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(12) United States Patent (10) Patent No.: US 9,161,545 B2 Levy Et Al USOO916.1545B2 (12) United States Patent (10) Patent No.: US 9,161,545 B2 Levy et al. (45) Date of Patent: *Oct. 20, 2015 (54) PSEUDOZYMAAPHIDISAS A BIOCONTROL Avis, T.J. and Belanger, R.R. (2002) “Mechanisms and Means of AGENT AGAINST VARIOUS PLANT Detection of Biocontrol Activity of Pseudozyma Yeasts Against Plant Pathogenic Fungi.” FEMSYeast Res 2(1):5-8. PATHOGENS Avis, T.J., et al. (2001) "Molecular and Physiological Analysis of the Powdery Mildew Antagonist Pseudozyma flocculosa and Related (71) Applicant: Yissum Research Development Fungi.” Phytopathology 91(3):249-254. Company of the Hebrew University of Begerow, D. and Bauer, R. (2000) “Phylogenetic Placements of Jerusalem Ltd., Jerusalem (IL) Ustilaginomycetous Anamorphs. As Deduced From Nuclear LSU rDNA Sequences.” Mycol. Res. 104:53-60. (72) Inventors: Marganit Levy, Rehovot (IL); Aviva Boekhout, T. (1995) “Pseudozyma Bandoni emend Boekhout, A Genus for Yeast-Like Anamorphs of Ustilanginales,” J. Gen. Appl. Gafni, Rishon LeZion (IL) Microbiol. 41:359-366. Buxdorf, K. etal. (2013) “The Epiphytic Fungus Pseudozyma aphidis (73) Assignee: Yissum Research Development Induces Jasmonic Acid- and Salicylic Acid/Noneypressor of PRI Company of the Hebrew University of Independent Local and Systemic Resistance.” Plant Pathol. Jerusalem Ltd., Jerusalem (IL) 161:2014-2022. Dik, A.J., et al. (1998) “Comparison of Three Biological Control (*) Notice: Subject to any disclaimer, the term of this Agents Against Cucumber Powdery Mildew (Sphaerotheca fuliginea) patent is extended or adjusted under 35 In Semi-Commercial-Scale Glasshouse Trials.” Eur. J. Plant Pathol. U.S.C. 154(b) by 0 days. 104(413-423). Henninger, W. and Windisch, S. (1975) “A New Yeast This patent is Subject to a terminal dis Sterigmatomyces, S. aphidis sp. n.” Arch. Microbiol. 105:49-50. claimer. International Search Report PCT/IL2011/000420 (2011), pp. 1-4. Kitamoto, D. etal. (1993)"Surface Active Properties and Antimicro (21) Appl. No.: 14/173,535 bial Activities of Mannosylerythritol Lipids. As Biosurfactants Pro duced by Candida Antarctica.” J. Biotechnol. 29.91-93. (22) Filed: Feb. 5, 2014 Konishi, M. et al. (2007) “Production of Different Types of Man nosylerythritol Lipids. As Biosurfactants by the Newly Isolated Yeast (65) Prior Publication Data Strains Belonging to the Genus Pseudozyma.” Appl. Microbiol. Biotechnol. 75:521-531. US 2014/O141976 A1 May 22, 2014 Motita, T. and Konishi, M. (2007) “Physiological Differences in the Formation of the Glycolipid Biosurfactants, Mannosylerythritol Lipids, Between Pseudozyma antarctica and Pseudozyma aphidis.” Related U.S. Application Data Appl. Microbiol. Biotechnol. 74:307-315. (63) Continuation of application No. 13/700,751, filed as Paulitz, T.C. and Belanger, R.R. (2001) “Biological Control in application No. PCT/IL2011/000420 on May 31, Greenhouse Systems.” Annu Rev. Phytopathol. 