Bioactive Fungi

Abstract

The present invention relates to fungi, in particular fungal endophytes of Daldinia spp., compounds produced by the fungi, uses of the fungi, uses of compounds produced by the fungi and similar compounds, and related methods.

Claims

1. A Daldinia spp. fungus substantially purified or isolated from a plant of the species Pittosporum bicolor.

2. A substantially purified or isolated fungus of Daldinia sp. (U254) as deposited at the National Measurement Institute under accession number V15/028236.

3. (canceled)

4. A method for producing an organic compound, said method comprising culturing a Daldinia spp. fungus in a culture medium under conditions suitable to produce said organic compound and recovering an organic compound produced by the fungus from fungal cells, from the culture medium, or from air space associated with the culture medium or fungus.

5. (canceled)

6. The method according to claim 4, wherein the culture medium is selected from one or more of oatmeal agar (OA), half strength potato dextrose agar (HPDA), potato dextrose agar (PDA), endophyte agar (ENDO), Murashige and Skoog with 20% sucrose (MS-SUC), half V8 juice/half potato dextrose agar (V8PDA), water agar (WA) and yeast malt extract agar (YME).

7. (canceled)

8. The method according to claim 4, wherein said organic compound is a volatile organic compound.

9. The method according to claim 4, wherein said organic compound is selected from one or more of: (a) the group consisting of: acetaldehyde, pentane, ethanol, 3-pentanol, acetone, 2,3-butanedione, isobutanol, ethyl acetate, isovaleraldehyde, 5,5-dimethyl-1,3-cyclopentadiene, 5,5-dimethyl-1,3-cyclopentadiene, bicyclo[4.1.0]hept-2-ene, 1-methyl-1,4-cyclohexadiene, (Z)-3-methyl-1,3,5-hexatriene, toluene, 4-methylphenol, 3-methyl-1-butanol, 2-methyl-1-butanol, (1Z)-3-methyl-1,3,5-hexatriene, (2Z)-3-methyl-1,3,5-hexatriene, p-xylene, styrene, dimethylcyclopentadiene, 1,4-cyclohexadiene, 4-heptyn-2-ol, 4-ethyl-1-octyn-3-ol, 2,2,4,6,6-pentamethyl-heptane, phenylethyl alcohol, guaiene, [1S-(1,4,7)]-1,2,3,4,5,6,7,8-octahydro-1,4,9,9-tetramethyl-4,7-methanoazulene, (E)-2-pentene, (Z)-2-pentene, 1-methyl-cyclohexene, 3-methyl-cyclohexene, tricyclene, -pinene, 1-isopropyl-3-methylcyclohexane, p-menth-3-ene, 1-methyl-4-(1-methylethyl)-cyclohexene, 2-carene, p-menth-1-ene, cymene, terpenes and C7 aliphatic/aromatic unsaturated hydrocarbons; and (b) a compound characterisable by about a base peak selected from the group consisting of a m/z of: 43.2, 59.1, 67.0, 68.1, 71.1, 79.0, 79.1, 81.0, 82.0, 91.0, 95.0, 97.0, 98.0, 108.0, 109.9, 115.0, 124.0, 132.9 and 192.8, or a base peak which is 0.1 of any of the foregoing, when analysed by mass spectrometry.

10. (canceled)

11. (canceled)

12. (canceled)

13. (canceled)

14. An organic compound when used in biofumigation or bioprotection, said organic compound being substantially identical to an organic compound produced by culturing a Daldinia spp. fungus in a culture medium under conditions suitable to produce said organic compound, or an analogue thereof.

15. A composition for use as a fumigant comprising at least one organic compound, or a biocidal composition including at least one organic compound, wherein the at least one organic compound is substantially identical to one or more compounds produced by culturing a Daldinia spp. fungus in a culture medium under conditions suitable to produce said organic compound, or an analogue thereof.

16.

17. The organic compound according to claim 14, wherein the at least one organic compound is a volatile organic compound.

18. The organic compound according to claim 14, wherein the at least one organic compound is selected from one or more of: (a) the group consisting of: acetaldehyde, pentane, ethanol, 3-pentanol, acetone, 2,3-butanedione, isobutanol, ethyl acetate, isovaleraldehyde, 5,5-dimethyl-1,3-cyclopentadiene, 5,5-dimethyl-1,3-cyclopentadiene, bicyclo[4.1.0]hept-2-ene, 1-methyl-1,4-cyclohexadiene, (Z)-3-methyl-1,3,5-hexatriene, toluene, 4-methylphenol, 3-methyl-1-butanol, 2-methyl-1-butanol, (1Z)-3-methyl-1,3,5-hexatriene, (2Z)-3-methyl-1,3,5-hexatriene, p-xylene, styrene, dimethylcyclopentadiene, 1,4-cyclohexadiene, 4-heptyn-2-ol, 4-ethyl-1-octyn-3-ol, 2,2,4,6,6-pentamethyl-heptane, benzaldehyde, phenylethyl alcohol, guaiene, [1S-(1,4,7)]-1,2,3,4,5,6,7,8-octahydro-1,4,9,9-tetramethyl-4,7-methanoazulene, (E)-2-pentene, (Z)-2-pentene, 1-methyl-cyclohexene, 3-methyl-cyclohexene, tricyclene, a-pinene, 1-isopropyl-3-methylcyclohexane, p-menth-3-ene, 1-methyl-4-(1-methylethyl)-cyclohexene, 2-carene, p-menth-1-ene, cymene, terpenes, C7 aliphatic/aromatic unsaturated hydrocarbons, 4-hydroxy-2-butanone, butyric acid, methylenecyclohexane, 4-methylcyclohexane, 1,3-cycloheptadiene, butyraldehyde, spiro[2.4]hepta-4,6-diene and acetoin; and (b) a compound characterisable by about a base peak selected from the group consisting of a m/z of: 43.2, 59.1, 67.0, 68.1, 71.1, 79.0, 79.1, 81.0, 82.0, 91.0, 95.0, 97.0, 98.0, 108.0, 109.9, 115.0, 124.0, 132.9 and 192.8, or a base peak which is 0.1 of any of the foregoing, when analysed by mass spectrometry.

