added. The measures calculated for the MP included tree extension, consistency index, retention index and rescheduled consistency index (TL, Cl, RI, RCI). In both analyzes, the alignment gaps and missing data were eliminated from the database (full deletion option) and the branch reliability was assessed by computing 1,000 bootstrap replicates.

[044] Of the 55 Nodulisporium-related isolates, the size of the rRNA gene sequence (5.8S / ITS) varied between 436 and 664 base pairs, of which 371 were included in the final database for analysis. In the NJ analysis, the ideal phenogram showed a sum of the branch lengths of 0.525. The MP analysis produced 8,631 more parsimonious phenograms (TL = 211, Cl = 0.654 RI = 0.916, RCI = 0.569, for the parsimony informational sites). NJ and MP analyzes produced phenograms with similar topology and bootstrap values. Therefore, only the PM phenogram is presented (1 of 8,631, Figure 2).

Petition 870190074672, of 8/2/2019, p. 21/68

13/41 [045] The isolates tended to cluster according to the teleomorph of the Nodulisporium, Hypoxylon and Daldinia species. The DR1 of Nodulisporium sp. agglomerated with the Hypoxylon species, with an 80% bootstrap support. This group formed a cluster with other Nodulisporium and Hypoxylon isolates, with a 14% bootstrap support (Cycle 1). This cluster was next to another group of Hypoxylon isolates with a 41% bootstrap support (Cycle 2). A large group of Daldinia isolates formed the next related cluster with a 37% bootstrap support (Cycle 3).

Example 4 - Insecticidal bioactivity of Nodulisporium so. (DR1) and other endophytic fungi [046] In vitro bioassays have been developed to test the bioactivity of Nodulisporium sp. (DR1) and other endophytic fungi (Endophytes A to U) against a variety of stored grain pest insects, Tribolium castaneum (rice beetle and other grains), Rhyzopertha dominica (cereal beetle) and Cryptolestes ferrugineus ( scarab). The bioassays were performed in 90 mm split Petri dishes. The split plates included an impermeable barrier crossing the center of each one, which divides them entirely into two halves, with only volatile compounds capable of crossing the partition (that is, without direct contact between the endophyte and its liquid exudates with the test insect). The isolates were inoculated into Petri dishes with PDA by placing a 6 mm agar plug with mycelium actively growing 13 mm from the edge of the plate (that is, in one half of the plate). The isolates were allowed to grow at 25 ° C (in the dark) for 6 days. Subsequently, the insect pests were inoculated in the other half of the plate by placing 3 insects in their respective input (T. castaneum - wheat flour and yeast, R. dominica whole wheat seed, C. ferrungineus - oat flakes). The plates were sealed with LDPE plastic film (approximately 0.01 mm thick) and

Petition 870190074672, of 8/2/2019, p. 22/68

14/41 covered in aluminum foil (ie in the dark). After 3, 7 and 10 days, insect mortality was determined by assessing insect movement as an indicator of mortality. Mortality was calculated by comparing the number of dead insects to the total number on the plate and expressed by the percentage of mortality. The Activity Spectrum was calculated for each isolate by adding the number of insect species that were fully controlled (that is, 100% mortality). The Biocidal Activity Ranking was calculated for each isolate by averaging the percentage of mortality for all insects and all points in time. The data were analyzed using ANOVA as performed on GenStat, version 14. The experiment was completely randomized with 4 replicates for each endophyte.

[047] The DR1 of Nodulisporium sp. exhibited broad-spectrum biocidal activity against the three pest insects, completely controlling C. ferrugineus and R. dominica (100% mortality) and severely affecting T. castaneum survival (89% mortality) (Table 1) . The biocidal activity of Nodulisporium sp. (DR1) was rapid, with biocidal activity against insect pests observed within 3 days, and total control within 7 to 10 days. Nodulisporium sp. (DR1) exhibited the highest Biocidal Activity Ranking (1) of all endophytes tested, since it had the highest average mortality.

Petition 870190074672, of 8/2/2019, p. 23/68

Table 1 - Biocidal activity of fungal endophytes (including Nodulisporium sp. DR1) against stored grain pest insects after 3, 7 and 10 days of exposure. Biocidal activity represented by% mortality and given by a heat map (dark gray (++++): 100% mortality, medium dark gray (+++): 67% to 99% mortality, medium gray (+ +): 33% to 66% mortality, light gray (+): 1 to 33% mortality, white (-): 0% mortality).

Cryptolestes ferrugineus Tribolium castaneum Rhyzopertha dominica

Day 3 Day 7 10th day Day 3 Day 7 10th day Day 3 Day 7 10th day Activity Spectrum Average of Mortality Biocidal Activity Ranking DR1 33% 0% (-) 44% (4.4.) 2 72% 1 Endophyte A 0% (-) [llilll ....... 0% (-) 22% (+) IlilBIll llifiiill 11% (+) 44% (-H-) 2 44% 17 Endophyte B 33% {4-4-) ΙίΙίΙΙι 0% (-) 8% ω 3 70% 2 Endophyte C 0% (-) 33% - lilÍlll IIIIBIII 56% (± ± 1 IIIIIIIIIIIllllli 11% (±) 72¾ 1 49% 14

