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WO 2015/077278 Al 28 May 2015 (28.05.2015) W 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 2015/077278 Al 28 May 2015 (28.05.2015) W P O P C T (51) International Patent Classification: (81) Designated States (unless otherwise indicated, for every A 63/00 (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/US20 14/066296 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, 19 November 2014 (19.1 1.2014) KZ, LA, LC, LK, LR, LS, LU, LY, MA, MD, ME, MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, NO, NZ, OM, (25) Filing Language: English PA, PE, PG, PH, PL, PT, QA, RO, RS, RU, RW, SA, SC, (26) Publication Language: English SD, SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM, TN, TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW. (30) Priority Data: 61/906,676 20 November 2013 (20. 11.2013) US (84) Designated States (unless otherwise indicated, for every kind of regional protection available): ARIPO (BW, GH, (71) Applicant: NOVOZYMES BIOAG A/S [DK/DK]; GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, ST, SZ, Krogshoejvej 36, DK-2880 Bagsvaerd (DK). TZ, UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, RU, (72) Inventor; and TJ, TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, DK, EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU, (71) Applicant (for B W only): KANG, Yaowei [US/US]; 225 LV, MC, MK, MT, NL, NO, PL, PT, RO, RS, SE, SI, SK, Windsor Dr., Christiansburg, Virginia 24073 (US). SM, TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, (72) Inventors: TRAN, Anh; 4806 GolfView Dr., Roanoke, GW, KM, ML, MR, NE, SN, TD, TG). Virginia 24019 (US). LELAND, Jarrod; 1802 Shadow Declarations under Rule 4.17 : Lake Rd, Blacksburg, Virginia 24060 (US). — as to applicant's entitlement to apply for and be granted a (74) Agents: SLADEK, Todd L. et al; Novozymes North patent (Rule 4.1 7(H)) America, Inc., 60 E. 42nd St., Suite 700, New York, New York 10165 (US). Published: — with international search report (Art. 21(3)) (54) Title: COMPOSITIONS AND METHODS COMPRISING CHROMOBACTERIUM FOR CONTROLLING PLANT NEMAT ODE PESTS AND PLANT INSECT PESTS (57) Abstract: Disclosed herein are pest con trolling compositions comprising a novel strain of Chromobacterium spp. Further dis closed are methods of using such composi MWU300 tions for controlling plant pests and other in vasive pests. MWU205 MWU328 *— NRRL B-503SO — C b ct er r subtsugae 00 l l © Fig o o COMPOSITIONS AND METHODS COMPRISING CHROMOBACTERIUM FOR CONTROLLING PLANT NEMATODE PESTS AND PLANT INSECT PESTS FIELD OF THE INVENTION Disclosed herein are compositions comprising a novel strain of Chromobacterium for controlling plant pests and other invasive pests. Further disclosed are methods of using such compositions for controlling crop damaging plant nematode pests and insect pests in agricultural environments. BACKGROUND OF THE INVENTION Pests, such as nematodes, insects, and acari (mites and ticks) are a major problem for the agriculture industry, limiting productivity, often significantly. Although chemical solutions have been used to control pests, excessive use of chemicals leaves residues in soil, water, and air, and has additional adverse effects on non-target organisms and the ecological balance. In addition, pests can develop resistance to chemical pesticides, limiting their effectiveness and application. Public concern over potential health hazards of chemical pesticides and the increase in cost of chemical pesticides has also led to the exploration of more eco-friendly pest management tactics. Microbial pesticides have been developed for use as an alternative, or in some cases as a supplement, to chemical pesticides. Microbial pesticides are living organisms (e.g., bacteria and fungi) that intervene in the life cycle of pests (by killing or disabling the pest). Examples of microbial pesticides include microbial nematicides, and in particular, nematophagous bacteria including strains of Chromobacterium spp. Tian, B.; Yang, J.; Zhang, K. (2007). Bacteria used in the biological control of plant- parasitic nematodes: populations, mechanisms of action, and future prospects . FEMS Microbiol Ecol 6 1 : 197-213 discusses a group of important natural enemies of nematode pests, nematophagous bacteria. Soby, S.D.; et al, (2013). Chromobacterium vaccinii sp. nov., isolated from native and cultivated cranberry (Vaccinium macrocarpon Ait.) bogs and irrigation ponds. International Journal of Systematic and Evolutionary Microbiology 63: 1840-1846 discusses a number of Gram-negative, motile, mesophilic, violacein-producing bacteria isolated from the soils and roots of Vaccinium macrocarpon