19. (canceled)

20. The organic compound according to claim 14, wherein the at least one organic compound is selected from one or more of: the group consisting of: acetaldehyde, 3-pentanol, isovaleraldehyde, 3-methyl-1-butanol, 2-methyl-1-butanol, 1,4-cyclohexadiene, 4-hydroxy-2-butanone, butyric acid, methylenecyclohexane, 4-methylcyclohexane, 1,3-cycloheptadiene, butyraldehyde, spiro[2.4]hepta-4,6-diene and acetoin.

21. The organic compound according to claim 14, wherein the at least one organic compound is selected from one or more of the group consisting of acetaldehyde, 3-pentanol, isovaleraldehyde and acetoin.

22. The organic compound according to claim 14, wherein the at least one organic compound includes a combination of isovaleraldehyde and/or acetoin with 3-pentanol, and/or a combination of any one or more of isovaleraldehyde, acetoin and 3-pentanol with any one or more of acetaldehyde, 1,4-cyclohexadeine, 2-methyl-1-butanol, 1,3-cycloheptadeine and 4-methylcyclohexene.

23. A method for inhibiting an insect or a micro-organism comprising exposing the insect or micro-organism to the organic compound according to claim 14.

24. The method according to claim 23, wherein the insect is a pest of stored grain.

25. The method according to claim 23, wherein the insect is of the species selected from one or more of Tribolium castaneum, Rhyzopertha dominica, Cryptolestes ferrugineus and Oryzaephilus suinamensis.

26. The method according to claim 23, wherein 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.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0083] FIGS. 1A-1F depict a phylogenetic tree showing the phylogenetic relationships among Daldinia sp (U254) and D. eschscholtzii, D. concentrica, D. childiaelpyrenaica, D. vemicosa/loculatalnovae-zelandiae, D. petriniae, along with their respective allies. Isolate U254 is represented by a solid black square in FIG. 1B.

[0084] FIG. 2 shows the percentage mortality of Tribolium castaneum following exposure to Daldinia sp. (U254) in insect assays, when grown on different media (bars from left to right: HPDAhalf strength potato dextrose agar, OAoatmeal agar, PDA, ENDOendophyte agar, MS-SUCMurashige and Skoog with 20% sucrose, V8PDAhalf V8 juice/half PDA, WAwater agar, YMEyeast malt extract agar). Bars represent the least significant difference (LSD) at a significance level of 5%.

DETAILED DESCRIPTION OF THE EMBODIMENTS

Example 1Endophyte Identification

[0085] A broad-based endophyte discovery program was undertaken across Victoria and the Northern Territory, Australia. Over 1000 endophytic fungi were isolated from over 100 plant species. Preliminary screens for bioactivity identified approximately 250 isolates of varying degrees of bioactivity.

Example 2Isolation of Daldinia sp. (U254)

[0086] A single endophytic fungal isolate of Daldinia sp. (U254) was collected from a cool temperate rainforest within the Yarra Ranges of Victoria, Australia. The isolate was collected as an endophyte of foliar tissue from Pittosporum bicolor in the Yarra Ranges.

[0087] The host plant, Pittosporum is comprised of around 200 species, and is widely distributed across Australasia, Oceania, East Asia and parts of Africa. A number of species are extensively cultivated globally and grown as ornamental plants. Pittosporum bicolor is a shrub or tree of commonly 3 to 10 metres in height when mature. Leaves are alternate, usually narrowly ovate to narrowly oblong, commonly 3 to 8 centimetres long and 5 to 18 millimetres wide. Leaf margins are flat to recurved. The leaf apex is subacute to obtuse. The lower leaf surface is usually densely hairy and rarely glabrescent with age. Petioles are commonly 2 to 3 millimetres in length. Flowers are usually terminal, solitary or a few together. Sepals are commonly 3 to 6 millimetres long, with few to many hairs. Petals are commonly 8 to 11 millimetres long, yellow with purple-maroon markings. Ovaries are hairy. Capsules are globose, usually 5 to 8 millimetres long, hairy, grey, and with valves dark on the inner face. Seeds are few to many, and red in colour.

Example 3Morphological Characterisation of the Daldinia sp. (U254)

[0088] Colonies of Daldinia sp. (U254) were grown on oatmeal agar (OA) and reached the edge of a 9 centimetre Petri dish in 3-5 days. They were at first whitish, felty, azonate, with diffuse margins, becoming grey with dark grey, green and white tones; reverse cream to yellow.

[0089] Co-indiogenous structures of normal Daldinia types were not observed, instead the vegetative mycelium differentiated into a network of stromatic structures, composed of black, thick-walled, incrusted hyphae, producing thick-walled chlamydospore-like structures, remaining sterile. The hyphae were branched, hyaline and smooth.