15/41

Petition 870190074672, of 8/2/2019, p. 24/68

Cryptolestes ferrugineus Tribolium castaneum

Rhyzopertha dominica

Day 3 Day 7 10th day Day 3 Day 7 10th day Day 3 Day 7 10th day Activity Spectrum Average of Mortality Biocidal Activity Ranking Endophyte D 0% (-) 11% (+) 44% 11% (+) (+) (w) 2 50% 12 Endophyte E 0% (-) 0% 0% (-) 56% (++) llliilli! 'ίβ / Λ 56% (-H-) 1 44% 17 Endophyte F 11% (+) 0% (-) IIIIHIII (+) 3 70% 2 Endophyte G 0% (-) 73% 0% (-) 0% (-) 58% 3 56% 7 Endophyte H 0% (-) 11% (+) 33% 0% (-) 0% (-) 2 49% 13 Endophyte I 11% ......... (+) ......... 11% (±) 11% .......... (+) ......... 11% ........ (+) ......... iiiiiil 11 * + ») 2 48% 15

16/41

Petition 870190074672, of 8/2/2019, p. 25/68

Cryptolestes ferrugineus Tribolium castaneum

Rhyzopertha dominica

Day 3 Day 7 10th day Day 3 Day 7 10th day Day 3 Day 7 10th day Activity Spectrum Average of Mortality Biocidal Activity Ranking Endophyte J 0% (-) 0% (-) 11% (+) 3 68% 4 Endophyte K 0% (-) 0% (-) 22% ......... (+) ......... 11% (+) 28% (+) IlilÈII ...... tttl ...... 2 42% 19 Endophyte L 0% (-) 0% (-) 11% 11% (+) 33% 2 47% 16 Endophyte M 0% (-) 22% (+) 0% (-) Iliiilll Ml 2 56% 8 Endophyte N 0% (-) 0% (-) 67% 0% (-) 8% (+) 2 52% 11 Endophyte O Q% (-) 22% (±) 56% 0% (-) 11% ........ ω ........ 2 54% 9

17/41

Petition 870190074672, of 8/2/2019, p. 26/68

Cryptolestes ferrugineus Tribolium castaneum Rhyzopertha dominica

Day 3 Day 7 10th day Day 3 Day 7 10th day Day 3 Day 7 10th day Activity Spectrum Average of Mortality Biocidal Activity Ranking Endophyte P 11% 33-3% (++) 11% (+) 7% (+) (+++) 3 60% 5 Endophyte Q iiiieii 'ίβΚΛ 0% (-) IIIIBII ΙΙΙβ 0% (-) lllilli 0% (-) 1 41% 20 Endophyte R 0% (-) 11% (+) 33% (4.4.} 0% (-) IIIIBII (+) 22% IIIM ^ (44.4) 1 38% 21 Endophyte S 0% (-) 0% (-) 44% ....... (+. +) ...... 11% ......... (+) ...... „ 44% 2 54% 9 Endophyte T 0% (-) 11% (+) $ 7% IMIllli 11% (+) (+++ ·) 1 59% 6 U endophyte ιιιιβιι ΙΙΙΙΙΜ 11% (+) IIIIBII 0% (-) 33% 1··· 33% 1··· 0% (-) 0 21% 22 33% (+ - +) 56% ΙΙΙ11ΪΙΙ F Pr. 0.002 <0.001 0.004 0.393 0.005 <0.001 0.097 <0.001 <0.001

18/41

Petition 870190074672, of 8/2/2019, p. 27/68

Cryptolestes ferrugineus Tribolium castaneum

Rhyzopertha dominica

Day 3 Day 7 10th day Day 3 Day 7 10th day Day 3 Day 7 10th day Activity Spectrum Average of Mortality Biocidal Activity Ranking LSD (5%) 25% 43% 28% 37% 35% 15% 23% 32% 32%

19/41

Petition 870190074672, of 8/2/2019, p. 28/68

20/41

Example 5 - Volatoloma of Nodulisporium sp. (DR1) [048] The gases in the head space above Nodulisporium sp. (DR1). The isolate was grown under macroaerophilic conditions, which consisted of growing the fungus on ramps of PDA and Yeast Malt Extract (YME) in ampoules with 20 ml head space, at an agar: air ratio of 1: 2.5. The ampoules were sealed with a screw cap with PTFE septum and left to grow for 10 days at room temperature.

[049] A solid phase microextraction (SPME) was performed in the head space to capture the volatile substances produced by the endophytes. A StableFlex fiber (Supelco) coated with (i) 75 pm of CAR / PDMS or (ii) 30 to 50 pm of DVB / CAR / PDMS was used to absorb volatile substances from the headspace of the ampoules. The automated sampling was performed by a Gerstel multipurpose sampler using the private software Maestro. The fiber was conditioned at 250 ° C for 60 min before the start of activities and for 30 minutes between each sample. For each sample, the fiber was inserted into the ampoule and incubated at room temperature for 7 minutes to absorb volatile substances, after which the fiber was inserted into an injection port without dividing an Agilent 7890 GC System where the contents were thermally desorbed (250 ° C for 6 min) in a capillary column (Agilent DB-624, 30 mx 250 pm id., 1.4 pm film thickness or Agilent DB-5MS, 30 mx 250 pm id., 0, 25 pm film thickness). The column oven was programmed according to the following: 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 quadripolar mass selective detector (mass spectrometer, MS) operating in electron impact ionization mode at 70 eV. The transfer line temperature was maintained at 280 ° C during the chromatographic run. The source temperature was 280 ° C. The acquisitions were

Petition 870190074672, of 8/2/2019, p. 29/68

21/41 performed over a mass range from mz 29 to 330, with a sweep time of 200 ms.