39:103-133. Rau, U. etal. (2005) “Formation and Analysis of Mannosylerythritol 2011, now Pat. No. 8,975,213. Lipids Secreted By Pseudozyma aphidis.” Appl. Microbiol. (60) Provisional application No. 61/350,217, filed on Jun. Biotechnol. 66:551-559. 1, 2010. Rau, U. etal. (2005) “Downstream Processing of Mannosylerythritol Lipids Produced by Pseudozyma aphidis.” Eur, J. Lipid Sci. Technol. (51) Int. Cl. 107:373-380. AOIN 63/04 Singh, P. and Cameotra, S.S. "Potential Applications of Microbial (2006.01) Surfactants in Biomedical Sciences.” TRENDS Biotechnol. (52) U.S. Cl. 22(3): 142-146. CPC ...................................... A0IN 63/04 (2013.01) Spadaro, D. and Gullino, M.L. (2004) “State of the Art and Future (58) Field of Classification Search Prospects of the Biological Control of Postharvest Fruit Diseases.” None Intl. J. Food Microbiol. 91:185-194. See application file for complete search history. Urquhart, E.J. and Punja, Z.K. (2002) “Hydrolytic Enzymes and Antifungal Compounds Produced by Tilletiopsis Species, Phyl (56) References Cited losphere Yeasts That Are Antagonists of Powdery Mildew Fungi.” Can. J. Microbiol. 48(3):219–229. U.S. PATENT DOCUMENTS Primary Examiner — Alton Pryor 2013, O184154 A1 7/2013 Levy et al. (74) Attorney, Agent, or Firm — AuerbachSchrot LLC; Jeffrey I. Auerbach; William C. Schrot FOREIGN PATENT DOCUMENTS (57) ABSTRACT WO WO 2004/O2O647 3, 2004 WO WO 2011, 151819 12/2011 Biocontrol agents are provided, which protect plant and plant material from pests and pathogens, and promote the growth of OTHER PUBLICATIONS plants. Also provided are compositions comprising the same, Allen, T.W., et al. (2004) “Effect of Foliar Disease on the Epiphytic methods for protecting plant and plant material and promot Yeast Communities of Creeping Benigrass and Tall Fescue.” Can. J. ing growth in plants, and uses of said biocontrol agent in the Microbiol. 50(10):853-860. preparation of a pesticidal composition and a growth-promot Avis, T.J. and Belanger, R.R. (2001) "Specificity and Mode of Action ing composition. of the Antifungal Fatty Acid cis-9-Heptadecenoic Acid Produced by Pseudozyma flocculosa.” Appl. Environ. Microbiol. 67(2):956-960. 29 Claims, 16 Drawing Sheets U.S. Patent Oct. 20, 2015 Sheet 1 of 16 US 9,161,545 B2 U.S. Patent Oct. 20, 2015 Sheet 2 of 16 US 9,161,545 B2 D - 2 - ) ---- ) ---- ) - C - CD esoTrì6oT eO?oTe3uteºE zTr? U.S. Patent Oct. 20, 2015 Sheet 3 of 16 US 9,161,545 B2 U.S. Patent Oct. 20, 2015 Sheet 4 of 16 US 9,161,545 B2 O O 20 30 Exp. t. UV (min) U.S. Patent Oct. 20, 2015 Sheet 5 of 16 US 9,161,545 B2 U.S. Patent Oct. 20, 2015 Sheet 6 of 16 US 9,161,545 B2 U.S. Patent Oct. 20, 2015 Sheet 7 of 16 US 9,161,545 B2 8 264. O O 0. A. EA PST XCC XCW C AB 3C Fig. 7C U.S. Patent Oct. 20, 2015 Sheet 8 of 16 US 9,161,545 B2 2. 