Example 4Molecular Characterisation of the Daldinia sp. (U254)

[0090] A phylogenetic analysis of Daldinia sp. (U254) was undertaken by sequence homology comparison of the 5.8S-ITS ribosomal gene region. The isolate was grown on OA, from which mycelia were harvested and used for DNA extraction using the Qiagen Blood and Tissue Kit according to manufacturer's instructions (QIAGEN, Germany). The ribosomal gene region was amplified using a KAPA2G Robust PCR kit with the universal primers ITS5 (5-GGAAGTAAAAGTCGTAACAAGG-3) and ITS4 (5-TCCTCCGCTTATTGATATGC-3). The PCR conditions were as follows: 94 C., 5 minutes (1 cycle); 94 C., 30 seconds; 50 C., 30 seconds; 72 C., 2 minutes (35 cycles); 72 C., 7 minutes (1 cycle). The amplicon was then submitted to Macrogen (Seoul, South Korea) for purification and sequencing.

[0091] Sequence homology searching against the NCBI nucleotide database (Blastntype specimen parameter) identified closely related fungi to isolate U254, of which Daldinia species were the closest match (e.g. 96%) (Table 1). These sequences, and sequences of other Daldinia species (type specimens) were collected and aligned in MEGA5 using the Muscle algorithm (sourced from Stadler et al.). Based on the sequence alignment, phylogenetic relationships were inferred using Maximum Likelihood (ML) and Maximum Parsimony (MP) analyses. For ML analysis, phenograms were obtained using the nearest-neighbour-interchange method, applying the Tamura-Nei model. For MP analysis, phenograms were obtained using the Subtree-Pruning-Regrafting algorithm (search level 3). 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. The phylogenetic trees had similar topology to Stadler et al. (2014), forming 5 distinct Daldinia clades that comprised Daldinia eschscholtzii (A), Daldinia concentrica (B), Daldinia vernicosa (C1), Daldinia novae-zelandiae (C2), Daldinia loculata/loculatoides (C3), Daldinia childe/pyrenaica (D), and Daldinia petrinae (E). The Daldinia sp. (U254) clustered in the Daldinia loculatoides and Daldinia loculata clade (clade C3), along with Daldinia grandis and Daldinia gelatinosa (red squareDaldinia sp. U254) (FIG. 1). Despite the low bootstrap support (consistent with Stadler et al., 2014) isolate U254 is clearly a Daldinia species, and likely belongs to the Daldinia loculatoides/loculata clade. As it does not produce anamorphic structures in vitro like other species in the clade, it is a new Daldinia species.

TABLE-US-00001 TABLE 1 Comparison of ribosomal gene sequences of Daldinia sp. (U254) with other closely related species (type specimens) from NCBI sequence homology search. NCBI Max E Query Accession Genus Species Score Value Coverage Identity gb|JX658510.1| Daldinia palmensis 835 0 87% 96% gb|JX658517.1| Daldinia raimundi 830 0 87% 96% gb|JX658479.1| Daldinia dennisii 830 0 87% 96% gb|JX658441.1| Daldinia decipiens 752 0 88% 93% gb|JX658537.1| Daldinia barkalovii 623 5e176 72% 93% gb|KC968938.1| Hypoxylon fraxinophilum 623 5e176 100% 86% gb|KC968922.1| Hypoxylon liviae 593 4e167 99% 85% gb|KC968930.1| Hypoxylon gibriacense 584 3e164 98% 85% gb|KC968934.1| Hypoxylon laminosum 573 6e161 98% 85%

Example 5Bioactivity of Daldinia sp. (U254)

[0092] In vitro bioassays were established to test the bioactivity of Daldinia sp. (U254) against a range of insect pests (Tribolium castaneum) and plant pathogenic fungi (Botrytis cinerea, Alternaria alternata and Rhizoctonia cerealis). The bioassays used a 9 centimetre split Petri plate, which contained an impermeable septum through the centre of the plate, which completely separated the plate into two halves. This only permitted volatile compounds to pass over the septum to act against the test organism, and prevented any direct contact between the endophyte (or its liquid exudates) and the test organism.

[0093] For the insect assay Daldinia sp. (U254) was inoculated on to Petri plates containing OA, half potato dextrose agar (HPDA), potato dextrose agar (PDA), endophyte agar (ENDO), Murashige and Skoog with 20% sucrose (MS-SUC), half V8 juice/half PDA (V8PDA), water agar (WA) and yeast malt extract agar (YME). An agar plug containing actively growing mycelia from the endophyte was placed approximately 13 millimetres from the edge of the plate (i.e. on one half of the plate). The endophytic fungus was allowed to grow at room temperature (in the dark) for 6 days. Subsequently, the insect pests were inoculated on to the other half of the plate by placing four insects onto filter paper and a food source (wheat meal). Plates were sealed with low-density polyethylene (LDPE) plastic film (approximately 0.01 millimetres thick) and covered in aluminium foil (i.e. kept in the dark). After 3, 7, 10, 14, 18 and 25 days the viability was assessed by measuring the activity of the insect (non-activedead; activealive).

[0094] Measurements were compared to the control and expressed as percentage mortality versus the control (FIG. 2). Data were analysed using ANOVA as performed in GenStat, version 14. The experiment was fully randomised with 4 replicates. Daldinia sp. (U254) caused mortality rates of 100% for Tribolium castaneum following 3 days exposure to the endophyte when grown on OA. The insecticidal bioactivity of Daldinia sp. (U254) was significantly greater on OA than all other media at 3 and 7 days exposure, with HPDA providing equivalent activity at 10 to 25 days exposure (maximum insecticidal activity94%).