[050] The initial identification of volatile substances produced by Nodulisporium sp. (DR1) was performed by comparing libraries using standard chemical databases. Secondary confirmatory identification was performed by comparing mass spectral data of authentic patterns to data on fungal volatile substances. All chemical names in this report follow the nomenclature of standard chemical databases. In all cases, uninoculated control ampoules were also analyzed, and the compounds found in them were subtracted from those that appeared in the ampoules supporting the fungal development. The experimental identification of fungal volatile substances was based on observed mass spectral data compared to data in these chemical databases and to authentic standards (where possible).

[051] The GC-MS analysis (0 to 65 minutes) identified 54 volatile metabolites produced by Nodulisporium sp. (DR1) when grown for 10 days in PDA and YME at room temperature (Table 2). These compounds represented a variety of structural classes including monoterpenoids (predominant), alcohols, esters, aldehydes and sesquiterpenoids. The volatoloma of Nodulisporium sp. (DR1) was more complex when grown in PDA than in YME, with 54 compounds identified in PDA, compared to 49 in YME. Similarly, seven more compounds were produced at a higher concentration in PDA than in YME.

Petition 870190074672, of 8/2/2019, p. 30/68

Table 2 - GC-MS head space analysis of volatile compounds produced by Nodulisporium sp. (DR1) when

grown in PDA and YME medium for 10 days at am biente Name OK (min) Correspondence between libraries BP (m / z) Significant Secondary Ions mz (BP% ratio) PDA YME * Acetaldehyde 3.45 3 44.1 43 (44.3), 42 (13.89) + + *Ethanol 5.35 3 45.1 46.1 (24.49), 43.1 (14.42) +++ +++ * lsoprene 5.42 3 67.0 53.1 (33.81), 65.1 (12.97) + + mz67,68,53 @ 6.14min 6.07 67.0 68 (85.3), 53 (40.1) + + Acetone 6.12 2 43.1 58.1 (66.27) + + * Methyl acetate 6.96 3 43.1 74.1 (51.13) + + mz59,42,60,41 @ 9.60min 9.51 59.1 42.1 (43.59), 60.1 (30.15), 41.2 (19.08) + + * Ethyl acetate 11.03 3 43.1 61.1 (39.57), 70.1 (33.77) + + Trichloromethane 11.7 2 83.0 85 (66.4), 86.9 (10.84), 47.2 (10.71) + + mz43,41,42 @ 13,48min 13.4 43.2 41.2 (70.95), 42.1 (64.33) ++ ++ 1,3-Cyclopentadiene, 5.5- dimethyl-1 16.9 1 79.0 77.1 (48.88), 94 (43.42) + 5 Butanoic acid, methyl ester 17.6 2 74.1 71.1 (66.04), 43.2 (39.95), 86.9 (35.79) + +

22/41

Petition 870190074672, of 8/2/2019, p. 31/68

Name OK Correspondence BP Significant Secondary Ions PDA YME mz79,77,94 @ 18,53 18.4 79.0 77.1 (58.47), 94 (57.44), 91.1 (35.01) + + 1,3,5-Hexathriene, 3-methyl-, (Z) - 18.9 2 79.1 77 (58.65), 94 (56.92), 91 (34.56) + 5 *Toluene 19.3 3 91.0 92 (51.12), 65 (10.14) + 5 * 3-Methyl-1-butanol 20.2 3 55.0 70.1 (80.81), 42.1 (57.06), 43 (47.19) ++ ++ * 2-Methyl-1-butanol 20.4 3 56.0 57.1 (89), 70.1 (70.36), 41.1 (45.55) ++ + 1,3,5-Hexathriene, 3-methyl-, (Z) -1 20.7 2 79.1 77 (73.45), 94 (66.91), 91 (30.89) + + Isobutyl acetate 21.1 1 43.1 56.1 (51.03), 73.1 (33.04) + + Similar to methyl ester of 2-methylbutanoic acid 21.1 1 88.0 57.1 (52), 41.1 (17.09) ++ + 3- Acid methyl ester methylbutanoic 21.1 2 74.1 85 (36.42), 59.1 (21.27), 43.1 (16.63) ++ ++ 1,3,5-Hexathriene, 3-methyl-, (Z) -2 21.4 2 79.0 77 (61.57), 94 (54.52) + + Benzene, 1-ethyl-3-methyl- 27.2 1 105.0 90.9 (41.34), 120 (37.46), 77.1 (29.74) + + a-Terpineno 28.1 2 121.0 93 (87.86), 136 (60.3), 91 (53.62) + 5

