# Patent

## Title
Compositions and methods for modulating plant parasitic nematodes

## Abstract

Provided is a method for modulating soybean cyst nematode infestation of a plant comprising applying to the plant an effective amount of a composition comprising a Chromobacterium, Burkholderia, and/or Flavobacterium strain to modulate the soybean cyst nematode infestation. The composition may be applied directly to the seed or in a substrate for growing the plant, such as in the soil.

## Background
CROSS REFERENCE TO RELATED APPLICATIONS
This application is a continuation-in-part application of U.S. application Ser. No. 13/280,311, filed Oct. 24, 2011, which claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Application No. 61/406,569, filed Oct. 25, 2010. This application is also a continuation-in-part of U.S. application Ser. No. 13/843,971, filed Mar. 15, 2013, which is a continuation-in-part of U.S. application Ser. No. 13/034,575, filed Feb. 24, 2011, which claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Application Nos. 61/308,287, filed Feb. 25, 2010, and 61/406,541, filed Oct. 25, 2010. This application is also a continuation-in-part of International Application No. PCT/US13/30631, filed Mar. 13, 2013, which claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Application Nos. 61/609,937, filed Mar. 13, 2012, and 61/733,730, filed Dec. 5, 2012. All of the applications are hereby incorporated by reference.


TECHNICAL FIELD
Disclosed herein are pesticidal Chromobacterium, Burkholderia, and Flavobacterium strains and compositions, as well as their methods of use for controlling plant parasitic nematodes, particularly, soybean cyst nematodes.
BACKGROUND
Nematodes are non-segmented, bilaterally symmetric, worm-like invertebrates that possess a body cavity and complete digestive system but lack respiratory and circulatory systems. Their body wall contains a multilayer cuticle, a hypodermis with four longitudinal cords, and internal musculature (Chitwood, D. J. (2003). Nematicides. Encyclopedia of Agrochemicals , vol 3. J. R. Plimmer. New York, John Wiley & Sons. 3: 1104-1115). Their body contents are mostly occupied by digestive and reproductive systems. Nematodes may be classified as either parasitic or free living. Parasitic nematodes may be classified by their hosts (e.g., plant parasites). Free living nematodes may be classified according to their feeding habits and include the following groups: (1) omnivores; (2) bacterial feeders; (3) fungal feeders and (4) predators.
Plant parasitic nematodes generally feed on underground parts of plants, such as roots, bulbs, and tubers as well as above ground parts of the plants, such as leaves and stems Annual crop losses caused by plant-parasitic nematodes have been estimated to exceed US $100 billion (Koenning, S. R., Overstreet, C. et al. (1999). Journal of Nematology 31: 587-618). Examples of plant parasitic nematodes include but are not limited to nematodes belonging to Meloidogyne spp. (e.g., root-knot nematodes); Pratylenchus spp. (e.g., lesion nematodes); Heterodera spp. (e.g., cyst nematodes); Globodera spp. (cyst nematodes); Ditylenchus spp. (e.g., stem and bulb nematodes); Tylenchulus spp. (e.g., citrus nematodes), Xiphinema spp. (e.g., dagger nematodes); Radopholus spp. (burrowing nematodes); Rotylenchulus spp. (e.g. reniform nematodes); Helicotylenchus spp. and Scutellonema spp. (e.g.spiral nematodes); Belonolaimus spp. (e.g., sting nematodes); Bursaphelenchus spp. (e.g. pine wilt nematodes); Hoplolaimus spp. (lance nematodes); Longidorus spp. (needle nematodes); Nacobbus spp. (false root-knot nematodes); and Aphelenchoides spp. (foliar nematodes). The most efficient means for controlling nematodes is via nematicides that inhibit egg hatching, juvenile motility and/or plant infectivity. The development of chemical control for plant-parasitic nematodes is challenging because of both environmental and physiological reasons: (1) most phytoparasitic nematodes live in a confined area in soil near the roots and hence, delivery of a chemical nematicide is difficult and (2) the outer surface of nematodes is a poor biochemical target, and is impermeable to many organic molecules (Chitwood, D. J. (2003). Nematicides. Encyclopedia of Agrochemicals , vol 3. J. R. Plimmer. New York, John Wiley & Sons. 3: 1104-1115). Moreover, delivery of toxic compounds by an oral route is nearly impossible because most plant parasitic nematode species ingest material only after they have penetrated and infected plant roots. Therefore, nematicides have tended to be broad-spectrum toxins with high volatility or with other chemical and physical properties promoting their motility in soil.
According to Sasser and Freckman (In J. A. Veech and D. W. Dickson (Eds.), Vistas on Nematology, 1987, (pp. 7-14). Society of Nematologists, Hyattesville), crop losses by nematodes range from 8 to 20% on major crops around the world. Plant parasitic nematodes can cause considerable crop damage with annual losses estimated at $87 billion worldwide (Dong, L. Q. and Zhang, K. Q. (2006) Plant Soil 288: 31-45). Fumigants such as methyl bromide are very effective in controlling both soil-borne plant diseases and nematodes but due to the high mammalian toxicity, ozone depleting effects and other residual effects, the use of methyl bromide has already been banned in various countries and its complete withdrawal from the market is planned by international agreement (Oka, Y., Nacar, S. et al. (2000). Phytopathology 90:710-715). Chemical alternatives such as methyl iodide, 1,3-Dichloropropene, and chloropicrin also have issues with mammalian and environmental safety. Chemical non-fumigant nematicides are being phased out and banned. Most recently, the US-EPA announced that aldicarb will be phased out.
Accordingly, there is currently a need for additional nematicides; in particular, those with maximal nematode-specific toxicity. In addition, in light of the ever-increasing resistance of plant pathogens to synthetic pesticides, and the environmental concerns associated with chemical pesticide use, there is a need for new, naturally-occurring pesticides (e.g., biopesticides) to which plant pathogens have not developed resistance, and which have minimal environmental effects.
SUMMARY
The present invention provides natural microbial pesticides for modulating infestation of a plant by plant parasitic nematodes.
In a particular embodiment, the present invention provides a method for modulating soybean cyst nematode (H. glycines) infestation of a plant comprising contacting the plant with an effective amount of a composition comprising Flavobacterium sp. H492 (NRRL Accession No. B-50584) to modulate the soybean cyst nematode infestation.
In another embodiment, the present invention provides a method for modulating soybean cyst nematode infestation of a plant comprising contacting the plant with an effective amount of composition comprising Burkholderia A396 (NRRL Accession No. B-50319) to modulate the soybean cyst nematode infestation.
In yet another embodiment, the present invention provides a method for modulating soybean cyst nematode infestation of a plant comprising contacting the plant with an effective amount of composition comprising Chromobacterium subtsugae sp. nov. (NRRL Accession No. B-30655) to modulate the soybean cyst nematode infestation.
