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Title:
USE OF NOOTKATONE FOR CONTROLLING PHYTOPATHOGENIC MICROBES
Document Type and Number:
WIPO Patent Application WO/2018/210870
Kind Code:
A1
Abstract:
The application relates to the methods for preventing, treating, or reducing an infection by phytopathogenic, facultative saprophytic or saprotrophic microbes (e.g. phytopathogenic fungi) in crop plants (pre-harvest), or in a crop plant material, or on surfaces in contact with the crop plant material, comprising: contacting them with a composition containing nootkatone and optionally an additional active ingredient; or comprising applying said composition to a vector pest. The application further relates to the compositions comprising nootkatone and optionally an additional active ingredient.

Inventors:
ARUNAN GOMATHI V (IN)
Application Number:
PCT/EP2018/062603
Publication Date:
November 22, 2018
Filing Date:
May 15, 2018
Export Citation:
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Assignee:
EVOLVA SA (CH)
ARUNAN GOMATHI V (IN)
International Classes:
A01N35/06; A01N31/16; A01N65/00; A01P1/00; A01P3/00; A23B7/154; A23B9/26; A61L2/18; B65B55/18
Domestic Patent References:
WO2015120412A12015-08-13
WO2018051344A12018-03-22
WO2017191138A12017-11-09
Foreign References:
US20150250166A12015-09-10
US20050187289A12005-08-25
US20150007368A12015-01-01
US20120246767A12012-09-27
US7442785B22008-10-28
US20040249219A12004-12-09
US6531303B12003-03-11
US6689593B22004-02-10
US20100151519A12010-06-17
Other References:
K. DANCEWICZ ET AL: "Deterrent activity of (+)-nootkatone and its derivatives towards the peach potato aphid (Myzus persicae Sulzer)", PROGRESS IN PLANT PROTECTION, vol. 52, no. 5, 1 January 2012 (2012-01-01), pages 221 - 225, XP055374348
DA-SONG YANG: "Chemical constituents from Ampelopsis cantoniensis and their anti- angiogenic activities Evolutionary ecology of alpine plants, especially the Qinghai-Tibet Plateau area View project Yong-Ping Yang Chinese Academy of Sciences", CHINESE TRADITIONAL AND HERBAL DRUGS, vol. 45, no. 7, 1 April 2014 (2014-04-01), pages 900 - 905, XP055487176, DOI: 10.7501/j.issn.0253-2670.2014.07.002
KATHARINA M SCORA ET AL: "Effect of volatiles on mycelium growth of Penicillium digitatum, P. italicum, and P. ulaiense", J. BASIC MICROBIOL., vol. 38, no. 5-6, 1 November 1998 (1998-11-01), pages 405 - 413, XP055487197
TING YANG ET AL: "Metabolic engineering of geranic acid in maize to achieve fungal resistance is compromised by novel glycosylation patterns", METABOLIC ENGINEERING, vol. 13, no. 4, 4 February 2011 (2011-02-04), US, pages 414 - 425, XP055487218, ISSN: 1096-7176, DOI: 10.1016/j.ymben.2011.01.011
TATJANA STEVIC ET AL: "Chemical composition and inhibitory activity of selected essential oils against fungi isolated from medicinal plants", LEKOVITE SIROVINE, vol. 34, no. 34, 1 January 2014 (2014-01-01), pages 69 - 80, XP055487224, ISSN: 0455-6224, DOI: 10.5937/leksir1434069S
RAMANDEEP KAUR ET AL: "Antifungal Potential of Inula racemosa Root Extract and Vetiveria zizanioides Root Essential Oil against Some Phytopathogenic Fungi", INDIAN JOURNAL OF ECOLOGY, vol. 43, no. 2, 19 October 2016 (2016-10-19), pages 631 - 636, XP055487241, DOI: http://indianecologicalsociety.com/society/wp-content/themes/ecology/pdf/Indian-Journal-of-Ecology-43-Special-issue-2-2016.pdf
P RAJI ET AL: "INHIBITORY EFFECT OF PLANT EXTRACTS AND PLANT OILS ON XANTHOMONAS ORYZAE PV ORYZAE, THE BACTERIAL BLIGHT PATHOGEN OF RICE Associate", INTERNATIONAL JOURNAL OF APPLIED AND NATURAL SCIENCES (IJANS), vol. 5, no. 2, 1 February 2016 (2016-02-01), pages 71 - 76, XP055487249
"Disease measurements in plant pathology", TRANSACTIONS OF THE BRITISH MYCOLOGICAL SOCIETY, vol. 31, no. 3-4, June 1948 (1948-06-01), pages 343 - 345
LENNOX, C. L.; SPOTTS, R. A., PLANT DIS., vol. 87, 2003, pages 645 - 649
BARDAS G. A ET AL., PEST MANAGEMENT SCIENCE, vol. 66, 2010, pages 967 - 973
LEROUX, P, BOTRYTIS: BIOLOGY, PATHOLOGY AND CONTROL, 2004, pages 195 - 222
FOOD SCI TECH INT, vol. 12, no. 4, 2006, pages 353 - 359
GIONFRIDDO ET AL.: "Elimination of Furocoumarins in Bergamot Peel Oil", PERFUMER & FLAVORIST., vol. 29, 2004, pages 48 - 52
FERREIRA MAIA ET AL.: "Plant-based insect repellents: a review of their efficacy, development and testing", MALARIA JOURNAL, vol. 10, no. l-ll, 2011
KEJLOVA ET AL.: "Phototoxicity of bergamot oil assessed by in vitro techniques in combination with human patch tests", TOXICOL IN VITRO, vol. 21, 2007, pages 1298 - 1303, XP022340072, DOI: doi:10.1016/j.tiv.2007.05.016
"Safety Assessment of Citrus-Derived Peel Oils as Used in Cosmetics", COSMETIC INGREDIENT REVIEW EXPERT PANEL FINAL REPORT, 30 September 2014 (2014-09-30), pages 1 - 31
TAKAHASHI ET AL., BIOTECHNOL BIOENG, vol. 97, no. 1, 2007, pages 170 - 181
HARTLEY ET AL., GENOME RES, vol. 10, 2000, pages 1788 - 1795
Attorney, Agent or Firm:
REES, Kerry (GB)
Download PDF:
Claims:
WHAT IS CLAIMED IS:

1. A method of preventing, treating, or reducing an infection by phytopathogenic, facultative saprophytic or saprotrophic microbes on a surface and/or in a pre-harvest propagated plant or propagated plant material, comprising contacting the surface and/or the propagated plant or propagated plant part with a composition comprising nootkatone.

2. The method according to claim 1 , wherein the propagated plant or part thereof is a root, seed, a leaf, a seedling, a shoot, a bud, a fruit, a blossom, a flower, a nut, or a mushroom.

3. The composition according to claim 1 or 2, comprising at least about 0.0001%, at least about 0.001%, at least about 0.01%, at least about 0.1%, at least about 1%, at least about 2%, at least about 5%, at least about 7.5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, or greater by weight nootkatone.

4. The method according to any of the preceding claims, wherein the composition comprises at least one additional active ingredient that is a fungicide, fungistatin, bactericide, bacteriostatin, or pesticide.

5. The method according to claim 4, wherein the at least one additional active ingredient is active against a phytopathogenic, facultative saprophytic or saprotrophic fungus.

6. The method according to claim 4 or 5, wherein the at least one additional active ingredient is active against a phytopathogenic, facultative saprophytic or saprotrophic filamentous fungus.

7. The method according to any of claims 4 - 6, wherein the at least one additional active ingredient is active against a phytopathogenic, facultative saprophytic or saprotrophic yeast.

8. The method according to any of claims 4 - 7, wherein the at least one additional active ingredient is active against a phytopathogenic, facultative saprophytic or saprotrophic bacteria.

9. The method according to any preceding claim, wherein the composition is formulated in a solid form, a liquid form, a suspension or an emulsion.

10. The method according to any preceding claim, wherein the method of application is spraying, atomizing, dusting, scattering, brushing on, submerging, dipping, coating, pouring, or rubbing.

11. The method according to any preceding claim, wherein the method of application is at least partially enclosing in a bag, net, container or plastic wrap.

12. The method according to any preceding claim, wherein the composition is applied before sowing or planting.

13. The method according to any preceding claim, wherein the composition is applied before or during flowering.

14. The method according to any preceding claim, wherein the composition is applied before harvesting.

15. The method according to any preceding claim, wherein the composition is applied during a period of quarantine.

16. The method according to any preceding claim, wherein the composition is applied during a period of high humidity and/or warmth.

17. The method according to any preceding claim, wherein propagated plant material comes from a propagated plant that is a cereal, a beet, a leguminous plant, an oil crop, a cucurbit, a fibre plant, a citrus plant, a vegetable plant, a plant in the laurel family, maize, canola, tobacco, a nut tree, coffee, sugar cane, tea, grape, hop, a plantain, a latex plant, or an ornamental plant.

18. The method according to any preceding claims, wherein the surface is a work surface, a conveyor belt, a door, a wall molding, a wall, a sheet of glass, a surface of a vehicle, a surface of equipment, a surface of packaging material, or a surface of a tool used to handle, process, or transport one or more pre-harvest food materials.

19. A method of preventing, treating, or reducing an infection by phytopathogenic, facultative saprophytic, or saprotrophic microbes in a pre-harvest propagated plant or propagated plant material, comprising applying a composition comprising nootkatone to a pest.

20. The method of claim 19, wherein the pest is a mite or a caterpillar.

21. The method of claim 20, wherein the mite is a cheese mite or a flour mite.

22. The method of claim 19, wherein the pest is a sap-sucking insect.

23. The method of claim 22, wherein the sap-sucking insect is an aphid or a thrips.

24. A composition for preventing, treating or reducing an infection by phytopathogenic, facultative saprophytic or saprotrophic microbes in a propagated plant, or propagated plant part, comprising nootkatone and an additional active ingredient.

25. The composition according to claim 24, wherein the at least one additional active ingredient is a fungicide, fungistatin, bactericide, bacteriostatin, or pesticide.

26. The composition according to claim 24 or 25, wherein the at least one additional active ingredient is active against a phytopathogenic, facultative saprophytic or saprotrophic fungus.

27. The composition according to any of claims 24 - 26, wherein the at least one additional active ingredient is active against a phytopathogenic, facultative saprophytic or saprotrophic filamentous fungus.

28. The composition according to any of claims 24 - 27, wherein the at least one additional active ingredient is active against a phytopathogenic, facultative saprophytic or saprotrophic yeast.

29. The composition according to any of claims 24 - 28, wherein the at least one additional active ingredient is active against a phytopathogenic, facultative saprophytic or saprotrophic bacteria.

30. The composition according to any of claims 24 - 29, wherein the composition is formulated in a solid form, a liquid form, a suspension or an emulsion.

31. The composition according to any of claims 24 - 30, comprising at least about 0.0001%, at least about 0.001%, at least about 0.01%, at least about 0.1%, at least about 1%, at least about 2%, at least about 5%, at least about 7.5%, at least about 10%, at least about 15%), at least about 20%>, at least about 25%>, or greater by weight nootkatone.

32. The composition according to any of claims 24 - 31, wherein the nootkatone is present in an amount that is greater than about 10 ppm, about 25 ppm, about 50 ppm, about 62.5 ppm, about 100 ppm, about 125 ppm, about 150 ppm, about 200 ppm, about 250 ppm, or about 500 ppm, or about 1000 ppm, or about 5000 ppm of the composition.

33. The composition according to any of claims 24-32, wherein the additional active ingredient is resveratrol.

34. Use of the composition according to claims 24 to 33 for preventing, treating, or reducing an infection by phytopathogenic, facultative saprophytic or saprotrophic microbes on a surface and/or in a pre-harvest propagated plant or propagated plant material.

Description:
USE OF NOOTKATONE FOR CONTROLLING PHYTOPATHOGENIC MICROBES

BACKGROUND OF THE INVENTION

Field of the Invention

[0001] The present invention relates to novel crop protection against fungal and/or bacterial infections and food preservation. The active ingredients according to the invention are distinguished by particularly good tolerance by plants and favorable ecological properties.

Description of Related Art

[0002] Modern agriculture has a focus on maximising productivity from the available land whilst minimalising environmental impact. However, significant portions of crops are lost annually due to pests and spoilage. Pest-control solutions should ideally be effective, environmentally friendly, sustainable and safe.

[0003] Nootkatone, a derivative of vaiencene, is a sesquiterpene naturally found in citrus oils, such as orange and grapefruit, and other plant matter. Vaiencene is formed by cyclization of the acyclic pyrophosphate terpene precursor, farnesyl diphosphate (FPP), and its oxidation results in the formation of nootkatone. While ofte used as a flavorant and fragrance, nootkatone has been shown to have additional bioactiviti.es, such pesticidal and insecticidal properties. Such properties from a natural compound, such as nootkatone, are quite desirable because the utility of synthetic chemical agents for combatting pests, insects, and the like suffers both from the perspective of effectiveness (i.e., development of resistance) but also from that of perception. Many people dislike the "chemical" smell of synthetic chemical agents and still others dislike the idea of releasing synthetic chemical agents into the environment. In addition, some synthetic chemical agents are poisonous to humans, as well.

[0004] The rising global human population is placing increasing stress on the current means of food production and agricultural land use. There is an environmental need to maximize the efficient use of land allocated to farming and commercial forestry, while simultaneously reducing the impact of modern farming and forestry practices to the surrounding natural environments. It has been estimated that the largest loss of food material is that of planted, sown, or otherwise nurtured crops and plant propagation material that are not fit for harvesting. This applies to material harvested for direct consumption or incorporation into human foodstuffs, and to material harvested for storage (i.e., seeds for propagation), for direct consumption or incorporation into foodstuffs for livestock, domesticated, companion or farmed animals, birds and fish.

[0005] One specific cause of food material loss is sap-sucking insects. Sap-sucking insects puncture cell walls of buds, fruit, leaves, shoots and stems of plants and suck up plant cell contents following injection of enzymes to assist in extraction. Not only does this feeding process damage plants, but sap-sucking insects can transmit diseases to the plants during feeding. Such pests are capable of very rapid infestation and are the cause of significant damage and loss in the agricultural system, in part, by creating surface lesions making the plant or plant part more susceptible to infection.

[0006] Other insect pests are also associated with significant pre- and post-harvest losses of crops, especially in developing countries. For example, caterpillars of certain members of the order Lepidoptera (i.e., the larvae of moths and butterflies) present significant economic challenges to humans by damaging or ruining crops. From an economic standpoint, species of the Tortricidae, Noctuidae, and Pyralidae families, such as armyworms, corn earworms, Pieris brassicae, cloth moths, and cotton bollworms are particularly problematic.

[0007] In addition, exposure of plants pre- or post-harvest to phytopathogenic, facultative saprophytic, or saprotrophic microbes represents another significant cause of food material loss. Some of these microbes cause rot in hundreds of important crops before harvest, such as apples, grapes, strawberries, raspberries, tomatoes, lettuce, broccoli, peas, and beans, as well as cut flowers.

[0008] Therefore, in light of the current challenges to crop protection, there is a growing need for effective, sustainable, environmentally friendly compositions and methodologies to protect crops and propagated materials.

SUMMARY OF THE INVENTION

[0009] Provided herein are effective natural compositions and methods of their use in crop protection. [0010] The inventors have surprisingly found that microbially-produced terpenes (such as nootkatone) are useful in the prevention and treatment of microbial infections of propagated plants and propagated plant materials.

[0011] In a first aspect, the invention provides a method of preventing, treating, or reducing an infection by phytopathogenic, facultative saprophytic or saprotrophic microbes on a surface and/or in a pre-harvest propagated plant or propagated plant material, comprising contacting the surface and/or the propagated plant or propagated plant part with a composition comprising nootkatone. In one embodiment of the first aspect, the propagated plant or part thereof is a root, seed, a leaf, a seedling, a shoot, a bud, a fruit, a blossom, a flower, a nut, or a mushroom. In one embodiment of the first aspect, at least about 0.0001%, at least about 0.001%, at least about 0.01%, at least about 0.1%, at least about 1%, at least about 2%), at least about 5%, at least about 7.5%, at least about 10%>, at least about 15%, at least about 20%, at least about 25%, or greater by weight nootkatone. In one embodiment of the first aspect, the composition comprises at least one additional active ingredient that is a fungicide, fungistatin, bactericide, bacteriostatin, or pesticide. In one embodiment of the first aspect, the at least one additional active ingredient is active against a phytopathogenic, facultative saprophytic, or saprotrophic fungus. In one embodiment of the first aspect, the at least one additional active ingredient is active against a phytopathogenic, facultative saprophytic, or saprotrophic filamentous fungus. In one embodiment of the first aspect, the at least one additional active ingredient is active against a phytopathogenic, facultative saprophytic, or saprotrophic yeast. In one embodiment of the first aspect, the at least one additional active ingredient is active against a phytopathogenic, facultative saprophytic, or saprotrophic bacteria. In one embodiment of the first aspect, the composition is formulated in a solid form, a liquid form, a suspension, or an emulsion. In one embodiment of the first aspect, the method of application is spraying, atomizing, dusting, scattering, brushing on, submerging, dipping, coating, pouring, or rubbing. In one embodiment of the first aspect, the method of application is at least partially enclosing in a bag, net, container, or plastic wrap. In one embodiment of the first aspect, the composition is applied before sowing or planting. In one embodiment of the first aspect, the composition is applied before or during flowering. In one embodiment of the first aspect, the composition is applied before harvesting. In one embodiment of the first aspect, the composition is applied during a period of quarantine. In one embodiment of the first aspect, the composition is applied during a period of high humidity and/or warmth. In one embodiment of the first aspect, propagated plant material comes from a propagated plant that is a cereal, a beet, a leguminous plant, an oil crop, a cucurbit, a fibre plant, a citrus plant, a vegetable plant, a plant in the laurel family, maize, canola, tobacco, a nut tree, coffee, sugar cane, tea, grape, hop, a plantain, a latex plant, or an ornamental plant. In one embodiment of the first aspect, the surface is a work surface, a conveyor belt, a door, a wall molding, a wall, a sheet of glass, a surface of a vehicle, a surface of equipment, a surface of packaging material, or a surface of a tool used to handle, process, or transport one or more pre-harvest food materials.

[0012] In a second aspect, the invention provides a method of preventing, treating, or reducing an infection by phytopathogenic, facultative saprophytic, or saprotrophic microbes in a pre-harvest propagated plant or propagated plant material, comprising applying a composition comprising nootkatone to a pest. In one embodiment of the second aspect, the pest is a mite. In one embodiment of the second aspect, the mite is a cheese mite or a flour mite. In one embodiment of the second aspect, the pest is a sap-sucking insect. In one embodiment of the second aspect, the sap-sucking insect is an aphid or a thrips.

[0013] In a third aspect, the invention provides a composition for preventing, treating or reducing an infection by phytopathogenic, facultative saprophytic or saprotrophic microbes in a propagated plant, or propagated plant part, comprising nootkatone and an additional active ingredient. In one embodiment of the third aspect, the at least one additional active ingredient is a fungicide, fungistatin, bactericide, bacteriostatin, or pesticide. In one embodiment of the third aspect, the at least one additional active ingredient is active against a phytopathogenic, facultative saprophytic or saprotrophic fungus. In one embodiment of the third aspect, the at least one additional active ingredient is active against a phytopathogenic, facultative saprophytic or saprotrophic filamentous fungus. In one embodiment of the third aspect, the at least one additional active ingredient is active against a phytopathogenic, facultative saprophytic or saprotrophic yeast. In one embodiment of the third aspect, the at least one additional active ingredient is active against a phytopathogenic, facultative saprophytic or saprotrophic bacteria. In one embodiment of the third aspect, the composition is formulated in a solid form, a liquid form, a suspension or an emulsion. In one embodiment, the composition of the third aspect includes at least about 0.0001%, at least about 0.001%), at least about 0.01%, at least about 0.1%>, at least about 1%, at least about 2%, at least about 5%, at least about 7.5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, or greater by weight nootkatone. In one embodiment of the third aspect, the nootkatone is present in an amount that is greater than about 10 ppm, about 25 ppm, about 50 ppm, about 62.5 ppm, about 100 ppm, about 125 ppm, about 150 ppm, about 200 ppm, about 250 ppm, about 500 ppm, about 1000 ppm, or about 5000 ppm of the composition. In another embodiment of the third aspect, the additional active ingredient is resveratrol. Use of the composition of the third aspect is further contemplated for preventing, treating, or reducing an infection by phytopathogenic, facultative saprophytic, or saprotrophic microbes on a surface and/or in a pre-harvest propagated plant or propagated plant material.

[0014] These and other features and advantages of the present invention will be more fully understood from the following detailed description taken together with the accompanying claims. It is noted that the scope of the claims is defined by the recitations therein and not by the specific discussion of features and advantages set forth in the present description.

DESCRIPTION OF DRAWINGS

[0015] FIG. 1 shows a biosynthetic pathway for nootkatone.

[0016] FIG. 2 shows percentage of food losses of fruit and vegetables worldwide.

[0017] FIG. 3 shows the percent inhibition by nootkatone on B. cinerea (strawberry isolate) mycelial growth.

[0018] FIG. 4 illustrates the EC 50 of nootkatone for B. Cinerea (strawberry isolate) mycelial growth inhibition.

[0019] FIG. 5 shows the percent inhibition by nootkatone on B. cinerea (grape isolate) mycelial growth.

[0020] FIG. 6 illustrates the EC 50 of commercial fungicides for B. cinerea (grape isolate) mycelial growth inhibition.

[0021] FIG. 7 illustrates the observation for B. Cinerea (grape isolate) after 22 days in 125 ppm and 250 ppm of nootkatone-treated plates, the magnified image shows the development of mature/big sclerotia suggesting a stress response. Bar = 500 μιη.

[0022] FIGS. 8A and 8B show the percent inhibition by nootkatone at lower concentrations on T. roseum (apple isolate) mycelial growth (FIG. 8A) and the percent inhibition by nootkatone at higher concentrations on T. roseum (apple isolate) mycelial growth (FIG. 8B).

[0023] FIG. 9 illustrates the EC 50 of nootkatone for T. roseum mycelial growth inhibition.

[0024] FIG. 10 shows the structure of the toxin alternariol.

[0025] FIG. 11 shows the percent inhibition by nootkatone on A. alternata mali (apple) mycelial growth observed on day 5.