23/41

Petition 870190074672, of 8/2/2019, p. 32/68

Name OK Correspondence BP Significant Secondary Ions PDA YME Styrene 28.6 1 104.0 103 (42.47), 78 (34.55) + + Felandreno 29.5 2 93.0 91 (52.81), 92 (34.69), 77 (31.84) + + * α-Pineno 30 3 93.0 91 (41.98), 92 (36.23), 77 (22.44) + + Cyclohexane, 1,2,4tris (methylene) - 30.6 2 91.0 105 (64.52), 78 (58.05), 120 (45.81) + + mz79,67,93 @ 32,89min 32.8 79.0 67 (53.42), 68.3 (52.52), 93 (46.77) + + Similar to β-Pineno 33.4 2 93.0 69.1 (38.9), 91 (21.96), 41.1 (15.4) ++ + mz105,120 @ 33.86 33.8 105.0 120 (33.3) + + * a-Felandreno 34.4 3 93.0 91 (55.66), 77 (30.27), 136 (22.92) + + Benzene, cyclopropyl-2 34.9 2 117.0 118 (70), 115 (45) ++ + a-Terpineno 35.2 2 121.0 93 (87.86), 136 (60.3), 91 (53.62) + + mz111,71,125 @ 35,42min 35.4 111.0 71 (46.62), 124.9 (42.29), 55 (29.07) + + * Limonene 35.8 3 93.0 68.1 (107.1), 67.1 (81.57), 79 (42.63) ++ + * p-Cimeno 36.1 3 119.0 134 (28.79), 91 (22.18) +++ +++ * Eucalyptol 36.5 3 81.0 108 (81.85), 110.9 (66.95), 71 (66.91) ++ ++ Benzene, cyclopropyl-3 36.8 2 117.0 118 (74.92), 115 (43.99), 91 (33.83) ++ +

24/41

Petition 870190074672, of 8/2/2019, p. 33/68

Name OK Correspondence BP Significant Secondary Ions PDA YME Cyclohexane, 1,2,4- tris (methylene) -p1 37.5 2 105.0 91 (91.21), 120 (82.6), 79 (44.73) ++ + Cyclohexane, 1,2,4tris (methylene) -p2 37.8 2 105.0 120 (78.18), 91 (65.73), 77 (38.79) + + Cyclohexane, 1,2,4tris (methylene) -p3 37.9 2 105.0 120 (82.94), 91 (70.33), 77 (39.96) + + Cyclohexane, 1,2,4- tris (methylene) -p4 38.1 2 105.0 120 (82.2), 91 (70.61), 77 (39.83) + + Cyclohexane, 1.2.4tris (methylene) -p5 38.2 2 105.0 91 (78.85), 120 (75.07), 77.1 (47.04) + + mz115,116 @ 38,36min 38.3 115.0 116 (86.99) + + Cyclohexane, 1,2,4tris (methylene) -p6 38.5 2 115.0 91 (36.19), 120.1 (33.6), 77 (18.44) + + * Terpinolene 39.1 3 121.0 93 (101.08), 136 (97.92), 91 (53.55) + + 1,3-Cylohexadiene, 1- methyl-4- (1-methylethyl) - 39.2 2 121.0 93 (68.19), 136 (52.39) + +

25/41

Petition 870190074672, of 8/2/2019, p. 34/68

Name OK Correspondence BP Significant Secondary Ions PDA YME Benxene, (2-methyl-1- propenyl) - 40.1 2 132.0 117 (87.34), 115 (50.94), 91 (31.97) ++ ++ 2,4,6-Octatriene, 3,4- dimethyl- 42.4 1 121.1 136 (46.47), 105 (44.88), 91 (34.74) + + * Phenylethyl alcohol 44 3 91.0 92 (50.08), 121.9 (23.13), 64.9 (12.21) + + Azuleno 46.5 1 104.9 107 (94.41), 147 (75.67), 93 (73.38) + 5 * a-Terpineol 46.7 3 59 93 (90.1), 121 (55.1), 136 (43.8) + +

- The fragmentation pattern corresponds to compound spectra in the NIST library (> 75%)

- The fragmentation pattern corresponds to compound spectra in the NIST library (> 90%)

- The fragmentation pattern corresponds to authentic pattern spectra * - Fragmentation pattern compared to an authentic pattern + - significant peak; ++ - main peak; +++ - dominant peak

26/41

Petition 870190074672, of 8/2/2019, p. 35/68

27/41

Example 6 - Insecticidal activity of compounds from Nodulisporium sp. (DR1) [052] A total of 76 chemical standards were evaluated for their insecticidal activity against the stored grain pest Tribolium castaneum. These chemical patterns represented compounds in the volatoloma of Nodulisporium sp. (DR1) or were structural analogs of these compounds. The bioassays were performed in 90 mm split Petri dishes (as in example 4) (Figure 3). The insect pest was inoculated in one half of the Petri dish by placing 4 insects in the input (wheat flour and yeast). Then, a chemical standard was divided into aliquots (5 μΙ_, except isoprene - 10 μΙ_) on the other half of the petri dish on filter paper. The plates were immediately sealed with Parafilm®, covered in aluminum foil (that is, in the dark) and kept at room temperature. Insect mortality was monitored daily, assessing insect movement as an indicator of mortality. Mortality was calculated by comparing the number of dead insects to the total number on the plate and expressed by the percentage of mortality. The data were analyzed using ANOVA as performed on GenStat, version 14. The experiment was completely randomized with 5 replicates for each compound.

[053] A total of 4 compounds exhibited high insecticidal activity against T. castaneum, producing 100% mortality after 24 to 48 h of exposure (Table 3). These compounds included isoprene (10 μΙ_), mentofuran, (-) - menthol and 2-methyl-3buten-2-ol (Table 4). These four compounds were either monoterpenoids or alcohols. In addition, only one of these compounds has been confirmed within the Nodulisporium sp. (DR1), isoprene (10 μΙ_). A total of 12 compounds exhibited average insecticidal activity (36% to 99% mortality after 24 to 76 h of exposure), while 20 compounds exhibited low insecticidal activity (1% to 35%

Petition 870190074672, of 8/2/2019, p. 36/68

28/41 mortality after 24 to 96 h of exposure). A total of 40 compounds did not exhibit insecticidal activity.