In a particular embodiment, the composition of the invention is a whole cell broth, cell fraction, filtrate, supernatant, compound, and/or extract of the bacterial strain. In another embodiment, the composition is a formulation comprising the bacterial strain and inerts.
In certain embodiments, contacting the plant includes application of the composition to a seed of the plant prior to planting. In additional embodiments, contacting the plant is accomplished by adding the composition to the substrate in which the plant is grown (e.g., soil) either before, at the time of, or after planting the seed (e.g., by soil drenching or irrigation).
In an embodiment of the invention, the methods are capable of reducing the number of soybean cyst nematodes in the soil or plant, or on the plant, compared to a plant that not been contacted with the composition. Modulation can also include promotion of plant growth and/or increase in the yield of seeds produced by the plant, compared to a plant that has not been contacted with the composition.
In a particular embodiment of the invention, the plant is a soybean plant.


DETAILED DESCRIPTION
While the compositions and methods disclosed herein are susceptible to various modifications and alternative forms, exemplary embodiments will herein be described in detail. It should be understood, however, that there is no intent to limit the invention to the particular forms disclosed, but on the contrary, the intention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the invention as defined by the appended claims.
Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is included therein. Smaller ranges are also included. The upper and lower limits of these smaller ranges are also included therein, subject to any specifically excluded limit in the stated range.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are now described.
It must be noted that as used herein and in the appended claims, the singular forms “a,” “and” and “the” include plural references unless the context clearly dictates otherwise.
Definitions
As defined herein, “derived from” means directly isolated or obtained from a particular source or alternatively having identifying characteristics of a substance or organism isolated or obtained from a particular source. In the event that the “source” is an organism, “derived from” means that it may be isolated or obtained from the organism itself or medium used to culture or grow said organism.
As defined herein, “whole broth culture” or “whole cell broth” refers to a liquid culture containing both cells and media. A whole cell broth can also be obtained from bacteria grown on a plate, by harvesting the cells from the plate and suspending them in water or other liquid. The terms “whole broth culture” and “whole cell broth” are used interchangeably.
As defined herein, “supernatant” refers to the liquid remaining when cells grown in broth or harvested in another liquid from an agar plate and are removed by centrifugation, filtration, sedimentation, or other means well known in the art.
As defined herein, “filtrate” refers to liquid from a whole broth culture that has passed through a membrane.
As defined herein, “extract” refers to a liquid substance removed from cells by a solvent (water, detergent, buffer, organic solvent) and separated from the cells by centrifugation, filtration, phase partition or other method.
As defined herein, “metabolite” refers to a compound, substance or byproduct of fermentation of a microorganism, or supernatant, filtrate, or extract obtained from a microorganism that has pesticidal and particularly, nematicidal activity.
As defined herein, an “isolated compound” is essentially free of other compounds or substances, e.g., at least about 20% pure, preferably at least about 40% pure, more preferably about 60% pure, even more preferably about 80% pure, most preferably about 90% pure, and even most preferably about 95% pure, as determined by analytical methods, including but not limited to chromatographic methods and electrophoretic methods.
As defined herein, “carrier” is an inert, organic or inorganic material, with which the active ingredient is mixed or formulated to facilitate its application to a plant, seed or other object to be treated, or to facilitate its storage, transport and/or handling.
As defined herein, “modulate”, is used to mean to alter the amount of pest infestation, plant growth, root extension, seed germination or rate of spread of pest infestation, rate of plant growth, rate of root extension, and/or rate of seed germination. Modulation includes reduction of pest infestation, increase in plant growth, increase in root extension, increase in seed germination, reduction of the rate of spread of pest infestation, increase in the rate of plant growth, increase in the rate of root extension, and/or increase in rate of seed germination.
As defined herein, “pest infestation” is the presence of a pest in an amount that causes a harmful effect including a disease or infection in a host population or emergence of an undesired pest in a growth system.
As defined herein, “pesticide” is a substance derived from a biological product or chemical substance that increase mortality or inhibit the growth rate of plant pests and includes but is not limited to nematicides, algaecides, herbicides, insecticides, plant fungicides, plant bactericides, and plant viricides.
As defined herein, a “plant parasitic nematode” is a nematode that feeds on and causes injury on any part of a plant.
As defined herein, “a non-parasitic nematode” is a nematode that exists independently in the soil and does not cause damage or injury to any part of a plant.
As defined herein, “substrate” is a surface or medium in which a plant grows. Substrate includes, but is not limited to, soil, an artificial growth surface or medium, water, and sediment.
Chromobacterium 
A strain of Chromobacterium sp., particularly a strain of Chromobacterium subtsugae and more particularly, a strain of Chromobacterium subtsugae sp. nov. and even more particularly a strain of Chromobacterium subtsugae sp. nov. having the identifying characteristics of NRRL Accession No. B-30655, is described in U.S. Pat. No. 7,244,607. Methods of growing the Chromobacterium sp. and making a composition of Chromobacterium sp. and its products (e.g., a whole-cell broth) are described, for example, in U.S. Pub. No. 2012-0100236.
Burkholderia 
A non-Burkholderia cepacia complex, non-Burkholderia plantari, non-Burkholderia gladioli, Burkholderia sp., in particular, Burkholderia A396 sp. nov. rinojensis (NRRL Accession No. B-50319), that is non-pathogenic to vertebrates, such as birds, mammals and fish, is described in, for example, U.S. Pub. No. 2011-0207604 and U.S. App. No. 13/843,971. Methods of growing the Burkholderia sp. and making a composition of Burkholderia sp. and its products (e.g., a whole-cell broth) are also described in U.S. Pub. No. 2011-0207604 and U.S. application Ser. No. 13/843,971.
Flavobacterium 
Flavobacterium sp., a gram negative bacterium, is a member of the Flavobacteriaceae family. In particular, the Flavobacterium species is Flavobacterium sp. H492 (NRRL Accession No. B-50584), as described in PCT App. No. PCT/US13/30631. Methods of growing the Flavobacterium sp. and making a composition of the Flavobacterium sp. and its products (e.g., a whole-cell broth) are also described in PCT App. No. PCT/US13/30631.
Compositions
Compositions comprise a strain from a Chromobacterium sp., Burkholderia sp., and/or Flavobacterium sp. The compositions may be a substantially pure culture, whole broth culture, liquid culture, suspension, cell fraction, supernatant, filtrate, extract, or compound of the bacterial strain, or combinations of the foregoing which in particular have nematicidal activity.
The compositions set forth above can be formulated in any manner. Non-limiting formulation examples include, but are not limited to, emulsifiable concentrates (EC), wettable powders (WP), soluble liquids (SL), aerosols, ultra-low volume concentrate solutions (ULV), soluble powders (SP), microencapsulation, water dispersed granules, flowables (FL), microemulsions (ME), nano-emulsions (NE), seed treatments, etc. In any formulation described herein, percent of the active ingredient is within a range of about 0.01% to 99.99%.
