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WO 2014/160354 Al 2 October 2014 (02.10.2014) P O P C T

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WO 2014/160354 Al 2 October 2014 (02.10.2014) P O P C T (12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (19) World Intellectual Property Organization International Bureau (10) International Publication Number (43) International Publication Date WO 2014/160354 Al 2 October 2014 (02.10.2014) P O P C T (51) International Patent Classification: (81) Designated States (unless otherwise indicated, for every C12N 1/20 (2006.01) kind of national protection available): AE, AG, AL, AM, AO, AT, AU, AZ, BA, BB, BG, BH, BN, BR, BW, BY, (21) International Application Number: BZ, CA, CH, CL, CN, CO, CR, CU, CZ, DE, DK, DM, PCT/US2014/026371 DO, DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, (22) International Filing Date: HN, HR, HU, ID, IL, IN, IR, IS, JP, KE, KG, KN, KP, KR, 13 March 2014 (13.03.2014) KZ, LA, LC, LK, LR, LS, LT, LU, LY, MA, MD, ME, MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, NO, NZ, (25) Filing Language: English OM, PA, PE, PG, PH, PL, PT, QA, RO, RS, RU, RW, SA, (26) Publication Language: English SC, SD, SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM, TN, TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, (30) Priority Data: ZW. 61/783,395 14 March 2013 (14.03.2013) US 61/783,573 14 March 2013 (14.03.2013) US (84) Designated States (unless otherwise indicated, for every kind of regional protection available): ARIPO (BW, GH, (71) Applicant: GEORGIA STATE UNIVERSITY RE¬ GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, SZ, TZ, SEARCH FOUNDATION, INC. [US/US]; P.O. Box UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, RU, TJ, 3999, Atlanta, Georgia 30302 (US). TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, DK, EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU, LV, (72) Inventors: PIERCE, George E.; 112 Rosebury Drive, MC, MK, MT, NL, NO, PL, PT, RO, RS, SE, SI, SK, SM, Canton, Georgia 301 15 (US). CROW, Sidney; 2033 Cliff TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, GW, Creek Court, Smyrna, Georgia 30080 (US). KM, ML, MR, NE, SN, TD, TG). (74) Agents: GILES, P. Brian et al; Meunier Carlin & Curf- Declarations under Rule 4.17 : man, LLC, Suite 500, 817 W. Peachtree Street NW, At lanta, Georgia 30308 (US). — of inventorship (Rule 4.17(iv)) [Continued on next page] (54) Title: INHIBITING OR REDUCING FUNGAL GROWTH (57) Abstract: Provided are methods and compositions for inhibiting or reducing fungal growth. The methods comprise exposing a location to a composition comprising one or more enzymes, one or more bacteria, and/or an enzymatic extract, wherein the one or more enzymes, one or more bacteria, and/or the enzymatic extract isolated from one or more bac teria are exposed to location in a quantity sufficient to inhibit or reduce fungal growth. Figure 2 w o 2014/160354 A l 111 111 II III Hill II II I III llll I I Hill II I II Published: INHIBITING OR REDUCING FUNGAL GROWTH CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 61/783,395, filed March 14, 2013, and U.S. Provisional Application No. 61/783,573, filed March 14, 2013, which are hereby incorporated herein by reference in their entirety. BACKGROUND Fungi can be detrimental to many different facets of life. For example, fungi (e.g., mildew or mold) can negatively affect aesthetics or human living conditions, e.g., through degradation/deterioration of material, through contamination, by making material, e.g., wood, appear undesirable, or through production of undesirable toxins. By way of another example, fungi can be detrimental on fruits and vegetables, as entire harvests of a fruit or vegetable could be wiped out by the growth of a fungus, e.g., through contamination and/or production of undesirable toxins. Many fungi respond to ethylene, often with spore germination being a fungal ethylene response mechanism. While some fungi are known to produce ethylene, many more fungi do not synthesize ethylene but can still respond to ethylene. Methods targeting the response to ethylene or production of ethylene in fungi could therefore be targeted to inhibit or reduce fungal growth. SUMMARY Provided herein are methods and compositions for inhibiting or reducing fungal growth. The methods comprise exposing the plant or plant part to one or more bacteria, one or more enzymes, an enzymatic extract isolated from one or more bacteria, or any combination thereof, in a quantity sufficient to inhibit or reduce fungal growth at the location. The one or more bacteria can be selected from the group consisting of genus Rhodococcus, genus Brevibacterium, genus Pseudonocardia, genus Nocardia, genus Pseudomonas, and combinations thereof. The one or more enzymes can be selected from the group consisting of nitrile hydratases, amidases, asparaginases, ACC deaminases, cyanoalanine synthase-like enzymes, monooxygenases, dioxygenases, cyandiases, and