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

Mortality Duration N ^Q of Compounds No activity 0% 40 Low activity 1% to 35% 24 to 96 h 20 Average activity 36% to 99% 24 to 72 h 12 High activity 100% 24 to 48 h 4

Petition 870190074672, of 8/2/2019, p. 37/68

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

Treatment % of mortality Confirmed / Analog Structural Class Day 1 Day 2 Day 3 Day 4 Control (Water) 0.0 0.0 0.0 0.0 2-Pentanol 0.0 0.0 0.0 0.0 Analog Alcohol (-) - β-Ρΐηβηο 0.0 0.0 0.0 0.0 Analog Monoterpenoid 2-Methylbutyl acetate 0.0 0.0 0.0 0.0 Analog Ester 2- isovalerate methylbutyl 0.0 0.0 0.0 0.0 Analog Ester Hexanal 0.0 0.0 0.0 0.0 Analog Aldehyde 3-octane 0.0 0.0 0.0 0.0 Analog Ketone 1-methyl-1-cyclohexene 0.0 0.0 0.0 0.0 Analog Ester 2-heptanone 0.0 0.0 0.0 0.0 Analog Ketone Methyl 2-methylbutyrate 0.0 0.0 0.0 0.0 Analog Ester Methyl acetate 0.0 0.0 0.0 0.0 Analog Ester Ethyl acetate 0.0 0.0 0.0 0.0 Analog Ester

29/41

Petition 870190074672, of 8/2/2019, p. 38/68

Treatment % of mortality Confirmed / Analog Structural Class Methanol 0.0 0.0 0.0 0.0 Analog Alcohol Ethanol 0.0 0.0 0.0 0.0 Confirmed Alcohol Amyl alcohol 0.0 0.0 0.0 0.0 Analog Alcohol Acetone 0.0 0.0 0.0 0.0 Analog Ketone (+) - Carvona 0.0 0.0 0.0 0.0 Analog Monoterpenoid (-ι -) - β-Ρίηβηο 0.0 0.0 0.0 0.0 Analog Monoterpenoid (-) - a-Pineno 0.0 0.0 0.0 0.0 Analog Monoterpenoid Eugenol 0.0 0.0 0.0 0.0 Analog Monoterpenoid S-Limonene 0.0 0.0 0.0 0.0 Analog Monoterpenoid (-) - lsoledeno 0.0 0.0 0.0 0.0 Analog Sesquiterpenoid (-) - írans-Cariofeleno 0.0 0.0 0.0 0.0 Analog Sesquiterpenoid α-Terpineol 0.0 0.0 0.0 0.0 Confirmed Monoterpenoid γ-Terpinene 0.0 0.0 0.0 0.0 Analog Monoterpenoid α-Humeleno 0.0 0.0 0.0 0.0 Analog Sesquiterpenoid m-Cimeno 0.0 0.0 0.0 0.0 Analog Monoterpenoid o-Cimeno 0.0 0.0 0.0 0.0 Analog Monoterpenoid p-Cimeno 0.0 0.0 0.0 0.0 Confirmed Monoterpenoid

30/41

Petition 870190074672, of 8/2/2019, p. 39/68

Treatment % of mortality Confirmed / Analog Structural Class with Nerolidol 0.0 0.0 0.0 0.0 Analog Sesguiterpenoid Ethyl Caproate 0.0 0.0 0.0 0.0 Analog Ester Isopropyl Butriate 0.0 0.0 0.0 0.0 Analog Ester Eitla Butyrate 0.0 0.0 0.0 0.0 Analog Ester Propylbenzene 0.0 0.0 0.0 0.0 Analog Hydrocarbon Ethyl isovalerate 0.0 0.0 0.0 0.0 Analog Ester Methyl Caproate 0.0 0.0 0.0 0.0 Analog Ester Methyl isovalerate 0.0 0.0 0.0 0.0 Analog Ester Ethylbenzene 0.0 0.0 0.0 0.0 Analog Hydrocarbon Mirceno 0.0 0.0 0.0 0.0 Analog Monoterpenoid Isoamyl isobutyrate 0.0 0.0 0.0 0.0 Analog Ester (+) - írans-p-m and nt-2-e in 0.0 0.0 0.0 2.9 Analog Monoterpenoid a-Terpineno 0.0 2.9 2.9 2.9 Confirmed Monoterpenoid Sabineno 2.9 2.9 2.9 2.9 Analog Monoterpenoid Caproic acid 0.0 0.0 2.9 2.9 Analog Carboxylic acid o-Cresol 0.0 0.0 0.0 4.0 Analog Hydrocarbon (+) - Pineno 0.0 4.0 4.0 4.0 Analog Monoterpenoid