The compositions may be in the form of a liquid, gel or solid. A solid composition can be prepared by suspending a solid carrier in a solution of active ingredient(s) and drying the suspension under mild conditions, such as evaporation at room temperature or vacuum evaporation at 65° C. or lower. A composition may comprise gel-encapsulated active ingredient(s). Such gel-encapsulated materials can be prepared by mixing a gel-forming agent (e.g., gelatin, cellulose, or lignin) with a culture or suspension of live or inactivated bacterial strain of the present invention, or a cell-free filtrate or cell fraction of the bacterial culture or suspension, or a spray- or freeze-dried culture, cell, or cell fraction or in a solution of pesticidal compounds used in the method of the invention; and inducing gel formation of the agent.
The composition may additionally comprise a surfactant to be used for the purpose of emulsification, dispersion, wetting, spreading, integration, disintegration control, stabilization of active ingredients, and improvement of fluidity or rust inhibition. In a particular embodiment, the surfactant is a non-phytotoxic non-ionic surfactant which preferably belongs to EPA List 4B. In another particular embodiment, the nonionic surfactant is polyoxyethylene (20) sorbitan monolaurate. The concentration of surfactants may range between about 0.1-35% of the total formulation; a preferred range is about 5-25%. The choice of dispersing and emulsifying agents, such as non-ionic, anionic, amphoteric and cationic dispersing and emulsifying agents, and the amount employed is determined by the nature of the composition and the ability of the agent to facilitate the dispersion of the compositions of the present invention.
The composition set forth above may contain one or more of a Chromobacterium sp., Burkholderia sp., or Flavobacterium sp. and may be combined with another microorganism and/or pesticide (e.g., nematicide, fungicide, insecticide, herbicide). The microorganism may include, but is not limited to, an agent derived from Bacillus sp. (e.g., B. firmus, B. thuringiensis, B. pumilus, B. licheniformis, B. amyloliquefaciens, B. subtilis), Paecilomyces sp. (P. lilacinus), Pasteuria sp. (P. penetrans), Pseudomonas sp., Brevabacillus sp., Lecanicillium sp., Ampelomyces sp., Pseudozyma sp., Streptomyces sp. (S. bikiniensis, S. costaricanus, S. avermitilis), Trichoderma sp., Gliocladium sp., avermectin, Myrothecium sp., Paecilomyces spp., Sphingobacterium sp., Arthrobotrys sp., Chlorosplrnium sp, Neobulgaria sp, Daldinia sp, Aspergillus sp, Chaetomium sp, Lysobacter sp, Lachnum papyraceum, Verticillium suchlasporium, Arthrobotrys oligospora, Pochonia chlamydosporia (synonym: Verticillium chlamydosporium), Hirsutella minnesotensis, Hirsutella rhossiliensis, Pleurotus ostreatus, Omphalotus olearius, Lampteromyces japonicas, Brevudimonas sp., and Muscodor sp.
The pesticide may be a natural oil, oil product or chemical pesticide. In particular, the agent may be a natural oil or oil-product having nematicidal, fungicidal and/or insecticidal activity (e.g., paraffinic oil, tea tree oil, lemongrass oil, clove oil, cinnamon oil, citrus oil (including but not limited to bitter orange, orange, lemon) rosemary oil, pyrethrum, allspice, bergamot, blue gum, camomile, citronella, common jasmine, common juniper, common lavender, common myrrh, field mint, freesia, gray santolina, herb hyssop, holy basil, incense tree, jasmine, lavender, marigold, mint, peppermint, pot marigold, spearmint, ylang-ylang tree, and saponins.
The chemical pesticide may be a single site anti-fungal agent which may include but is not limited to benzimidazole, a demethylation inhibitor (DMI) (e.g., imidazole, piperazine, pyrimidine, triazole), morpholine, hydroxypyrimidine, anilinopyrimidine, phosphorothiolate, quinone outside inhibitor, quinoline, dicarboximide, carboximide, phenylamide, anilinopyrimidine, phenylpyrrole, aromatic hydrocarbon, cinnamic acid, hydroxyanilide, antibiotic, polyoxin, acylamine, phthalimide, benzenoid (xylylalanine), a demethylation inhibitor selected from the group consisting of imidazole, piperazine, pyrimidine and triazole (e.g., bitertanol, myclobutanil, penconazole, propiconazole, triadimefon, bromuconazole, cyproconazole, diniconazole, fenbuconazole, hexaconazole, tebuconazole, tetraconazole), myclobutanil, and a quinone outside inhibitor (e.g., strobilurin). The strobilurin may include but is not limited to azoxystrobin, kresoxim-methoyl or trifloxystrobin. In yet another particular embodiment, the anti-fungal agent is a quinone, e.g., quinoxyfen (5,7-dichloro-4-quinolyl 4-fluorophenyl ether). The anti-fungal agent may also be derived from a Reynoutria extract. The chemical pesticide can also be a multi-site non-inorganic, chemical fungicide. For example, the chemical fungicide may be chloronitrile, quinoxaline, sulphamide, phosphonate, phosphite, dithiocarbamate, chloralkythios, phenylpyridin-amine, or cyano-acetamide oxime.
Nematicides may include, but are not limited to, avermectin nematicides (e.g., abamectin); botanical nematicides (e.g., carvacrol); carbamate nematicides (e.g., benomyl carbofuran, carbosulfan, cloethocarb); oxime carbamate nematicides (e.g., alanycarb, aldicarb aldoxycarb, oxamyl tirpate); fumigant nematicides (e.g., carbon disulfide, cyanogen, 1,2-dichloropropane, 1,3-dichloropropene, dithioether, methyl bromide, methyl iodide, sodium tetrathiocarbonate); organophosphorus nematicides, which includes, but are not limited to, organophosphate nematicides (e.g., diamidafos, fenamiphos, fosthietan, phosphamidon); organothiophosphate nematicides (e.g., cadusafos, chlorpyrifos, dichlofenthion dimethoate ethoprophos, fensulfothion, fosthiazate, heterophos, isamidofos, isazofos, phorate, phosphocarb, terbufos, thionazin, triazophos); phosphonothioate nematicides (e.g., imicyafos, mecarphon); and other nematicides (e.g., acetoprole, benclothiaz, chloropicrin, dazomet, DBCP, DCIP, fluensulfone, furfural, metam, methyl isothiocyanate, xylenols, spirotetramat).
The compositions disclosed herein can also be used in combination with other growth promoting agents such as synthetic or organic fertilizers (e.g., di-ammonium phosphate in either granular or liquid form), compost teas, seaweed extracts, plant growth hormones such as IAA (indole acetic acid) used in a rooting hormone treatment for transplants either alone or in combination with plant growth regulators such as IBA (indole butyric acid) and NAA (naphthalene acetic acid), and growth promoting microbes, such as Bacillus spp., Pseudomonads, Rhizobia, and Trichoderma spp.
