US007579183B1 (12) United States Patent (10) Patent No.: US 7,579,183 B1 Hua (45) Date of Patent: Aug. 25, 2009 (54) SAPROPHYTIC YEAST, PICHIA ANOMALA Symp. on Pistachios and Almonds (1998) (Eds) L. Ferguson and D. Kester—Acta Horticulturae No. 470, Int. Soc. for Horticultural Sci (75) Inventor: Sui-Sheng T. Hua, Orinda, CA (US) ence, Leuven, Belgium. Hua, S.-S. T. J.L. Baker and M. Flores-Espiritu, “Interactions of (73) Assignee: The United States of America as Saprophytic Yeasts with a nor Mutant of Aspergillus flavus.” Applied represented by the Secretary of and Environmental Microbiology (1999) 65(6):2738-2740. Hua, S., “Biocontrol Approach to Reduce Aspergillus flavus Popula Agriculture, Washington, DC (US) tion in Tree Nut Orchards.” In: Proceedings of California Conference on Biological Control (2000) (Ed) M.S. Hoddle University of Cali (*) Notice: Subject to any disclaimer, the term of this fornia, Riverside. patent is extended or adjusted under 35 Hua, S.-S. T., "Potential Use of Saprophytic Yeasts to Control U.S.C. 154(b) by 222 days. Aspergillus flavus in Almond and Pistachio Orchards.” In: Proceed ings of the Third International Symposium of Pistachios and (21) Appl. No.: 11/607,713 Almonds (2002) (Eds) I.Battle, I. Hormaza, and M.T. Espiau—Acta Horticulture, No. 591 Int. Society for Horticultural Science, Leuven, (22) Filed: Dec. 1, 2006 Belgium. Hua, S.-S. T., "Application of a yeast, Pichia anomala strain WRL (51) Int. Cl. 076 to control Aspergillus flavus for reducing aflatoxin in pistachio CI2N L/6 (2006.01) and almond.” Biological Control of Fungal and Bacterial Plant Patho gens, Jun.9-12, 2004, Trentino, Italy. IOBC Bulletin: 27(8): 291-294. AOIN 63/00 (2006.01) Hua, S.-S. T. S. Kuang, L. Chang, and K. Bundy-Mills, “Identifica AOIN 63/04 (2006.01) tion of Fungal andYeast isolates from Almond and Pistachio Orchard (52) U.S. Cl. ..................... 435/255.2:435/938: 504/117 by Analysis of Nuclear Large Subunit (26S) Ribosomal DNA (58) Field of Classification Search ....................... None Sequences.” In: Proceedings of Aflatoxin and Fumonisin Elimination See application file for complete search history. Workshop, Sacramento, CA. Oct. 24-28, 2004. Janisiewics, W.J., and L. Korsten, “Biological Control of Postharvest (56) References Cited Diseases of Fruits.” Annual Review Phytopathology (2002) 40:411 441. FOREIGN PATENT DOCUMENTS Payne, G.A., “Aflatoxin in Maize.” Critical Reviews in Plant Sciences WO WO 97/.45787 ck 9, 1999 (1992) 10(5):423-440. Wilson, C.L. and M.E. Wisniewski. “Biological Control of Postharvest Diseases of Fruits and Vegetables: An Emerging Tech OTHER PUBLICATIONS nology.” Annual Reviews Phytopathology (1989) 27:425-441. Hua, S.-S.T., "Biocontrol of Aspergillus flavus by Saprophytic Yeast: Commission of the European Community. “Commission Directive Progress from Laboratory Bioassay to Field Trial.” Proceedings of 98/53/EC laying down the sampling methods and the methods of the 3rd Fungal Genomics, 4th Fumonisin, and 16th Aflatoxin Elimi analysis for the official control of the levels for certain contaminants nation Workshops (Oct. 13-15, 2003) p. 85. in foodstuffs” (Jul. 1998) Official Journal of the European Commu nity L201/93-101. * cited by examiner Buck, J.W. “Combinations of Fungicides with Phylloplane Yeasts Primary Examiner Vera Afremova for Improved Control of Botrytis cinerea on Geranium Seedlings.” Phytopathology (2004) 94(2): 196-202. (74) Attorney, Agent, or Firm—Howard V. Owens; Leslie Cotty, P.J. P. Bayman, D.S. Egel, and K.S. Elias, "Agriculture, Shaw; John Fado Aflatoxins and Aspergillus.” In: The Genus Aspergillius, (1994) (Eds) K.A. Powell, A. Renwick, and J.F. Perberdy, Plenn Press, New (57) ABSTRACT York pp. 1-21. A biologically pure culture of a yeast of the species Pichia Diener, U.L., et al., “Epidemiology of Aflatoxin Formation of anomala (WRL-076). The yeast is identified as NRRL Aspergillus flavus.” Annual Review Phytopathology (1987) 25:249 Y-30842 and is capable of inhibiting growth of a deleterious 27O. microorganism on a plant or portion thereof to which a bio Ellis, W.O., J.P. Smith, and B.K. Simpson, "Aflatoxins in Food: Occurrences, Biosynthesis, Effects on Organisms, Detection, and logically effective amount of a culture of the yeast is applied. Methods of Control.” Critical Reviews in Food Science and Nutrition Further disclosed is a composition for use in protection of (1991) 30 (3):403-439. agricultural crops. Hua, S.