31/41

Petition 870190074672, of 8/2/2019, p. 40/68

Treatment

Ethyl 2-methylbutyrate (R) -Carvone

R + Limonene

3-Penten-2-one

3-Careno a-Felandreno (-) - Linalool

Terpinolene

1,4-Cineol m-Cresol

Methyl isobutyrate

Ethyl isobutyrate

Irans-2-Hexenal isobutyric acid

Aoetaldehyde

3-methyl-1 butanol

Terpinen-4-ol

Petition 870190074672, of 8/2/2019, p. 41/68% mortality

4.0

0.0

0.0

6.9

0.0

0.0

4.0

0.0

0.0

0.0

14.7

22.0

15.2

4.0

36.0

45.0

3.3

4.0

0.0

5.0

6.9

0.0

0.0

4.0

0.0

0.0

0.0

14.7

22.0

15.2

16.0

36 (0

45.0

35.3

4.0

5.0

5.0

6.9

0.0

0.0

8.0

5.0

10.0

14.7

22.0

22.1

16.0

36.0 ioii

49.3

4.0

5.0

5.0

6.9

8.0

8.0

8.0

10.0

13.3

13.3

14.7

22.0

22.1

24.0 li · «li

52.7

Confirmed / Analog

Analog

Analog

Confirmed

Analog

Analog

Analog

Analog

Confirmed

Analog

Analog

Analog

Analog

Analog

Analog

Confirmed

Confirmed

Confirmed

Structural Class

Ester

Monoterpenoid

Monoterpenoid

Ketone

Monoterpenoid

Monoterpenoid

Monoterpenoid

Monoterpenoid

Monoterpenoid

Hydrocarbon

Ester

Ester

Carboxylic acid

Aldehyde

Aldehyde

Alcohol

Monoterpenoid

32/41

Treatment % of mortality Confirmed / Analog Structural Class Isobuty acetate 44.8 44.8 63.0 63.0 Confirmed Éstor Isoamyl alcohol 49.3 64.7 64.7 64.7 Analog 3-methyl-2-butanone 70.0 70.0 70.0 70.0 Confirmed Áloool Eucalyptus 44.0 78.0 78.0 78.0 Analog Ketone Isoborneol (s) 05.3 80.0 80.0 80.0 Confirmed Monoterpenoid 2-methyl-l-butanol 80.0 80.0 80.0 80.0 Analog Hydrocarbon Alcohol / n-butyl 50.7 80.7 30.7 80.7 Analog Monoterpenoid p-Gresol (s) 80.0 82.9 82.9 82.9 Analog Áloool Canfenofs) 80.0 86.7 90.0 93.3 Analog Monoterpenoid llllb 0 · h / 10Γ * 1 ElOlCl 100.0 ÍW> ........ Analog Alcohol ... («) ..................................... 100.0 100.0 100.0 .......................... 1W 100.0 F Pr. <0.001 <0.001 <0.001 <0.001 LSD (5%) 19.59 18.68 19.42 19.92

33/41

Petition 870190074672, of 8/2/2019, p. 42/68

34/41

Example 7 - Insecticidal dose response of a key compound (isoprene) of Nodulisporium sp. (DR1) [054] An insecticidal dose response bioassay has been developed to assess isoprene against the stored grain pest T. castaneum. The bioassay was carried out in Schott flasks of 1 L. The insect pest was inoculated in the bioassay by placing 9 to 14 insects in the input (wheat flour and yeast). Then, the isoprene was divided into aliquots in the bioassay in volumes ranging from 20 to 250 pL, ensuring that there was no direct contact with the insect. The bottles were immediately sealed with Parafilm® and kept at room temperature. Insect mortality was monitored after 1, 6, 8 and 11 days of exposure, evaluating insect movement as an indicator of mortality. Mortality was calculated by comparing the number of dead insects to the total number in the bioassay and expressed by the percentage of mortality. The data were analyzed using ANOVA as performed on GenStat, version 14. The experiment was completely randomized with 4 replicates.

[055] Isopropene exhibited biocidal activity against T. castaneum with volumes in the range of 20 to 250 μΙ_ (Table 5). The fastest biocidal activity was observed after a 1-day exposure, with volumes ranging from 100 to 250 μ _ (59.2% to 100.0% mortality). The lowest volume that exhibited 100% mortality in the shortest period of time (1-day exposure) was 150 μΙ_, while 100 μΙ_ exhibited 100% mortality after a 6-day exposure.

Table 5 - Dose response (20 to 250 μΙ_) of isoprene against T.

castaneum

Volume (μΙ) Day 1 6th Day 8 Day 11 20 0.0% 9.5% 23.6% 44.3% 50 0.0% 43.6% 68.6% 80.7% 100 59.2% 100.0% 100.0% 100.0%

Petition 870190074672, of 8/2/2019, p. 43/68

35/41

Volume (pL) Day 1 6th Day 8 Day 11 150 100.0% 100.0% 100.0% 100.0% 200 100.0% 100.0% 100.0% 100.0% 250 100.0% 100.0% 100.0% 100.0% F Pr. <0.001 <0.001 <0.001 <0.001 LSD (5%) 11.7% 25.8% 26.4% 15.5%

Example 8 - Insecticidal synergy of isoprene with other key compounds of Nodulisporium sp. (DR1) [056] An insecticidal synergy bioassay has been developed to assess the combinatory effect of isoprene with other compounds (2-methyl-1-butanol; 3-methyl-2-butanone; n-butyl alcohol; eucalyptol) of Nodulisporium sp volatoloma . (DR1) against the stored grain pest T. castaneum. Bioassays were performed in 500 mL Schott flasks. The insect pest was inoculated in the bioassay by placing 10 to 14 insects in the input (wheat flour and yeast). Then, the biocidal compounds were divided into aliquots in the bioassay at sublethal doses (20 pL / compound), ensuring that there was no direct contact with the insect. The bottles were immediately sealed with Parafilm® and kept at room temperature. Insect mortality was monitored after an exposure of 5 to 24 hours, evaluating the movement of insects as an indicator of mortality. Mortality was calculated by comparing the number of dead insects to the total number in the bioassay and expressed by the percentage of mortality. The data were analyzed using ANOVA as performed on GenStat, version 14. The experiment was completely randomized with 4 replicates for each combination of compounds.