Furthermore, the compositions can be used in combination with seed-coating agents. Such seed coating agents include, but are not limited to, ethylene glycol, carboxymethyl cellulose, methyl cellulose, polyethylene glycol, chitosan, carboxymethyl chitosan, peat moss, resins and waxes. The compositions may be applied using methods known in the art. Specifically, these compositions may be applied to and around plants or plant parts. Plants are to be understood as meaning in the present context all plants and plant populations such as desired and undesired wild plants or crop plants (including naturally occurring crop plants). Crop plants can be plants which can be obtained by conventional plant breeding and optimization methods or by biotechnological and genetic engineering methods or by combinations of these methods, including the transgenic plants and including the plant cultivars protectable or not protectable by plant breeders' rights. Plants include all parts and organs of plants above and below the ground, such as shoot, leaf, flower and root, examples which may be mentioned being leaves, needles, stalks, stems, flowers, fruit bodies, fruits, seeds, roots, tubers and rhizomes. The plants include, but are not limited to, harvested material, and vegetative and generative propagation material, for example cuttings, tubers, rhizomes, offshoots and seeds. Plants that may be treated include but are not limited to: (A) major edible food crops, which include but are not limited to (1) cereals (e.g., African rice, barley, durum wheat, einkorn wheat, emmer wheat, finger millet, foxtail millet, hairy crabgrass, Indian barnyard millet, Japanese barnyard millet, maize, nance, oat, pearl millet, proso millet, rice, rye, sorghum, Sorghum spp., rye, spelt wheat); (2) fruits (e.g., abiu, acerola, achacha, African mangosteen, alpine currant, ambarella, American gooseberry, American persimmon, apple, apricot, arazá, Asian palmyra palm, Asian pear, atemoya, Australian desert raisin, avocado, azarole, babaco, bael, banana, Barbados gooseberry, bergamot, betel nut, bignay, bilberry, bilimbi, binjai, biriba, bitter orange, black chokeberry, black mulberry, black sapote, blackberry, blue-berried honeysuckle, borojó, breadfruit, murmese grape, button mangosteen, cacao, calamondin, canistel, cantaloupe, cape gooseberry, cashew nut, cassabanana, cempedak, charichuelo, cherimoya, cherry, cherry of the Rio Grande, cherry plum, Chinese hawthorn, Chinese white pear, chokeberry, citron, cocona, coconut, cocoplum, coffee, coffee Arabica, coffee robusta, Costa Rica pitahaya, currants, custard apple, date, date-plum, dog rose, dragonfruit, durian, elderberry, elephant apple, Ethiopian eggplant, European nettle tree, European wild apple, feijoa, fig, gac, genipapo, giant granadilla, gooseberry, goumi, grape, grapefruit, great morinda, greengage, guava, hardy kiwi, hog plum, horned melon, horse mango, Indian fig, Indian jujube, jabuticaba, jackberry, jackfruit, Japanese persimmon, Japanese wineberry, jocote, jujube, kaffir lime, karanda, kei apple, kepel apple, key lime, kitembilla, kiwi fruit, korlan, kubal vine, kuwini mango, kwai muk, langsat, large cranberry, lemon, Liberian coffee, longan, loquat, lychee, malay apple, mamey sapote, mammee apple, mango, mangosteen, maprang, marang, medlar, melon, Mirabelle plum, miracle fruit, monkey jack, moriche palm, mountain papaya, mountain soursop, mulberry, naranjilla, natal plum, northern highbush blueberry, olive, otaheite gooseberry, oval kumquat, papaya, para guava, passionfruit, pawpaw, peach, peach-palm, pear, pepino, pineapple, pitomba Eugenia luschnathiana, pitomba talisia esculenta, plantain, plum, pomegranate, pomelo, pulasan, purple chokeberry, quince, rambutan, ramontchi, raspberry, red chokeberry, red currant, red mulberry, red-fruited strawberry guava, rhubarb, rose apple, roselle, safou, salak, salmonberry, santol, sapodilla, satsuma, seagrape, soncoya,sour cherry, soursop, Spanish lime, Spanish tamarind, star apple, starfruit, strawberry, strawberry guava, strawberry tree, sugar apple, Surinam cherry, sweet briar, sweet granadilla, sweet lime, tamarillo, tamarind, tangerine, tomatillo, tucuma palm, Vaccinium spp., velvet apple, wampee, watermelon, watery rose apple, wax apple, white currant, white mulberry, white sapote, white star apple, wolfberry (Lyceum barbarum, L. chinense), yellow mombin, yellow pitaya, yellow-fruited strawberry, guava); (3) vegetables (e.g., ackee, agate, air potato, Amaranthus spp., American groundnut, antroewa, armenian cucumber, arracacha, arrowleaf elephant ear, arrowroot, artichoke, ash gourd, asparagus, avocado, azuki bean, bambara groundnut, bamboo, banana, barbados gooseberry, beet, beet root, bitter gourd, bitter vetch, bitterleaf, black mustard, black radish, black salsify, blanched celery, breadfruit, broad bean, broccoli, brussels sprout, Buck's horn plantain, buttercup squash, butternut squash, cabbage, caigua, calabash, caraway seeds, carob, carrot, cassabanana, cassava, catjang, cauliflower, celeriac, celery, celtuce, chard, chayote, chickpea, chicory, chilacayote, chili pepper (Capsicum annuum, C. baccatum, C. chinense, C. frutescens, C. pubescens), Chinese cabbage, Chinese water chestnut, Chinese yam, chives, chufa sedge, cole crops, common bean, common purslane, corn salad, cowpea, cress, cucumber, cushaw pumpkin, drumstick tree, eddoe, eggplant, elephant foot yam, elephant garlic, endive, enset, Ethiopian eggplant, Florence fennel, fluted gourd, gac, garden rocket, garlic, geocarpa groundnut, good king henry, grass pea, groundut, guar bean, horse gram, horseradish, hyacinth bean, iceplant, Indian fig, Indian spinach, ivy gourd, Jerusalem artichoke, jicama, jute, kale, kohlrabi, konjac, kurrat, leek, lentil, lettuce, Lima bean, lotus, luffa, maca, maize, mangel-wurzel, mashua, moso bamboo, moth bean, mung bean, napa cabbage, neem, oca, okra, oldham's bamboo, olive, onion, parsnip, pea, pigeon pea, plantain, pointed gourd, potato, pumpkins, squashes, quinoa, radish, rapeseed, red