[0026] FIG. 12 shows the percent inhibition by nootkatone on A. sp. (apple) mycelial growth observed on day 10.

[0027] FIG. 13 shows the percent inhibition by nootkatone on Colletotrichum gloeosporioides mycelial growth observed on day 8.

[0028] FIG. 14 illustrates the EC 50 of nootkatone for Colletotrichum gloeosporioides mycelial growth inhibition.

[0029] FIG. 15 shows the percent inhibition by nootkatone on Fusarium sp. mycelial growth observed on day 5.

[0030] FIG. 16 shows the percent inhibition by nootkatone on Fusarium sp. mycelial growth observed on day 10.

[0031] FIG. 17A shows a comparison of a control plate versus 50 ppm NKT treated plate versus a 250 ppm NKT treated plate.

[0032] FIG. 17B shows the observation for Fusarium sp. that after 20 days in 250 ppm nootkatone-treated plates, white, non-pigmented mycelia developed, whereas control plates growth purple mycelia.

[0033] FIGS. 18A-C show the effect of control (FIG.18A) and nootkatone (FIGS. 18B- C) on Botrytis cinerea (grape isolate) spore germination.

[0034] FIGS. 19A and 19B illustrate spore germination in control plates and Colletotrichum gloeosporioides plates treated with nootkatone (NKT). Plate 1 = control at 16 hr; Plate 2 = C. gloeosporioides treated with 7.8 ppm NKT at 16 hr; Plate 3 = C. gloeosporioides treated with 15.6 ppm NKT at 16 hr; Plate 4 = C. gloeosporioides treated with 31.2 ppm NKT at 16 hr; Plate 5 = C. gloeosporioides treated with 62.5 ppm NKT at 16 hr; Plate 6 = C. gloeosporioides treated with 125 ppm NKT at 16 hr; Plate 7 = C. gloeosporioides treated with 250 ppm NKT at 16 hr; Plate 9 = C. gloeosporioides treated with 15.6 ppm NKT at 48 hr; Plate 10 = C. gloeosporioides treated with 31.5 ppm NKT at 48 hr; and Plate 11 = C. gloeosporioides treated with 62.5 ppm NKT at 48 hr.

[0035] FIG. 20 shows spore germination inhibition by nootkatone on C. Gloeosporioides compared to carbendazim measured by OD at 600 nm.

[0036] FIGS. 21A and B illustrate spore germination in Rhizopus sp. in control plates and 50 ppm fluconazole (FIG. 21A) treated plates versus 125 ppm and 250 ppm nootkatone (FIG. 2 IB) at 16 hr.

[0037] FIGS. 22A-C shows control apples (FIG. 22A) versus nootkatone treated apples at 62.5 ppm (FIG. 22B) and 125 ppm (FIG. 22C) concentrations prior to inoculation with C. Gloeosporioides spores.

[0038] FIGS. 23A-L shows control (FIGS. 23A-C), nootkatone (FIGS. 23G-L), and carbendazim (FIGS. 23D-F) treated oranges.

[0039] FIGS. 24A-K show photographs of sprayed (SP) and dipped (D) oranges on observation Day 7 following treatment with control, carbendazim (carben), and nootkatone (NKT). Dl and D2 indicate that fruit was dipped for 1 or 2 minutes, respectively.

[0040] FIGS. 25A-D show photographs of sprayed and dipped oranges on observation Day 12 following treatment with 62.5 ppm NKT (FIGS. 25A and 25C) and 500 ^LImL Amistar TOP ® (FIGS. 25B and 25D).

[0041] FIGS. 26A-B show photographs of sprayed oranges on observation Day 15 following spray treatment with 125 ppm NKT (FIG. 26A) and 500 μΙ7ι Ι. Amistar TOP ® (FIG. 26B). The circle indicates mycelia inside the fruit.

[0042] FIGS. 27A-C show Alternaria spp. on cherry (FIG. 27A), musk melon (FIG. 27B), and apple (FIG. 27C).

[0043] FIGS. 28A-B show percent mycelial growth inhibition of Alternaria sp. under control conditions and following treatment with NKT alone, resveratrol (RESV) alone, and NKT + resveratrol (NKT+RESV) (FIG. 28A) and corresponding comparative Potato Dextrose Agar (PDA) plates (FIG. 28B).

[0044] FIGS. 29A-F illustrate Alternaria sp. spore germination at 24 hours under control conditions (FIG. 29A) and treated with NKT at 125 ppm (FIG. 29B), 62.5 ppm (FIG. 29C), 10 ppm carbendazim (FIG. 29D), and NKT+RESV at 62.5 ppm each (FIG. 29E) and 125 ppm (FIG. 29F).

[0045] FIGS. 30A-B show percent mycelial growth inhibition of Botryodiplodia theobromae following treatment with NKT at 50 ppm, 125 ppm, and 250 ppm, and Tebuconazole at 2 ppm, 10 ppm, and 50 ppm (FIG. 30A) and corresponding comparative PDA plates (FIG. 30B).

[0046] FIGS. 31A-F show spore germination of B. theobromae (BT) at 24 hours in a control plate (FIG. 31 A) and in plates treated with NKT at 500 ppm (FIG. 3 IB) and 250 ppm (FIG. 33C), 10 ppm Tebuconazole (FIG. 3 ID), and NKT+RESV at 500 ppm each (FIG. 3 IE) and 250 ppm each (FIG. 3 IF).

[0047] FIGS. 32A-D show images of Monilinia laxa conidia (FIG. 32A), conidia stained with blue lactophenol blue (FIG. 32B), M. laxa in cherry (FIG. 32C), and front and back side views of laxa on PDA plates (FIG. 32D).

[0048] FIGS. 33A-B show percent mycelial growth inhibition of M. laxa by NKT+RESV treatment (FIG. 33A) and corresponding comparative mycelial growth on PDA plates (FIG. 33B).

[0049] FIG. 34 shows post-harvest management of control, NKT+RESV treated, and Amistar TOP® treated sweet cherries.

[0050] FIGS. 35A-B show physical properties of sweet cherries following treatment with Amistar TOP® (FIG. 35A) and NKT+RESV (FIG. 35B).

[0051] FIGS. 36A-B show Aspergillus flavus (FIG. 36A) and Aspergillus niger (FIG. 36B) on PDA plates.

[0052] FIGA. 37A-E show spore germination in A. flavus plates at 24 hours under control conditions (FIG. 37A) and treated with NKT at 500 ppm (FIG. 37B) and 250 ppm (FIG. 37C) and Fluconazole at 500 ppm (FIG. 37D) and 250 ppm (FIG. 37E). Arrows indicate individual spores.

[0053] FIGS. 38A-B show percent inhibition of Aspergillus conidia growth at 72 hours after NKT and Fluconazole treatment (FIG. 38A) and corresponding images of Aspergillus conidia in a 96-well plate (FIG. 38B) treated with various concentrations of nootkatone (NKT) or Fluconazole. Optical density was measured at 600 nm to determine growth inhibition in the 96 well plate. [0054] FIGS. 39A-B show inhibitory effect of NKT on A. niger conidia (FIG. 39A), and microscopic images of a control plate (untreated) of A. niger conidia and A. niger conidia plates after 24 hours treatment with NKT at 500 ppm, 250 ppm, and 125 ppm (FIG. 39B).

[0055] FIGS. 40A-B show mycelial growth inhibition of A. niger by NKT and Tebuconazole treatment at 116 hours (FIG. 40A) and corresponding images of mycelial growth on PDA plates following treatment with NKT at 250 ppm and 62.5 ppm (FIG. 40B).

[0056] FIGS. 41A-G illustrate spore germination of Blue mold 24 hours after treatment with control (FIG. 41 A), NKT+RESV at 250 ppm (FIG. 41B), 125 ppm (FIG. 41C), and 62.5 ppm (FIG. 41D), and NKT at 250 ppm (FIG. 41E), 125 ppm (FIG. 41F), and 62.5 ppm (FIG. 41G).

[0057] FIGS. 42A-H show images of Colletotrichum gloeosporioides on various fruit and vegetables.

[0058] FIGS. 43A-C illustrate C. gloeosporioides conidia germination under control conditions (FIG. 43A) and following treatment with NKT (250 ppm) (FIG. 43B) and NKT (125 ppm) (FIG. 43C).

[0059] FIGS. 44A-E illustrate C. gloeosporioides conidia germination under control conditions (FIG. 44A) and following treatment with NKT at 62.5 ppm (FIG. 44B), 31.25 ppm (FIG. 44C), 15.625 ppm (FIG. 44D), and 7.8 ppm (FIG. 44E).

[0060] FIGS. 45A-D show conidia germination in a survival assay where conidia taken from control or treatment plates were replated on untreated plates. FIG. 45A is a control PDA plate. Conidia taken from 250 ppm NKT (FIG. 45B) and 125 ppm NKT (FIG. 45C) plates show minimal germination. FIG. 45D shows percentage of conidia germination inhibition measured by optical density at 600 nm of C. gloeosporioides treated with NKT or carbendazim.

[0061] FIGS. 46A-D illustrate Magnaporthe grisea conidia germination inhibition under control conditions (FIG. 46A) and following 24 hours of treatment with NKT+RESV at 250 ppm (FIG. 46B), 125 ppm (FIG. 46C), and 62.5 ppm (FIG. 46D).

[0062] FIGS. 47A-D illustrate M. grisea conidia germination inhibition at 24 hours under control conditions (FIG. 47A) and following treatment with by NKT at 250 ppm (FIG. 47B), 125 ppm (FIG. 47C), and 62.5 ppm (FIG. 47D). [0063] FIGS. 48A-E illustrate Botrytis cinerea spore germination inhibition at 18 hours under control conditions (FIG. 48A) and after NKT treatment at 250 ppm (FIG. 48B), 125 ppm (FIG. 48C), and 62.5 ppm (FIG. 48D), and NKT_ RESV at 62.5 ppm (FIG. 48E).

[0064] FIG. 49 shows B. cinerea mycelial growth inhibition by NKT, RESV, and NKT+RESV.

[0065] FIGS. 50A-B show inhibition of mycelial growth rate by NKT+RESV, NKT, and RESV from Day 1 to Day 5 (FIG. 50A) and from Day 5 to Day 10 (FIG. 50B).

[0066] FIGS. 51A-D show typical results of treating caterpillars on bushes in the open air with 0.25% NKT (v/v) aerosol spray. The caterpillars were frequently observed over 6 hours. All displayed the following timeline of response. FIG. 51A shows a caterpillar immediately prior to treatment. FIG. 5 IB shows the caterpillar immediately after treatment. FIG. 51C shows the caterpillar lhr after treatment. All caterpillars became inactive within the first hour after treatment and most died within 30 to 45 minutes following treatment. FIG. 5 ID shows the caterpillar 6 hrs after treatment. All caterpillars died and darkened within 6 hrs after treatment.

[0067] FIGS. 52A-E show typical effects of feeding caterpillars with collected leaves sprayed with 0.06% NKT. FIG. 52A shows caterpillars at the time of introduction to treated leaves, where all caterpillars were observed to be active. FIG. 52B shows the caterpillars of the negative control group at 6 hrs after introduction to untreated leaves, and all caterpillars were observed to be active and feeding. FIG. 52C shows the caterpillars introduced to treated leaves at 6 hrs after introduction to untreated leaves. All caterpillars displayed restricted motility and many became completely inactive. FIG. 52D shows the caterpillars of the negative control at 24 hrs after introduction to untreated leaves. All caterpillars were active and feeding. FIG. 52E shows caterpillars introduced to treated leaves at 24 hrs after introduction to untreated leaves. All caterpillars on treated leaves died.

[0068] FIG. 53 is a GC-FID chromatogram overlay of Frutarom® nootkatone (i.e., citrus-derived nootkatone) and the nootkatone (NxV) used for studies described herein (see Examples below).

[0069] FIG. 54 is a GC-MS NIST library match of an unknown peak in Frutarom® nootkatone. The peak was identified as limonene. No limonene nor bergapten was found in the nootkatone used in the present application.

[0070] FIG. 55 shows spore germination of Uncinula necator under control conditions. [0071] FIG. 56 shows spore germination of Uncinula necator treated with different concentrations of nootkatone.

[0072] Skilled artisans will appreciate that elements in the Figures are illustrated for simplicity and clarity and have not necessarily been drawn to scale. For example, the dimensions of some of the elements in the Figures can be exaggerated relative to other elements to help improve understanding of the embodiment(s) of the present invention.

DETAILED DESCRIPTION

[0073] All publications, patents and patent applications cited herein are hereby expressly incorporated by reference in their entirety for all purposes.

[0074] Before describing the present invention in detail, a number of terms will be defined. As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. For example, reference to "an active ingredient" means one or more active ingredients.

[0075] It is noted that terms like "preferably," "commonly," and "typically" are not utilized herein to limit the scope of the claimed invention or to imply that certain features are critical, essential, or even important to the structure or function of the claimed invention. Rather, these terms are merely intended to highlight alternative or additional features that can or cannot be utilized in a particular embodiment of the present invention.

[0076] For the purposes of describing and defining the present invention it is noted that the term "substantially" is utilized herein to represent the inherent degree of uncertainty that can be attributed to any quantitative comparison, value, measurement, or other representation. The term "substantially" is also utilized herein to represent the degree by which a quantitative representation can vary from a stated reference without resulting in a change in the basic function of the subject matter at issue.

[0077] As used herein, the term "about" refers to ±10% of any particular value.

[0078] As used herein, the terms "or" and "and/or" are utilized to describe multiple components in combination or exclusive of one another. For example, "x, y, and/or z" can refer to "x" alone, "y" alone, "z" alone, "x, y, and z," "(x and y) or z," "x or (y and z)," or "x or y or z." [0079] As used herein, the term "pest" refers to arthropod pests, such as, insects and arachnids, such as mites, that can eat, propagate on or in, live in or on propagated plant material and/or food materials. For example, a pest can include a sap-sucking insect. As another example, a pest can be a cheese mite or a flour mite. Further examples of pests include weevils, aphids, thrips, beetles, grain borers, and grain moths.

[0080] As used herein, the term "food materials" refers to pre-harvest and post-harvest propagated plants and propagated plant parts or materials, meats, dairy products, and derivatives thereof. Food materials can include processed foods, prepared foods, and food ingredients. For example, food materials include flours, food additives, grain derivatives, sugars, amino acids, and the like. Food materials are typically meant for consumption by humans or other animals. One example of food materials is cultivated crops.

[0081] As used herein, the term "consumable product" includes any product that is consumable by humans and/or animals (for example, horses, cows, sheep, goats, chickens, dogs, or cats). In certain embodiments, consumable products can include, but are not limited to all food materials, nutraceuticals, pharmaceuticals (i.e., any product placed in the mouth), cereal products, rice products, tapioca products, bread products, meat and meat products, jellies, jams, egg products, milk and dairy products, cheese products, butter and butter substitute products, milk substitute products, soy products, edible oils and fat products, beverages, carbonated beverages, fruit drinks, fruit juices, beverage powder, milk based beverage powder, food extracts, plant extracts, meat extracts, and combinations thereof. In some embodiments, consumable products can include propagated plant materials or parts thereof (for example, hay, flour, cotton, and linen). In one embodiment, in instances where the term food material is used, the term consumable product can be used, and/or the terms food material and consumable product can be used interchangeably.

[0082] As used herein, the term "pre-harvest" refers to a period of time prior to harvesting of a food material. For example, in the context of a plant or cultured food material, pre-harvest includes any time starting with when a seed is planted to when a fully mature crop is ready for harvest.

[0083] As used herein, the term "active ingredient" refers to a chemical compound or mixture of chemical compounds capable of treatment of at least one of a pest or a phytopathogenic microbe on any propagated plant, propagated plant material, or other surface, materials and substances disclosed herein. As such, the active ingredient may have properties including but not limited to insecticidal, pesticidal, antimicrobial, antibacterial, and/or antifungal properties against microbes capable of infecting, growing and reproducing on propagated plants, portions thereof, or propagated plant material. For example, the active ingredients may be effective against phytopathogenic microbes including but not limited to microorganisms from the following classes: Ascomycetes (for example, Glomerella, Colletotrichum, Trichothecium, Venturia, Podosphaera, Erysiphe, Monilinia, Mycosphaerella, Uncinula, Leotiomyceta); Basidiomycetes (for example, the genera Hemileia, Rhizoctonia, Puccinia); Fungi imperfecti (for example, Botrytis, Helminthosporium, Rhynchosporium, Fusarium, Septoria, Cercospora, Alternaria, Pyricularia and, in particular, Pseudocercosporella herpotrichoides); Oomycetes (for example, Phytophthora, Peronospora, Bremia, Pythium, Plasmopara); Firmicutes (Bacilli, Clostridia, Mollicutes); Proteobacteria (Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, Deltaproteobacteria, Epsilonproteobacteria, Zetaproteobacteria).

[0084] As used herein, the term "antimicrobial" is understood as being effective in preventing or reducing infection of one or more food materials by microscopic organisms such as phytopathogenic microbes or facultative saprophytic microbes. Active ingredients can have antifungal and/or antibacterial effects.

[0085] As used herein, the term "antifungal" is understood as being effective in preventing or reducing a fungal infection and includes fungicides that kill fungi or fungal spores, and fungistatics that inhibit the growth and/or reproduction of fungi.

[0086] As used herein, the term "antibacterial" is understood as being effective in preventing or reducing a bacterial infection and includes bactericides that kill bacteria and bacteriostatics that inhibit the reproduction of bacteria.

[0087] "Phytopathogenic or saprophytic microscopic" organisms and phytopathogenic microbes are used interchangeably and encompass, but are not limited to, fungi, bacteria, oomycetes, and phytoplasma that infect, grow and reproduce on one or more food materials. As used herein, "phytopathogenic microbes" may be pathogenic to propagated plants, may be lysotrophic, or may be facultative saprophytic capable of infecting stressed or dying propagated plants, possibly in combination with plant pathogens. Examples of phytopathogenic, facultative saprophytic or saprotrophic microbes include but are not limited to microorganisms from the following classes: Ascomycetes (for example Glomerella, Colletotrichum, Trichothecium, Venturia, Podosphaera, Erysiphe, Monilinia, Mycosphaerella, Uncinula, Leotiomyceta); Basidiomycetes (for example the genera Hemileia, Rhizoctonia, Puccinia); Fungi imperfecti (for example Botrytis, Helminthosporium, Rhynchosporium, Fusarium, Septoria, Cercospora, Alternaria, Pyricularia and, Pseudocercosporella herpotrichoides); Oomycetes (for example Phytophthora, Peronospora, Bremia, Pythium, Plasmopara); Firmicutes (Bacilli, Clostridia, Mollicutes); Proteobacteria (Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, Deltaproteobacteria, Epsilonproteobacteria, Zetaproteobacteria); Phytomyxea (for example Plasmodiophora and Spongospora); Phytoplasma, Spiroplasma, Penicillium glaucum, Botrytis vulgaris, and Oilium fructigenum.

[0088] As used herein, the term "prevention or treatment of phytopathogenic microbial infections" is used interchangeably with "prevention or treatment of facultative saprophytic microbial infections" and refers to a process by which a population of microbes capable of infecting and damaging one or more pre-harvest food materials are at least one of: killed, growth-inhibited, or inhibited from reproducing, on an object, a surface, or one or more pre- harvest food materials. In this context, prevention or treatment of phytopathogenic microbes may include any manner of treatment performed to reduce the population of phytopathogenic microbes. Examples of treatments include applying a composition containing nootkatone to the microbes. Treatment of phytopathogenic microbes may include a second or subsequent treatment to prevent recovery of the population of phytopathogenic microbes.

[0089] As used herein, the terms "surface" or "object to be treated" interchangeably refer to any pre-harvest food material surface area and/or any other surface material that phytopathogenic microbes may attempt to infect, or are surfaces and objects which could act as vectors for the transportation of phytopathogenic microbes between infected and uninfected pre-harvest food materials or surfaces. Examples of surfaces include, without limitation, work surfaces, conveyor belts, doors, wall moldings, walls, sheets of glass, or any surface of a vehicle, equipment, packaging material or tool used to handle, process, or transport one or more pre-harvest food materials, such as propagated plants or propagated plant material, plant fruit, or plant seed.

[0090] As used herein, the terms "an environment rich in propagated plant material," "agricultural areas," "forestry areas," and "locations with high concentrations of propagated plant material or crops or parts thereof susceptible to damage by phytopathogenic microbes" are used interchangeably and refer to one or more environments capable of harboring high concentrations of phytopathogenic microbes. These areas may be outdoors or at least partially enclosed such as an indoor or sheltered environment capable of sustaining a different microclimate to the external environment. Examples of such areas and environments include, but are not limited to, an agricultural field, a field of crops, an arable field, a greenhouse, an orchard, a polytunnel, an area for mushroom cultivation, an area of commercial flower cultivation, a commercial forest, a hydroponics facility, parks, gardens, flower shows, and indoor or outdoor storage areas for pre-harvest propagated plants or propagated plant material, plant fruit, or plant seed, including but not limited to a granary, a flower shop, a potato shed, a food processing factory, or containers used to transport or store propagated plant material, including but not limited to bags, boxes, crates, nets, jars, tubs, sacks, silos, conveyor belts, trailers, storage bins, refrigerators, freezers, packaging material, or plastic wrap.

[0091] As used herein, the term "propagated plant" includes any crop or plant that is deliberately sown, planted, transplanted, cultivated or nurtured by humans. It may refer, for example, to whole plants, field crops, fruit or nut trees, seedlings, young plants, or plant seeds. The term "propagated plant material," encompasses "material to be harvested," and the "commercially relevant portion of a crop or plant" and refers, for example, to plant extracts, shoots, sprouts, leaves, cuttings, roots, tubers, bulbs, rhizomes, grain, fruits, seeds, nuts, and flowers or other plant parts of cosmetic, aesthetic, or commercial value. Examples of contemplated crops include but are not limited to mushrooms, fruit trees, and fruit plants (citrus fruit trees, lemon trees, lime trees, orange trees, grapefruit trees, apple trees, apricot trees, pear trees, plum trees, cherry trees, grape vines, nectarine trees, peach trees, tangerine trees, raspberry canes, blueberry bushes, pineapple plants, banana trees, strawberry plants, tomato plants, pepper plants, chili bushes), cereal crops (wheat, barley, rye, oats, hay, rice, quinoa, millet, sorghum and related species), beet (sugar and fodder beet), leguminous plants (beans, lentils, peas, soya beans), oil crops (oilseed rape, mustard, poppies, olive trees, sunflower plants, coconut trees, castor plants, cocoa trees, groundnuts, oil palms), cucurbits (pumpkin plants, cucumber plants, melon plants), fiber plants (cotton, flax, hemp, jute), vegetables (spinach, lettuce, asparagus, cabbages, carrots, onions, potatoes, broccoli, kale, chard, eggplant), the laurel family (avocado, Cinnamonum, camphor), or plants such as maize, canola, tobacco, nuts, coffee bush, sugar cane, tea, hops, the plantain family and latex plants, and also ornamentals (flowers, shrubs, deciduous trees, conifers, roses, tulips, daffodils, orchids, lilies, chrysanthemums, gerberas, primrose, iris, carnation, lilac, sunflower). [0092] As used herein, the term "effective concentration" refers to a concentration of an active ingredient (such as nootkatone or a derivative thereof) within a composition such that when the composition is applied to a pre-harvest food material, such as a propagated plant or propagated plant material, or to a relevant surface, at least one of a pest or a population of phytopathogenic microbes is at least one of killed, growth-inhibited, or inhibited from reproducing.