[0095] For the plant pathogenic fungus assay, Daldinia sp. (U254) was inoculated on to split Petri plates containing PDA. An agar plug containing actively growing mycelia from the endophyte was placed approximately 13 millimetres from the edge of the plate (i.e. on one half of the plate). The endophytic fungus was allowed to grow at room temperature (in the dark) for 6 days. Subsequently, the plant pathogenic fungi were inoculated on to the other half of the plate by placing an agar plug containing actively growing hyphae approximately 13 millimetres from the edge of the plate. Plates were sealed with LDPE plastic film (approximately 0.01 millimetres thick) and covered in aluminium foil. After 2, 5, 7, and 11 days the viability was assessed by measuring the radial growth of the plant pathogenic fungus.

[0096] Measurements were compared to the control and expressed as percentage inhibition versus the control (Table 2). Data were analysed using ANOVA as performed in GenStat, version 14. The experiment was fully randomised with 3 replicates. Daldinia sp. (U254) completely inhibited the growth of Botyrtis cinerea and Rhizoctonia cerealis at all time points, while the growth of Alternaria alternata was inhibited by a minimum of 76.3%.

TABLE-US-00002 TABLE 2 Percentage inhibition of Daldinia sp. (U254) in plant pathogenic fungus assays against 3 plant pathogenic fungi, Botrytis cinerea, Alternaria alternata and Rhizoctonia cerealis. Pathogen Day 2 Day 5 Day 7 Day 11 Botrytis cinerea 100.0% 100.0% 100.0% 100.0% Alternaria alternata 100.0% 85.7% 76.3% 81.8% Rhizoctonia cerealis 100.0% 100.0% 100.0% 100.0%

Example 6Volatolome of Daldinia sp. (U254)

[0097] Gases were analysed in the head space above cultures of Daldinia sp. (U254). The isolate was cultured under microaerophilic conditions, which consisted of growing the fungus on OA and YME slopes in 20 ml glass vials, with an agar:air ratio of 1:2.5. Vials were sealed with a screw cap lid with polytetrafluoroethylene (PTFE) septum, and grown for 9 days at room temperature.

[0098] A head space solid phase microextraction (SPME) was performed to capture volatiles produced by Daldinia sp. (U254). A StableFlex fibre (Supelco) coated with 75 micrometre Carboxen/PDMS was used to absorb volatiles from the head space of vials. Automated sampling was performed by an Gerstel Multi Purpose Sampler using the proprietary Maestro software. The fibre was conditioned at 270 C. for 60 minutes prior to commencement of activities and for 30 minutes between each sample. For each sample the fibre was inserted into the vial and incubated at room temperature for 7 minutes to absorb volatiles, after which the fibre was inserted into a splitless injection port of an Agilent 7890 gas chromatography (GC) system where the contents were thermally desorbed (250 C. for 6 minutes) onto an Agilent DB-624 capillary column (25 metres250 micrometres id., 1.4 micrometre film thickness, column 1) coupled to an Agilent inert fused silica 2 metres250 micrometres id (no film) (column 2) via a purged union controlled by an Agilent Auxiliary Electronic Pressure Control module. The column oven was programmed as follows: 35 C. (3 minutes), 3 C./minute to 200 C., then 25 C./minute to 250 C. (2 minutes). The carrier gas was helium with a constant flow rate of 1 millilitre/minute for column 1 and 1.8 millilitre/minute for column 2. The GC was interfaced with an Agilent 7000 GC/MS triple quadrupole 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 to 330, with a scan time of 200 milliseconds.

[0099] Initial identification of the volatiles produced by Daldinia sp. (U254) was made through library comparison using standard chemical databases. Secondary confirmatory identification was made by comparing mass spectral data of authentic standards with data of the fungal volatiles. All chemical names in this report follow the nomenclature of the standard chemical databases. In all cases, uninoculated control vials were also analysed and the compounds found therein were subtracted from those appearing in the vials supporting fungal growth. Tentative identification of the fungal volatiles was based on observed mass spectral data as compared to those in these chemical databases and those of authentic standards (where possible).

[0100] The GC-MS analysis (0 to 65 minutes) identified 31 volatile metabolites produced by Daldinia sp. (U254) when grown for 9 days on OA and YME at room temperature (Table 3). The metabolites produced by Daldinia sp. (U254) were representative of a number of structural classes, including alcohols (e.g. ethanol, 3-methyl-1-butanol), aldehydes (e.g. acetaldehyde, isovaleraldehyde), esters (e.g. ethyl acetate), terpenes and C7 aliphatic/aromatic unsaturated hydrocarbons and their associated derivatives (C7, e.g. 1-methylcyclohexene, 1-methyl-1,4-cyclohexadiene, toluene). The C7 compounds represent the major structural class within the volatolome, composing approximately one third of the compounds produced.