amaranth, rhubarb, ribbed gourd, rice bean, root parsley, runner bean, rutabaga, sago palm, salsify, scallion, sea kale, shallot, snake gourd, snow pea, sorrel, soybean, spilanthes, spinach, spinach beet, sweet potato, taro, tarwi, teasle gourd, tepary bean, tinda, tomato, tuberous pea, turnip, turnip-rooted chervil, urad bean, water caltrop trapa bicornis, water caltrop trapa natans, water morning slory, watercress, welsh onion, west African okra, west Indian gherkin, white goosefoot, white yam, winged bean, winter purslane, yacón, yam, yard-long bean, zucchinietables); (4) Food crops (e.g., abiu, acerola, achacha, ackee, African mangosteen, African rice, agate, air potato, alpine currant, Amaranthus app., Ambarrella, American gooseberry, American groundnut, American persimmon, antroewa, apple, apricot, arazá, Armenian cucumber, arracacha, arrowleaf elephant ear, arrowroot, artichoke, ash gourd, Asian palmyra palm, Asian pear, asparagus, atemoya, Australian desert raisin, avocado, azarole, azuki bean, babaco, bael, bambara groundnut, bamboo, banana, barbados gooseberry, barley, beet, beetroot, bergamot, betel nut, bignay, bilberry, bilimbi, binjai, biriba, bitter gourd, bitter orange, bitter vetch, bitterleaf, black chokeberry, black currant, black mulberry, black mustard, black radish, black salsify, black sapote, blackberry, blanched celery, blue-berried honeysuckle, borojó, breadfruit, broad bean, broccoli, Brussels sprout, Buck's horn plantain, buckwheat, Burmese grape, buttercup squash, butternut squash, button mangosteen, cabbage, cacao, caigua, calabash, calamondin, canistel, cantaloupe, cape gooseberry, caraway seeds, carob, carrot, cashew nut, cassava, catjang, cauliflower, celeriac, celery, celtuce, cempedak, chard, charichuelo, chayote, cherimoya, cherry, cherry of the Rio Grande, cherry plum, chickpea, chicory, chilacayote, chili pepper (Capsicum annuum, C. baccatum, C. chinense, C. frutescens, C. pubescens), Chinese cabbage, Chinese hawthorn, Chinese water chestnut, Chinese white pear, Chinese yam, chives, chokeberry, chufa sedge, citron, cocona, coconut, cocoplum, coffee, coffee (Arabica and Robusta types), cole crops, common bean, common purslane, corn salad, Costa Rica pitahaya, cowpea, cress, cucumber, currants, cushaw pumpkin, custard apple, date, date-plum, dog rose, dragonfruit, drumstick tree, durian, durum wheat, eddoe, eggplant, einkorn wheat, elderberry, elephant apple, elephant foot yam, elephant garlic, emmer wheat, endive, enset, Ethiopian eggplant, European nettle tree, European wild apple, feijoa, fig, finger millet, florence fennel, fluted gourd, foxtail millet, gac, garden rocket, garlic, genipapo, geocarpa groundut, giant granadilla, good king henry, gooseberry, goumi, grape, grapefruit, grass pea, great morinda, greengage, groundnut, grumichama, guar bean, guava, hairy crabgrass, hardy kiwi, hog plum, horned melon, horse gram, horse mango, horseradish, hyacinth bean, iceplant, Indian barnyard millet, Indian fig, Indian jujube, Indian spinach, ivy gourd, jabuticaba, jackalberry, jackfruit, jambul, Japanese barnyard millet, Japanese persimmon, Japanese wineberry, Jerusalem artichoke, jocote, jujube, jute, kaffir lime, kale, karanda, kei apple, kepel apple, key lime, kitembilla, kiwifruit, kohlrabi, konjac, korlan, kubal vine, kurrat, kuwini mango, kwai muk, langsat, large cranberry, leek, lemon, lentil, lettuce, Liberian coffee, lima bean, longan, loquat, lotus, luffa, lychee, maca, maize, malay apple, mamey saptoe, mammee apple, mangel-wurzel, mango, mangosteen, maprang, marang, mashua, medlar, melon, Mirabelle plum, miracle fruit, monk fruit, monkey jack, moriche palm, moso bamboo, moth bean, mountain papaya, mountain soursop, mulberry, mung bean, mushrooms, nance, napa cabbage, naranjilla, natal plum, neem, northern highbush blueberry, oat, oca, oil palm, okra, oldman's bamboo, olive, onion, orange, otaheite gooseberry, oval kumquat, papaya, para guava, parsnip, passionfruit, pawpaw, pea, peach, peach-palm, pear, pearl millet, pepino, pigeon pea, pineapple, Pitomba (Eugenia luschnathiana, Talisia esculenta), plantain, plum, pointed gourd, pomegranate, pomelo, potato, proso millet, pulasan, pumpkins and squashes, purple chokeberry, quince, quinoa, radish, rambutan, ramontchi, rapeseed, raspberry, red amaranth, red chokeberry, red currant, red mulberry, red-fruited strawberry guava, rhubarb, ribbed gourd, rice, rice bean, root parsley, rose apple, roselle, runner bean, rutabaga, rye, safou, sago palm, salak, salmonberry, salsify, santol, sapodilla, Satsuma, scallion, sea kale, seagrape, shallot, snake gourd, snow pea, soncoya, sorghum, Sorghum spp., sorrel, sour cherry, soursop, soybean, Spanish lime, Spanish tamarind, spelt wheat, spilanthes, spinach, spinach beet, star apple, starfruit, strawberry, strawberry guava, strawberry tree, sugar apple, sugar beet, sugarcane, surinam cherry, sweet briar, sweet granadilla, sweet lime, sweet potato, tamarillo, tamarind, tangerine, taro, tarwi, teasle gourd, tef, tepary bean, tinda, tomatillo, tomato, tuberous pea, tucuma palm, turnip, turnip-rooted chervil, urad bean, Vaccinium spp., velvet apple, wampee, water caltrop (Trapa bicornis, T. natans), water morning glory, watercress, watermelon, watery rose apple, wax apple, welsh onion, west African okra, west Indian gherkin, wheat, white currant, white goosefoot, white mulberry, white sapote, white star apple, white yam, winged bean, winter purslane, wolfberry (Lycium barbarum, L. chinense), yacon, yam, yangmei, yard-long bean, yellow mombin, yellow pitaya, yellow-fruited strawberry guava, zucchini; (B) Other edible crops, which includes but is not limited to (1) Herbs (e.g., Absinthium, alexanders, basil, bay laurel, betel nut, camomile, chervil, chili pepper (Capsicum annuum, C. baccatum, C. chinense, C. frutescens, C. pubescens), chili peppers, chives, cicely, common rue, common thyme, coriander, cress, culantro, curly leaf parsley, dill, epazote, fennel, flat leaf