[0093] As used herein, the term "nootkatone" refers to a compound seen in Figure 1 that may be synthesized, isolated, and purified from of a mixture of products produced in a host modified to express enzymes of the nootkatone biosynthetic pathway or that can be produced from naturally occurring sources, such as citrus plants. "Nootkatone" also refers to a mixture of chemical compounds containing or enriched for the nootkatone compound and derived from a modified host, such as a microorganism, or isolated or derived from plant extracts. "Nootkatone" further refers to derivatives and analogs thereof. For example, the nootkatone compound contemplated for use herein may be produced in vivo through expression of one or more enzymes involved in the nootkatone biosynthetic pathway in a recombinant yeast or in vitro using isolated, purified enzymes involved in the nootkatone biosynthetic pathway, such as those described in U.S. Patent Application Publication Nos. 2015/0007368 and 2012/0246767. Therefore, nootkatone as defined herein can differ chemically from other sources of nootkatone, such as extracts from plants and derivatives thereof, or may include such plant extracts and derivatives thereof.

[0094] As used herein, the term "nootkatone ex valencene" refers to nootkatone derived from oxidation of valencene that was produced by fermentation, such as by microorganisms harboring one or more valencene synthases and/or other molecules that catalyze formation of valencene. Further, nootkatone ex valencene refers to a combination of chemical compounds derived from oxidation of a valencene-containing fermentation product produced by culturing microorganisms harboring one or more valencene synthases and/or other molecules that catalyze formation of valencene. Nootkatone ex valencene can be purified to maximize the percent of nootkatone relative to other chemical compounds. For example, nootkatone ex valencene can be less than about 50%, about 50%, about 60%>, about 70%>, about 80%>, about 90%, or about 98% nootkatone.

[0095] Disclosed herein are nootkatone-containing compositions and methods of using the compositions that are effective at treating and preventing pest infestations and/or infections of phytopathogenic or facultative saprophytic microbes in a pre-harvest context. [0096] Some embodiments of the current disclosure aim to prevent or treat pest infestation or to reduce the frequency or prevalence of pest infestation of propagated plants, trees, flowers, fruit, propagated plant material or agricultural produce being grown for use in seed, food, or feed. In further embodiments, fields, flower beds, greenhouses, or other locations with high concentrations of propagated plant material susceptible to infestation by pests can be treated with nootkatone-containing compositions so as to reduce the frequency of infection or severity of damage to propagated plants and propagated plant material.

[0097] Some embodiments of the current disclosure aim to prevent or treat phytopathogenic or facultative saprophytic microbial infections, or to reduce the frequency or prevalence of phytopathogenic or facultative saprophytic microbial infections of propagated plants, trees, flowers, fruit, propagated plant material or agricultural produce being grown for use in seed, food, or feed. In further embodiments, fields, flower beds, greenhouses, or other locations with high concentrations of propagated plant material susceptible to infection by phytopathogenic or facultative saprophytic microbes can be treated with nootkatone- containing compositions so as to reduce the frequency of infection or severity of damage to propagated plants and propagated plant materials pre-harvest.

[0098] Some embodiments of the current disclosure aim to prevent, treat, or delay onset of pest infestation or to reduce the frequency, prevalence, or progression of pest infestation of cultivated crops pre-harvest.

[0099] Some embodiments of the current disclosure aim to prevent, treat, or delay onset of infection by phytopathogenic or facultative saprophytic microbes or to reduce the frequency, prevalence, or progression of infection by phytopathogenic or facultative saprophytic microbes of cultivated crops pre-harvest.

[00100] Some embodiments of the current disclosure aim to prevent, treat, or delay onset of pest infestation or to reduce the frequency, prevalence, or progression of pest infestation of surfaces.

[00101] Some embodiments of the current disclosure aim to prevent, treat, or delay onset of infection by phytopathogenic or facultative saprophytic microbes or to reduce the frequency, prevalence, or progression of infection by phytopathogenic or facultative saprophytic microbes of surfaces.

[00102] In another embodiment, the current disclosure provides methods and uses for a composition comprising nootkatone suitable for treating a surface, a food material, or locations with high concentrations of propagated plants or propagated plant parts susceptible to damage by phytopathogenic or facultative saprophytic microbial infections, for delaying the onset of or reducing the frequency of phytopathogenic or facultative saprophytic microbial-induced damage to propagated plants or propagated plant material pre-harvest, such as during cultivation.

[00103] Similarly, some embodiments of the current disclosure are intended to increase or maximize the commercial value of a propagated plant or propagated plant material by applying a nootkatone-containing composition to the propagated plant or propagated plant material to at least one of either prevent infection of the propagated plant or propagated plant material with a population of phytopathogenic or facultative saprophytic microbes, or treat such an infection resulting in maintained and/or improved propagated plant health and/or aesthetic appearance of the propagated plant.

[00104] In yet other embodiments, the disclosure aims to prevent or treat phytopathogenic or facultative saprophytic microbial infections, or to reduce the frequency or prevalence of phytopathogenic or facultative saprophytic microbial infections of seeds. Damage to seeds caused by phytopathogenic microorganisms can occur as early as storage of the seeds or when the seeds are introduced into the soil, or during and immediately after germination of the seeds. This phase is critical since the roots and shoots of the growing plants are particularly sensitive and even minor damage can lead to deformation, delayed development or to the death of the whole plant. In one embodiment, the compositions of the present disclosure can be applied to the seed, the medium to be implanted with the seed, or both through, for example, irrigation water.

[00105] Of particular importance in the present disclosure is the recognition of nootkatone's ability to reduce or prevent the occurrence of transmission of phytopathogenic or facultative saprophytic microbial diseases by sap-sucking insects by combatting both the microbial diseases and insects simultaneously. In this aspect, nootkatone has proven to be both an effective antimicrobial and an effective pesticide. The advantage of using a single compound with both biocidal activities is that a greater degree of disease control can be achieved within a shorter amount of time and in a single treatment of cultivated crops infested with sap-sucking insects and/or infected with the indicated microbial diseases. As a result, the double-acting compound can prevent the rapid spread of disease in areas of intensive plant propagation, such as in modern farms where large areas are almost exclusively populated by a single variety of a single species of propagated plant. Moreover, the advantage of a single active compound that combats both sap-sucking insects and the indicated microbial diseases can be particularly important in developing countries where fewer resources are available to spend on biocidal agents.

[00106] In one embodiment, the use of nootkatone provides a sustainable and biodegradable alternative to current active agents against phytopathogenic or facultative saprophytic microbes.

[00107] In several aspects of the invention, nootkatone (i) may be present in a formulation or kit with at least one additional (ii) active ingredient, pesticide, insecticide, fungicide or bactericide. Such compositions may be formulated for separate, simultaneous or successive administration. For separate or successive administration, (i) and (ii) may, for example, be provided as a kit. Without being restricted by specific examples, the second active ingredient may be, for example any suitable class of phytopathogenic pesticides such as azoxystrobin, myclobutanil, propiconazole, thiophanate methyl, ziram, hypochlorites, chloramines, dichloroisocyanurate and trichloroisocyanurate, wet chlorine, chlorine dioxide, peroxides, peracetic acid, potassium persulfate, sodium perborate, sodium percarbonate, and urea perhydrate, iodine, concentrated alcohols (such as ethanol, 1-propanol, called also n-propanol and 2-propanol, isopropanol and mixtures thereof), phenolic substances, hexachlorophene, triclosan, trichlorophenol, tribromophenol, pentachlorophenol, cationic surfactants, benzalkonium chloride, cetyl trimethylammonium bromide or chloride, didecyldimethylammonium chloride, cetylpyridinium chloride, benzethonium chloride chlorhexidine, glucoprotamine, octenidine dihydrochloride, colloidal silver, silver nitrate, copper sulfate, phosphoric acid, sulfuric acid, organochlorides, organophosphates, carbamates, pyrethroids, neonicotinoids, ryanoids.

[00108] In some embodiments, compositions comprising nootkatone or a derivative thereof, may be administered in a sprayable composition.

[00109] In some embodiments, compositions comprising nootkatone or a derivative thereof may be administered within water applied to propagated plants, such as in water used for irrigation or utilized in a predominantly aqueous growth media for the plant, such as in hydroponic growth.

[00110] In some embodiments, compositions comprising nootkatone or a derivative thereof, may be administered as a preventative treatment to prevent "colour break" or other damage to propagated plants or propagated plant material. [00111] In some embodiments, compositions comprising nootkatone may be administered to a surface on or within a vehicle including but not limited to an agricultural vehicle, forestry vehicle, or vehicle for transporting propagated plants, propagated plant produce, or agricultural or forestry equipment.

[00112] Surfaces to be treated for phytopathogenic or facultative saprophytic microbes can be any part of a propagated plant, propagated plant material, agricultural area, vehicles, or any agricultural, forestry, horticultural, food processing or food industry work surface, food handler, equipment, tools or storage containers. Such surfaces may comprise plant stems, shoots, buds, leaves, flowers, fruit, wood, metal, plastic, gloves, sheets of wrapping plastic, cotton, wool, silk, satin, or any fabric suitable for use in agriculture, forestry, floristry, food processing, food transport or food storage.

[00113] In some embodiments, nootkatone compositions may be combined with existing technologies to treat and/or prevent phytopathogenic or facultative saprophytic microbial infections. For example, a fabric suitable for wrapping propagated plant material for transport or storage may be contacted, impregnated or coated with a composition comprising nootkatone. An example of such a fabric is a thin transparent sheet or film (typically about 12 μιη to 8 μιη thick), herein referred to as a "plastic film," such as but not limited to plastic wrap or food wrap. Such sheets may be made from polyvinyl chloride, low density polyethylene, or polymers of glucose, including thin transparent sheets made from regenerated cellulose. Another example of a fabric that may be contacted, impregnated or coated with a composition comprising nootkatone is an "agrotextile," which is defined herein as a fabric typically having a knitted, woven, or nonwoven structure suitable for use in agriculture, horticulture, floristry, forestry, animal or bird husbandry, commercial or domestic use in gardens, greenhouses, and the like. The fibres or sheets used to manufacture agrotextiles include polypropylene, polyethylene, biodegradable plastic, or any inexpensive plant fibre such as jute or coir.

[00114] Agrotextiles are used as a ground cover and typically applied after sowing or planting a crop or ornamental plant. Agrotextiles can be air, water vapour, water, and/or light permeable and are effective at elevating ground temperature and retaining water content. Thus farmers, greenhouse owners and domestic users deploy agrotextiles to create a microclimate highly favourable for accelerated plant growth and for earlier planting or sowing with reduced risk of frost. However, agrotextiles make the conventional use of pesticides problematic and the created microclimate conditions of higher temperature and higher humidity may also encourage the growth of pests and in particular of phytopathogenic, facultative saprophytic or saprotrophic microbes. Hence, it is an objective of some embodiments of the current invention to provide an agrotextile coated with or including nootkatone and capable treating phytopathogenic, facultative saprophytic or saprotrophic microbes. Examples of agrotextiles coated with or including nootkatone may be at least a portion of a forcing cover, polytunnel, plant cover, fruit cover, insect screen, shade screen, blanching screen, ventilation screen, agro bag, crop net, fruit net, or nut bag. The provision of shade by a suspended agrotextile is beneficial for some propagated plants, but also for air drying some types of propagated plant material including but not limited to fruits (such as grapes, tomatoes, peppercorn, spices, herbs) and flowers (such as hops, lavender, sunflowers, roses and orchids). Agro bags, fruit nets and plastic film coated with or impregnated with a composition including nootkatone can also reduce the damage phytopathogenic, facultative saprophytic or saprotrophic microbes can cause to harvested crops or harvested propagated plant material. In this way, an agrotextile or plastic film coated, contacted or impregnated with a composition comprising stilbene is suitable for use in agriculture, horticulture, forestry, gardens, greenhouses, areas used to store or transport propagated plant material, food processing or kitchens. Further combinations of nootkatone-containing compositions and agricultural or food industry devices and methods are contemplated as described herein elsewhere.

COMPOSITIONS

[00115] The active ingredients contemplated herein are used in the form of compositions. The active ingredients can be applied to a food material, such as a propagated plant or propagated plant material before, simultaneously with, or after the harvest. The active ingredients can be applied, if desired, together with other carriers conventionally used in the art of formulation, surfactants or other additives which aid application. Suitable carriers and additives can be solid or liquid and are the substances expediently used in the art of formulation, for example natural or regenerated mineral materials, solvents, dispersants, wetting agents, adhesives, thickeners, binders or fertilizers.

[00116] Nootkatone-containing compositions contemplated herein can be formulated for direct application to a surface, a food material, and/or an environment rich in propagated plant material to reduce the population or as a prophylactic to prevent the growth of the population or spread of the population to other locations of phytopathogenic, facultative saprophytic or saprotrophic microbes, by exposing the subject to the nootkatone-containing composition. In addition, nootkatone-containing compositions contemplated herein can be formulated for application as a dip, such as by dispensing into or onto a zone or area of water in which the articles to be treated may be immersed. For example, seeds and/or seedlings can be dipped in a nootkatone-containing composition prior to planting. A further manner of application includes coating/impregnating surfaces and/or articles with nootkatone-containing compositions.

[00117] Generally and without limitation, compositions contemplated herein can be in the form of an aqueous liquid, an oil-based liquid, a concentrated liquid, a gel, a foam, an emulsion, a slurry, a paint, a clear coat, a wax, a block, a pellet, a puck, a granule, a powder, a capsule, a vesicle, an effervescent tablet, slow release tablet, an impregnated dissolvable sheet or film, an impregnated material, and combinations thereof.

[00118] In certain embodiments, a composition may be formulated as a liquid or aerosol formulation suitable for application in a spray, a roll on, a dip, detergents, carpet cleaner, durable water repellence formulations.

[00119] In certain embodiments, a composition may be formulated for application by dispensing into or onto an area of water suitable for use as an immersion dip or volume of washing water into which articles to be treated and/or plants may be at least partially submerged. In this context, the composition can be provided as an aerosol, a solution, an emulsion, an oil, a spray, a gel, a powder, a foam, a block, a pellet, a puck, a granule, a vesicle, a powder, a capsule, and combinations thereof.

[00120] In certain embodiments, a composition may be formulated comprising a portion of material such as a tissue, pad, cloth, sponge or sheet impregnated, immersed or coated with a liquid composition comprising nootkatone at a concentration of between 0.0001 - 5% by volume of the liquid composition or at a concentration of about 1 to about 10,000 parts per million (ppm). For example, a liquid composition can contain about 10, or about 25, or about 50, or about 75, or about 100, or about 200, or about 300, or about 400, or about 500 ppm nootkatone. The material can be impregnated, immersed and/or coated with a liquid composition including nootkatone at a concentration of between 0.0001 - 5% by volume of the liquid composition or at a concentration of about 1 to about 10,000 parts per million (ppm). For example, a material can be impregnated, immersed and/or coated with a liquid composition including nootkatone at a concentration of 10, or about 25, or about 50, or about 75, or about 100, or about 200, or about 300, or about 400, or about 500 ppm nootkatone. In certain aspects, the portion of material is a disposable thin sheet of material such as a tissue, a wet wipe, or a wet pad, similar to those sold under the Swiffer®, Pledge®, Windex®, Clorox® brands.

[00121] In other embodiments of the invention, compositions contemplated herein can contain a carrier and at least about 0.0001%, or at least about 0.001%, or at least about 0.01%), or at least about 0.1%>, or at least about 1%, or at least about 2%, or at least about 5%, or at least about 7.5%, or at least about 10%, or greater than about 10%, or greater than about 15%, or greater than about 20%, or greater than about 25%, or greater than about 50% by weight stilbene. In some applications, nootkatone can be present in an amount that is greater than about 60%, about 70%, about 80%, about 90%, about 95% or about 99% by weight of the composition. In one example, the provided compositions contain nootkatone in an amount at or about 0.0001% to at or about 2%, or about 0.001% to at or about 5%, or about 0.01% to at or about 75% by weight of the composition. In another example, a composition may contain nootkatone in an amount of from at or about 1% to at or about 50% by weight of the composition. In another example, a composition may contain nootkatone in an amount of from at or about 5% to at or about 40% by weight of the composition. In another example, a composition may contain nootkatone in an amount of from at or about 10% to at or about 30% by weight of the composition. In another example, a composition may contain nootkatone in an amount of from at or about 15% to at or about 25% by weight of the composition. In another example, a composition may contain nootkatone in an amount of from at or about 1% to at or about 90% by weight of the composition. In another example, a composition may contain nootkatone in an amount of about 10%, or about 15%, or about 20%), or about 25%, or about 30%, or about 50% by weight of the composition. In another example, a composition may contain nootkatone in an amount of up to about 99% or more by weight of the composition.

[00122] In certain embodiments, compositions contemplated herein can contain a carrier and at least about 1 ppm to about 1,000 ppm of the nootkatone (one ppm is equivalent to 1 milligram of something per liter of water (mg/L) or 1 milligram of something per kilogram soil (mg/kg)). In some embodiments, the compositions comprise at least about 1 ppm, or at least about 10 ppm, or at least about 20 ppm, or at least about 25 ppm, or at least about 50 ppm, or at least about 62.5 ppm, or at least about 100 ppm, or at least about 125 ppm, or at least about 150 ppm, or at least about 200 ppm, or at least about 250 ppm, or at least about 500 ppm or 1,000 ppm or greater of nootkatone. In some applications, nootkatone can be present in an amount that is greater than about 10 ppm, about 25 ppm, about 50 ppm, about 62.5 ppm, about 100 ppm, about 125 ppm, about 150 ppm, about 200 ppm, about 250 ppm, or about 500 ppm, or about 1000 ppm, or about 5000 ppm of the composition.

[00123] In one particular embodiment, a contemplated nootkatone-containing composition is provided as a concentrate. For example, a nootkatone-containing composition may be provided as a 20X, or a 1 OX, or a 5X, or a 3X concentrate that can be diluted by an end user with an appropriate solvent to achieve a IX working concentration. Alternatively, a nootkatone-containing composition may be provided to an end user at a IX working concentration. However, any concentration is contemplated for use herein. For example, compositions provided as concentrates can be used without dilution at all or may be diluted from a highly concentrated concentrate (e.g., about 20X to about 100X) to some multiple of concentration higher than IX, such as 2X, 2.5X, 3X, etc. or can be used at a more dilute concentration, such as 1/2X, 1/4X, 1/lOX, etc. While concentrates are more preferred as commercially available goods, the industry and end consumers typically apply dilute compositions to food materials, such as propagated plants and propagated plant material and materials and surfaces.

[00124] In another embodiment, a contemplated composition may be seen in Table No. 1, where ingredients can be measured in percent volume per volume, percent weight per volume, or percent by weight.

[00125] Table No. 1. Contemplated composition formulation

[00126] In certain embodiments, compositions contemplated herein may include nootkatone and one or more additional active ingredients. The one or more additional active ingredients may be effective against at least one of pests of propagated plants, phytopathogenic microbes, facultative saprophytic microbes, and/or saprophytic microbes. In some aspects, the one or more additional active ingredient may have toxicity for insects, or bacteria or fungi. In some aspects, the additional active ingredients may be a bactericide, bacteriostatic, fungicide, fungistatic, microbicide, microbistatic, pesticide, herbicide, insecticide, and larvicide. In some aspects, the additional active ingredients may have highly selective toxicity for a specific sap-sucking insect, such as aphids or thrips.

[00127] In another embodiment, an additional active ingredient can be lipid-soluble so that it can be released over an extended period of time, such as, for example, approximately 2 months.

[00128] Further examples of additional active ingredients include plant essential oil compounds or derivatives thereof. Examples include aldehyde C16 (pure), a-terpineol, amyl cinnamic aldehyde, amyl salicylate, anisic aldehyde, benzyl alcohol, benzyl acetate, cinnamaldehyde, cinnamic alcohol, carvacrol, carveol, citral, citronellal, citronellol, p- cymene, diethyl phthalate, dimethyl salicylate, dipropylene glycol, eucalyptol (cineole) eugenol, is-eugenol, galaxolide, geraniol, guaiacol, ionone, menthol, methyl salicylate, methyl anthranilate, methyl ionone, methyl salicylate, a stilbene (such as resveratrol), a- pheliandrene, pennyroyal oil perillaldehyde, 1- or 2-phenyl ethyl alcohol, 1- or 2-phenyl ethyl propionate, piperonal, piperonyl acetate, piperonyl alcohol, D-pulegone, terpinen-4-ol, terpinyl acetate, 4-tert butylcyclohexyl acetate, thyme oil, thymol, metabolites of trans- anethole, vanillin, and ethyl vanillin.

[00129] In another embodiment, a contemplated composition may include a nootkatone to additional active ingredient ratio of about 1 : 10, or about 1 :8, or about 1 :6, or about 1 :4, or about 1 :2, or about 1 : 1, or about 2: 1, or about 4:1, or about 6: 1, or about 8: 1, or about 10: 1.

[00130] In other embodiments, compositions contemplated herein can include nootkatone in combination with one or more additives, such as a fragrance, a preservative, a propellant, a pH buffering agent, a UV blocker, a pigment, a dye, a surfactant, an emulsifier, a solvent, a salt, an acid, a base, an emollient, a sugar, and combinations thereof. Additional additives include disinfectants, such as quaternary ammonium compounds, phenol-based antimicrobial agents, and botanical oils with disinfectant properties.