-S., J.L. Baker, and O.K. Grosjean, “Improvement of the Quality and Value of Pistachios and Almonds Through Preharvest 23 Claims, 9 Drawing Sheets Biocontrol of Aspergillus flavus.” In: Proceeding of the International (1 of 9 Drawing Sheet(s) Filed in Color) U.S. Patent Aug. 25, 2009 Sheet 1 of 9 US 7,579,183 B1 A. flavus A F827 FG s U.S. Patent Aug. 25, 2009 Sheet 2 of 9 US 7,579,183 B1 U.S. Patent Aug. 25, 2009 Sheet 4 of 9 US 7,579,183 B1 | cN Y- Co. co ri (N d r C C C C v0 X SnMel WJO O U.S. Patent Aug. 25, 2009 Sheet 5 of 9 US 7,579,183 B1 Effect of Yeast on Colonization of A. flavus on Wonded Pistachio 30.0% - 25.0% 20.0% 15.0% 10.0% S.O. 0.0% - --- Control Yeast treated F.G. 5 U.S. Patent Aug. 25, 2009 Sheet 7 Of 9 US 7,579,183 B1 & Enlarged View U.S. Patent Aug. 25, 2009 Sheet 8 of 9 US 7,579,183 B1 Water activity is adjusted to 0.96 by adding PEG 8000 to the growth medium FIG. 8 U.S. Patent Aug. 25, 2009 Sheet 9 Of 9 US 7,579,183 B1 ??eq6u??duuespuequ'auu?eau1 6"OIH (u/no)eleoe US 7,579,183 B1 1. 2 SAPROPHYTIC YEAST, PICHIAANOMALA FIG. 6. is a graph of the reduction of Aspergillus flavus spores by yeast on wounded pistachio nut-fruits. Spores were SUMMARY OF THE INVENTION enumerated by plating the samples on nutrient agar plates. FIG. 7... is a photo of the biocontrol of Aspergillus flavus According to an embodiment of the invention is a biocon isolated from corn kernels by Pichia anomala WRL-076. trol agent comprising Pichia anomala WRL-076. Preferably FIG.8. is a photo of the biocontrol of Aspergillus flavus by the biocontrol agent comprises Pichia anomala WRL-076 Pichia anomala WRL-076 at low wateractivity. Polyethylene deposit number NRRLY-30842. glycol 8000 was used to adjust the water in the medium. A further embodiment of the invention is a biologically FIG. 9. is a graph of the effect of yeast on the population of pure culture of a yeast of the species Pichia anomala WRL 10 Burkholderia cepacia. Yeast cells were sprayed to the pista 076 deposit number NRRLY-30842, or composition thereof, chio trees. Nut-fruit samples were collected and enumerated the culture capable of competitively inhibiting growth of a for the number of bacteria. deleterious micro-organism on an agricultural crop to which a biologically effective amount of the culture is applied. DETAILED DESCRIPTION OF THE INVENTION Pichia anomala, is referred to herein as strain WRL-076. 15 A further embodiment of the invention is a composition The present invention is of a new yeast species Pichia comprising the biocontrol agent WRL-076 and a carrier. Any anomala which can be applied to agricultural produce to carrier that permits the biocontrol agent to be delivered to a reduce pre-harvest and postharvest decay via competitive target plant in a manner Such that the biocontrol agent remains viable and pathogenic may be employed in the composition. inhibition of a wide range of micro-organisms. A further embodiment is a method ofusing Pichia anomala Statement of Deposit to inhibit aflatoxin producing microorganisms on affected A biologically pure culture of Pichia anomala was depos agricultural crops. ited May 9, 2005 under terms of the Budapest Treaty with the According to still further features in the described embodi Agricultural Research Service Culture Collection (NRRL) ments, the portion of a plant is selected from the group con 25 National Center for Agricultural Utilization Research, Agri sisting of a stone fruit, a pome fruit, a citrus fruit, grapes, a cultural Research Service, U.S. Department of Agriculture, Vegetable, a flower bulb, an herb, a grain, a root, a leaf, a grain 1815 North University Street, Peoria, Ill. 61604 USA and and berries. given the accession number NRRL Y-30842. The microor According to further embodiments the yeast is Supplied in 30 ganism deposit was made under the provisions of the “Budap an active or dormant physiologic state. est Treaty on the International Recognition of the Deposit of According to still further features in the described pre Microorganisms for the Purposes of Patent Procedure”. All ferred embodiments the yeast is Supplied in a physical form restrictions on the availability to the public of these deposited selected from a liquid Suspension, an emulsion, a powder, microorganisms will be irrevocably removed upon issuance granules, a lyophylsate and a gel. 35 of a United States patent based on this application. For the According to still further features in the described embodi purposes of this invention, any isolate having the identifying ments the chemical antibiotic is a fungicide or an antimicro characteristics of strains NRRL Y-30842, including subcul bial agent or a pesticide. tures and variants thereof which have the identifying charac teristics and activity as described herein are included. BRIEF DESCRIPTION OF THE DRAWINGS 40 Specifically, the present invention can be used to reduce the incidence and/or severity of bacterial and fungal pathogens of The file of this patent contains at least one drawing nuts, grapes, citrus fruit, pome fruit, stone fruit, Strawberries, executed in color.