parsley, ginseng, gray santolina, herb hyssop, holy basil, hop, jasmine, kaffir lime, lavender, lemon balm, lemon basil, lemon grass, lovage, marjoram, mint, oregano, parsley, peppermint, perilla, pot marigold, rooibos, rosemary, sage, shiny-leaft buckthorn, sorrel, spearmint, summer savory, tarragon, Thai basil, valerian, watercress, wild betel, winter savory, yerba mate); (2) Spices (e.g., ajowan, allspice, anise, bay laurel, black cardamom, black mustard, black pepper, caper, caraway seeds, cardamom, chili pepper (Capsicum annuum, C. baccatum, C. chinense, C. frutescens, C. pubescens), chili peppers, cinnamon, clove, common juniper, coriander, cumin, fennel, fenugreek, garlic, ginger, kaffir lime, liquorice, nutmeg, oregano, pandan, parsley, saffron, star anise, turmeric, vanilla, white mustard); (3) Medicinal plants (e.g., absinthium, alfalfa, aloe vera, anise, artichoke, basil, bay laurel, betel leat, betel nut, bilberry, black cardamom, black mustard, black pepper, blue gum, borojó, camomlie, caper, cardamom, castor bean, chili peppers, Chinese yam, chives, cola nut, common jasmine, common lavender, common myrrh, common rue, cilantro, cumin, dill, dog rose, epazote, fennel, fenugreek, gac, garlic, ginger, gray santolina, gum Arabic, herb hyssop, holy basil, horseradish, incense tree, lavender, lemon grass, liquorice, lovage, marijuana, marjoram, monk fruit, neem, opium, oregano, peppermint, pot marigold, quinine, red acacia, red currant, rooibos, safflower, sage, shiny-leaf buckthorn, sorrel, spilanthes, star anise, tarragon, tea, turmeric, valerian, velvet bean, watercress, white mustard, white sapote, wild betel, wolfberry (Lycium barbarum, L. chinense), yerba mate); (4) Stimulants (e.g., betel leaf, betel nut, cacao, chili pepper (Capsicum annuum, C. baccatum, C. chinense, C. frutescens, C. pubescens), chili peppers, coffee, coffee (Arabica, Robusta), cola nut, khat, Liberian coffee, tea, tobacco, wild betel, yerba mate); (5) Nuts (e.g., almond, betel nut, Brazil nut, cashew nut, chestnut, Chinese water chestnut, coconut, cola nut, common walnut, groundnut, hazelnut, Japanese stone oak, macadamia, nutmeg, paradise nut, pecan nut, pistachio nut, walnut); (6) Edible seeds (e.g., black pepper, Brazil nut, chilacayote, cola nut, fluted gourd, lotus, opium, quinoa, sesame, sunflower, water caltrop (Trapa bicornis, T. natans); (7) Vegetable oils (e.g., black mustard, camelina, castor bean, coconut, cotton, linseed, maize, neem, niger seed, oil palm, olive, opium, rapeseed, safflower, sesame, soybean, sunflower, tung tree, turnip); (8) Sugar crops (e.g., Asian palmyra palm, silver date palm, sorghum, sugar beet, sugarcane); (9) Pseudocereals (e.g., Amaranthus spp., buckwheat, quinoa, red amaranth); (10) Aphrodisiacs (e.g., borojó, celery, durian, garden rocket, ginseng, maca, red acacia, velvet bean); (C) Non food categories, including but not limited to (1) forage and dodder crops (e.g., agate, alfalfa, beet, broad bean, camelina, catjang, grass pea, guar bean, horse gram, Indian barnyard millet, Japanese barnyard millet, lespedeza, lupine, maize, mangel-wurzel, mulberry, niger seed, rapeseed, rice bean, rye); (2) Fiber crops (e.g., coconut, cotton, fique, hemp, henequen, jute, kapok, kenaf, linseed, manila hemp, New Zealand flax, ramie, roselle, sisal, white mulberry); (3) Energy crops (e.g., blue gum, camelina, cassava, maize, rapeseed, sorghum, soybean, Sudan grass, sugar beet, sugarcane, wheat); (4) Alcohol production, (e.g., barley, plum, potato, sugarcane, wheat, sorghum); (5) Dye crops (e.g., chay root, henna, indigo, old fustic, safflower, saffron, turmeric); (6) Essential oils (e.g., allspice, bergamot, bitter orange, blue gum, camomile, citronella, clove, common jasmine, common juniper, common lavender, common myrrh, field mint, freesia, gray santolina, herb hyssop, holy basil, incense tree, jasmine, lavender, lemon, marigold, mint, orange, peppermint, pot marigold, spearmint, ylang-ylang tree); (6) Green manures (e.g., alfalfa, clover, lacy Phacelia, sunn hemp, trefoil, velvet bean, vetch); (7) Erosion prevention (e.g., bamboo, cocoplum); (8) Soil improvement (e.g., lupine, vetch); (9) Cover crops (e.g., Alfalfa, lacy Phacelia, radish); (10) Botanical pesticides (e.g., jicama, marigold, neem, pyrethrum); (11) Cut flowers (e.g., carnation, chrysanthemum, daffodil, dahlia, freesia, gerbera, marigold, rose, sunflower, tulip); (12) Ornamental plants (e.g., African mangosteen, aloe vera, alpine currant, aster, black chokeberry, breadfruit, calamondin, carnation, cassabanana, castor bean, cherry plum, chokeberry, chrysanthemum, cocoplum, common lavender, crocus, daffodil, dahlia, freesia, gerbera, hyacinth, Japanese stone oak, Jasmine, lacy Phacelia, lotus, lupine, marigold, New Zealand flax, opium, purple chokeberry, ramie, red chokeberry, rose, sunflower, tulip, white mulberry); (D) Trees which include but are not limited to abelia, almond, apple, apricot, arborvitae nigra american, arborvitae, ash, aspen, azalea, baldcypress, beautybush, beech, birch, black tupelo, blackberry, blueberry, boxwood, buckeye, butterfly bush, butternut, camellia, catalpa, cedar, cherry, chestnut, coffeetree, crab trees, crabapple, crapemyrtle, cypress, dogwood, douglasfir, ebony, elder American, elm, fir, forsythia, ginkgo, goldenraintree, hackberry, hawthorn, hazelnut, hemlock, hickory, holly, honeylocust, horsechestnut, hydrangea, juniper, lilac, linden, magnolia, maple, mockorange, mountainash, oak, olive, peach, pear, pecan, pine, pistache, planetree, plum, poplar, pivet, raspberry, redbud, redcedar, redwood, rhododendron, rose-of-sharon, sassafras, sequoia, serviceberry, smoketree, soapberry, sourwood, spruce, strawberry tree, sweetshrub, sycamore, tuliptree, viburnum, walnut, weigela, willow, winterberry, witchhazel, zelkova; and (E) Turf, which includes, but is not limited to, Kentucky bluegrass, tall fescue, Bermuda grass, zoysia grass, perennial ryegrass, and fine fescues (e.g. creeping red, chewings, hard, or sheep fescue).