[00131] In other embodiments, nootkatone-containing compositions can include a carrier, such as an aqueous liquid carrier, water, a saline, a gel, an inert powder, a zeolite, a cellulosic material, a microcapsule, an alcohol such as ethanol, a hydrocarbon, a polymer, a wax, a fat, an oil, and the like. Other examples of carriers include agrotextiles and plastic film or food wrap. Some carriers include time release materials where a nootkatone-containing composition may be released over a period of hours, or days, or weeks. Additional carriers include agricultural substances, such as, a natural fertilizer, a chemical fertilizer, mulch, compost, top soil, potting soil, vermiculite or other soil amendments, or agricultural waste products, and mixtures thereof. Solid carriers which are used for example for dusts and dispersible powders are typically comprised of ground natural minerals, such as calcite, talc, kaolin, montmorillonite or attapulgite. To improve the physical properties, it is also possible to add highly-disperse silica or highly-disperse absorptive polymers. Suitable paniculate adsorptive carriers for granules are porous types, for example pumice, brick grit, sepiolite or bentonite, and suitable non-sorptive carrier materials are, for example, calcite or sand. Moreover, a large number of pregranulated materials of inorganic or organic nature can be used, such as, in particular, dolomite or comminuted plant residues.

[00132] Carriers may be added to a composition in an amount of about 10%, or about 15%, or about 20%, or about 25%, or about 30%, or about 50% by weight of the composition. In some applications, a carrier can be present in an amount that is at or greater than about 60%, about 70%, about 80%, about 90%, about 95%, or about 99% by weight of the composition.

[00133] Solvents suitable for incorporation into compositions according to some aspects of the current invention include but are not limited to aromatic hydrocarbons, preferably the fractions Cs to C 12 , for example xylene mixtures or substituted naphthalenes, phthalic esters, such as dibutyl phthalate or dioctyl phthalate, aliphatic hydrocarbons, such as cyclohexane or paraffins, alcohols and glycols and also their ethers and esters, such as ethanol, ethylene glycol, ethylene glycol monomethyl ether or ethylene glycol monoethyl ether, ketones, such as cyclohexanone, strongly polar solvents, such as N-methyl-2-pyrrolidone, dimethyl sulfoxide or dimethylformamide, and free or epoxidized vegetable oils, such as epoxidized coconut oil or soya oil; water or solvents derived from natural products.

[00134] Depending on the nature of the active ingredients to be formulated, suitable surface-active compounds are non-ionic, cationic and/or anionic surfactants having good emulsifying, dispersing and wetting properties. Surfactants are also to be understood as meaning mixtures of surfactants.

[00135] Additives which aid application of compositions according to some aspects of the invention include natural or synthetic phospholipids from the series of the cephalins and lecithins, for example phosphatidylethanolamine, phosphatidylserine, phosphatidylglycerine, or ly so lecithin. [00136] Table Nos. 2-7 below provide examples of dispensable compositions where nootkatone is the active ingredient.

[00137] Table No. 2. Formulation Examples of Wettable Powders

[00138] The active ingredient is mixed thoroughly with the additives and the mixture is ground thoroughly in a suitable mill. This gives wettable powders which can be diluted with water to give suspensions of any desired concentration.

[00139] Table No. 3. Formulation Examples of an Emulsion Concentrate

[00140] Emulsions of any desired dilution which can be employed in crop protection can be prepared from this concentrate by dilution with water.

[00141] Table No. 4. Formulation Examples of Dusts

[00142] Ready-to-use dusts are obtained by mixing the active ingredient with the carrier and grinding the mixture in a suitable mill. Such powders can also be used for dry seed treatment.

[00143] Table No. 5. Formulation Examples of Extruder Granules

[00144] The active ingredient is mixed with the additives, ground and moistened with water. This mixture is extruded and subsequently dried in a stream of air.

[00145] Table No. 6. Formulation Examples of Coated Granules

[00146] In a mixer, the finely ground active ingredient is applied uniformly to the kaolin which has been moistened with polyethylene glycol. This gives dust-free coated granules.

[00147] Table No. 7. Formulation Examples of a Suspension Concentrate

[00148] This gives a suspension concentrate from which suspensions of any desired dilution can be prepared by dilution with water. Such dilutions can be used for treating live plants and plant propagation material by means of spraying, pouring-on or immersion and for protecting them against sap-sucking insect infestation and microbial infection.

METHODS

[00149] According to some embodiments of the current invention, compositions comprising active ingredients may be applied once per day, once per week, twice per week, once per two weeks, once per month, once per two months, once per three months, or once per lifecycle of the object or food material to which the composition is being applied. Compositions according to aspects of the current invention may be employed as pure active ingredients or, preferably, together with the auxiliaries conventionally used in the art of formulation and are therefore processed in a known manner to give, for example, emulsion concentrates, spreadable pastes, ready-to-spray or ready-to-dilute solutions, dilute emulsions, wettable powders, soluble powders, dusts, granules, or encapsulations, for example in polymeric materials. The methods of application, such as spraying, atomizing, dusting, scattering, brushing on, submerging, coating, pouring or rubbing, and the type of composition are selected to suit the intended aims and prevailing circumstances.

[00150] A preferred method of applying a composition including an active ingredient is to apply the composition to the aerial parts of the plant, especially the foliage (foliar application). Number and rates of application depend on the biological and climatic environment of the pest and/or microbial pathogen. Nootkatone or a composition containing nootkatone can also be directly applied to seed kernels, grains, plants, or fruits for the purposes of coating, either by soaking the roots, seed kernels, grains, plants, or fruits in succession with a liquid preparation of an active ingredient or by coating them with a moist or dry preparation which already comprises the active ingredients. In addition, other types of application to plants are possible in specific cases, for example the targeted treatment of buds or fruit-bearing parts of the plant.

[00151] In an embodiment, propagated plant material or propagated plants can be treated in combination with a biocide, for example, when plant breeding to produce resistant varieties.

[00152] In yet another embodiment, propagated plant material or propagated plants can be treated during a period of high humidity and/or warmth.

[00153] In other embodiments, propagated plant material or propagated plants can be treated prior to harvest and then subjected to at least one subsequent treatment after harvest (e.g., a pre-harvest and post-harvest treatment), prior to transportation or during a period of quarantine or storage. In some aspects the subsequent treatment may be the same as the first, or may comprise a different treatment including but not restricted to any of refrigerating, freezing, heating to 30°C or higher, treatment with red light, treatment with ultra-violet light, treatment with a soap spray, applying a composition including, for example, organochlorides, organophosphates, carbamates, pyrethroids, neonicotinoids, ryanoids (such as DDT, methoxychlor, diazinon, oxamyl, carbofuran, methomyl, dinotefuran, cyfluthrin, tetramethrin, acetamiprid, thiamethoxam, chlorantraniliprole or flubendiamide), and combinations thereof. For example, in one aspect of the invention, an agricultural area or environment rich in propagated plant material is treated with a composition comprising nootkatone or a derivative thereof, and is then subjected to a subsequent treatment with a composition comprising a stilbenoid (such as resveratrol) or a derivative thereof.

[00154] Advantageous amounts per application of the compositions comprising active ingredients (active ingredient = a.i.) are typically 1 g to 1 kg of a.i./ha, in particular 10 g to 100 g of a.i./ha, particularly preferably 15 g to 70 g of a.i./ha. For the coating of propagated plants and propagated plant material, including but not limited to roots, seed kernels, grains, fruits or harvested seed, the rates of application are typically 0.0001 g - 100 g of a.i./Liter of solution, 0.001 g - 75 g of a.i./L of solution, 0.01 g - 50 g of a.i./L of solution, 0.1 g - 25 g of a.i./L of solution, 1.0 g - 10 g of a.i./L of solution, preferably 0.01 g - 1.0 g of a.i./L of solution, or 0.1 g - 10 g of a.i./L of solution or 0.0001 g - 100 g of a.i. per 1000 kg, 0.001 g - 75 g of a.i. per 1000 kg, 0.01 g - 50 g of a.i. per 1000 kg, 0.1 g - 25 g of a.i. per 1000 kg, 1.0 g - 10 g of a.i. per 1000 kg, or preferably 0.01 g - 1.0 g of a.i. per 1000 kg of plant material, or 0.1 g - 10 g of a.i. per 1000 kg of roots, seed kernels, grains, plants, or fruits, or other propagated plant material.

[00155] In some embodiments, contemplated methods include treating agricultural or forestry areas, greenhouses or other artificial microclimates such as within human dwellings or under sheets of agrotextiles. In some embodiments, treatment with a composition including a nootkatone may be performed at a temperature between 0 and 25 C, preferably between 5 C and 20 C. In some embodiments, treatment with a composition comprising a nootkatone may be performed prior to the growing season, or in the first two months of the growing season, or once the minimum overnight temperature exceeds at least 7 C, preferably at least IO C.

[00156] Various methods according to some aspects of the current invention may be employed to contact propagated plants, propagated plant material, surfaces, areas rich in propagated plant parts, agricultural areas susceptible to infection by phytopathogenic, facultative saprophytic or saprotrophic microbes with nootkatone-containing compositions. Such methods may include addition of nootkatone-containing compositions to water in which the propagated plant material, surface or area to be treated may be rubbed, wiped, brushed, or sprayed.

[00157] Nootkatone can be applied, such as by directly pouring the composition into the water or placing a composition dispenser within a sink, rain collection receptacle, tank, irrigation channel, hand pump spray or any other appropriately sized receptacle such that the surface, plant, portion of a plant, object or environment to be treated comes into contact with the nootkatone at an effective concentration of, for example, between 1 and 2,000 ppm. For example, an effective concentration of nootkatone can be at least about 1 ppm, or at least about 10 ppm, or at least about 20 ppm, or at least about 25 ppm, or at least about 50 ppm, or at least about 62.5 ppm, or at least about 100 ppm, or at least about 125 ppm, or at least about 150 ppm, or at least about 200 ppm, or at least about 250 ppm, or at least about 500 ppm or 1,000 ppm or greater. The plant, portion of a plant, surface, object or environment to be treated may be exposed to any of the contemplated of nootkatone-including compositions for about 1 second to about 24 hours before rinsing or treatment with uv light, or the applied nootkatone-containing composition may be left without active removal to degrade naturally. In a preferred embodiment of one aspect of the current invention, the plant, portion of a plant, surface, object or environment to be treated is exposed to an effective amount of nootkatone, such as, at concentration of 250 ppm, for 15 seconds.

[00158] In a further embodiment, methods of application to a subject, surface or plant material of an effective concentration of nootkatone by liquid, spray, powder, or wash is preferably performed in a commercial or domestic area for growing plants such as an agricultural field, forest, flowerbed, a polytunnel, greenhouse, conservatory, office, home, and/or dwelling.

Dispensers/ Applicators

[00159] In some embodiments, dispensers or applicators for dispensing or applying a composition contemplated herein are intended to be reused. For example, upon dispensing a nootkatone-containing composition, the dispenser or applicator can be refilled. In other embodiments, a dispenser or applicator is a single -use device or substance that functions as a nootkatone composition carrier that is itself dispensed or degraded. For example, a dispenser or applicator (i.e., carrier) can be an agricultural substance for distribution in, on, and/or around agricultural areas, such as a natural fertilizer, a chemical fertilizer, mulch, compost, top soil, potting soil, vermiculite or other soil amendments, or agricultural waste products. Such agricultural substances may themselves be dispensed or applied by spreaders and other means as known in the art.

[00160] Topical compositions disclosed herein may be dispensed using a dispenser or applicator including one or more of a spray bottle, a brush, a dropper, a sponge, a soft-tipped marking device with reservoir, pressurized dispenser, an aerosol can, a roll on bottle, a wipe, a tissue, a duster, and other devices suitable for application to surfaces, objects, propagated plants or areas rich in propagated plant material. For example, propagated plants, propagated plant material, surfaces, areas rich in propagated plant parts, agricultural areas susceptible to infection by phytopathogenic, facultative saprophytic or saprotrophic microbes may be sprayed, brushed, wiped, dipped, and/or soaked with a nootkatone-containing composition.

[00161] In one embodiment, compositions contemplated herein may be applied to one or more surfaces using an applicator having a reservoir for carrying a composition in a wet form and/or a dry form. Examples of applicators that may be used include an aerosol container with a spray nozzle with or without a spray straw to focus delivery of the composition, a spray gun, an impregnated sheet, film, and/or matrix where the composition is released onto the surface by a releasing agent, such as water or other carrier. Additional examples include a pump sprayer, a trigger sprayer, a pressurized spraying device, a sponge, a squeegee, an airbrush, a brush, or a roller. The composition may alternatively be applied by spraying or dispersing over at least a portion of an agricultural area susceptible to infection phytopathogenic, facultative saprophytic or saprotrophic microbes, including but not limited to spraying from a tractor, irrigation spray, helicopter, or crop duster or airplane.

[00162] Another aspect of the current invention includes pretreatment of surfaces, objects, propagated plants, propagated plant material, surfaces, areas rich in propagated plant parts, agricultural areas susceptible to infection by phytopathogenic, facultative saprophytic or saprotrophic microbes with nootkatone-containing compositions to prevent said microbes from spreading and/or increasing in population size. This may be accomplished by coating the surfaces or objects with compositions that resist removal from the surface and contain an amount of nootkatone, such as a paint, a clear coat, a wax, an oil, an adhesive, a resin, a cleaning solution, and combinations thereof. Another approach includes lining the surfaces, objects, or areas rich in propagated plant material, with one or more nootkatone-impregnated materials, such as thermoplastic or thermoset sheets, plastic wrap, paperboard, or cardboard impregnated with nootkatone. For example, a nootkatone-impregnated agrotextile may be used to at least partially enclose a plant growing area (including but not limited to a greenhouse or flower bed) or a transport container or receptacle, including boxes, bins, cartons, etc., or storage area for plants or portions of plants, such as barns, elevators, etc.

[00163] In an embodiment, a propagated plant part or propagated plant material is pretreated by at least partially enclosing in a plastic container, plastic wrap or plastic film impregnated, coated or contacted with a nootkatone-containing composition so as to reduce the susceptibility to infection by phytopathogenic, facultative saprophytic or saprotrophic microbes. The plastic container, plastic wrap or plastic film or may be at least partially air and/or water permeable. The plastic container, plastic wrap or plastic film may be a bioplastic or biodegradable plastic, preferably also comprising a biodegradable plasticizing agent. The pretreatment or preventative composition comprising a biodegradable plastic, biodegradable plasticizer and nootkatone is preferred in aspects of the invention relating to methods of farming, transportation and storage of propagated plants and/or propagated plant materials with the highest sustainability and minimal environmental impact of waste materials.

[00164] In a further embodiment, when the dispenser is a disposable thin sheet of material such as a tissue, a wet wipe, or a wet pad, such dispensers may be used to treat individual propagated plant parts or propagated plant materials by physically removing at least a portion of a population of phytopathogenic, facultative saprophytic or saprotrophic microbes, for example, by wiping. At the same time as the physical removal of the phytopathogenic, facultative saprophytic or saprotrophic microbes, a protective residue, layer, or film of nootkatone-containing composition is deposited on the treated surface to prevent reinfection. In this way, treatment may be two-fold: physical removal and chemical disinfection and/or inhibition. Such sheets of material may be prepackaged for use such as in resealable, liquid- impervious pouches.

[00165] A further treatment approach is to construct surfaces, objects, or storage or transport receptacles with nootkatone-impregnated or nootkatone-coated materials, such as plastics, wood, cloth, textiles, composites, or porous materials to prevent re-infection of propagated plants, propagated plant parts and agricultural equipment between harvests, plantings, or other suitable interval. Such an approach is particularly suitable for construction of furniture, greenhouses, agrotextiles, gloves, crates, boxes, vases, pots or bags suitable for growing, transporting, handling, or displaying of propagated plants or propagated plant materials. The approaches disclosed herein can be used alone or in any combination.

EXAMPLES

[00166] The Examples that follow are illustrative of specific embodiments of the invention, and various uses thereof. They are set forth for explanatory purposes only and are not taken as limiting the invention. In particular, these examples establish the effectiveness of the disclosed compositions against phytopathogenic, facultative saprophytic, or saprotrophic microbes. Therefore, treatment in pre-harvest crop cultivation context will be effective against such microbes.

[00167] An antimicrobial effect is present if the actions of the active ingredients show a significantly lower disease incidence, disease severity, or index of infection than the untreated plants. The microbiological infection (index of infection) is assessed as described in (Disease measurements in plant pathology, Transactions of the British Mycological Society, 31( 3-4):343-45, June 1948). For example, disease incidence can be represented by the percentage or proportion of diseased plants, leaves, stalks, fruits, blossoms or other parts in a sample of plants (percentage = {number of infected plants/total number of plants assessed} x 100). Assessment of disease incidence can be suitable for determining early stage infection and can provide a general indication of the prevalence in a given population of plants. Disease severity can be another way to represent a microbial infection and can be more appropriate in diseases like rusts, downy and powdery mildew, leaf spots or other similar diseases. Disease severity can be represented by the percentage of the plant covered by a symptom, lesion or damage caused by the disease (for example, 1, 5, 10, 20 or 50% of leaf area infected). Disease severity can be put on a scale of 0-10 (i.e., 0 = no disease on leaf and pods; 1 = small brown spot covering <1% leaf area (pin point spots on fruit); 3 = brown sunken spots 1-10% leaf area (< 1% fruit area); 5 = brown spots 11-25%) leaf area (1-10% fruit areas); 7 = circular brown sunken spots 26-50%) leaf area (11-25%) fruit area); 9 = circular to irregular >51% leaf area (>26% fruit area).

[00168] A) Crop protection by nootkatone.

[00169] Example Al Action against Puccinia recondita in wheat in a greenhouse

[00170] 7-day-old wheat plants are sprayed to drip point with a spray mixture prepared from a formulated active ingredient or a spray mixture not containing the active ingredient (control). After 24 hr, the treated and untreated plants are infected with a conidia suspension of the Puccinia recondita fungus. Treated and untreated plants are subsequently incubated for 7 days in a greenhouse at a relative atmospheric humidity of 90-100% and 20° C. Ten days post-infection, the incidence and/or severity of the microbiological infestation is assessed. The treated plants show a significant lower index of infection than the untreated plants.

[00171] Example A2 Action Against Erysiphe graminis in Wheat in the Open

[00172] In a field trial (10 m 2 ), winter wheat cv. "Bernina" in the growth phase is sprayed with a mixture prepared with a wettable powder of the active ingredient or a spray mixture not containing the active ingredient (control). Infection is allowed to occur naturally. 10 days post-infection, microbiological infestation is assessed. The treated plants show a significant lower index of infection than the untreated plants.

[00173] Example A3 Action Against Cercospora nicotianae in Tobacco Plants

[00174] 6-week-old tobacco plants (cv. "Burley") are sprayed to drip point with a spray mixture prepared from a formulated active ingredient or a spray mixture not containing the active ingredient (control). After 24 hr, the treated and untreated plants are infected with a spore suspension of Cercospora nicotianae (Ciba No. 295; max. 150,000 per ml), and subsequently incubated for 5 days at 20-22°C and a relative atmospheric humidity of 70- 90%. 10 days post-infection, the incidence and/or severity of fungal infestation is assessed and compared with the infestation on untreated plants. The treated plants show a significant lower index of infection than the untreated plants.

[00175] Example A4 Action Against Penicillium italicum in Oranges by coating

[00176] 40 oranges cv. "Valencia" are sprayed with a spray mixture prepared with the wettable powder of the active ingredient or a spray mixture not containing the active ingredient (control). After 24 hr, the treated and untreated oranges are infected with a conidia suspension of the Penicillium italicum fungus. Treated and untreated oranges are subsequently incubated for 7 days at a relative atmospheric humidity of 90-100%) and 20° C. 10 days post-infection, the incidence and/or severity of microbiological infestation is assessed. The treated plants show a significant lower index of infection than the untreated oranges.

[00177] Example A5 Action Against Sphaerotheca fuliginea in Cucumber by coating

[00178] 40 cucumbers (Cucumis sativus) are dipped in a liquid containing active ingredient or in a similar liquid not containing the active ingredient (control). After 24 hr, the treated and untreated cucumbers are infected with a conidia suspension of the Sphaerotheca fuliginea fungus. Treated and untreated cucumbers are subsequently incubated for 7 days at a relative atmospheric humidity of 90-100% and 20° C. 10 days post-infection, the incidence and/or severity of microbiological infestation is assessed. The treated plants show a significant lower index of infection than the untreated cucumbers.

[00179] B) Crop protection by nootkatone.

[00180] Example B 1 Action Against Erysiphe graminis in Wheat

[00181] In a field trial (10 m 2 ), winter wheat cv. "Kanzler" in growth phase 31-32 is sprayed with a spray mixture prepared with a wettable powder of the active ingredient. Infection was permitted to occur naturally. The treated plants show a significant lower index of infection than the untreated plants.

[00182] Example B2 Action Against Pyricularia oryzae in Rice in the Open

[00183] On a 12 m 2 plot, rice plants are sprayed with a spray mixture prepared from a formulated active ingredient or a spray mixture not containing the active ingredient (control). Infection is permitted to occur naturally. For evaluation, the leaf area infested with the Pyricularia oryzae fungus is measured 44 days post-application. The treated plants show a significant lower index of infection than the untreated plants.

[00184] Example B3 Action against Cercospora nicotianae in Tobacco Plants

[00185] 6-week-old tobacco plants (cv. "Burley") are sprayed to drip point with a spray mixture prepared from a formulated active ingredient or a spray mixture not containing the active ingredient (control). After 24h, the treated and untreated plants are infected with a spore suspension of Cercospora nicotianae (Ciba No. 295; max. 150,000 per ml), and subsequently incubated for 5 days at 20-22°C and a relative atmospheric humidity of 70- 90%). 10 days post-infection, the incidence and/or severity of fungal infestation is assessed and compared with the infestation on untreated plants. The treated plants show a significant lower index of infection than the untreated plants.

[00186] C) Crop protection by nootkatone.

[00187] Example CI Action Against Pseudoperonospora cubensis in Cucumber plants

[00188] 16- to 19-day-old cucumber plants ("Wisconsin") are sprayed to drip point with a spray mixture prepared from a formulated active ingredient or a spray mixture not containing the active ingredient (control). After 24h, the treated and untreated plants are infected with a sporangia of Pseudoperonospora cubensis (strain 365, Ciba; max. 5000 per ml), and the treated plants are subsequently incubated for 1-2 days at 18-20° C. 10 days post- infection, the incidence and/or severity of microbiological infestation is assessed. The treated plants show a significant lower index of infection than the untreated plants.