5 2 5 T. P. inoculat. (d) Fig. 8 U.S. Patent Oct. 20, 2015 Sheet 9 of 16 US 9,161,545 B2 Wh. Plan. DetaC. Leav. PA PA U.S. Patent Oct. 20, 2015 Sheet 10 of 16 US 9,161,545 B2 dHO 1 O 108 autoclav. Fig. 9D Fig. 9E U.S. Patent Oct. 20, 2015 Sheet 11 of 16 US 9,161,545 B2 2O . s 100 8) n 60 us 9 9 40 20 O rio 12 S T. P. infect. (d) Fig. 9F U.S. Patent Oct. 20, 2015 Sheet 12 of 16 US 9,161,545 B2 O 22 28 34 38 T. P. Infect. (d) Fig. 10A U.S. Patent Oct. 20, 2015 Sheet 13 of 16 US 9,161,545 B2 &S &S O.O :S Fig. 11A :S 1 OO &S. & S. 80 :& & S. 60 :S. S. S. &, 40 S. 2O . , S 8::::::::::*::::-------------------------essarrassessessessessessessessessesselaers''''''''''''''''''' 20 23 29 37 44 '''''''' Fi 11B D. aft. Appl. 9 100 w. 10/ml sp. P. aphidis w/o 108/ml sp. P. aphidis Fig.11C U.S. Patent Oct. 20, 2015 Sheet 14 of 16 US 9,161,545 B2 Cont. Treat. PR PN2 ACT PR1 PN2 ACT Fig. 12A Treat. Cont. PR1 PDF1.2 PR1 PDF1.2 Fig. 12B U.S. Patent Oct. 20, 2015 Sheet 15 of 16 US 9,161,545 B2 Fig. 13A U.S. Patent Oct. 20, 2015 Sheet 16 of 16 US 9,161,545 B2 Fig. 13B T. P. inoculat. (d) US 9,161,545 B2 1. 2 PSEUDOZYMAAPHDISASA BOCONTROL group of yeast related to the Ustilaginales Boekhout, T. AGENT AGAINST VARIOUS PLANT (1995) General and Applied Microbiology 41(359-366). PATHOGENS They are mostly epiphytic (derive moisture and nutrients from the air and rain) or saprophytic (grow on and derive their CROSS-REFERENCE TO RELATED nourishment from dead or decaying organic matter), and they APPLICATIONS are non-pathogenic to plants and animals Avis, T. J. and Belanger, R. R. (2002) FEMS Yeast Res 2(1):5-81. This application claims priority to U.S. patent application Pseudozyma rugulosa and P. flocculosa have recently been Ser. No. 13/700,751 (filed Mar. 8, 2013), PCT/IL2011/ found to exhibit biological activity against the different pow 000420 (filed May 31, 2011; expired) and 61/350,217 (filed 10 dery mildews with which they are associated Dik, A.J., et al. Jun. 1, 2010), each of which applications is herein incorpo (1998) Eur. J. Plant Pathol. 104(413-4231. Pflocculosa has rated by reference in its entirety. been found to secrete an unusual fatty acid that displays FIELD OF THE INVENTION antibiotic activity against several pathogens Avis, T. J. and 15 Belanger, R. R. (2001) Appl Environ Microbiol 67(2): 956 The present invention relates to biocontrol agent derived 960; Avis, T. J., et al., (2001) Phytopathology 91(3):249 from Pseudozyma Aphidis effective against various plant 254. On the other hand, Avis et al. Avis, T. J., et al., (2001) pathogens. More particularly, the invention provides a com Phytopathology 91 (3):249-254 found no colony collapse of position comprising a bactericidal, fungicidal and pesticidal powdery mildew (Sphaerotheca fuliginea (Schlechtend.:Fr.) fungal biocontrol agent derived from P. Aphidis, which is also Pollacci) and no production of antifungal fatty acids by useful in enhancing plant growth, Vitality and pathogen resis Pseudozyma aphidis isolated from aphid Secretions (isolate tance, and extending the lifespan or shelf-life of produce and CBS 517.83). Paphidis is a close relative of P. rugulosa other organic products. The invention also relates to methods Begerow, D. and Bauer, R. (2000) Mycol. Res. 104(53-60), for accomplishing the same. which was first isolated from aphid secretions Henninger, W. 25 and Windisch, S. (1975) Arch. Microbiol. 105(1):47-48 but BACKGROUND OF THE INVENTION has also been found on plant surfaces Allen, T. W., et al., (2004) Can.
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