Methods of Production
Chromobacterium sp., Burkholderia sp., and/or Flavobacterium sp. may be cultivated in nutrient medium using methods known in the art. The organisms may be cultivated by shake flask cultivation, small scale or large scale fermentation (including but not limited to continuous, batch, fed-batch, or solid state fermentations) in laboratory or industrial fermentors performed in suitable medium and under conditions allowing cell growth. The cultivation may take place in suitable nutrient medium comprising carbon and nitrogen sources and inorganic salts, using procedures known in the art. Suitable media may be available from commercial sources or prepared according to published compositions.
After cultivation, a substantially pure culture or whole cell broth comprising said strain, or cell fraction, supernatant, filtrate, or compound (e.g., metabolite and/or extract) of or derived from said Chromobacterium sp., Burkholderia sp., and/or Flavobacterium sp. may be used in formulating a pesticidal composition. Alternatively, after cultivation, the compounds and/or metabolites may be extracted from the culture broth.
The extract may be fractionated by chromatography. Chromatographic fractions may be assayed for toxic activity against, for example, plant parasitic nematodes, such as Heterodera glycines (soybean cyst nematode), using methods known in the art. This process may be repeated one or more times using the same or different chromatographic methods.
Uses
The compositions, cultures, supernatants, metabolites and pesticidal compounds set forth above may be used as pesticides and in particular, may be used as nematicides, alone or in combination with one or more pesticidal substances set forth above and applied to plants, substrates for growing plants, or seeds as set forth herein.
Specifically, nematodes that may be controlled using the method set forth above include, but are not limited to, plant parasitic nematodes such as root-knot, reniform, cyst, and lesion nematodes, including but not limited to Meloidogyne sp., Tylenchorhynchus sp., Belonolaimus sp., Hoplolaimus sp., Helicotylenchus sp., Pratylenchus sp., Rotylenchulus sp., Heterodera sp. (e.g., Heterodera glycines), Globodera sp., Trichodorus sp., Paratrichodorus sp., Xiphinema sp., and Criconema sp. In particular, the plant parasitic nematodes may include but are not limited to seed gall nematodes (Afrina wevelli), bentgrass nematodes (Anguina agrostis), shoot gall nematodes (Anguina spp.), seed gall nematodes (Anguina spp., A. amsinckiae, A. balsamophila; A. tritici), fescue leaf gall nematodes (A. graminis), ear-cockle (or wheat gall) nematodes (Anguina tritici), bud and leaf (or foliar) nematodes (Aphelenchoides spp., A. subtenuis), begonia leaf (or fern, spring crimp, strawberry foliar, strawberry nematodes, summer dwarf) nematodes (A. fragariae), fern nematodes (A. olesistus), rice nematodes (A. oryzae), currant nematodes (A. ribes), black currant (or chrysanthemum) nematodes (A. ritzemabosi), chrysanthemum foliar or leaf nematodes (A. ritzemabosi), rice white-tip (or spring dwarf, or strawberry bud) nematodes (A. besseyi), fungus-feeding (mushroom) nematodes (Aphelenchoides composticola), Atalodera spp. (Atalodera lonicerae, Atalodera ucri), spine nematodes (Bakernema variabile), sting nematodes (Belonolaimus spp., B. gracilis, B. longicaudatus), pine wood nematodes (Bursaphalenchus spp., B. xylophilus, B. mucronatus), sessile nematodes (Cacopaurus spp., C. epacris, C.pestis), amaranth cyst nematodes (Cactodera amaranthi), birch cyst nematodes (C. betulae), cactus cyst nematodes (C. cacti), estonian cyst nematodes (C. estonica), Thorne's cyst nematodes (C. thornei), knotweed cyst nematodes (C. weissi), ring nematodes (Criconema spp.), spine nematodes (Criconema spp., C. civellae, C. decalineatum, C.spinalineatum), ring nematodes (Criconemella axeste, C. curvata, C. macrodora, C. parva), ring nematodes (Criconemoides spp., C. citri, C. simile), spine nematodes (Crossonema fimbriatum), eucalypt cystoid nematodes (Cryphodera eucalypti), bud, stem and bulb nematodes (Ditylenchus spp., D. angustus, D. dipsaci, D. destructor, D. intermedius), Mushroom spawn nematodes (D. myceliophagus), awl nematodes (Dolichodorus spp., D. heterocephalus, D. heterocephalous), spear nematodes (Dorylaimus spp.), stunt nematodes (Geocenamus superbus), cyst nematodes (Globodera spp.), yarrow cyst nematodes (G. achilleae), milfoil cyst nematodes (G. millefolii), apple cyst nematodes (G. mali), white cyst potato nematodes (G. pallida), golden nematodes (G. rostochiensis), tobacco cyst nematodes (G. tabacum), Osborne's cyst nematodes (G. tabacum solanacearum), horsenettle cyst nematodes (G. tabacum virginiae), pin nematodes (Gracilacus spp., G. idalimus), spiral nematodes (Helicotylenchus spp., H. africanus, H. digonicus, H. dihystera, H. erythrinae, H. multicinctus, H. paragirus, H. pseudorobustus, H. solani, H. spicaudatus), sheathoid nematodes (Hemicriconemoides spp., H. biformis, H. californianus, H. chitwoodi, H. floridensis, H. wessoni), sheath nematodes (Hemicycliophora spp., H. arenaria, H. biosphaera, H. megalodiscus, H. parvana, H. poranga, H. sheri, H. similis, H. striatula), cyst nematodes (Heterodera spp.), almond cyst nematodes (H. amygdali), oat (or cereal) cyst nematodes (H. avenae), Cajanus (or pigeon pea) cyst nematodes (H. cajani), Bermuda grass (or heart-shaped, or Valentine) cyst nematodes (H. cardiolata), carrot cyst nematodes (H. carotae), cabbage cyst nematodes or brassica root eelworm (H. cruciferae), nutgrass (or sedge) cyst nematodes (H. cyperi), Japanese cyst nematodes (H. elachista), fig (or ficus, or rubber) cyst nematodes (H. fici), galeopsis cyst nematodes (H. galeopsidis), soybean cyst nematodes (H. glycines), alfalfa root (or pea cyst) nematodes (H. goettingiana), buckwheat cyst nematodes (H. graduni), barley cyst nematodes (H. hordecalis), hop cyst nematodes (H. humuli), Mediterranean cereal (or wheat) cyst nematodes (H. latipons), lespedeza cyst nematodes (H. lespedezae), Kansas cyst nematodes (H. longicolla), cereals root eelworm or oat cyst nematodes (H. major), grass cyst nematodes (H. mani), lucerne cyst nematodes (H. medicaginis), cyperus (or motha) cyst