[00189] D) Crop protection by nootkatone.

[00190] Action against Phytophythora infestans in tomato plants infested with aphids

[00191] Twenty to thirty apterous adult aphids are introduced to 8-week-old tomato plants. Fourteen days after the aphids are introduced, the infested plants are sprayed to drip point with a spray mixture prepared from a formulated combination of active ingredients resveratrol and nootkatone or a spray mixture not containing the active ingredients (control). After 24h, the treated and untreated plants are infected with a spore suspension of Phytophythora infestans (max. 150,000 per ml), and subsequently incubated for 5 days at 20- 22°C and a relative atmospheric humidity of 70-90%. 10 days post-infection, the incidence and/or severity of fungal infestation is assessed and compared with the infestation on untreated plants. The treated plants show a significant lower index of infection than the untreated plants.

Example No. 1. Studies on effect of nootkatone against fungal pathogens causing post-harvest damage in fruits and vegetables.

INTRODUCTION

[00192] Loss in farming can be due to many factors, and one of the major factors is postharvest disease, which can occur at any time during postharvest handling, from harvest to consumption. According to a study done from 2012-2014, on "Assessment of Quantitative Harvest and Post-Harvest Losses of Major Crops and Commodities in India" by ICAR- CIPHET, the percentage of post-harvest losses is given below:

[00193] Table No. 8. Percentage of post-harvest losses per crop

[00194] According to the report, post-harvest loss of fruits and vegetables is as much as 16% with a value of up to $6.3 billion. According to the Food and Agriculture Organization of the United Nations, in the fruits and vegetables sector (see Fig. 2), losses in agricultural production dominate for all three industrialized regions, mostly due to post-harvest fruit and vegetable grading caused by quality standards set by retailers.

[00195] Particularly for fruits and vegetables, it is important to consider reduction in quantity and quality, as some diseases may not render produce unsaleable, but still can reduce product value. Botrytis cinerea infection alone causes rot in 200 economically important crops. In addition to economic loss, many rot diseases pose a potential health risk. For example, mycotoxin contamination is a major concern when a diseased fruit/vegetable is used for further food processing.

[00196] Fungi are mostly responsible for the post-harvest diseases of fruits and vegetables. The most common postharvest disease causing fungi are Penicillium, Alternaria, Fusarium, Botrytis, CoUetotrichum, and Geotrichum, though there are many others. One of the major groups of post-harvest disease is "quiescent" or "latent" post-harvest disease, where the pathogen initiates infection of the host usually before harvest, but remains dormant until the physiological status of the host tissue becomes favorable for the infection to precede. Examples of quiescent infections include anthracnose of various tropical fruit caused by CoUetotrichum spp. and grey mold of strawberry caused by Botrytis cinerea. The other major group of post-harvest diseases arises from infections initiated during and after harvest. Often these infections occur through surface wounds created by mechanical or insect injury. Common post-harvest diseases resulting from wound infections include blue and green mold (caused by Penicillium spp.) and transit rot (caused by Rhizopus).

[00197] Table No. 9 cites some of the important fungal pathogens, the diseases they cause and their host (fruits or vegetables):

[00198] Table No. 9. Fungal Pathogens and Related Diseases

spergi us niger B ac mo Mango; n ons

[00199] Many of the chemical pesticides used for field applications cannot be used for post-harvest applications due to their toxicity. Moreover, many pathogens are developing resistance to the chemical fungicides available in the market. Therefore, it is important to find effective economical and natural solutions to preserve perishable fruits and vegetables.

Materials and methods

[00200] The test pathogens: Colletotrichum gloeosporioides, Fusarium sp. Alternaria alternata apple pathotype mali (A. mali), Alternaria alternata, Botrytis cinerea, Trichothecium roseum, Penicillium spp. and Rhizopus sp. were all isolated from infected fruits. Each pathogen was isolated in house.

[00201] Isolation of pathogens: Infected fruits were surface sterilized with 0.1% mercuric chloride for 2 minutes and then washed in sterile distilled water thrice by soaking for 2 minutes. The infected portions were excised and moisture was removed by placing in sterile tissue paper. These bits of tissue were kept on potato dextrose agar (PDA) Petri plate with streptomycin 100 ppm to avoid bacterial contamination and incubated for 3-8 days at 25 ± 0.5°C. The hyphal tips, which grew out from the infected tissue were isolated and sub- cultured onto PDA and brought into pure culture.

[00202] Pathogens Isolated:

Colletotrichum gloeosporioides - isolated from infected mango fruit var neelam Fusarium sp. - isolated from infected caster seeds

Alternaria alternata apple pathotype mali (A. mali), - isolated from infected apple leaf, second isolate from infected apple Botrytis cinerea - isolated from infected strawberry fruits

Botrytis cinerea - isolated from infected grapes

Trichothecium roseum - isolated from infected apple fruit

Rhizopus sp. isolated from infected tomato fruit

[00203] Pathogenicity test: The pathogenicity of isolated pathogens was verified by inoculating the fungus on fruits and by analysing the symptoms caused.

[00204] Preparation of media: 39 grams of PDA from Hi media (MH096-500G) was dissolved in 900 mL of double-distilled water and mixed well. Media pH was checked with filter paper and adjusted to 5.0-5.5 with 1 N NaOH and 1 N HC1.

[00205] Preparation of compound and assay plates: Nootkatone (NxV, see Examples below) produced by with 98% purity was used in all studies. Nootkatone at 250 mg/ml in ethanol was used as stock solution and dilutions either in water or in media were made. In use, the required quantity of compound was added to media at 45°-50°C and mixed vigorously to make a miscible solution of nootkatone in media, which was immediately poured (18-20 mL PDA per plate) in sterile Petri plates with an 8 cm diameter. Control plates with only media and control plates with 0.1% ethanol (to compensate ethanol concentration used for nootkatone dilutions of 250 ppm) were prepared. Poisoned food plate method was used to test the inhibitory effect of compound on the growth of fungi.

[00206] Inoculum preparation and inoculation: Test PDA plates were inoculated with actively growing mycelial discs of 0.5 cm diameter, cut with a cork borer from fungal culture growing on PDA plate. Test plates with nootkatone, commercial comparators, and respective control plates were incubated at 24-26°C.

[00207] Observations: Radial mycelial growth was measured, when hyphae in control touched the edge of the Petri plate, i.e., the diameter of the culture is 8 cm in control. The diameter of the growth of the pathogens were measured with a glass/transparent scale by looking against light. Growth was recorded as the actual growth from the edge of the disc. Growth measurements were made in four directions. If growth did not form a smooth circle and grew with undulating margins (mostly due to compound effect), then the least and highest regions were measured to average out the rate of growth.

[00208] Calculations: Four directions were selected with least, medium, and highest diameter growth. The average of three to four measurements was calculated for each plate. The inoculated disc size was subtracted from the diameter of mycelial growth. Once again, the average of three replicate plates was calculated with Standard Error (SE).

[00209] Formula for % of inhibition:

{(growth in control - growth in treated) / growth in control} X 100

[00210] Calculation of Effective concentration 50 (EC 50 ): The EC 50 was calculated from a dose response curve fitting with logistic model using Curve expert 1.4.

RESULTS

[00211] Overall, nootkatone exhibited varied percentages of inhibition on the mycelial growth of CoUetotrichum gloeosporioides, Fusarium sp., Alternaria alternate apple pathotype mali (A. mali), Botrytis cinerea, and Trichothecium roseum. Consolidated results of all the pathogens tested for radial mycelial growth inhibition by NKT are shown in Table No. 10 below.

[00212] Table No. 10. Mycelial growth inhibition of Pathogens by Nootkatone.

Effect of Nootkatone on two strains of Botrytis cinerea

[00213] Nootkatone was observed to be highly effective in inhibiting radial mycelial growth of two resistant strains of B. cinerea with an EC 50 of 140-145 ppm.

[00214] Botrytis cinerea belongs to Ascomycota division of fungi. Botrytis cinerea is a Necrotrophic fungi. B. cinerea is classified by Fungicide resistance action committee in PATHOGEN RISK LIST (December 2013) as high risk pathogen because it develops fast resistance against many fungicides. Moreover, B. cinerea rot occurs in -200 economically important crops including cabbage, lettuce, broccoli, beans, grape, strawberry, raspberry, blackberry, roses, dendrobium (orchid flowers) and many more.

[00215] Both strains of B. cinerea were detected as resistant strains to existing fungicides. B. cinerea strawberry isolate is resistant to carbendazim (Car R ) (Lennox, C. L., and Spotts, R. A. 2003. Plant Dis. 87:645-649) and moderately resistant to conazoles (Azole MR ) (Leroux, P, 2004 in Botrytis: biology, pathology and control 195-222). Mutation at 198 AA from GAG (Glu) to GCG (Ala) in the tubulin beta chain, which causes resistance to carbendazim, was observed in this strain.

[00216] Effect of Nootkatone on B. cinerea (strawberry isolate): Nootkatone exhibited about 40% inhibition at 125 ppm & 85% inhibition at 250 ppm on the mycelial growth of B. cinerea (strawberry isolate) when compared with control. The positive control, ketaconazole, did not inhibit the mycelial growth of B. cinerea {see Fig. 3). The EC 50 of nootkatone for B. cinerea (strawberry isolate) is 145 μg/mL (shown in Fig. 4). The EC 50 of conventional fungicides for B. cinerea (strawberry isolate) were determined for comparison: carbendazim is more than 2000 μg/mL, and ketoconazole is 10 μg/mL.

[00217] A separate experiment to determine the effects of higher concentrations of nootkatone on B. cinerea (strawberry isolate) was performed similar to that above. The results are shown in Table No. 11.

[00218] Table No. 11. Mycelial growth inhibition of B. cinerea (strawberry isolate) by nootkatone.

[00219] Effect of Nootkatone on B. cinerea (grape isolate): Nootkatone exhibited about 43% inhibition at 125 ppm & 73% inhibition at 250 ppm on the mycelial growth of B. cinerea (grape isolate) when compared with control. The comparator ketaconazole inhibits the mycelial growth of B. Cinerea (grape isolate) to 50%> at 10 ppm {see Fig. 5). EC 50 values were determined as shown in Fig. 6. Fungicide resistance in the B. cinerea (grape isolate) was determined to be Car R , Azoxystrobin R , Azole s , and Iprodione s . One interesting observation for B. Cinerea (grape isolate) was that after 22 days, in 125 ppm and 250 ppm of NKT treated plates, mature/big sclerotia were observed {see Fig. 7). Sclerotia are dormant structures that form during times of stress, e.g., lack of food, excessive heat, or exposure to toxic chemicals. However, in the controls only small primordial structures of sclerotia were observed.

[00220] EC50 for B. cinerea (grape isolate) by compounds: EC50 for nootkatone was 140 μg/mL, for iprodione was 1.8 μg/mL, for carbendazim was more than 500 μg/ml, for tebuconazole was 200 ng/mL, and for azoxystrobin was more than 500 μg/mL (tested without alternate respiration inhibitor).

[00221] EC 50 for B. cinerea (grape isolate) by compounds to determine cut off (to assess the sensitivity): EC 50 for iprodione (pure) - 10.0 μg/mL, for carbendazim - 10.0 μg/mL of benzimidazole, for azoxystrobin - 10.0 μg/mL and for tebuconazole - 300 ng/mL (Lennox, C. L., and Spotts, R. A. 2003. Plant Dis. 87:645-649, Bardas G. A, et al, 2010. Pest Management Science 66: 967-973 and Leroux, P, 2004 in Botrytis: biology, pathology and control 195-222).

[00222] Baseline sensitivity: based on the EC 50 of commercial compounds tested as an index to determine resistance, B. cinerea (grape isolate) is Car R , Azoxystrobin R , Azole s , and Iprodione s . Fungicides of the class Methyl Benzimidazole Carbamates (MBC) inhibit β- tubulin assembly during mitosis. Compounds of this group include benomyl, carbendazim, fuberidazole, and thiabendazole. Resistance is common in many fungal species. Several target site mutations, mostly E198A/G/K, F200Y are present in the β-tubulin gene. A mutation at 198AA of the tubulin gene from glutamic acid to alanine confers resistance to carbendazim - sequence analysis of the tubulin gene in the grape isolate confirmed presence of the mutation.

[00223] Conclusion

[00224] Nootkatone is a highly effective inhibitor of radial mycelial growth of two conventional fungicide resistant strains of B. cinerea with an EC 50 of 140-145 ppm. Nootkatone must have a different mechanism of action than conazole, strobilurins, benzimidazole, and iprodione. As a result of these observations, nootkatone-containing compositions can be used to treat diseases caused by resistant strains of B. cinerea in the field as well as post-harvest. Effect of Nootkatone on Trichothecium roseum

[00225] Nootkatone was observed to be highly effective in inhibiting radial mycelial growth of T. roseum with an EC 50 of 25 ppm. This is the lowest EC 50 observed for any of the five pathogens tested.

[00226] Trichothecium roseum is characterized by its flat and granular colonies which are initially white and develop to be light pink in color. T. roseum is distinctive from other species of the genus with its characteristic zig-zag patterned chained conidia, and two celled tear drop shaped conidia with attachment mark.

[00227] Diseases caused by T. roseum on fruits and vegetables: Sporadic occurrences are reported of T. roseum infection on many plants and but it is mostly observed in association with other pathogens. T. roseum causes fruit rot on grapes, oranges, apples, tomatoes, muskmelons, watermelons, bananas, peaches, prunes, nectarines, plums, corn, ear rot on maize, and it also affects mushrooms.

[00228] Toxins produced by T. roseum in food material and its effect: T. roseum produces numerous toxins including trichothecenes, mycotoxicin T-2, and neosolaniol, which can be found in apples, including red delicious, Fuji, and Ralls cultivars. T. roseum toxins can penetrate apple fruit tissues. Mycotoxicin T-2 is tetracyclic sesquiterpenoid 12,13- epoxytrichothene ring system, which is very stable and not degraded during storage/milling and cooking/processing of food. Mycotoxicin T-2 has an LD 50 of approximately 1 mg/kg for humans. Longer exposure in human leads to the development of alimentary toxic aleukia (ATA) and within 9 weeks, the bone marrow will slowly degenerate. It is a cytotoxic compound.

[00229] Radial mycelial growth inhibition of T. roseum by Nootkatone: Nootkatone exhibited 58% inhibition and 88% inhibition at 25 ppm and 250 ppm, respectively (see Fig. 8A). A treatment at higher concentrations of nootkatone is shown in Fig. 8B. The EC 50 of nootkatone against radial mycelial growth of T. roseum was 23 ppm (23 μg/mL; see Fig. 9). The comparator, carbendazim, inhibited the mycelial growth of T. roseum to 50% at 5.0 ppm.

Effect of Nootkatone on Alternaria alternata mali [00230] Alternaria alternata mali damages all pomes (apples, pears, etc.), mangoes, stone fruit, cucurbits, tomatoes, eggplants, capsicum, and brassicas. A. alternata is the most common organism causing rotten apples. Disease severity depends on the wounds on the apple. Pathogens are present in the field.

[00231] Pathogen identification: Mycelia at early stages are white, with profuse growth. Mycelia turn pale olivaceous to olivaceous-brown colour colonies at maturity due to production of conidiophores. A. alternata produces multicellular conidia in chains in an acropetal manner. Conidia are light olivaceous to dark brown in colour, with varied shapes from obclavate to mostly ellipsoidal, muriform having tapered apex with 1 to 3 longitudinal and 2-10 transverse septa.

[00232] Toxins produced by A. alternata in food material and its effect: A. alternata produces many toxins namely, altemariol, altertoxins I, II, III, tenuazonic acid, and tenuazonic acid. Altemariol {see Fig. 10) is a topoisomerase I and II poison. Occurrence of altemariol in apple juice, cranberry juice, grape juice, prune nectar, raspberry juice, red wine, and lentils has been reported. Tenuazonic acid was reported to be toxic to several animal species, e.g. mice, chicken, and dogs.

[00233] Radial mycelial growth inhibition of A. alternata mali by Nootkatone: 250 ppm NKT (98% pure) inhibited A. alternata mali to 57%>, on day 5 at 4.5 cm diameter growth of pathogen {see Fig. 11). Inhibition was reduced to 46%> on 10 th day when the pathogen reached full Petri plate (8.5 cm) size {see Fig. 12). This may be due to volatiles being diffused as the days of incubation increased.

Effect of Nootkatone on Colletotrichum gloeosporioides

[00234] Colletotrichum gloeosporioides causes a disease called anthracnose in more than 500 plants and causes damage in crop yield. C. gloeosporioides causes bitter rot in pomes (apples and pears), peaches, bananas, mangoes, papayas, and strawberries and in solanaceous vegetables such as eggplant, capsicum bell pepper, chilies, and cucurbit and legumes and smudge in onions.

[00235] Pathogen identification: Colletotrichum gloeosporioides is isolated from infected mangoes in house. It produces cottony mycelia with white colour. At maturity, conidiomata develop and form a fruit body called an acervulus, which is black in colour. When conidia are produced from conidiophores, they turn a salmon colour. Conidia are rod- shaped with length of about 10-15 μιη and a width of about 5-8 μιη.

[00236] Radial mycelial growth inhibition of C. gloeosporioides by Nootkatone

[00237] Radial mycelial growth of C. gloeosporioides was inhibited by Nootkatone to 61% and 71% at 125 ppm and 250 ppm, respectively {see Fig. 13). EC50 was calculated as 93 ppm (93 μg/mL) by non-linear curve-fitting in curve expert software {see Fig. 14). At 5 ppm Ketaconazole exhibited 50% inhibition of radial mycelial growth of C. gloeosporioides.

[00238] Effect of Nootkatone on Fusarium spp.

[00239] Fusarium causes rot during storage. It affects many fruits and most damage is caused in bananas and pineapples. It affects many vegetables such as cucurbits, tomatoes, eggplants, capsicum, asparagus, potatoes, corn, onions, and garlic. Fusarium causes damping off, seed germination inhibition, and damage in corn and maize {Sholberg, P.L., Conway, W.S., 2001. Postharvest pathology) .

[00240] Pathogen identification of Fusarium sp.: Fusarium produces cottony mycelia with white colour, pink colour pigmentation appear at the reverse side of the plate. After four to 6 days of growth, multicellular, sickle-shaped macro conidia and micro-conidia are formed.

[00241] Toxins produced by Fusarium sp. in food material and their effect:

Fusarium sp. produces many toxins including fumonisins, zearalenone, and Aflatoxin Bl . Fumonisins are carcinogenic metabolites. In China, a high incidence of oesophageal cancer in humans has been attributed to fumonisins contamination. Zearalenone (ZEA) is estrogenic in nature and mostly affects the urogenital system. It produces a condition known as hyperestrogenism in pigs and has also been implicated in some incidents of precocious puberty changes in children. Aflatoxin Bl is a potent liver toxin causing hepatocarcinogenesis, hepatic necrosis, cirrhosis, and acute liver damage in affected animals, feed with aflatoxin contaminated food {Food Sci Tech Int 2006; 12(4):353-359).

[00242] Radial mycelial growth inhibition of Fusarium sp. by Nootkatone: Radial mycelial growth of Fusarium sp. was inhibited by 250 ppm of Nootkatone (98%> pure) up to 78% on the 5 th day of treatment at 4.5 cm diameter growth of mycelia in control {see Fig. 15). Inhibition reduced to 67% on the 10 th day {see Fig. 16), when the mycelia reached full Petri plate (8 cm) in size in the control. Fig. 17A shows a comparison of a control plate versus 50 ppm NKT treated plate versus a 250 ppm NKT treated plate. EC 50 of nootkatone was 95 ppm (95 μg/ml) against Fusarium sp. At 5 ppm ketaconazole exhibited 67% inhibition of radial mycelial growth of Fusarium sp.

[00243] Of particular interest, observations were recorded up to 20 days from starting the experiment/incubation at 5 day intervals. The mycelial in the control plate of Fusarium sp. produced pigments that were pinkish orange on 5 th day to the 10 th day and turned purple after 15 days and continued to stay as purple mycelia. In the control, along with pigmentation, sporulation also started and produced virulent spores. In contrast, Fusarium sp. grown on PDA plates with Nootkatone at 250 ppm remained almost white with a slight tint of purple in the center and with a 20% reduction in sporulation {see Fig. 17B).

Example No. 2. Studies on effect of nootkatone against fungal pathogens causing damage in fruits and vegetables - spore germination inhibition.

Overview

[00244] The ability of nootkatone to inhibit fungal pathogen spore germination was assessed in Colletotrichum gloeosporioides, Fusarium sp., Alternaria alternata apple pathotype mali (A. mali), Botrytis cinerea, Trichothecium roseum, Penicillium spp. and Rhizopus spp.

MATERIALS AND METHODS

[00245] Isolation of pathogens: Infected fruits were surface sterilized with 0.1 % mercuric chloride for 2 minutes and then washed in sterile distilled water thrice by soaking for 2 minutes. The infected portions were excised and moisture was removed by placing in sterile tissue paper. These bits of tissue were kept on potato dextrose agar (PDA) Petri plate with streptomycin 100 ppm to avoid bacterial contamination and incubated for 3-8 days at 25 ± 0.5°C. The hyphal tips, which grew out from the infected tissue were isolated and sub- cultured onto PDA and brought into pure culture.

[00246] Pathogens Isolated:

Colletotrichum gloeosporioides - isolated from infected mango fruit var neelam Fusarium sp. - isolated from infected caster seeds Alternaria alternata apple pathotype mali (A. mali), - isolated from infected apple leaf, second isolate from infected apple

Botrytis cinerea - isolated from infected strawberry fruits

Botrytis cinerea - isolated from infected grapes

Trichothecium roseum - isolated from infected apple fruit

Rhizopus sp. isolated from infected tomato fruit

[00247] Preparation of media:

[00248] Potato Dextrose Agar (PDA) from Hi media (MH096-500G) was used throughout these studies. 39 grams of PDA was dissolved in 900 ml of double distilled water and mixed well. Media pH was checked with pH paper and pH was adjusted to 5.0-5.5 with 1 N NaOH and 1 N HC1.