[057] A synergistic insecticidal effect was observed when isoprene was combined with 2-methyl-1-butanol, 3-methyl-2-butanone, n-butyl alcohol and eucalyptol in sublethal doses (20 pL @) (Table 6) . In contrast, isoprene did not exercise

Petition 870190074672, of 8/2/2019, p. 44/68

36/41 insecticidal effect when applied alone with the same volumes (20 and 40 μΙ_). The synergistic insecticidal activity of isoprene with 2-methyl-1-butanol, 3-methyl-2-butanone or n-butyl alcohol was rapid, with a 100% mortality observed after 5 hours, whereas isoprene with eucalyptol exhibited a 100% mortality after 24 hours. It is believed that the enhanced bioactivity of isoprene when applied in sublethal doses together with other bioactive compounds can be attributed to different modes of action of the compound, since they have different structures and are of various chemical classes (alcohols, ketones and monoterpenoids).

Table 6 - Synergistic effect (insecticide) of isoprene and other key compounds of Nodulisporium sp. (DR1).

Compound Volume (μΙ) 5 hours 24 hours Isoprene 20 0.0% 0.0% Isoprene 40 2.3% 2.3% Isoprene + 2-methyl-1-butanol 20 + 20 98.1% 100.0% Isoprene + 3-methyl-2-butanone 20 + 20 100.0% 100.0% Isoprene + n-butyl alcohol 20 + 20 100.0% 100.0% Isoprene + Eucalyptol 20 + 20 67.3% 100.0% F Pr. <0.001 <0.001 LSD (5%) 10.4% 28.0%

Example 9 - Fungicidal activity of isoprene, alone and in synergy with other key compounds of Nodulisporium sp. (DR1) [058] A fungicidal bioassay has been developed to evaluate the effect of isoprene against the vegetable pathogenic fungus Fusarium verticillioides when applied alone and in synergy with other compounds (2-methyl-1-butanol; 3-methyl-2-butanone;

Petition 870190074672, of 8/2/2019, p. 45/68

37/41 acetaldehyde; eucalyptol; linalool; sabinene) of Nodulisporium sp. (DR1). The pathogen was inoculated into a third of the Petri dish by placing a plug of hyphae agar actively growing in PDA. Then, the isoprene was divided into aliquots (10 μΙ_) in another third of the petri dish on filter paper. In the synergistic 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 dish on filter paper. The plates were immediately sealed with Parafilm® and kept at room temperature. After 4 days, the growth of the pathogen was determined by measuring the colony radius (in two alternative planes). The measurements were compared to the control and expressed as a percentage of inhibition in relation to the control plate (no growth = 100%). Compound combinations that exhibited 100% inhibition were evaluated for fungistatic or fungicidal activity by placing the original plug on a fresh PDA plate. The data were analyzed using ANOVA as performed on GenStat, version 14. The experiment was completely randomized with 4 replicates for each combination of compounds.

[059] Isopropene exerted a fungicidal effect when applied alone that inhibited development by 40.9% (Table 7). When isoprene was combined with acetaldehyde, the development of F. verticillioides was inhibited by 100%, while isoprene with linalool inhibited growth by 99.9%. When hyphal plugs of these combinations of compounds were transferred to fresh PDA, there was no development in the treatment with isoprene-acetaldehyde, thus indicating fungicidal bioactivity, whereas development in the treatment with isoprene-linalool was observed, thus indicating fungistatic bioactivity. With regard to synergistic experiments, it is believed that the enhanced bioactivity of isoprene when applied in sublethal doses together with other bioactive compounds can be attributed to different modes of action of the compound, since they have

Petition 870190074672, of 8/2/2019, p. 46/68

38/41 diverse structures and are of varied chemical classes (alcohols, ketones, aldehydes and monoterpenoids).

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

Compound Volume (pL) Day 4 Activity Isoprene 10 40.9% AT Isoprene + 2-methyl-1-butanol 10 + 10 69.1% AT Isoprene + 3-methyl-2-butanone 10 + 10 57.8% AT Isoprene + Acetaldehyde 10 + 20 100.0% Fungicide Isoprene + Eucalyptol 10 + 10 88.4% AT Isoprene + Linalool 10 + 10 99.9% Fungistatic Isoprene + Sabinene 10 + 10 32.1% AT F Pr. <0.001 LSD (5%) 15.0%

Example 10 - Bactericidal effect of isoprene, alone and in synergy with other key compounds of Nodulisporium volatoloma alone. (DR1) [060] A bactericidal bioassay has been developed to assess 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 Nodulisporium sp. (DR1). The pathogen was inoculated into a third of the Petri dish tracing a streak of bacterial cells from a culture actively growing (overnight) on nutrient agar (NA). Then, the isoprene was divided into

Petition 870190074672, of 8/2/2019, p. 47/68

39/41 aliquots (10 μΙ_) in another third of the petri dish on filter paper. In the synergistic 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 dish on filter paper. An untreated control was included, which did not consist of any compounds. The plates were immediately sealed with Parafilm® and kept at room temperature. After 3 days, the growth of the pathogen was evaluated visually and classified based on the following scale: 2 - growth equivalent to that of the control; 1 - less growth than the control; 0 - no growth. The combinations of compounds that exhibited 100% inhibition were evaluated for bacteriostatic or bactericidal activity by subculture from the original streak on a fresh NA plate. The data were analyzed using ANOVA as performed on GenStat, version 14. The experiment was completely randomized with 4 replicates for each combination of compounds.