TABLE-US-00003 TABLE 3 GC-MS headspace analysis of the volatile compounds produced by Daldinia sp. (U254) when grown on OA and YME for 9 days at room temperature. Relative Compound Match RT BP (m/z) Significant Minor Ions (% of BP) Intensity Acetaldehyde 3 3.51 44.1 43 (44.3), 42 (13.89) + Pentane 2 4.95 43.1 42.1 (63.35), 41.1 (44.56), 57.1 (26.32) + Ethanol 3 5.39 45.1 46.1 (24.49), 43.1 (14.42) +++ mz67, 68, 53@6.14 min 6.14 67.0 68.1 (85.3), 53.1 (40.02) + Acetone 2 6.21 43.1 58.1 (66.27) + mz59, 42, 60, 41@9.60 min 9.62 59.1 42.1 (43.59), 60.1 (30.15), 41.2 (19.08) + Ethyl acetate 3 11.03 43.1 61.1 (39.57), 70.1 (33.77) + mz43, 41, 42@13.48 min 13.50 43.2 41.2 (70.95), 42.1 (64.33) + Isovaleraldehyde 3 13.96 44.0 43.0 (93.80), 41.2 (89.88), 58.1 (81.88) + mz79, 77, 94@15.33 min 15.33 79.0 94 (59), 77 (56.52), 91.1 (38.45) + mz79, 77, 94@16.67 min 16.66 79.0 77 (52.78), 94.1 (44.48), 91 (21.11) + mz79, 77, 94@17.04 min 17.04 79.0 77.1 (48.88), 94 (43.42) ++ mz79, 77, 94@17.97 min 17.97 79.0 94 (58.92), 77 (58.26), 91 (35.98) + mz79, 77, 94@18.53 min 18.53 79.0 77.1 (58.47), 94 (57.44), 91.1 (35.01) +++ mz79, 77, 94@19.02 min 19.02 79.1 77 (58.65), 94 (56.92), 91 (34.56) +++ Toluene 3 19.42 91.0 92 (51.12), 65 (10.14) +++ mz79, 91, 77@20.01 min 20.02 79.0 91 (1306.57), 77 (963.93), 94 (48.68) ++ 3-Methyl-1-butanol 3 20.29 55.0 70.1 (80.81), 42.1 (57.06), 43 (47.19) ++ 2-Methyl-1-butanol 3 20.47 56.0 57.1 (89), 70.1 (70.36), 41.1 (45.55) + mz79, 77, 94@20.87 min 20.87 79.1 77 (73.45), 94 (66.91), 91 (30.89) +++ mz79, 77, 94@21.55 min 21.55 79.0 77 (61.57), 94 (54.52) +++ 1-Methyl-1,4-cyclohexadiene 3 22.03 79.1 77 (75.27), 94 (55.41), 91 (28.26) ++ mz91, 92@23.09 min 23.10 91.0 92 (55.2), 0 (0) +++ Phenol, 4-methyl- 2 24.47 108.0 107 (62.04), 79 (12.57), 77 (9.94) ++ mz108, 79, 77@26.18 min 26.19 108.0 79 (63.92), 77 (38.91) + Styrene 2 28.73 104.0 103 (42.47), 78 (34.55) + Benzaldehyde 3 34.68 105.0 106 (95.77), 77 (65.67), 51 (16.49) + mz95, 67, 110@36.60 min 36.61 95.0 67.1 (41.38), 110 (29.01) + mz79, 108@38.88 min 38.91 79.0 108 (87.72), 77.1 (51.5) + mz97, 69, 125@42.54 min 42.54 97.0 69.2 (32.09), 125 (21.95) + Phenylethyl alcohol 3 44.11 91.0 92 (50.08), 121.9 (23.13), 64.9 (12.21) + 1 - Fragmentation pattern matches spectra of compound in NIST library (>75%) 2 - Fragmentation pattern matches spectra of compound in NIST library (>90%) 3 - Fragmentation pattern matches spectra and retention time of authentic standard + - significant peak; ++ - major peak; +++ - dominant peak

[0101] For large scale trapping, the fungus was inoculated onto 120 standard OA plates and incubated in the dark for 3 days at 25 C. An 18.5 litre desiccator was baked at 150 C. for 3 days and allowed to cool inside a laminar flow cabinet. The fungal plates were stacked without lids inside the desiccator in an overlapping manner so as to prevent them from touching the agar of the plates, while allowing maximum air exchange. The desiccator was sealed using LDPE plastic film and wrapped in aluminium foil (i.e. in the dark). The tap opening in the lid was closed using a silicone rubber stopper in which two holes were drilled. Two pieces of Teflon tubing were passed through these holes, one leading all the way to approximately 2 centimetres above the desiccator bottom, and a shorter one to approximately 5 centimetres from the desiccator top acting as the air inlet. A Supelco VOST Stack Sampling Tube (Tenax TA:Petroleum Charcoal, 2:1) was attached to the inlet (outside the desiccator) to purify the air entering the system. To the outlet, a 305 centimetre glass column filled with approximately 100 grams of anhydrous sodium sulphate was attached to remove excess moisture from the gas phase. A second Sampling Tube was attached to the end of the glass column to trap volatiles produced by the fungal cultures (Fungal Volatiles Trap, FVT). Negative pressure was applied to the whole system to result in a flow rate of approximately 40-60 millilitres/minute, channeling the culture headspace through the second trap to adsorb any volatiles present. The system was left undisturbed for 7 days at room temperature, after which the trap was removed and placed inside the oven of an Agilent 7890 GC and connected to the GC APC (auxiliary pressure control) module. High purity Nitrogen was flushed through the trap at an initial rate of approximately 25 millilitres/minute. A piece of stainless steel tubing was connected to the outlet of the trap, leading into a glass vial sitting on top of a metal block partially submerged in liquid nitrogen that acted as a cold trap. The FVT was dry purged at ambient temperature for 30 minutes, then heated to 200 C. to thermally desorb the volatiles, which were recovered in the cold trap. The FVT was then reconnected to the desiccator for another 7 days and the process repeated. With this setup, approximately 300-450 milligrams of mixed hydrocarbon volatiles were recovered per week. The recovered extracts were combined and analysed on an Agilent J&W DB-624 60 m (length)0.53 millimetre (inner diameter)3 micrometre (film thickness) (column 1) connected to a short inert column as described above (column 2). Carrier gas was helium at a constant flow rate of 3.8 millilitre/minute for column 1 and 1.8 millilitre/minute for column 2. Temperature programming was 110 C. for 11 minutes, then 20 C./minute to 250 C. (7 minutes). The GC was interfaced with an Agilent 7000 GC/MS as described above. Automated sampling was performed by a Gerstel MultiPurpose Sampler using the proprietary Maestro software. An aliquot of 0.2 microlitre recovered volatiles was injected into the split/splittless injection port using a 5:1 split ratio.