[00249] Yeast Peptone Dextrose Broth (YPD) from Sigma Aldrich was used throughout these studies. 50g of YPD was dissolved in in 900 ml of double distilled water and mixed well. Media pH was checked with pH paper and pH was adjusted to 5.0-5.5 with IN NaOH and IN HC1.

[00250] Preparation of compound and assay plates:

[00251] Nootkatone of 98% purity was used in all these studies. Nootkatone at 250 mg/ml in ethanol was used as stock solution and dilutions either in water or in liquid media were made. Spore germination was carried out in 96 well microtitre plates. Each well was filled with 100 of compound with 2X concentration. For example, if the required test concentration is 250 ppm the 2X concentration used was 500 ppm. To this, an equal volume of spore (conidia are the asexual spores) suspension with 4xl0 4 conidia was added. A commercial comparator was used based the selected fungal species, and the test concentration was used based on literature. A micro-dilution method was used to find out the susceptibility of conidial germination. The 96 well plate was incubated at 25 °C in a moist chamber to prevent evaporation of test solutions in the 96 well plate.

[00252] Inoculum preparation and inoculation:

[00253] In all the present study for all the fungal species tested, asexual spores, i.e., conidia were used to assess the spore germination inhibition. For each species spore germination efficiency was carried out in sterile milliQ water, YPD with 10-50% range and 0.5-2% sugar solution were tested in the same sequential order. In the controls, the efficiency of spore germination was achieved 80-90% before carrying out an assay to test the efficacy of nootkatone.

[00254] Observations:

[00255] Conidia were observed at 16 hrs, 24 hrs and 48 hrs under microscope for the specific spore germination stages and nootkatone's effect on these stages and recorded by photography. At certain times, optical density (OD) at 600 nm was measured in a plate reader.

[00256] Calculations:

[00257] Approximate conidia germinated and ungerminated were calculated by sample observations under microscope. % germination of inhibition was calculated as {(growth in control - growth in treated) / growth in control} X 100 either from microscopic observation or from OD at 600 nm. Observations by microscope were relied upon to calculation inhibition in the spore germination inhibition study.

RESULTS

[00258] Nootkatone exhibited varied percentages of inhibition on the spore germination of CoUetotrichum gloeosporioides, Fusarium sp., Alternaria alternata apple pathotype mali (A. mali), Botrytis cinerea (grape isolate), Trichothecium roseum, and Rhizopus sp.

[00259] Consolidated results of all the pathogens tested for spore germination inhibition by NKT is represented in Table No. 12 below.

[00260] Table No. 12. Spore germination inhibition (in %) of pathogens by Nootkatone.

inhibition inhibition

No

250 Not tested 80 85 100 10 inhibition

Effect of Nootkatone on Botrytis cinerea (grape isolate) spore germination

[00261] Nootkatone reduced the germ tube length though it could not arrest the spore germination completely at 125 and 250 ppm with 50 and 85% reduction in the germ tube length for B. cinerea (grape isolate). Observations are seen in Fig. 18.

Effect of Nootkatone on Trichothecium roseum spore germination

[00262] Only 50% inhibition was observed at 125 ppm nootkatone for T. roseum. No inhibition at concentrations lower than 125 ppm nootkatone was observed. Chemical comparator carbendazim at 10 ppm also exhibited only 50% inhibition only by reducing the length of the germ tube.

[00263] Conidia of most of the tested fungal species germinate in sterile water. But T. roseum conidia did not germinate. In sterile water, a few attempts were made with different concentrations of spores with repeated washings with water to remove self- inhibitors. These attempts at germination were not successful. To germinate T. roseum spores, a 0.5-2%> sugar solution and 50% YPD (half- strength) were also tested. Half strength YPD showed 95-98% germination, while a 2% sugar solution led to 80% germination. Further studies of testing effect of nootkatone and chemical comparator carbendazim were conducted in half strength YPD.

Effect of Nootkatone on Alternaria alternata mali spore germination

[00264] Only 25% inhibition was observed up to 250 ppm nootkatone for A. mali.

Effect of Nootkatone on Colletotrichum gloeosporioides spore germination

[00265] Nootkatone is highly effective in inhibiting the spore germination of Colletotrichum gloeosporioides, at 125 ppm and 250 ppm 100%) inhibition was observed. Even at a lower concentration of 7.5 ppm, none of the spores germinated effectively. Spores testing was carried out in 10% YPD, as spores in control showed highest germination in 10% YPD rather than in water. The assay was carried out in a 96 well plate as described in general methods. [00266] Optical density was measured at 600 nm in plate reader on 4 day of starting of assay. Inhibition was calculated by subtracting OD of test with OD of control. Spore germination at 16 and 48 hr in control and nootkatone treated plates is seen in Fig. 19A and 19B. The percent inhibition compared to control is shown in Fig. 20.

[00267] Discussion and conclusion drawn from spore germination experiments with

C. gloeosporioides: The anthracnose and bitter rot in apple causative organism C. gloeosporioides was inhibited by Nootkatone very effectively. Compared to carbendazim at the same concentrations, Nootkatone exhibited 20-35% higher rate of inhibition, calculated by OD 600. These results indicate that nootkatone can be used as an effective control of C. gloeosporioides post-harvest and in crop management.

[00268] Observations under microscope revealed many malformations and deviations in the conidial germination pattern of C. gloeosporioides. The first stage of spore germination is adhesion of spore to polystyrene surface under in vitro testing, which is parallel to the leaf or fruit surface under natural colonization. Spores adhered to the polystyrene surface will not come out of the well and they cannot be removed by pipetting. Nootkatone at 125 and 250 ppm prevented the spores from adhering to the polystyrene surface of 96 well plate. All most all spores were removed from the well by pipette. This observation indicates that nootkatone can be used to control disease caused by C. gloeosporioides as post-harvest management and in crop management. Indeed, these results suggest that nootkatone composition can prevent conidia attachment to the surfaces of consumable products.

[00269] In Colletotrichum spp., glycoproteins contribute to the hydrophobicity of the spore surface and are involved in the initial rapid attachment of conidia. Glycoproteins combine with extracellular matrix (ECM) secretion of Colletotrichum spp. and form a strong adhesive. Without being bound by theory, it is possible that hydrophobicity of the spores has been disrupted by nootkatone at higher concentrations as the compound itself is hydrophobic or some mechanism which affects ECM and or glycoprotein secretion.

[00270] The second stage of spore germination is division of single cell spores into two cells. Germ tubes extend from the end of the cells. The third stage is germ tube elongation and culmination into an aspersorium (a specialized structure globular, obtuse in shape with high amount of glycerol and turgor pressure surrounded by highly melanised wall). This is the strongest part of the whole germination and helps in the penetration into the plant tissue. Most of fungal pathogens like Rhizopus sp. can enter through wounds of the plant tissue and are not capable of penetrating through the intact tissue, whereas Colletotrichum is able to penetrate intact tissue with the help of the aspersorium. So, failure to produce an aspersorium makes the pathogen less virulent.

[00271] At all the lower concentrations, most of the conidia germinated were without aspersorium. In addition, some of the germ tubes were curved which indicates loss of polarity in the growth. At 62.5 ppm nootkatone affected germ tube morphology, with swollen and frequently septated cells, which is in contrast to smooth long cells as seen in control germ tubes.

[00272] After 48 hr, the malformations remained and continued to be severe in effect. The end of the germ tubes was highly branched with fan like structures. At even the lowest concentration tested, a majority of the spores did not show germination with germ tube culminating into aspersoria. This shows that at concentrations as low as 7.8 ppm, nootkatone highly impaired the germination and spores treated with nootkatone cannot cause damage to intact fruit or plant tissues.

Effect of Nootkatone on Fusarium sp. spore germination

[00273] No inhibition by nootkatone on the spore germination of Fusarium sp. was observed.

Effect of Nootkatone on Rhizopus sp. spore germination

[00274] Rhizopus spp. are common in fruit rots as saprophytic pathogens. The present isolate was obtained from rotten tomatoes. Nootkatone exhibited good inhibition on spore germination of Rhizopus sp. about 80% and 50% inhibition was observed in 250 ppm and 125 ppm treatments, respectively. Fluconazole at 50 ppm showed 100% inhibition on the Rhizopus sp. Spore germination at 16 hr in control and nootkatone treated plates is seen in Fig. 21.

Example No. 3. Application of nootkatone for management of bitter rot of apples induced by Colletotricheum gloeosporioides spore inoculum.

Overview

[00275] Fungi infections can be very visible and affect fruit value. Therefore, nootkatone compositions were tested to determine whether preventative (prophylactic) or curative (after initiation of infection) treatments with the compositions were more effective. [00276] Methods: "Red delicious" apples were collected without pesticides. Apples were washed in tap water and wiped with a paper towel. Their surfaces were sterilized in 0.2% mercuric chloride and rinsed in sterile distilled water thrice to remove traces of HgCl 2 . Five spots of 1.5 cm diameter were marked and gently pricked with a needle of a 2 ml syringe to create mechanical damage. In apples under natural conditions, pathogens from outside enter through mechanical damage.

[00277] Control assays - Spores used for infection were checked for germination in YPD broth. All the pathogens germinated to 90% and above. Control apples with only distilled water spray were maintained and no infection was observed.

[00278] Spores of C. gloeosporioides were harvested from PDA plate of 10-15days old cultures. The fruit body of the cultures, namely acervuli, were picked and immersed in water for 15 minutes to get the mucilage dissolved and the spores separated. C. gloeosporioides spore suspensions were filtered through sterile cheese cloth to remove mycelial bits. Infection of the apples was carried out by spraying spore suspensions of 10-13 x 10 4 C. gloeosporioides spores (counted by hemocytometer) on apples.

[00279] Nootkatone was dissolved in ethanol at 250 mg/ml concentration. Reference compound: carbendazim 50%> wettable powder dissolved in water.

Treatments

[00280] Apples were soaked for one minute in solutions/suspensions of test compounds and concentrations. Control apples, only infected and treated with water, and apples without infection dipped in 2% ethanol. Nootkatone was tested at 62.5, 125, 250 and 500 ppm. Carbendazim was tested at 500 ppm.

[00281] Preventative treatment: apples were sprayed with spores after 48 hrs of compound treatment.

[00282] Curative treatment: apples were sprayed with spores and left for 48 hrs for the spore to adhere and germinate and are gently dipped in the treatment composition.

[00283] Apples were placed on sterile filter paper in plastic trays and covered with aluminum foil with whole for air circulation. Incubation was done in an environmental chamber with 95% humidity at 23°C.

[00284] Results are shown in Table Nos. 13 and 14 and Fig. 22.

[00285] Table No. 13. Effectiveness of Nootkatone as a Preventative Spray. Treatments (nootkatone,

% area

"NKT" - Preventative - affected

concentrations

Control 15.7

NKT 62.5 ppm 1.3

NKT 125 ppm 0

NKT 250 ppm 0

NKT 500 ppm 0

Carbendazim 500 ppm 4.0

[00286] Table No. 14. Effectiveness of Nootkatone as a Curative Spray.

[00287] NKT was effective on "bitter rot of apple" (C. gloeosporioides) at a concentration of 62.5 ppm on the 10 th day of inoculation, less than its EC50 of 95 ppm, and better than carbendazim at 500 ppm.

[00288] A preventative spray was found to be more effective than a curative spray (100% control at 125 ppm preventative nootkatone spray vs. 70% control with 125 ppm curative treatment). A 62.5 ppm NKT concentration was the lowest concentration tested that showed 95% protection. NKT at concentrations of 250 ppm and 500 ppm was effective in a curative spray.

Example No. 4. Application of nootkatone for protection of oranges.

Overview

[00289] Oranges are naturally susceptible to fungal infections. This example examined whether nootkatone could protect oranges from natural fungal infections.

[00290] Example 4.1 [00291] Oranges were bought from an organic store without any pesticide spray. The oranges were washed in sterile water and placed into a growth chamber for drying overnight. The next day, the oranges were dipped into treatment solutions (125 ppm NKT, 250 NKT ppm, or 500 ppm carbendazim). Control fruits were dipped in 0.1% ethanol in water (1 mL in 1 liter) to compensate for the ethanol concentration in the NKT treatments. A dry, autoclaved tissue paper was spread in the tray and not sprinkled with water. The oranges were kept for incubation in the environmental chamber at 25°C and 85% humidity.

[00292] Observations: Scoring of infected areas and isolation of pathogens were carried out on the 10, 14, and 18 th days of treatment.

[00293] Results: Nootkatone concentrations of 125 ppm and 250 ppm resulted in fruit with only 1.7% spoilage, which was edible and very good. In two out of three fruits, not even a single spot was seen. In one fruit at 250 ppm nootkatone concentration, a hypersensitive reaction to fungal infection was observed. Drying and browning of infected skin was observed. In contrast, an average of 47% spoilage was observed in fruits treated with 500 ppm of carbendazim, and the fruits were inedible. Control fruits were spoiled over 40-80%) of their surface with average of 57% spoilage and were inedible. Results are seen in Fig. 23.

[00294] Organisms isolated from the control and carbendazim-treated oranges included Penicillium sp., Alternaria sp., CoUetotrichum gloeosporioides, Geotrichum candidum/ Galactomyces citri-aurantii (a known human pathogen which causes bronchial trichosis, eye infections, oral infections, joint infections, and others), one fungus under observation, and one bacteria (listed in the order of prevalence in each treatment group). Only Penicillium spp. were isolated from the nootkatone treated oranges.

[00295] Present day treatments of oranges include treatment with conventional fungicides, packing fruit in corrugated containers, cold storage for extended periods, chlorine washes, soda ash treatments, and water rinses. But, none of the present fungicides are registered as specially for post-harvest applications due to tolerance limit issues. Therefore, nootkatone can be a particularly welcome post-harvest treatment of oranges.

[00296] Example 4.2

[00297] Purpose of study: To examine the efficacy of nootkatone in curing oranges from infection (the study material has visible disease symptoms) & confining damage further. Further, this study was designed to investigate treatment efficacy differences due to method of treatments. [00298] Valencia oranges not previously treated with pesticide spray and with symptoms of Alternaria rot and Phytophthora rot were bought, washed in sterile water, and placed into a growth chamber for drying for a day. The next day, the oranges were either dipped into or sprayed with treatment solutions (62.5 ppm NKT, 125 ppm NKT, 250 ppm NKT, 500 μΐνΐ. Amistar Top® (a combination of Azocystrobin and Difenoconazole), Thyme oil (100 μΐνΐ, of solution), or carbendazim at 1 and 2 g/L. Control fruit was dipped in 0.1% ethanol in water (1 mL in 1 liter) to compensate for the ethanol in NKT treatment. Oranges were dipped for either 1 or 2 minutes. Under spraying conditions, oranges were drenched, then allowed to sit for 5 minutes before being placed on a new sheet of filter paper. The oranges were kept for incubation in the environmental chamber at 25°C and 85% humidity.

[00299] Observations: Scoring of infected areas and isolation of pathogens was carried out on the 7 th , 12 th , 15 th , and 20 th days of treatment. On day 15 specifically, oranges classified as soft (suspected of rotting) and with small brown patches were cut open and observations were made about internal conditions. Generally speaking, the dominant disease appeared to be Penicillium mold (green mold caused by Penicillium digitatum and blue mold caused by P. italicam). Phytophthora rot was observed and Alternaria rot appeared to be confined to the calyx attachment and spread inside. Stem end rot by Lasiodiplodia theobromae was observed in small patches.

[00300] Results:

[00301] Day 7: No infection was observed in oranges when treated with 62.5 ppm, 125 ppm, and 250 ppm NKT (dipped or sprayed - method of treatment was not a factor). Similarly, oranges treated with 2 g/L carbendazim, 500 μΏΙ ^ of Amistar TOP ® , and 100 thyme oil showed no signs of infection. Following treatment with 1 g/L carbendazim, two out of four oranges showed Penicillium mold (i.e., P. digitatum and P. italicam) and Alternaria rot was observed in one of four similarly treated oranges. Treatment with 0.1% ethanol in water resulted in one out of four oranges being damaged by blue and green mold (see FIGS. 24 A- K). Mild infection of Alternaria/Phytophthora observed in many fruits - further course of infection progression was followed.

[00302] Day 12: No infection on oranges was observed following treatment with 125 ppm and 250 ppm NKT treatment (dipped and sprayed), carbendazim 2 g/L, 500 μΏΙ ^ Amistar TOP ® , or 100 thyme oil (no images shown). Both dipping and spraying with 62.5 ppm NKT resulted in the rotting of one orange in each group of four fruits (see FIGS. 25A-C). Amistar TOP showed brown dots suggestive of a hypersensitive reaction (see FIGS. 25D). Oranges treated with 1 g/L carbendazim and control oranges were completely covered with blue and green mold (no images shown).

[00303] Day 15: All oranges treated with 250 ppm NKT and 100 thyme oil looked fresh and showed no signs of infection (no images shown). There were no hypersensitive reactions (i.e., browning, blemish, shrinkage, or scaring) observed in the oranges treated with 250 ppm NKT. Two oranges treated with 125 ppm NKT were soft and when cut open did not show any mycelia. But, one orange smelled rotten, and the color of the pulp had changed (see FIG. 26A). Amistar TOP ® -treated fruit showed growth of Alternaria spp. mycelia inside the fruit. But on the top, it was a small brown spot and fruit was soft. (FIG. 26B).

[00304] Day 20: The same status as of day 15 was observed on day 20 (no images shown).

[00305] Conclusion:

[00306] Nootkatone at 125 and 250 ppm concentrations protects exposed oranges from rot up to 20 days. The tests used were rigorous because the test oranges used were not farm fresh and showed symptoms of disease before starting treatment. In addition, the orange skins were mildly shrunken, as it was summer season. Nootkatone can be administered via spraying or dipping to achieve the same results, which are concentration dependent.

Example No. 5. Application of nootkatone for management of mite infestation of post-harvest consumable products.

Overview

[00307] Tyrophagus putrescentiae is a cosmopolitan mite species. Together with the related species T. longior, it is commonly referred to as the mold mite or the cheese mite or storage mite. The size of the mites is 200-500 μιη, and they cannot be seen by eye. It is a common pest of stored products, especially those with a high protein and fat content (meat, cheese, nuts and seeds, dried eggs, etc.). It feeds on the fungi that grow on the foodstuffs. These mites cause copra itch and breathing problems to humans in continuous contact with mites. These mites are also often reported as contaminant of dog foods.

[00308] Current treatment of storage mites is by methyl bromide (MB) fumigation of cheese rooms and storage grains in gunny bags. MB causes neurological symptoms and at higher toxicity exposures pulmonary injury and associated circulatory failure occurs in humans. Therefore, the present example explored the ability of nootkatone to treat or prevent infestation by cheese/storage mites.

[00309] To test whether nootkatone could repel or kill mites, initially one week old nootkatone assay plates with fungus growing on them were placed next to mite infected plates. Mites were observed to migrate to the one week old nootkatone plates, likely because they were attracted to the mycelia growing on the plates which the mites eat. After three days, the plates were observed under microscope. For each plate around 10 mites were observed. All mites found were dead at 72 hr in 100 ppm and 250 ppm nootkatone plates.

[00310] Next, 35 day old plates containing 125 ppm and 200 ppm NKT with fungal grown were allowed to be infected with mites. In the 100 ppm NKT plate, all mites were alive and active. In the 250 ppm NKT plate, out of 10 mites observed, 2 were found dead and others were very sluggish.

[00311] Based on these observations, a treatment of a consumable product or an associated surface with a composition containing 100 ppm nootkatone or greater can kill or repel storage mites for at least one week. The greater the concentration of nootkatone applied, the longer the treatment is effective against storage mites.

Example No. 6. Studies On Effect Of Nootkatone And Resveratrol Against Fungal Pathogens Causing Damage In Fruits And Vegetables.

Overview

[00312] Alternaria spp. can be isolated from infected cherries, and is a major pathogen found in fruit and vegetables. A. alternate {Alternaria black spot), which causes decay to most fresh fruit and vegetables pre- or post-harvest, has become a significant disease for fresh fruit {see FIGS. 27A-C). For example, Alternaria spp. appear to be the most common decay organism of post-harvest apple, where these species represented 81.9%. Among the different diseases caused by the genus Alternaria, blight disease is most dominant one that causes average yield loss in the range of 32-57%).

[00313] Cumulative Effect of Nootkatone and Resveratrol on Alternaria alternata mycelial growth [00314] Pathogen isolation: Alternaria alternata was isolated from sweet cherries. It presented with an orange color at the reverse side and green on the top of a Potato Dextrose Agar (PDA) plate (not shown).

[00315] Mycelial growth inhibition of alternata by nootkatone and resveratrol

[00316] After 5 days of treatment, mycelial growth of A. alternata was cumulatively inhibited by Nootkatone (NKT; 240 ppm) and resveratrol (RESV; 240 ppm) by 80%. NKT alone showed inhibition at a maximum of 52% at a concentration of 240 ppm, while RESV at 240 ppm showed only 45%. Tebuconazole, a commercially available fungicide, was used as a control (2.5 ppm) and inhibited alternata mycelial growth by 80% {see FIGS. 28A-B).

[00317] Effects of Nootkatone and Resveratrol on Alternaria alternata spore germination

[00318] Pathogen Identification: Alternaria alternata was isolated from sweet cherries, and mycelium on both sides of the Potato Dextrose Agar (PDA) plate presented green.

[00319] Inhibition of Conidial Germination

[00320] A. alternata was seeded on PDS plates and observations were conducted 24 hours later for control {see FIG. 29A), 125 ppm and 62.5 ppm NKT, and 10 ppm carbendazim {see FIGS. 29B-D). NKT at 250 ppm and 125 ppm controls Alternaria sp. conidia germination by 100%. Similarly, no spore germination was observed on PDA plates following treatment with NKT+RESV at 62.5 ppm {see FIGS. 29E-F). PDA plates treated with 62.5 ppm NKT alone germinated with small germ tubes {see FIGS. 29B-C).

[00321] Benzimidazole Resistance strain: Carbendazim (10 ppm) was tested on a benzimidazole resistant strain of A. alternata and no inhibition of conidia germination was observed. Mutations in the β-tubulin gene may be involved.

[00322] Effect of Nootkatone and Resveratrol on Botryodiplodia theobromae mycelial growth

[00323] Botryodiplodia theobromae, also referred to as Lasiodiplodia theobromae, causes damage at both pre- and post-harvest stages and affects more than 200 plant species. Post-harvest diseases include stem end rot, fruit and tuber rots, which affect crops including mangoes, oranges, avocados, papayas, bananas, cocoa, and yams. B. theobromae causes dieback, blights, and root rot in many plants and severely affects guava, coconut, and grapes. [00324] Pathogen isolation: B. theobromae was isolated from infected oranges and brought to pure culture by repeated sub-culturing by fungal tip isolation method.

