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(12) United States Patent (10) Patent No.: US 9.485,994 B2 Leveau Et Al

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(12) United States Patent (10) Patent No.: US 9.485,994 B2 Leveau Et Al US0094.85994B2 (12) United States Patent (10) Patent No.: US 9.485,994 B2 LeVeau et al. (45) Date of Patent: Nov. 8, 2016 (54) SYNERGY-BASED BIOCONTROL OF PLANT De Boer et al., “Anti-fungal properties of chitinolytic dune soil PATHOGENS bacteria”, Soil Biology & Biochemistry, 30(2): 193-203 (1998). De Boer et al., “Growth of chitinolytic dune soil beta-subclass (71) Applicant: The Regents of the University of Proteobacteria in response to invading fungal hyphae'. Applied and California, Oakland, CA (US) Environmental Microbiology, 67(8): 3358-3362 (2001). De Boer et al., “Collimonas fingivorans gen, nov... sp. nov., a (72) Inventors: Johannes Henricus Josephus Leveau, chitinolytic soil bacterium with the ability to grow on living fungal Davis, CA (US); Hung Kim Doan, hypahe”, IJSEM, 54(Pt3):857-864 (2004). Davis, CA (US) Fritsche et al., “Identification and characterization of genes under (73) Assignee: The Regents of the University of lying hitinolysis in Collimonas fungivorans Ter331. FEMS Micro California, Oakland, CA (US) biology Ecology, 66(1): 123-135 (2008). Hoppener-Ogawa et al., “Specific detection and real-time PCR (*) Notice: Subject to any disclaimer, the term of this quantification of potentially mycophagous bacteria belonging to the patent is extended or adjusted under 35 genus Collimonas in different soil ecosystems'. Appl Environ U.S.C. 154(b) by 0 days. Microbiol. 73(13):4191-4197 (2007). Hoppener-Ogawa et al., “Collimonas arenae sp. Nov. and Col (21) Appl. No.: 14/537,620 limonas pratensis sp. nov., isolated from (semi-)natural grassland (22) Filed: Nov. 10, 2014 soils”. International Journal of Systematic and Evolutionary Micro biology, 58: 414-419 (2008). (65) Prior Publication Data Hoppener-Ogawa et al., “Impact of Collimonas bacteria on com US 2015/O135371 A1 May 14, 2015 munity composition of soil fungi'. Environ Microbiol, 11(6): 1444 1452 (2009). Related U.S. Application Data Hoppener-Ogawa et al., “Mycophagous growth of Collimonas (60) Provisional application No. 61/902,046, filed on Nov. bacteria in natural soils, impact on fungal biomass turnover and 8, 2013. interactions with mycophagous Trichoderma fungi'. ISME J. 3(2):190-198 (2009). (51) Int. Cl. Kamilova et al., "Collimonas fungivorans, an unpredicted in vitro AOIN 63/00 (2006.01) but efficient in vivo biocontrol agent for the Suppression of tomato (52) U.S. Cl. foot and root rot'. Environmental Microbiology, 9(6): 1597-1603 CPC ..................................... A0IN 63/00 (2013.01) (2007). Leveau et al., "Genomic flank-sequencing of plasposon insertion (58) Field of Classification Search sites for rapid identification of functional genes”,Journal of Micro None biological Methods, 66(2): 276-285 (2006). See application file for complete search history. Leveau et al., The bacterial genus Collimonas: mycophagy, weath (56) References Cited ering and other adaptive solutions to life in oligotropic soil envi ronments, Environ Microbiol. 12(2):281-292 (2010). U.S. PATENT DOCUMENTS Mela et al., "Comparative genomics of the pIPO2/pSB102 family of environmental plasmids: sequence, evolution, and ecology of 4,245,432 A 1/1981 Dannelly pTer331 isolated from Collimonas fungivorans Ter331". FEMS 4,272.417 A 6, 1981 Barke et al. Microbiology Ecology, 66(1): 45-62 (2008). 4,808.430 A 2f1989 Kouno Mela et al., “Dual transcriptional profiling of a bacterial/fungal 5,645,831 A 7, 1997 Chilcott et al. confrontation: Collimonas fungivorans versus Aspergillus niger'. 5,876.739 A 3, 1999 Turnbladet al. ISME J 5(9): 1494-1504 (2011). 9,131,699 B2 9, 2015. Ano et al. Mela et al., “Comparative genomics of bacteria from the genus 2003/0176428 A1 9/2003 Schneidersmann et al. Collimonas: linking (dis)similarities in gene content to phenotypic variation and conservation”. Environmental Microbiology Reports FOREIGN PATENT DOCUMENTS 4(4): 424-432 (2012). Offre et al., Microdiversity of Burkholderiales associated with WO 98.21964 A1 5, 1998 mycorrhizal and nonmycorrhizal roots of Medicago truncatula, WO 98.21965 A1 5, 1998 WO 98.21966 A2 5, 1998 FEMS Microbiology Ecology, 65(2): 180-192 (2008). WO 98.21967 A1 5, 1998 Postma et al., "Soil suppressiveness and functional diversity of the WO 98.50422 A1 11, 1998 Soil microflora in organic farming systems'. Soil Biology & Bio WO 99.09819 A1 3, 1999 chemistry, 40(9): 2394-2406 (2008). WO 99.0982O A1 3, 1999 Uroz et al., “Efficient mineral weathering is a distinctive functional WO 99.10477 A1 3, 1999 trait of the bacterial genus Collimonas', Soil 2013Biology & WO OO 58442 A1 10, 2000 Biochemistry 41 (10): 21782186 (2009). WO 02/28.186 A2 4/2002 Uroz et al., “Structure and function of bacterial communities in WO O2/O80675 A1 10, 2002 aging soils: insights from the Mendocino Ecological Staircase'. 