[061] Isopropene had no bactericidal effect when applied alone. A bactericidal effect was only observed when the isoprene was combined with acetaldehyde and 2-methyl-1-butanol, however the data were not significant (Table 8). Isopropene with acetaldehyde was the only combination that completely inhibited the growth of P. syringae, while isoprene with 2-methyl-1-butanol reduced growth. When the colonies of these combinations of compounds were transferred to fresh NA, no growth was observed in the treatment with isoprene-acetaldehyde, thus indicating bactericidal bioactivity. With regard to synergistic experiments, it is believed that the enhanced bioactivity of isoprene when applied in sublethal doses together with other bioactive compounds can be attributed to different modes of action of the compound, since they have different structures and are of varying chemical classes (alcohols, ketones, aldehydes and monoterpenoids).

Petition 870190074672, of 8/2/2019, p. 48/68

40/41

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

Compound Volume (pL) Scale of Growth Activity Control 2 AT Isoprene 10 2 AT Isoprene + Acetaldehyde 10 + 20 0 Bactericide Isoprene + 2-methyl-1-butanol 10 + 10 1 AT Isoprene + 3-methyl-2-butanone 10 + 10 2 AT Isoprene + Eucalyptol 10 + 10 2 AT Isoprene + Linalool 10 + 10 2 AT Isoprene + Sabinene 10 + 10 2 AT F Pr. NS LSD (5%) NS

[062] As used in this document, unless the context dictates otherwise, the term "understand" and its variations, such as "comprising", "understands" and "understood", are not intended to exclude other additives, components, whole numbers or phases.

[063] Reference to any prior art in this specification is not, and should not be construed as, an acknowledgment or any form of suggestion that that prior art constitutes part of common knowledge in Australia or any other jurisdiction or that can be expected

Petition 870190074672, of 8/2/2019, p. 49/68

41/41 reasonably that this prior technique is confirmed, understood and considered relevant by those skilled in the art.

[064] Finally, it should be kept in mind that various changes, modifications and / or additions can be made without departing from the scope of the present invention as outlined in this document.

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 Center I 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, Μ. 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 characterization, 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 (17)

1. Biofumigant or biocidal composition CHARACTERIZED for including isoprene or an analog thereof. 2. Biofumigant or biocidal composition, according to claim 1, CHARACTERIZED by including an additional bioactive component selected from alcohols, ketones, aldehydes and monoterpenoids. 3. Biofumigant or biocidal composition, according to claim 1, CHARACTERIZED by the fact that the additional 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. Biofumigant or biocidal composition according to claim 3, CHARACTERIZED by the fact that the bioactive component includes acetaldehyde, either alone or in combination with 2-methyl-1-butanol. 5. Biofumigant or biocidal composition, according to one of claims 1 to 4, CHARACTERIZED by the fact that at least one of the bioactive components is derived from an endophyte. 6. Use of a composition that includes isoprene or an analog of the same CHARACTERIZED because it occurs in biofumigation or bioprotection. 7. Use, according to claim 6, CHARACTERIZED by the fact that the composition includes an additional 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, CHARACTERIZED by the fact that the bioactive component includes acetaldehyde, either alone or in combination with 2-methyl-1-butanol. Petition 870190074672, of 8/2/2019, p. 51/68 2/3 9. Method for inhibiting an insect or a micro-organism, the method being CHARACTERIZED in that it includes exposing the insect or micro-organism to a biocidal or biofumigant composition that includes isoprene or an analog thereof. 10. Method according to claim 9, CHARACTERIZED by the fact that the biofumigant or biocidal composition includes an additional 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. Method according to claim 10, CHARACTERIZED by the fact that the bioactive component includes acetaldehyde, either alone or in combination with 2-methyl-1-butanol. 12. Method according to any one of claims 9 to 11, CHARACTERIZED in that it includes inhibiting an insect that is a stored grain pest. 13. Method, according to claim 9, CHARACTERIZED by the fact that the insect is of the species selected from one or more of Tribolium castaneum, Rhyzopertha dominica, Cryptolestes ferrugineus and Oryzaephilus suinamensis. 14. Method, according to any one of claims 9 to 11, CHARACTERIZED by including inhibiting a microorganism and by the fact that the microorganism is a fungus selected from one or more of the genera Fusarium, Botrytis, Alternaria or Rhizoctonia or a bacteria of the genus Pseudomonas. 15. Method, according to claim 14, CHARACTERIZED by the fact that the microorganism is a fungus selected from one or more of Fusarium verticillioides, Botrytis cinerea, Alternaria alternata and Rhizoctonia cerealis. 16. Method, according to any one of claims 9 to 15, CHARACTERIZED because it includes inhibiting a microorganism and the fact that the microorganism is a bacterium of the species Pseudomonas syringae. Petition 870190074672, of 8/2/2019, p. 52/68 3/3 17. Use of a composition that includes isoprene or an analogue of the same CHARACTERIZED because it is a biofumigant for the disinfestation of stored grains.