[00325] Mycelial growth inhibition of B. theobromae by nootkatone on PDA plates (poisoned food plate method): Mycelial growth of B. theobromae was inhibited by nootkatone by 63% and 75% at 125 ppm and 250 ppm, respectively. NKT 250 ppm showed 10% greater growth inhibition of mycelia compared to 10 ppm Tebuconazole but 15% less growth inhibition than 50 ppm Tebuconazole (see FIGS. 30A-B). Resveratrol did not show any inhibition of B. theobromae (data not shown).

[00326] Effect of Nootkatone and Resveratrol on Botryodiplodia theobromae conidia germination

[00327] Treatment Conditions: Microscopic observations were conducted 24 hours after incubation with 250 ppm and 500 ppm concentrations of nootkatone (NKT) alone or NKT+RESV or 10 ppm Tebuconazole (positive control). Untreated PDA plates were used as a negative control.

[00328] Results: Untreated control plates showed no inhibition of B. theobromae (BT) conidia germination {see FIG. 31 A). No inhibition of conidia germination was observed following incubation with NKT alone at 500 ppm or 250 ppm {see FIGS. 31B-C) or with 10 ppm Tebuconazole (FIG. 3 ID). However, B. theobromae conidia germination was effectively controlled with combination treatment of NKT+RESV each at 500 ppm and 250 ppm {see FIGS. 31E-F).

[00329] Effect of Nootkatone and Resveratrol on Monilinia laxa.

[00330] Pathogen isolation: M. laxa primarily infects apples, pears, plums, peaches, and cherries, and is commonly observed in cherries in Europe {see FIGS. 32A-D). M. fructicola, by contrast, is a common cherry pathogen in the U.S. but not in Europe.

[00331] For this study, M. laxa was isolated from cherry fruit purchased off the shelf of air-conditioned store and left on table for 2 days to allow disease to progress.

[00332] Observations: Microscopic observations were conducted 24 hours after treatment with NKT, RESV, or NKT+RESV at concentrations of 30 ppm , 60 ppm, 90 ppm, 120 ppm, 180 ppm, and 240 ppm, or 10 ppm Tebuconazole (positive control). NKT+RESV at all concentrations inhibited M. laxa mycelial growth {see FIGS. 33A-B). Further, 98% of mycelial growth was inhibited by incubation with NKT at 60 ppm. Example No. 7. Preservation of cherries following treatment with compositions comprising Nootkatone.

[00333] Approximately half of the market losses in Western sweet cherry fruit have resulted from disease. The major market losses are caused by the seven fungal pathogens listed in Table No. 15 below.

[00334] Table No. 15 Spoilage microorganisms identified during testing.

[00335] Efforts to reduce fruit loss have used a variety of fungicides to little effect. For example, iprodione was used extensively as a postharvest treatment of sweet cherries until March 1996. The manufacturer restricted applications to no fewer than 7 days before harvest. For post-harvest applications, captan and Tebuconazole are used in the United States. However, captan is seldom used because residues on fruit are prohibited in several export markets. Tebuconazole, on the other hand, is considered a medium risk fungicide but resistance to this fungicide develops quickly. For example, Monilinia spp. resistance to Tebuconazole has been reported. But, post-harvest use of synthetic fungicides in European Union countries and Turkey is prohibited due to fungicide regulatory issues. GRAS (generally recognized as safe) and other natural fungicides and chemical-free storage methods for prolonging cherry fruit merchantability are being developed. However, additional compositions and methods for prolonging cherry fruit merchantability are still needed.

[00336] This example describes a bioassay in which cherries were exposed to a nootkatone-containing composition to determine susceptibility of naturally occurring fungi to nootkatone.

[00337] Methodology:

[00338] Organically grown cherries (no previous pesticide treatment) were purchased, of which 30-40% fruits were already spoiled. From these samples, fruits without symptoms of spoilage were picked, rinsed with sterile water and distributed uniformly in regards to size, shape, and ripening stage between treatments (22 fruits per treatment).

[00339] Treatments:

[00340] Stock solutions of nootkatone (NKT) and nootkatone combined with resveratrol (NTK+RESV) were prepared in ethanol to provide a range of concentrations (50 ppm, 75 ppm, and 150 ppm). Treatments were applied directly to cherries by dipping fruit in the respective treatment solutions for one minute. A commercial fungicide comprising a co- formulation of azoxystrobin and difenoconazole (available from Syngenta as Amistar TOP®) was diluted to 500 μΙ7Ι, in ethanol and used as a positive control. Azoxystrobin and difenoconazole are not registered for post-harvest treatment of cherries, but they are registered for use in post-harvest potatoes, citrus, and pome fruits. An essential oil (thyme oil diluted to 100 μΙ7Ι, in ethanol) was used as a second positive control representing a nature-derived alternative. A negative control was also employed, consisting of the ethanol solution used to make the nootkatone stock solutions of this series of experiments.

[00341] Assessments:

[00342] All sets of cherries were spread over a sterile tissue in a tray and incubated in an environmental chamber at 25 °C and 85% humidity for eight days. Following eight days of incubation, the cherries were examined for freshness, firmness, microbial growth and general signs of spoilage.

[00343] Several spoilage microorganisms and two bacteria were isolated from the rotten cherries during the course of the incubations and identified by culturing, purification, and microscopic characterization. The organisms identified during testing are shown in Table No. 16 below.

[00344] Table No. 16 Spoilage microorganisms identified during testing.

Pathogens Isolated

Monilinia laxa

Alternaria spp.

Cladosporium spp.

Penecillium expansum

Diamorphic fungus unclassified

RESULTS [00345] Referring to FIGS. 34A-D and 35A-B, in the untreated control, nearly 80% of fruit was spoiled on the 8th day of incubation (see FIG. 34A). In comparison, 19% of fruit treated with compositions comprising NKT at 50 ppm and 75 ppm (see FIG. 34D) control were spoiled on the 8th day of incubation (but fruit looked slightly dried up and shrunken). Only 23% of fruits treated with 100 μΤ/L thyme oil were spoiled on the 8th day of incubation incubation (but fruit looked slightly dried up and shrunken). Only 14% of fruits treated with Amistar TOP ® (at 500 μΤ/L in ethanol solution) were spoiled on the 8th day of incubation (see FIG. 34C) incubation (but fruit looked slightly dried up and shrunken). Similarly, only 14% of fruits treated with a combination of NKT+RESV at 50 ppm and 75 ppm each were spoiled on the 8th day of incubation. Close visual inspection of fruit led to the observation that skin wrinkling was seen with fruits treated with NKT alone and with Amistar TOP ® (FIG. 35A), but fruits treated with a combination of NKT+RESV appeared fresher, exhibiting tight, shiny skins (FIG. 35B). Results are shown in Table No. 17 below.

[00346] Table No. 17. Effects of Nootkatone Treatment.

[00347] Alternaria rot by two strains of Alternaria spp. caused the highest amount of spoilage in this study. Brown rot by Monilinia laxa was the second most dominant pathogen.

[00348] The test results indicate a dosage-dependent antifungal effect following treatment of fruits with compositions comprising nootkatone. The anti-spoilage effect of nootkatone was further enhanced by co-treatment with resveratrol, and it was further noted that fruits treated with a blend of nootkatone and resveratrol had a more fresh appearance indicated by a tighter and shinier skin. Based on these results, it is believed that above 90% of cherries can be protected from spoilage with a pre-harvest spray or immediately after harvest leading to an extended shelf life.

Example No. 8. Studies On Effect Of Nootkatone Against Aspergillus Spp.

[00349] The present example sought to determine the ability of nootkatone to inhibit Aspergillus spp. spore germination. Comparisons to known fungicides were also performed.

METHODOLOGY

[00350] 10% Yeast Peptone Dextrose (YPD) broth was prepared from 100% YPD by diluting the broth in sterilized water. A stock solution of ethanol-nootkatone (EVE -NKT) was prepared by dissolving 250 mg of NKT in 1 mL of ethanol. Fluconazole stock solution was prepared by dissolving 100 mg in 1 ml of DMSO, and Tebuconazole stock solution was prepared by dissolving 25 mg in 1 mL of MilliQ water (MQ). Working solutions were prepared by dissolving stock solutions in 10%> YDP to desired concentrations. NKT was used at concentrations of 500 ppm, 250 ppm, 125 ppm, 62.5 ppm, 32.5 ppm, and 15.5 ppm, Flucanozole was used at concentrations of 500 ppm, 250 ppm, 125 ppm, 62.5 ppm, and 31 ppm. Tebuconazole was used at concentrations of 125 ppm, 62.5 ppm, 31.25 ppm, 15.625 ppm, and 7.8 ppm.

[00351] Aspergillus spp. were isolated from airborne conidia on Potato Dextrose Agar (PDA) plates {see FIGS. 36A-B). Conidia were collected and suspended in 5 mL of 10%> YPD (for adhesion of conidia and suspension of conidia in the liquid medium) and microscopic observation was conducted to confirm cell number. One hundred microliters of 10% YPD were added per well in a 96 well plate with NKT and Fluconazole/Tebuconazole at 2X concentrations in the respective wells and media alone as a control. One hundred microliters of Aspergillus conidia were then added to respective wells, and controls were added with water, and incubated in a moist chamber at 25°C. Cells were observed under a microscope after 24 hours of incubation.

RESULTS [00352] Aspergillus flavus spore germination at 24 hrs Post-Treatment: NKT at concentrations of 500 ppm and 250 ppm inhibited about 100% and 80% of conidia germination, respectively. In contrast, fluconazole at concentrations of 500 ppm and 250 ppm failed to inhibit germ tube formation {see FIGS. 37A-E). NKT showed 82% inhibition of Aspergillus spp. conidia 48 hrs post-treatment (data not shown).

[00353] Aspergillus flavus conidia at 72 hrs Post-Treatment: OD measurements at 6000 nm were taken and percentage inhibition was calculated {see FIGS. 38A-B). NKT at 500 ppm and 250 ppm showed 82% and 80% inhibition of germ tube formation compared to fluconazole, which caused 35 to 20% inhibition.

[00354] Aspergillus niger conidia at 24 hrs Post-Treatment: NKT at 500 ppm and 250 ppm showed 100% inhibition of germ tube formation (microscopic observation). The combination of NKT+RESV at 62.5 ppm each inhibited germination of conidia, whereas neither NKT nor RESV alone at 62.5 ppm was able to inhibit germination. No inhibition by RESV was observed up to concentrations of 500 ppm (results not shown). Complete inhibition of conidia was observed following treatment with control Tebuconazole at 10 ppm {see FIGS. 39A-B).

[00355] Aspergillus niger mycelial growth at 116 hrs Post-Treatment: Mycelial growth was 93% inhibited by NKT 250 ppm treatment. Following 250 ppm NKT, the size of mycelial balls was less than control due to reduced length of hyphae. NKT at 125 ppm showed 73% inhibition of mycelial growth. Tebucanozale at 1 ppm resulted in 40% mycelial growth inhibition {see FIGS. 40A-B).

Example No. 9. Studies On Effect Of Nootkatone And Resveratrol Blue Mold.

[00356] Overview: Blue mold affects fruit such as apples and oranges and is one of the fastest spreading air-borne pathogens. The mold tends to invade more rapidly and predominate mixed infections accounting for 60-80% of decay. In addition to affecting fruit, blue mold also results in the spoilage of bread. Apple blue mold is a significant post-harvest disease that causes heavy economic losses due to decay in stored apples destined for the fresh market.

[00357] Treatments: Spore germination studies were generally performed as described in Example No. 8 for microscopic observation. [00358] Observations: After 24 hours, microscopic observations revealed that 250 ppm nootkatone (NKT) inhibited spore germination of blue mold by about 90% (see FIGS. 41B- G). Specifically, many spores germinated but with very short germ tubes. About seventy percent of spore germination was halted at 125 ppm NKT. The combination of NKT+RESV did not show much difference when compared to NKT treatment alone. Complete inhibition was observed following treatment with 12.5 ppm Tebuconazole (data not shown).

Example No. 10. Studies on Effect of Nootkatone on Colletotrichum gloeosporioides.

[00359] Pathogen isolation: Colletotrichum gloeosporioides was isolated from infected mangoes. C. gloeosporioides causes a disease called anthracnose in over 200 plant species and is a major pathogen all over the world (see FIGS. 42A-H). C. gloeosporioides causes bitter rot in pomes (apple and pear) and dragon fruit, and causes anthracnose in peaches, bananas, mangoes, papayas, strawberries, and solanaceous vegetables (i.e., eggplant, capsicum bell peppers, chilies, curcurbit, and legumes). C. gloeosporioides also causes smudge in onions.

[00360] Observations: Microscopic observations were made after 24 hours of incubation with NKT (7.8 ppm, 15.625 ppm, 31.25 ppm, 62.5 ppm, 125 ppm, and 250 ppm), carbendazim (7.8 ppm, 62.5 ppm, and 125 ppm), and control (ethanol only). In control samples, almost 95% conidia germinated and produced dark melanised appressoria with long germ tubes, which is the typical conidia germination of C. gloeosporioides (FIG. 43A). Conidia treated with NKT at 250ppm and 125ppm showed 100% inhibition germ tube formation (see FIGS. 43B-C). Various stages of conidia germination were inhibited by NKT at concentrations of 7.8-62.5 ppm (see FIGS. 44A-E). Conidia showed nodulated and shortened abnormal germ tubes. Loop-like germ tube formation and curved and short germ tube formation was also observed. It is possible that nootkatone treatment at higher concentrations disrupted the hydrophobicity of the conidia because nootkatone itself is hydrophobic, or some mechanism, which affects the extracellular matrix (ECM) and/or glycoprotein secretion may be the cause.

[00361] In a survival assay, conidia taken from the control plate and the 250 ppm and 125 ppm NKT plates were plated on new PDA plates (without NKT) to check for survival rates of conidia after 48 hours. The control plate showed extensive conidia survival (FIG. 45A), but only 3 and 10 conidia revived from approximately 500 conidia and formed colonies from the 250 ppm and 125 ppm NKT plates, respectively (see FIGS. 45B-C).

[00362] Optical Density Measurement: Using an assay similar to that described above, optical density was measured at 600 nm in a plate reader on 4 th day following start of assay. Inhibition was calculated by subtracting OD of test with OD of control. NKT at concentrations of 250 ppm and 125 ppm inhibited 100% of C. gloeosporioides germ tube formation (see FIGS. 45D). Conidia treated with 125 ppm of carbendazim exhibited only 65% inhibition of conidia germination, and appressoria were produced in the germ tubes (not shown).

Example No. 11. Studies On Effect Of Nootkatone And Resveratrol On

Magnaporthe grisea

[00363] Overview: The global turf and ornamental grass protection market has been estimated at $5.20 billion and is projected to reach $6.45 billion by 2021. A major rice disease that often causes great economic loss is rice blast (Magnaporthe grisea). It is estimated that out of the total yield loss due to disease in rice, 35% is caused by blast.

[00364] In February 2016, wheat blast was spotted in Bangladesh, its first report in Asia. Wheat is the second major food source in Bangladesh, after rice. The blast disease has, so far, caused up to 90% yield losses in more than 15,000 hectares.

[00365] Inhibition of M. grisea spore germination

[00366] Observations: Microscopic observations were made 24 hours after incubating M. grisea samples with NKT at concentrations of 62.5 ppm, 125 ppm, and 250 ppm, NKT+RESV at concentrations of 62.5 ppm, 125 ppm, and 250 ppm each, or 10 ppm carbendazim (positive control).

[00367] Results of M. grisea spore germination: Referring to FIGS. 46A-D, no germination of M. grisea conidia was observed following incubation with RESV+NKT at 250 ppm or 125 ppm, and conidia were highly vacuolated compared to control. NKT+RESV at 62.5 ppm inhibited conidia germination by 90%, and 10% of conidia contained short germ tubes without appressoria. Carbendazim at 10 ppm completely inhibited conidia germination (data not shown). [00368] Referring to FIGS. 47A-D, NKT at 250 ppm showed 100% inhibition of germ tube formation compared to control. At 125 ppm NKT, 50% conidia germination was observed with short germ tube formation (arrows). Germinated conidia culminated at non- melodized and vacuolated appressoria. Additionally, 125 ppm NKT treated conidia were bulged and twice as large as control (see FIGS. 47A versus 47C). NKT at 62.5 ppm showed nearly 100% germination with short germ tubes and bulged appressoria (see FIG. 47D).

Example No. 12. Studies On Effect Of Nootkatone And Resveratrol On Botrytis cinerea

[00369] Pathogen isolation: Strawberry fruit was purchased off the shelf of an air- conditioned store and left on a table for two days for disease to progress. B. cinerea strawberry isolate was carbendazim resistant (due to a mutation at 198 AA from GAG (Glu) to GCG(Ala) in the tubulin beta chain).

[00370] Observations: Microscopic observations were made after 18 hours of incubation of Botrytis cinerea with NKT, RESV, or NKT+RESV at concentrations of 62.5 ppm, 125 ppm, and 250 ppm. Tebuconazole was used as a positive control at a concentration of 10 ppm.

[00371] Results of B. cinerea spore germination: Referring to FIGS. 48A-E, NKT+RESV at 62.5 ppm inhibited conidia germination better than 125 ppm of NKT or RESV alone (data not shown). NKT alone at 250 ppm showed 100% inhibition of germ tube formation, while only 50% inhibition was observed following incubation with 62.5 ppm of NKT. Complete conidia germination was observed following incubation with 10 ppm Tebuconazole.

[00372] Inhibition of B. cinerea mycelial growth

[00373] Results: Referring to FIG. 49, the combined effect of NKT+RES (RESV) on B. cinerea radial mycelial growth inhibition was more pronounced than NKT or RESV alone. For example, incubation of NKT+RESV at 60 ppm each showed 17% and 18% better inhibition than 120 ppm of NKT or RESV alone. These results indicate that RESV creates an additive effect thereby reducing the quantity of NKT required, which is advantageous because production of NKT is costlier than producing RESV. [00374] Rate of growth per day: Mycelial growth (growth rate in centimeters per day) of B. cinerea was measured for the first five days and then from days five to ten. Incubation of the combination of NKT+RESV at 60 ppm each resulted in a reduction of mycelial growth rate from 0.3 cm/day on the 5 th day and to 0.07 cm/day on the 10 th day. The rate of growth retention by NKT and RESV alone at 60 ppm was about 10 and 5 folds higher, respectively, than NKT+RESV on 10 th day (see FIGS. 50A-B).

Example No. 13: Evaluation Of Nootkatone's Larvicidal Activity Against Caterpillars.

[00375] Overview: Some of the most damaging and therefore commercially relevant larvae are the larvae of Lepidoptera species. Caterpillars are, collectively, the larvae of moths and butterflies which are members of the order Lepidoptera. There are over 1 10,000 different species in this order. The aim of this experiment was to confirm that the larvicidal activity of Nootkatone extended to caterpillar larvae

METHODOLOGY

[00376] Test Formulations: Nootkatone (NKT) solutions at 0.25% concentrations were obtained by dissolving 98% pure NKT in 100%) EtOH (250 mg/mL) and diluted to achieve 0.25% solutions with filtered butane to permit application via an aerosol spray. For a negative control, 4 ml of 100% ethanol was added to filtered butane to give an appropriate volume of solution for the aerosol sprays used for each test.

[00377] Selection of specimens: All caterpillars selected for use in these experiments were 5-6 cm long and actively moving. Caterpillars in the treatment groups were sprayed outside (in the open air) on bushes with lOOmg NKT aerosol per caterpillar or with the same amount of negative control aerosol.

[00378] Treatment conditions: The whole experiment was carried out at 26±1°C. During and after treatment, caterpillars were permitted to move where they wished over the branches of the bushes.

[00379] Assessment / Observations: Caterpillars of the nootkatone-treated and negative control groups were frequently monitored over the 6 hours course of the experiment for whole body movements and categorised as sinuously motile, sluggishly motile (intermittent jerky movements on touch), coiling, other movements, immotile/paralysed, or dead. RESULTS

[00380] Most nootkatone-treated caterpillars were noted to be dead by 30 to 45 minutes after treatment. Of the surviving caterpillars, all caterpillars exhibited restricted movement and became inactive within 1 hour of treatment. By 6 hours post-treatment, all caterpillars were dead and many had darkened. In contrast, the caterpillars exposed to negative control were observed to be alive, exhibited normal motility and were feeding normally (see FIGS. 51A-D).

Example No. 14: Evaluation Of Nootkatone Residue On Larval Food Source For Feeding Reduction And Larvicidal Activity.

[00381] Overview: Some methods of preventing and/or treating larvae disclosed herein comprise the application of nootkatone to a larval food source or hatching location. The aim of this experiment was to confirm that the larvicidal activity of Nootkatone extends to untreated larvae coming into contact and/or eating larval food sources sprayed with Nootkatone.

METHODOLOGY

[00382] Collection of specimen: Live caterpillars were collected from the same bush.

[00383] Test Formulations: Nootkatone (NKT) solution at a concentration of 600 ppm was obtained by dissolving 98% pure NKT in 100% EtOH and diluting in filtered Butane (LPG) to permit application via an aerosol spray. For a negative control, 4 ml of 100% ethanol was added to filtered butane (LPG) to give an appropriate volume of solution for the aerosol sprays used for each test.

[00384] Selection of specimens: All caterpillars selected for use in these experiments were 5-6 cm long and actively moving. Eight caterpillars were placed in each treatment group and kept in individual plastic trays with lOOg leaves. The leaves in one treatment group were sprayed with 300 μg NKT aerosol. The leaves in the negative control group were sprayed with the same quantity negative control aerosol.

[00385] Treatment conditions: The entire experiment was carried out at 26±1°C, which was well within the natural temperature range commonly experienced by the species of caterpillar tested. During treatment, caterpillar larvae were maintained in identical clean plastic trays containing the relevant leaves. The trays were oversized such that the caterpillars could crawl away from the leaves if their instinct was to do so. [00386] Assessment / Observations: Caterpillars were under continuous observation and scored for motility, immobility and death at 0 hours, 6 hours and 24 hours following introduction to NK treated or control leaves. Typical displays of motility included whole body movements, sinuous motility, coiling, and leg movements. Cessation of feeding, sluggish motility and intermittent jerky movements on touch stimulation were typical early symptoms of immotility and/or paralysis.