69: WO 2012,087980 A1 6, 2012 pp. 265-274, Soil Biology & Biochemistry (2014). WO 2013, 178664 A1 12/2013 Primary Examiner — Alton Pryor OTHER PUBLICATIONS (74) Attorney, Agent, or Firm — Kilpatrick Townsend & Stockton LLP Axelrood et al., “Molecular characterization of bacterial diversity from British Columbia forest soils subjected to disturbance.” Cana (57) ABSTRACT dian Journal of Microbiology, 48(7):655-674 (2002). Chow, et al., “Molecular characterization of bacterial diversity in Methods and compositions are provided for control of Lodgepole pine (Pinus contoria) rhizosphere soils from British pathogenic fungal or oomycetous infection. Columbia forest soils differing in disturbance and geographic source.” FEMS Microbiology Ecology, 42(3): 347-357 (2002). 39 Claims, 5 Drawing Sheets U.S. Patent Nov. 8, 2016 Sheet 1 of 5 US 9,485,994 B2 F.G. 1 U.S. Patent Nov. 8, 2016 Sheet 2 of 5 US 9,485,994 B2 FIG. 2 U.S. Patent Nov. 8, 2016 Sheet 3 of 5 US 9,485,994 B2 Ca35 and Serenade W. W. W. W. W. W. --- Ca35 and Serenade Cal35 and Serenade 3. Cat35 and Serenade -o FIG. 3 U.S. Patent Nov. 8, 2016 Sheet 4 of 5 US 9,485,994 B2 Ca35 and Serenade -o- 88:88: 8: FIG. 4 U.S. Patent Nov. 8, 2016 Sheet S of 5 US 9,485,994 B2 FIG 5 US 9,485,994 B2 1. 2 SYNERGY-BASED BOCONTROL OF PLANT Collimonas or a product thereof is the seed of said plant. In PATHOGENS Some embodiments, the part of said plant contacted with bacteria of the genus Bacillus or a product thereof is the seed CROSS-REFERENCES TO RELATED of said plant. In some embodiments, the part of said plant APPLICATIONS contacted with bacteria of the genus Collimonas or a product thereof is the foliage of said plant. In some embodiments, the This application claims priority to U.S. Provisional Appli part of said plant contacted with bacteria of the genus cation 61/902,046, filed Nov. 8, 2013, the contents of which Bacillus or a product thereof is the foliage of said plant. are hereby incorporated in their entirety for all purposes. In Some embodiments, the contacting of a) comprises 10 contacting the plant with bacteria of the genus Collimonas BACKGROUND OF THE INVENTION and a product thereof. In some embodiments, the contacting of b) comprises contacting the plant with bacteria of the Fungal and oomycetous pathogens can cause significant genus Bacillus and a product thereof. In some embodiments, damage to a wide variety of commercially important plant the contacting of a) comprises contacting the plant with varieties including crops and ornamental plants. Such patho 15 bacteria of the genus Collimonas and a product thereof, and gens can kill plants, reduce yield, reduce plant strength (e.g., the contacting of b) comprises contacting the plant with decrease resistance to lodging), cause symptoms of mineral bacteria of the genus Bacillus and a product thereof. deficiency, and predispose plants to infection by other patho In some embodiments, the fungal or oomycetous infection gens. As such, there is great interest in developing compo comprises an infection by a fungal pathogen from a class sitions and methods for control of fungal and oomycetous selected from the group consisting of Plasmodiophoromy pathogens. cetes, Oomycetes, Chytridiomycetes, Zygomycetes, Asco Fungal and oomycetous pathogens are typically con mycetes, Basidiomycetes, Deuteromycetes, Sordariomy trolled by use of synthetic chemicals (e.g., fungicides). cetes, and combinations thereof. In some cases, the fungal or However, effective synthetic chemicals can be dangerous, oomycetous pathogen is from the class Oomycetes. In some toxic, and expensive. In some countries, certain fungicides 25 embodiments, said fungal or oomycetous infection com or anti-oomycetous chemicals have been restricted or prises infection by a fungal pathogen from a genus selected banned for these and other reasons. Therefore, there is great from the group consisting of Fusarium, Geotrichum, Asper interest in developing fungal control methods and compo gillus, Alternaria, Botryosphaeria, Colletotrichum, Magna sitions that do not rely on synthetic chemical fungicides or porthe, Verticillium, Cryphonectria, Botrytis, Monilinia, anti-Oomycetous compounds, or reduce the use of Such 30 Sclerotium, Rhizoctonia, and combinations thereof. In some chemicals. embodiments, said fungal or oomycetous infection com Biocontrol agents are a promising candidate for reducing prises infection by an Oomycete from a genus selected from or eliminating the need for Such chemicals. Biocontrol the group consisting of Pythium, Phytophthora, and com agents are typically microorganisms, such as bacteria, or one binations thereof. or more products thereof, that are applied to a plant or a plant 35 In some embodiments, said bacteria of the genus Bacillus propagation material (e.g., soil) to control a pathogen. comprise Bacillus subtilis. In some embodiments, said bac However, there are a limited number of commercially avail teria of the genus Bacillus comprise Bacillus subtilis var. able biocontrol agents. For these and other reasons, there amyloliquefaciens. In some embodiments, said bacteria of remains a need to further develop methods and compositions the genus Bacillus comprise Bacillus subtilis QST 713.
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