[0102] The GC-MS analysis (0 to 65 minutes) identified 19 volatile metabolites produced by Daldinia sp. (U254) when grown for 14 days on OA at room temperature (Table 4). The metabolites produced by Daldinia sp. (U254) were representative of a number of structural classes, including alcohols (e.g. 3-pentanol), esters (e.g. ethyl acetate), terpenes and C7 aliphatic/aromatic unsaturated hydrocarbons and their associated derivatives (C7, e.g. 1-methyl-1,4-cyclohexadiene). The C7 compounds represent the major structural class within the volatolome, composing approximately one third of the compounds produced.

TABLE-US-00004 TABLE 4 GC-MS headspace analysis of the volatile compounds recovered from Daldinia sp. (U254) when grown on OA 14 days at room temperature. Relative compound match RT BP significant minor ions (% of BP) intensity 2-Pentene (E) 2 5.23 55.1 70.1 (75.44), 42.1 (31.77), 41.1 (16.73) + 2-Pentene (Z) 2 5.3 55.1 70.1 (77.31), 42.1 (32.69), 41.1 (17.32) + Ethyl Acetate 2 6.52 43.1 61.0 (30.59), 70.1 (21.67), 45.1 (16.08) + 3-Pentanol 2 8.53 59.1 41.1 (20.09), 58.1 (14.1), +++ Cyclohexene, 1-methyl- 2 9.26 81.0 96.0 (37.96), 67.0 (29.77), 54.1 (22.78) ++ Cyclohexene, 3-methyl- 2 10.21 81.0 96.0 (48.13), 67.1 (46.95), 68.1 (33.88) ++ Toluene 3 10.58 91.0 92.0 (71.82), 65.0 (9.05) +++ 1-Methyl-1,4-cyclohexadiene 3 11.59 79.0 94.0 (67.69), 77.0 (65.84), 91.0 (40.22) +++ Tricyclene 2 15.21 93.0 91.0 (27.29), 92.0 (20.52), 79.0 (17.71) + -Pinene 2 15.34 93.0 92.0 (37.49), 91.0 (37.43), 77.0 (24.62) +++ 1-Cylohexane, 1-isopropyl-, 2 16.27 97.1 55.1 (84.53), 96.0 (67.06), 81.0 (38.4) +++ 3-methyl- p-Menth-3-ene 2 16.38 95.1 81.0 (77.71), 67.0 (39.15), 138 (33.05) ++ Cyclohexene, 1-methyl-4-(1- 1 16.49 95.0 81.0 (62.78), 67.0 (49.94), 68.1 (30.49) ++ methylethyl)- 2-Carene 1 17.16 121.0 93.0 (92.67), 136.0 (57.41), 91.0 (48.95) +++ p-Menth-1-ene 1 17.38 95.0 81.0 (71.64), 67.1 (59.54), 68.1 (38.41) +++ Cymene 2 17.48 119.0 134.0 (30.17), 91.0 (20.45), 117.0 (14.29) +++ 1 - Fragmentation pattern matches spectra of compound in NIST library (>75%) 2 - Fragmentation pattern matches spectra of compound in NIST library (>90%) 3 - Fragmentation pattern matches spectra and retention time of authentic standard + - significant peak; ++ - major peak; +++ - dominant peak

Example 7Insecticidal Activity of Compounds from the Volatolome of Daldinia sp

[0103] A total of 20 chemical standards were evaluated for their insecticidal activity against the stored grain pest, Tribolium castaneum. These chemical standards represented compounds in the volatolome of Daldinia sp. or were structural analogues of these compounds (Table 5). The bioassays were conducted in 90 millimetre split Petri plates (as per example 5). 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 microlitre) on to the other half of the Petri plate on filter paper. Plates were immediately sealed with Parafilm, covered in aluminium foil (i.e. in the dark) and maintained at room temperature. The mortality of insects was monitored daily by assessing insect movement as an indicator of mortality. The mortality was calculated by comparing the number of dead insects to the total number in the plate, and expressed as percentage mortality (Tables 6). Data were analysed using ANOVA as performed in GenStat, version 14. The experiment was fully randomised with 5 replicates for each endophyte.