RESULTS

[00387] Greatly reduced rates of feeding were observed in trays containing leaves treated with nootkatone. The caterpillars were observed trying to move away from the leaves. Within 6 hours post-exposure, all caterpillars introduced to nootkatone treated leaves exhibited very restricted movement and became inactive. By 24 hours post-exposure, all caterpillars in the trays containing nootkatone-treated leaves were seen to be dead, whilst all the caterpillars in the negative control were alive and feeding normally (see FIGS. 52A-E).

Example No. 15: Comparison of nootkatone with citrus-derived nootkatone.

Overview

[00388] Nootkatone, as defined herein, has a particular chemical profile indicative of its constituent chemical species. Other sources of nootkatone can have different chemical profiles and therefore actually represent different chemical compositions. GC-FID analyses of the nootkatone used in the studies described above (obtained from oxidation of fermentation-derived valencene, also known as, nootkatone ex valencene (NxV)) and a citrus fruit-derived nootkatone (also known as nootkatone ex citrus, which is derived from citrus fruit and available from Frutarom®, Corona, CA) are shown in Fig. 53. The nootkatone used in the studies described herein lacked valencene and demonstrated a lower amount of 11,12- epoxide than the Frutarom® nootkatone. Moreover, further analysis of an unknown peak from the Frutarom® nootkatone sample revealed that the Frutarom® sample contained limonene (see Fig. 54), whereas the nootkatone used in the present studies was limonene-free. These results underscore the different chemical profile of the nootkatone used herein (NxV) compared to commercially-available nootkatone derived from citrus, such as that provided by Frutarom®. [00389] These results are also in accord with the observation (not shown) that nootkatone obtained from fermentation-derived valencene does not contain bergapten (or bergaptine). Bergapten (5-methoxypsoralen or 5 -MOP) is a compound found in bergamot and citrus essential oils that causes phototoxicity in humans. (Gionfriddo et al. "Elimination of Furocoumarins in Bergamot Peel Oil," Perfumer & Flavorist., 2004; 29:48-52; Ferreira Maia et al. "Plant-based insect repellents: a review of their efficacy, development and testing," Malaria Journal, 2011; 10:Suppll-l 1; and Kejlova et al. "Phototoxicity of bergamot oil assessed by in vitro techniques in combination with human patch tests." Toxicol In Vitro. 2007; 21 : 1298-1303). In addition, GHS health warning statements for bergapten indicate that it can cause allergic skin reactions, allergy or asthma symptoms, or breathing difficulties if inhaled, and can cause genetic defects or cancer in animals. For such reasons, a Cosmetic Ingredient Review expert panel in assessing the safety of 14 citrus-derived peel oil ingredients concluded no more than 0.0015% (15 ppm) bergapten should be included in cosmetic products {see "Safety Assessment of Citrus-Derived Peel Oils as Used in Cosmetics," Cosmetic Ingredient Review Expert Panel Final Report, September 30, 2014: 1- 31).

[00390] Bergapten is present in naturally derived valencene (from citrus) and carries over through the chemical oxidation that forms nootkatone. Bergapten can be photo-activated to become a skin irritant. Therefore, bergapten-free nootkatone obtained from fermentation- derived valencene has particular advantages over plant-derived nootkatone when applied to consumable products to be handled by agricultural workers and the like.

Example No. 16: Production of Nootkatone ex Valencene.

[00391] Nootkatone ex valencene may be produced in vivo through expression of one or more enzymes involved in the nootkatone biosynthetic pathway in a recombinant yeast or in vitro using isolated, purified enzymes involved in the nootkatone biosynthetic pathway, such as those described in U.S. Patent Application Publication Nos. 2015/0007368 and 2012/0246767. The final conversion of valencene to nootkatone may be done enzymatically in vivo or in vitro, or may be performed by chemical oxidation (typically inorganic) in vitro.

[00392] Briefly, the valencene synthase gene (CVS) from Citrus sinensis cv. Valencia (Valencia orange) was cloned from RNA isolated from the juice vesicles of freshly harvested Valencia orange using the procedure previously described in Example 1 of U.S. Pat. No. 7,442,785.

[00393] First, Yep-GW-URA (Takahashi et al, (2007) Biotechnol Bioeng. 97(1): 170-181) was generated by inserting a gateway cloning cassette (RfB) with the form attRl-Cm R -ccdB gene-attR2 (Hartley et al, (2000) Genome Res. 10: 1788-1795) into the Smal restriction site of YEp352-URA (Bio-Technical Resources), which contains an URA3 selectable marker, an ADH1 promoter and an ADH1 terminator flanking, two BamHI sites (one 5' to the ADH1 promoter and the other 3' to the ADH terminator), a 2-micron ori, an ampicillin resistance gene and a colEl origin of replication. The resulting vector was designated YEp-CVS-URA.

[00394] The CVS gene (set forth in SEQ ID NO: 1, and encoding amino acid sequence is set forth in SEQ ID NO: 2) was then amplified from RNA isolated from the juice vesicles of freshly harvested Valencia orange to contain restriction sites for subcloning into the yeast shuttle expression vector Yep-GW-URA. Following digestion of Yep-GW-URA with EcoRI and Xbal, the amplified product was cloned into the yeast shuttle expression vector YEp- GW-URA.

[00395] The YEp-CVS-ura vector was maintained in S. cerevisiae by selecting on SD minimal medium lacking uracil at 28° C. The vector also was maintained in Escherichia coli by selecting for resistance to ampicillin on LB medium containing 100 μg/mL ampicillin.

[00396] To screen for production of valencene, the Saccharomyces cerevisiae yeast cell strains CALI5-1 (ura3, leu2, his3, trpl, Aerg9::HIS3, HMG2cat/TRPl ::rDNA, dppl, sue), ALX7-95 (ura3, his3, trpl, Aerg9::HIS3, HMG2cat/TRPl ::rDNA, dppl, sue) or ALXl l-30 (ura3, trpl, erg9def25, HMG2cat/TRPl ::rDNA, dppl, sue) were used.

[00397] The CALI5-1 strain (see U.S. published Appl. No. US20040249219; U.S. Pat. Nos. 6,531,303 and 6,689,593) has a Aleu2 deletion, which required the introduction of leucine into its media. ALX7-95 was derived from CALI5-1 by correcting the Aleu2 deficiency of CALI5-1 with a functional LEU2 gene (see U.S. published Appl. No. US2010/0151519).

[00398] ALXl l-30 was constructed from CALI5-1 in several steps from ALX7- 175.1 as described in US2010/0151519. Briefly, ALX7-95 HPS was obtained by transforming a plasmid containing the Hyoscyamus muticus premnaspirodiene synthase (HPS) into ALX7-95 strain. The YEp-HPS plasmid was obtained by cloning the gene for HPS into Yep-GW-URA to give YEp-HPS-ura (YEp-HPS). Then, an error prone PCR reaction of the ERG9 gene was performed, and the resulting DNA was transformed into ALX7-95 harboring YEpHPS. Transformants were plated on YP medium lacking ergosterol and screened for premnaspirodiene production. Those that produced high levels of premnaspirodiene were saved. One strain, ALX7- 168.25 [ura3, trpl, his3, erg9 def 25, HMG2cat/TRPl ::rDNA, dppl, sue, YEpHPS] was transformed with a PCR fragment of the complete HIS3 gene to create a functional HIS3 gene. Transformants were isolated that were able to grow in the absence of histidine in the medium. From this transformation, ALX7- 175.1 was isolated [ura3, trpl, erg9def25, HMG2cat/TRPl ::rDNA, dppl, sue YEpHPS]. Finally, the plasmid YEpHPS was removed by growing ALX7- 175.1 several generations in YPD (10 g/L yeast extract, 20 g/L peptone, 20 g/L glucose) and plating cells on YPD plates. Colonies were identified that were unable to grow on SD medium without uracil (0.67 Bacto yeast nitrogen base without amino acids, 2% glucose, 0.14% yeast synthetic drop-out medium without uracil). This strain was designated ALX11-30.

[00399] For screening for production of valencene by valencene synthase or mutants, the YEp-CVS-ura plasmid, containing the CVS gene or modified versions of the CVS gene, was transformed into the above yeast strains using the lithium acetate yeast transformation kit (Sigma- Aldrich). The ALX7-95 and ALX11-30 strains generally produced more valencene than the CALI5-1 strain. CALI5-1 was used for initial screening in vials (as described in Example 3) and production in fermenters. Subsequently, ALX7-95 or ALX11-30 was used for screening in vials and fermenters. Typically, ALX7-95 was used for screening in vials and ALX11-30 was used for fermenters.

[00400] Transformants were selected on SDE-ura medium (0.67% Bacto yeast nitrogen base without amino acids, 2% glucose, 0.14% yeast synthetic drop-out medium supplement without uracil, and 40 mg/L ergosterol as needed). Colonies were picked and screened for valencene production using the microculture assay described below.

[00401] Production of valencene was performed in a 3-L fermentation tank (New Brunswick Bio flow 110). One liter of fermentation medium was prepared and autoclaved in the fermentation tank (20 g (NH 4 ) 2 S0 4 , 20 g KH 2 P0 4 , 1 g NaCl, MgS0 4 .7H 2 0, 4 g Solulys corn steep solids (Roquette)). The following components were then added: 20 ml mineral solution (0.028% FeS0 4 .7H 2 0, 0.029% ZnS0 4 .7H 2 0, 0.008% CuS0 4 .5H 2 0, 0.024% Na 2 Mo0 4 .2H 2 0, 0.024% CoCl 2 .6H 2 0, 0.017% MnS0 4 .H 2 0, 1 mL HC1); 10 mL 50% glucose; 30 mL vitamin solution (0.001%> biotin; 0.012%> calcium pantothenate, 0.06%> inositol, 0.012% pyridoxine-HCl, 0.012% thiamine-HCl); 10 mL 10% CaCl 2 , and 20 mL autoclaved soybean oil (purchased from local groceries). For sterol-requiring strains, including CALI5-1 and ALX7-95, 50 mg/L cholesterol or 40 mg/L ergosterol was included in the medium.

[00402] The seed culture for inoculating the fermentation medium was prepared by inoculating 50 mL of SDE-ura-trp medium (see Example 3.C.2.) with CALI5-1, ALX7-95 or ALXl l-30 containing the YEp-CVS-ura plasmid. This culture was grown at 28° C. until early stationary phase (24-48 hr). One mL of this culture was inoculated into 500 mL of SDE-ura-trp medium and grown for 24 hr at 28° C. A 50 mL aliquot (5% inoculum) was used to inoculate the medium in the fermentation tank.

[00403] The fermentor was maintained at 28° C. The air flow was 1 wm and the d0 2 was maintained above 30% by adjusting the agitation. The pH was maintained at 4.5 using phosphoric acid and NaOH or ΝΗ 4 ΟΗ.

[00404] When the glucose concentration fell below 1 g/L, a feeding regimen was initiated such that the glucose in the fermentor was kept between 0 and 1 g/L. The glucose feed consisted of 60% glucose (w/v).

[00405] At the end of the fermentation, generally about 132 hours after inoculation, sodium sulfate was added to 10-15% final concentration as was an additional 50 mL soybean oil, and the contents of the fermentor were agitated for one hour. After allowing the fermentation vessel contents to settle, the oil was recovered by centrifugation and the valencene content in the oil was determined.

[00406] To assay valencene, 3 mL of suspension was placed in a vial to which 3 mL of acetone containing 20 mg/L cedrene was added. After vortexing, the mixture was extracted with 6 mL hexane containing 10 mg/L hexadecane followed by additional vortexing. The organic phase was transferred to a second vial for analysis by gas chromatography using cedrene and hexadecane as internal standards for extraction efficiency and injection, respectively. The CALI5-1, ALX7-95 or ALXl l-30 S. cerevisiae containing Yep-CVS-ura, and expressing valencene synthase, was found to produce valencene.

[00407] The valencene-containing soybean oil, produced by fermentation as described above, was concentrated and purified using wiped-film distillation at 100° C. and 350 mTorr to generate an oil that contained approximately 68% valencene by weight. This material was converted to nootkatone by two different methods described below.

A. Oxidation of Valencene to Nootkatone Using Chromium Trioxide [00408] The valencene distillate produced as described above was oxidized to nootkatone using chromium trioxide and pyridine in dichloromethane as follows. Chromium trioxide (369 g, 3.69 mol, 22 eq) was added in portions to a solution of pyridine (584 g, 7.4 mol, 44 eq) in 5 L of dichloromethane. The mixture was stirred for 10 minutes, 50 grams of valencene distillate (68% w/w, 0.167 mol, 1 eq) was added over four minutes, and the mixture was stirred at 22° C. for 18 hours. The liquor was drained from the vessel, and the solids were washed twice with 2 L of methyl tert-butyl ether (MTBE). The combined organic layers were further diluted with 2 L of MTBE and successively washed three times with 1.25 L of 5% sodium hydroxide, twice with 2 L of 5% hydrochloric acid, and once with 2 L of brine. The organic phase was dried over 200 grams of anhydrous sodium sulfate, filtered, and concentrated by evaporation to give 36.8 grams crude nootkatone (48% w/w, 0.081 mol, 48% yield).

B. Oxidation of Valencene to Nootkatone Using Silica Phosphonate- Immobilized Chromium (III) Catalyst

[00409] Silica phosphonate chromium (III) resin (48.9 g, PhosphonicS, Ltd.) was placed in a 5 L round bottom flask equipped with a condenser, thermowell, overhead stirrer, and sparge tube. Two (2) L of t-butanol and valencene distillate (68%, 500 g, 1.67 moles, 1 eq) were added, the contents were heated to 45° C, and the heterogeneous suspension was allowed to stir as oxygen was sparged through the solution (ca 1.5 L/min) and nitrogen flushed over the head-space. 70% t-butyl hydroperoxide in water (TBHP, 315 g, 2.45 moles, 1.47 eq) was added to the solution over 2 hr while the temperature of the reaction was heated and maintained at 60±5° C. The reaction was allowed to stir until >90% of the valencene was consumed, as determined by gas chromatography. The reaction was then allowed to cool to room temperature and the silica catalyst removed by filtration. The flask and resin were washed with 500 mL isopropanol. One (1) L of deionized water was added to the combined organic solution (t-butanol and isopropanol), and the mixture was concentrated under reduced pressure by evaporation to afford an amber colored oil. The oil was dissolved in 3 L of toluene and washed with 3.125 L of 15% sulfuric acid for 15 minutes with vigorous agitation. The aqueous layer was removed and re-extracted with 1 L of toluene. The combined toluene layers were then washed three times with 2.5 L of 1 M sodium hydroxide, twice with 500 mL saturated sodium chloride, and dried over anhydrous magnesium sulfate. After filtration, the solvent was removed under reduced pressure by evaporation to afford 378 g of viscous amber oil (33%) nootkatone by weight, 0.57 moles, 34% yield). Example No. 17: Susceptibility of Uncinula necator to treatment with nootkatone.

[00410] This study describes a laboratory bioassay in which spores of Uncinula necator were exposed to a nootkatone-containing composition to determine germination susceptibility to nootkatone.

[00411] The organisms used for testing are shown in Table No. 18 below. [00412] Table No. 18. Organisms used for testing.

Treatment

[00413] Stock solutions of nootkatone were prepared by dissolving 50 mg of nootkatone in 1 mL of ethanol. Stock solutions of sulphur were prepared at 2 mg/mL. Nootkatone test formulation was prepared at 10% active ingredient concentration and dissolved in milli Q water to get 50mg/ml. Sulphur was used as positive control and prepared from the commercial fungicide Sultaf 80 WG diluted in milli Q, just before use. The final test concentrations for nootkatone were 500, 250, 125, and 62.5 ppm, and sulphur was 2000 ppm concentration.

Spore suspension preparation

[00414] Leaves of grape vines affected with powdery mildew were collected. Conidial chains (asexual spores) of U necator were disturbed with a brush in milliQ water to obtain individual conidia. Spore numbers were adjusted to 2 XI 0 4 spores per millilitre with the help of a Haemocytometer (Neubauer chamber).

Method of testing

[00415] The spore germination assay was performed using a 96 well micro titre plate in a sterile environment. One hundred microliters of spore suspension were added to each well, followed by a test concentration of either nootkatone or sulphur. For negative controls, water alone was added and for solvent control, instead of test compounds, 5 μί/ι ί ethanol was added to the media. Compound control and media control without spores were also maintained. For each treatment, triplicate wells were maintained with about 1000 spores in each well. Plates were incubated at 25°C in a moist sterile chamber.

Assessments

[00416] Spores were observed after 20 hours of incubation in a moist chamber. Between 100 to 150 spores were observed using the microscope, and the spores were scored according to the following criteria:

Fully germinated - with well-developed germ tube

Not germinated - intact rod shaped spores without germ tubes or beaklike, aborted germ tubes

Spore germination inhibition by treatments was calculated in comparison with germination rates observed in the controls. The formula to calculate percentage spore germination inhibition compared to control is as follows:

% inhibition = ((% mean spore germination in control samples - % mean spore germination in treated samples)/ % mean spore germination in control) X 100

Results

[00417] The results are shown in Table No. 19.

[00418] Table No. 19. Effects of Nootkatone Treatment of spore germination of Uncinula necator.

[00419] The untreated control spores exhibited 42% germination after 20 hours incubation and were observed to display noticably longer germtubes. No significant inhibition was observed in the control ethanol-treated spores.

[00420] Photographs of spore germination of Uncinula necator in control and nootkatone treated samples are illustrated in Figures 55 and 56, respectively. Nootkatone at 63 and 125 ppm exhibited 82 and 87% inhibition, respectively, compared to control. Nootkatone at 500 ppm exhibited almost complete inhibition of spore germination. Almost all spores were intact, with just a few displaying a small beak-like germtube. By contrast, the commercial fungicide having Sulphur as active ingredient showed only 44% inhibition at 2000 ppm.

[00421] The test results indicate that there was good inhibition of spore germination by nootkatone at concentrations of 63 ppm and above. Based on these observations, nootkatone can be used to control powdery mildew of grapes in the fields.

[00422] Having described the invention in detail and by reference to specific embodiments thereof, it will be apparent that modifications and variations are possible without departing from the scope of the invention defined in the appended claims. More specifically, although some aspects of the present invention are identified herein as particularly advantageous, it is contemplated that the present invention is not necessarily limited to these particular aspects of the invention.

Sequence Listing: SEQ ID NO: 1 (Citrus valencene synthase) atgtcgtctggagaaacatttcgtcctactgcagatttccatcctagtttatggagaaac catttcctcaaaggtgcttctgatttcaagaca gttgatcatactgcaactcaagaacgacacgaggcactgaaagaagaggtaaggagaatg ataacagatgctgaagataagcctgtt cagaagttacgcttgattgatgaagtacaacgcctgggggtggcttatcactttgagaaa gaaatagaagatgcaatacaaaaattatgt ccaatctatattgacagtaatagagctgatctccacaccgtttcccttcattttcgattg cttaggcagcaaggaatcaagatttcatgtgat gtgtttgagaagttcaaagatgatgagggtagattcaagtcatcgttgataaacgatgtt caagggatgttaagtttgtacgaggcagcat acatggcagttcgcggagaacatatattagatgaagccattgctttcactaccactcacc tgaagtcattggtagctcaggatcatgtaac ccctaagcttgcggaacagataaatcatgctttataccgtcctcttcgtaaaaccctacc aagattagaggcgaggtattttatgtccatga tcaattcaacaagtgatcatttatacaataaaactctgctgaattttgcaaagttagatt ttaacatattgctagagctgcacaaggaggaac tcaatgaattaacaaagtggtggaaagatttagacttcactacaaaactaccttatgcaa gagacagattagtggagttatatttttgggatt tagggacatacttcgagcctcaatatgcatttgggagaaagataatgacccaattaaatt acatattatccatcatagatgatacttatgatg cgtatggtacacttgaagaactcagcctctttactgaagcagttcaaagatggaatattg aggccgtagatatgcttccagaatacatgaa attgatttacaggacactcttagatgcttttaatgaaattgaggaagatatggccaagca aggaagatcacactgcgtacgttatgcaaaa gaggagaatcaaaaagtaattggagcatactctgttcaagccaaatggttcagtgaaggt tacgttccaacaattgaggagtatatgcct attgcactaacaagttgtgcttacacattcgtcataacaaattccttccttggcatgggt gattttgcaactaaagaggtttttgaatggatct ccaataaccctaaggttgtaaaagcagcatcagttatctgcagactcatggatgacatgc aaggtcatgagtttgagcagaagagagg acatgttgcgtcagctattgaatgttacacgaagcagcatggtgtctctaaggaagaggc aattaaaatgtttgaagaagaagttgcaaa tgcatggaaagatattaacgaggagttgatgatgaagccaaccgtcgttgcccgaccact gctcgggacgattcttaatcttgctcgtgc aattgattttatttacaaagaggacgacggctatacgcattcttacctaattaaagatca aattgcttctgtgctaggagaccacgttccattt tga

SEQ ID NO: 2 (Citrus valencene synthase)

MSSGETFRPTADFHPSLWR HFLKGASDFKTVDHTATQERHEALKEEVRRMITDAE

DKPVQKLRLIDEVQRLGVAYHFEKEIEDAIQKLCPIYIDSNRADLHTVSLHFRLLRQ Q

GIKISCDVFEKFKDDEGRFKSSLINDVQGMLSLYEAAYMAVRGEHILDEAIAFTTTH L

KSLVAQDHVTPKLAEQINHALYRPLRKTLPRLEARYFMSMINSTSDHLYNKTLLNFA

KLDFNILLELHKEELNELTKWWKDLDFTTKLPYARDRLVELYFWDLGTYFEPQYAF

GRKIMTQLNYILSIIDDTYDAYGTLEELSLFTEAVQRWNIEAVDMLPEYMKLIYRTL L

DAFNEIEEDMAKQGRSHCVRYAKEENQKVIGAYSVQAKWFSEGYVPTIEEYMPIAL

TSCAYTFVITNSFLGMGDFATKEVFEWIS NPKVVKAASVICRLMDDMQGHEFEQK

RGHVASAIECYTKQHGVSKEEAIKMFEEEVANAWKDINEELMMKPTVVARPLLGTI

LNLARAIDFIYKEDDGYTHSYLIKDQIASVLGDHVPF