combinations thereof. The details of one or more aspects are set forth in the accompanying drawings and description below. Other features, objects, and advantages will be apparent from the description and drawings and from the claims. DESCRIPTION OF DRAWINGS Figures 1A and IB show a comparison, at Day 6, between commercially prepared peaches (with fungicide and wax treatments) (Fig. 1A) with non-processed peaches (containing no fungicide or wax coating) that have been placed in wrapping paper containing catalyst (Fig IB). The catalyst treated peaches were free of visible mold growth, whereas the fungicide treated peaches showed significant mold growth. Figure 2 shows exposure of Cladosporium sp. spores to R. rhodochrous cells grown under selected conditions. Figure 2A1/2B1 shows the results when a defined number of spores of Cladosporium sp. were retained on 0.2 micron filters and the filters containing the fungal spores were then placed onto various media as follows: Cladosporium sp. spores on fungal growth medium without Rhodococcus present (2A1) and on a medium for delayed ripening activity containing Rhodococcus cells (2B1); Figure 2A2/2B2 shows filter containing the Cladosporium sp. spores on a fungal spore recovery medium (2A2), without Rhodococcus present, and on partially induced medium (2B2) containing Rhodococcus cells, and Figure 2A3/2B3 shows a filter containing Cladosporium sp. spores on a Rhodococcus growth medium without Rhodococcus present ( A3) and on with Rhodococcus present on the same growth medium, which is partially inducing the Rhodococcus Cells (2B3). The induced R. rhodochrous DAP 96253 cells clearly inhibited the germination of the Cladosporium sp. spores. Figure 3 shows fungal inhibition after exposure to Rhodococcus for 6 days. Sectored plates were used such that media supporting Rhodococcus growth were used in selected sectors while media supporting fungal growth was placed in the other sectors. In Figures 3A and 3B, Rhodococcus were placed in a sector containing a growth medium which induced the Rhodococcus cells. In the other sectors, a defined number of Fusarium sp. spores were placed in a medium which supported Fusarium spore germination and mycelia growth. Fusarium spore germination was completely inhibited after 6 days. In Figures 3C and 3D, the Rhodococcus cells were placed in a medium which supported growth but which did not induce the Rhodococcus cells. As in Figures 3A and 3B, the Fusarium spores were placed onto a medium supportive of spore germination and mycelia growth. It is apparent from Figures 3C and 3D that non-induced Rhodococcus cells did not inhibit spore germination and fungal growth of Fusarium. The Fusarium spores were inoculated on SAB media in separate compartment on all the plates. Figure 4 shows the effect of Rhodococcus cells on Fusarium sp. sporulation. Figure 4A shows the growth of Fusarium exposed to induced cells Rhodococcus in phosphate buffer. Figure 4B shows a control of non-inducing, growth medium for non-induced Rhodococcus. Figures 4C and 4D shows the growth of Fusarium exposed to sub-induced Rhodococcus in phosphate buffer. Fully induced Rhodococcus cells inhibited the growth of Fusarium sp. Figure 5 shows a non-limiting depiction of a three-layer apparatus for inhibiting or delaying fungal growth. The outer layers provide structural integrity to the apparatus. The catalyst layer, as defined herein below, comprises one or more of the enzymes or one or more bacteria disclosed herein and is located between the outer layers. Figures 6A to 6C provide non-limiting depictions of various apparatuses for inhibiting or delaying fungal growth. These apparatuses comprise a catalyst layer, one or more layers intended to provide structural integrity, and one or more layers intended to be removed prior to use of the apparatus. Removal of one or more of these layers may, for example, expose an adhesive for attachment of the apparatus to another physical structure. Figures 7A and 7B show non-limiting depictions of an apparatus for inhibiting or delaying fungal growth. The apparatus comprises a catalyst immobilized on a layer of film and attached to a physical structure (e.g., a box suitable for storage/transportation of fruit). Figure 8 provides a non-limiting depiction of an apparatus for inhibiting or delaying fungal growth. The apparatus comprises a slotted chamber structure that permits the insertion and replacement of one or more catalyst module elements, as defined below. The outer layers of the physical structure may be composed of a material that permits air to flow into the catalyst. Figure 9 is a comparison of Control Germination and Growth of G. destructans Spores, with Spores Exposed to Non-Induced and to Induced Cells of R. rhodochrous DAP 96253, at 15° C. Figure 10 is a comparison of Control Germination and Growth of G. destructans Spores, with Spores Exposed to Non-Induced and to Induced Cells of R.
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