Ait. and Kalmia angustifolia L. plants and from irrigation ponds associated with wild and cultivated cranberry bogs in Massachusetts, USA. Phylogenetic analyses of 16S rRNA gene sequences placed these isolates in a clade with Chromobacterium species, but the specialized environment from which they were isolated, indicate that the cranberry and Kalmia isolates comprise a separate species of Chromobacterium, for which the name Chromobacterium vaccinii sp. nov. is proposed. Martin, P.A.W; Gundersen-Rindal, D.; Blackburn, M.; Buyer, J. (2007). Chrombacterium subtsugae sp. nov., a betaproteobacterium toxic to Colorado potato beetle and other insect pests . International Journal of Systematic and Evolutionary Microbiology 57: 993-999 discusses that Chromobacterium subtsugae sp. nov., a motile, Gram-negative, violet-pigmented bacterium isolated from Maryland forest soil, was found to be orally toxic to Colorado potato beetle larvae and other insects. Martin, P.A.W. ; Hirose, E.; Aldrich, J.R. (2007). Toxicity of Chromobacterium subtsugae to Southern Green Stink Bug (Heteroptera: Pentatomidae) and Corn Rootworm (Coleoptera: Chrvsomelidae) . J. Econ. Entomol. 100 (3): 680-684 discusses a new species of Chromobacterium, Chromobacterium subtsugae, found to be toxic to Colorado potato beetle, Leptinotarsa decemlineata, and describes the toxicity of these bacteria in laboratory assays to other pest insects: two corn rootworm species and southern green stink bug. U.S. Patent No.: 7,244,607 discloses compositions for controlling an insect population including an insect food stuff and an insecticidally-effective amount of at least one Gram negative bacteria, viable, dead or alive, and/or an extract thereof, where the composition is applied to an area accessible to the insects and results in insect death. The compositions are ideally suited for the control of fire ants, cockroaches, carpenter ants and termites. U.S. Patent No.: 7,037,494 discloses Chromobacterium subtsuga sp. nov., a new species of the genus Chromobacterium which possesses insecticidal activity, is described. The invention also relates to insecticidally-active metabolites obtained from the strain and to insecticidal compositions comprising cultures of the strain and/or supernatants, filtrates, and extracts obtained from the strain, and use thereof to control insect pests. U.S. Patent Application Publication No.: 2012/0100236 discloses bioactive compounds and metabolites derived from Chromobacterium species culture responsible for controlling pests, compositions containing these compounds, methods for obtaining these compounds and methods of using these compounds and compositions for controlling pests. U.S. Patent Application Publication No.: 2013/0074735 discloses a process for producing purple-blue natural pigment containing violacein and its derivative (deoxyviolacein) using Chromobacterium sp. NIIST-CKK-01 (MTCC 5522, NCIM 5341 ; Genbank Accession No. FJ982784). The method comprises the steps of maintaining and growing the bacterium in a specific medium under defined conditions of pH, temperature and agitation. At the end of incubation, pigment and biomass is separated from the culture broth, pigment is recovered from the biomass through solvent extraction and finally pigment is concentrated by drying. Published PCT Patent Application No.: WO 2012/140212 discloses combinations suitable for agricultural use can include (I) a nematode-antagonistic biocontrol agent and (II) one or more agents selected, independently of each other, from any one of (A) to (H): (A) at least one fungicide; (B) at least one insecticide; (C) at least one synthetic nematicide; (D) bacterium of the genus Bacillus; (E) Harpin; (F) Isoflavones; (G) Plant growth regulators; and/or (H) Plant activators. Published PCT Patent Application No.: WO 2012/037352 discloses compositions for reducing pathogens in soil, and to methods of using such compositions to treat soils. The composition comprises intracellular components of lysed, beneficial, crop and non-crop rhizosphere-inhabiting yeast cells and whole or lysed, beneficial, crop and non-crop rhizosphere-inhabiting bacteria cells. The composition can also include a fertilizer, and a carrier suitable for delivering the composition to the soil. EP Patent Application Publication No.: EP 0171381 discloses bacteria which are capable of proliferating in an environment which is infested with nematodes (such as Pseudomonads which colonize the surfaces of plant roots) under conditions of microbial competition, and which inhibit nematodes. This invention also relates to a method of using such bacteria to inhibit nematodes
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