TABLE-US-00005 TABLE 5 Insecticidal activity of volatile compounds (20) produced by Daldinia sp. (U254) and their structural analogues, against T. castaneum (Spiro[2.4]hepta-4,6-diene, Isovaleraldehyde, Acetoin (s), 3-Pentanol: 100% mortality; Acetaldehyde, Butyraldehyde, 1,4 Cyclohexadeine, 3-Methyl-1-butanol, 2-Methyl-1-butanol: 36-99% mortality; 4-Hydroxy- 2-butanone, Butyric acid, Methylenecyclohexane, 4-Methylcyclohexene, 1,3 Cycloheptadiene: 1-35% mortality; Control (Water), 2-Pentanol, Benzaldehyde, 1-Methyl- 1,4-cyclohexadiene, Toluene, 2,3 Butanedione, Ethanol: 0% mortality). % Mortality Confirmed/ Structural Day 1 Day 2 Day 3 Day 4 Analogue Class Control (Water) 0.0 0.0 0.0 0.0 2-Pentanol 0.0 0.0 0.0 0.0 Analogue Alcohol Benzaldehyde 0.0 0.0 0.0 0.0 Analogue Aldehyde 1-Methyl-1,4- 0.0 0.0 0.0 0.0 Confirmed Hydrocarbon cyclohexadiene Toluene 0.0 0.0 0.0 0.0 Confirmed Hydrocarbon 2,3 Butanedione 0.0 0.0 0.0 0.0 Confirmed Ketone Ethanol 0.0 0.0 0.0 0.0 Confirmed Alcohol 4-Hydroxy-2-butanone 4.0 4.0 4.0 4.0 Analogue Ketone Butyric acid 0.0 0.0 6.9 6.9 Analogue Carboxylic Acid Methylenecyclohexane 8.0 8.0 16.0 16.0 Analogue Hydrocarbon 4-Methylcyclohexene 4.0 13.7 17.7 17.7 Analogue Hydrocarbon 1,3 Cycloheptadiene 12.5 20.8 27.5 27.5 Analogue Hydrocarbon Acetaldehyde 36.0 36.0 36.0 36.0 Confirmed Aldehyde Butyraldehyde 31.5 39.5 39.5 39.5 Analogue Aldehyde 1,4 Cyclohexadeine 40.0 40.0 43.3 43.3 Confirmed Hydrocarbon 3-Methyl-1-butanol 45.0 45.0 50.0 50.0 Confirmed Alcohol 2-Methyl-1-butanol 80.0 80.0 80.0 80.0 Confirmed Alcohol Spiro[2.4]hepta-4,6-diene 87.1 100.0 100.0 100.0 Analogue Hydrocarbon Isovaleraldehyde 100.0 100.0 100.0 100.0 Confirmed Aldehyde Acetoin (s) 100.0 100.0 100.0 100.0 Analogue Hydrocarbon 3-Pentanol 100.0 100.0 100.0 100.0 Confirmed Alcohol F Pr. <0.001 <0.001 <0.001 <0.001 LSD (5%) 24.51 24.8 27.1 28.2

TABLE-US-00006 TABLE 6 Classification of the level of insecticidal activity of volatile compounds produced by Daldinia sp. and their structural analogues, against T. castaneum. Mortality Duration No. of Compounds No activity .sup.0% 6 Low activity 0-35% 24-96 hrs 5 Medium activity 36-99% 24-72 hrs 5 High activity 100% 24-48 hrs 4

Example 8Insecticidal Dose Response of Key Compounds from the Volatolome of Daldinia sp. (U254)

[0104] An insecticidal dose response assay was established to evaluate four key compounds (3-pentanol, isovaleraldehyde acetoin and acetaldehyde) against the stored grain pest, T. castaneum. The bioassays were conducted in 1 litre Schott bottles. The insect pest was inoculated into the bioassay by placing 9-12 insects onto feed (wheat flour and yeast). The biocidal compounds were then aliquoted into the bioassay at volumes ranging from 20-200 microlitres, ensuring no direct contact with the insect. Bottles were immediately sealed with Parafilm and maintained at room temperature. The mortality of insects was monitored daily for three 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 for each endophyte.

[0105] 3-Pentanol exhibited the highest biocidal activity against T. castaneum of all compounds tested, with any volume ranging from 20-200 microlitres showing 100% mortality after one day. Acetoin and Isovaleraldehyde exhibited biocidal activity against T. castaneum with volumes ranging from 20-200 microlitres, with volumes ranging from 50-200 microlitres showing 100% mortality after a 1 day exposure. Acetaldehyde exhibited biocidal activity against T. castaneum with volumes ranging from 20-200 microlitres, and volumes ranging from 100-200 microlitres showing 98.2-100.0% mortality after a 1 day exposure (Table 7).

TABLE-US-00007 TABLE 7 Dose response (20-200 microlitres) of four key compounds on T. castaneum after a 1 day exposure. Volume (L) Acetoin Isovaleraldehyde 3-Pentanol Acetaldehyde 0 0% 0% 0% 0% 20 3% 3% 100% 2.3% 50 100% 100% 100% 28.3% 100 100% 100% 100% 98.2% 150 100% 100% 100% 100% 200 100% 100% 100% 100% F Pr. <0.001 <0.001 * <0.001 LSD (5%) 3.42% 3.10% * 12.55

Example 9Bactericidal Effect of 3-Pentanol, Acetoin and Isovaleraldehyde, Alone and in Synergy with Other Key Compounds from the Volatolome of Daldinia sp. (U254)

[0106] A bactericidal bioassay was established to evaluate the effect of the most insecticidal candidate compounds against the plant pathogenic bacterium Pseudomonas syringae, when applied alone or in synergy with other compounds from the volatolome of Daldinia sp. 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). A candidate compound was then aliquoted (10 microlitresexcept Acetaldehyde: 20 microlitres due to high volatility) onto another third of the Petri plate on filter paper. In the synergy treatments, a second compound (acetaldehyde, 2-methyl-1-butanol, 1,4 cyclohexadiene, 1,3 cycloheptadiene, 4-methylcyclohexene) 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: 2equivalent growth to the control; 1less growth than the control; 0no growth. Compound combinations that exhibited 100% inhibition were assessed for bacteristatic or bactericidal activity by subculturing from the original streak on to a fresh NA plate. Data were analysed using ANOVA as performed in GenStat, version 14. The experiment was fully randomised with 4 replicates for each compound combination.

[0107] Acetaldehyde and isovaleraldehyde exhibited high bactericidal activity against P. syringae when used alone or in combination. Acetoin and 3-pentanol did not exhibit any activity against P. syringae when used alone, but exhibited high bactericidal activity when combined with acetaldehyde. Isovaleraldehyde, when combined with acetaldehyde, 2-methyl-1-butanol, 1,4 cyclohexadiene, 1,3 cycloheptadiene or 4-methylcyclohexene completely inhibited the growth of P. syringae (Table 8). It is thought that the enhanced bioactivity of isovaleraldehyde and acetaldehyde when applied with other bioactive compounds could be attributed to different modes of action of the compounds, as they have diverse structures and are from varying chemical classes (alcohols, aldehydes, hydrocarbons).

TABLE-US-00008 TABLE 8 Bactericidal and synergistic effect of key Daldinia sp. (U254) compounds on biocidal activity against P. syringae. Isovaleraldehyde Acetoin 3-Pentanol Volume Growth Growth Growth (L) Scale Activity Scale Activity Scale Activity Control 2 2 2 Acetoin/Isoval/3- 10 0.25 2 2 Pentanol Acetaldehyde 10 + 20 0 Bactericidal 0.5 Bactericidal 0 Bactericidal 1,4-Cyclohexadeine 10 + 10 0 Bactericidal 2 NA 2-Methyl-1-butanol 10 + 10 0 Bactericidal 1 1 1,3-Cycloheptadeine 10 + 10 0 Bactericidal 2 NA 4-Methylcyclohexene 10 + 10 0 Bactericidal 2 NA F Pr. <0.001 <0.001 * LSD (5%) 0.32 0.41 *

Example 10Fungicidal Activity of 3-Pentanol, Acetoin and Isovaleraldehyde, Alone and in Synergy with Key Compounds from the Volatolome of Daldinia sp. (U254)

[0108] A fungicidal bioassay was established to evaluate the effect of key compounds from the volatolome of Daldinia sp. against the plant pathogenic fungus, Fusarium verticillioides, when applied alone and in synergy with other compounds from the volatolome of Daldinia sp. The pathogen was inoculated on to one third of the Petri plate by placing an agar plug of actively growing hyphae onto PDA. Candidate compounds were then aliquoted (10 microlitres) on to another third of the Petri plate on filter paper. In the synergy treatments a second compound (acetaldehyde, 1,4-cyclohexadiene, 2-methyl-1-butanol, 1,3 cycloheptadiene, 4-methylcyclohexene) was added to the final third of the Petri plate on filter paper. Plates were immediately sealed with Parafilm and maintained at room temperature. After 4 days the growth of the pathogen was determined by measuring the radius of the colony (on two alternate planes). Measurements were compared to the control and expressed as percentage inhibition relative to the control plate (no growth=100%). Compound combinations that exhibited 100% inhibition were assessed for fungistatic or fungicidal activity by placing the original plug onto a fresh PDA plate. Data were analysed using ANOVA as performed in GenStat, version 14. The experiment was fully randomised with 4 replicates for each compound combination.

[0109] Isovaleraldehyde and acetaldehyde both exhibited high fungicidal activity against F. verticillioides in isolation and combination with any of the compounds tested, completely (100%) inhibiting the growth of the pathogen (Table 9). In isolation, 3-pentanol exhibited moderate fungicidal activity against F. verticillioides, while Acetoin exhibited no activity. Both compounds showed 100% inhibition when combined with Acetaldehyde, and varying rates of inhibition (7.4% to 74.9%) when combined with other compounds.

TABLE-US-00009 TABLE 9 Fungicidal and synergistic effect of key Daldinia sp. (U254) compounds on biocidal activity against F. verticillioides. Isovaleraldehyde Acetoin 3-Pentanol Volume % Inhibition % Inhibition % Inhibition (uL)* (v control) Activity (v control) Activity (v control) Activity Acetoin/Isoval/3- 10 100.0% Fungicidal 0% 47.3% Pentanol Acetaldehyde 10 + 20 100.0% Fungicidal 100.0% Fungicidal 100.0% Fungicidal 1,4-Cyclohexadeine 10 + 10 100.0% Fungicidal 35.5% NA 2-Methyl-1-butanol 10 + 10 100.0% Fungicidal 52.2% 74.9% 1,3-Cycloheptadeine 10 + 10 100.0% Fungicidal 61.0% NA 4-Methylcyclohexene 10 + 10 100.0% Fungicidal 7.4% NA F Pr. * <0.001 * LSD (5%) * 21.41% *

[0110] It is to be understood that various alterations, modifications and/or additions may be made without departing from the spirit of the present invention as outlined herein.

[0111] As used herein, except where the context requires otherwise, the term comprise and variations of the term, such as comprising, comprises and comprised, are not intended to be in any way limiting or to exclude further additives, components, integers or steps.

[0112] Reference to any prior art in the specification is not, and should not be taken as, an acknowledgment or any form of suggestion that this prior art forms part of the common general knowledge in Australia or any other jurisdiction or that this prior art could reasonably be expected to be ascertained, understood and/or regarded as relevant by a person skilled in the art.

REFERENCES

[0113] 1. Stadler, M., Lsse, T., Fournier, J., Decock, C., Schmieschek, B., Tichy, H. V., & Peroh, D. (2014). A polyphasic taxonomy of Daldinia (Xylariaceae). Studies in mycology, 77, 1-143.