nematodes (Heterodera mothi), rice cyst nematodes (H. oryzae), Amu-Darya (or camel thorn cyst) nematodes (H. oxiana), dock cyst nematodes (H. rosii), rumex cyst nemtodes (H. rumicis), sugar beet cyst nematodes (H. schachtii), willow cyst nematodes (H. salixophila), knawel cyst nematodes (H. scleranthii), sowthistle cyst nematodes (H. sonchophila), tadzhik cyst nematodes (H. tadshikistanica), turkmen cyst nematodes (H. turcomanica), clover cyst nematodes (H. trifolii), nettle cyst nematodes (H. urticae), ustinov cyst nematodes (H. ustinovi), cowpea cyst nematodes (H. vigni), corn cyst nematodes (H. zeae), rice root nematodes (Hirschmanniella spp., H. belli, H. caudacrena, H. gracilis, H.oryzae), lance nematodes (Hoplolaimus spp.), Columbia nematodes (H. columbus), Cobb's lance nematodes (H. galeatus), crown-headed lance nematodes (H. tylenchiformis), pseudo root-knot nematodes (Hypsoperine graminis), needle nematodes (Longidorus spp., L. africanus, L. sylphus), ring nematodes (Macroposthonia (Mesocriconema xenoplax), cystoid nematodes (Meloidodera spp.), pine cystoid nematodes (M floridensis), tadzhik cystoid nematodes (M tadshikistanica), cystoid body nematodes (Meloidoderita spp.), stunt nematodes (Merlinius spp., M. brevidens, M. conicus, M. grandis, M. microdorus), root-knot nematodes (Meloidogyne spp., M. acronea, M arenaria, M.artiellia, M. brevicauda, M. camelliae, M. carolinensis, M. chitwoodi, M. exigua, M. graminicola, M. hapla, M. hispanica, M. incognita, M. incognita acrita, M. indica, M. inornata, M. javanica, M. kikuyuensis, M. konaensis, M. mali, M. microtyla, M. naasi, M ovalis, M platani, M. querciana, M. sasseri, M tadshikistanica, M. thamesi), knapweed nematodes (Mesoanguina picridis), Douglas fir nematodes (Nacobbodera chitwoodi), false root-knot nematodes (Nacobbus aberrans, N. batatiformis, N. dorsalis), sour paste nematodes (Panagrellus redivivus), beer nematodes (P. silusiae), needle nematodes (Paralongidorus microlaimus), spiral nematodes (Pararotylenchus spp.), stubby-root nematodes (Paratrichodorus allius, P. minor, P. porosus, P. renifer), pin nematodes (Paratylenchus spp., P. baldaccii, P. bukowinensis, P. curvitatus, P. dianthus, P. elachistus, P. hamatus, P. holdemani, P. italiensis, P. lepidus, P. nanus, P. neoamplycephalus, P. similis), lesion (or meadow) nematodes (Pratylenchus spp., P. alleni, P. brachyurus, P. coffeae, P. convallariae, P. crenatus, P. flakkensis, P. goodeyi, P. hexincisus, P. leiocephalus, P. minyus, P. musicola, P. neglectus, P. penetrans, P. pratensis, P. scribneri, P. thornei, P. vulnus, P. zeae), stem gall nematodes (Pterotylenchus cecidogenus), grass cyst nematodes (Punctodera punctate), stunt nematodes (Quinisulcius acutus, Q. capitatus), burrowing nematodes (Radopholus spp.), banana-root nematodes (R. similis), rice-root nematodes (R. oryzae), red ring (or coconut, or cocopalm) nematodes (Rhadinaphelenchus cocophilus), reniform nematodes (Rotylenchulus spp., R. reniformis, R. parvus), spiral nematodes (Rotylenchus spp., R. buxophilus, R. christiei, R. robustus), Thorne's lance nematodes (R. uniformis), Sarisodera hydrophylla, spiral nematodes (Scutellonema spp., S. blaberum, S. brachyurum, S. bradys, S. clathricaudatum, S. christiei, S. conicephalum), grass root-gall nematodes (Subanguina radicicola), round cystoid nematodes (Thecavermiculatus andinus), stubby-root nematodes (Trichodorus spp., T. christiei, T. kurumeensis, T. pachydermis, T. primitivus), vinegar eels (or nematodes) (Turbatrix aceti), stunt (or stylet) nematodes (Tylenchorhynchus spp., T. agri, T. annulatus, T. aspericutis, T. claytoni, T.ebriensis, T. elegans, T. golden, T.graciliformis, T. martini, T. mashhoodi, T. microconus, T. nudus, T. oleraceae, T. penniseti, T. punensis), citrus nematodes (Tylenchulus semipenetrans), dagger nematodes (Xiphinema spp., X. americanum, X. bakeri, X. brasiliense, X. brevicolle, X. chambersi, X. coxi, X. diversicaudatum X. index, X. insigne, X. nigeriense, X. radicicola, X. setariae, X. vulgarae, X. vuittenezi). In an even more particular embodiment, the nematodes, include but are not limited to Meloidogyne incognita (root knot nematodes), as well as Globodera rostochiensis and Globodera pailida (potato cyst nematodes); Heterodera glycines (soybean cyst nematode); Heterodera schachtii (beet cyst nematode); and Heterodera avenae (cereal cyst nematode) and Rotylenchulus reniformis (reniform nematodes) (Pratylenchus spp.) (lesion nematodes), Belonolaimus sp. (sting nematode), Hopolaimus sp. (lance nematode), Bursaphalenchus spp., (e.g., pine wood nematodes), and Ditylenchus spp. (e.g., stem nematodes).
Application of an effective pesticidal controlling amount of a Chromobacterium sp., Burkholderia sp., and/or Flavobacterium sp. is provided. A substantially pure culture, whole cell broth, supernatant, filtrate, extract or compound of the bacterial strain may be applied, alone or in combination with another pesticidal substance, in an effective pest control or pesticidal amount. An “effective amount” is defined as the quantity of microorganism cells, supernatant, whole cell broth, filtrate, cell fraction or extract, metabolite and/or compound alone or in combination with another pesticidal substance that is sufficient to modulate plant parasitic nematode infestation. The amount that will be within an effective range can be determined by laboratory or field tests by those skilled in the art. In an embodiment of the invention, when the composition is applied directly to the seed, the effective amount is a concentration of about 0.01%-30% of the active ingredient (for example, the Chromobacterium sp., Burkholderia sp., or Flavobacterium sp.) per 100g of seed. In another embodiment, the effective amount is a concentration of about 0.05%-25%, or about 0.1%-20%, or about 0.5%-15%, or about 1%-10%, or about 2%-5% of the active ingredient per 100 g of seed. In yet another embodiment, the effective amount is a concentration of about 0.5% to 1% of the active ingredient per 100 g of seed. In another embodiment of the invention, when the composition is applied to the soil by, for example, in furrow, the effective amount is about 0.1-50 oz of the active ingredient per 1000 ft row. In other embodiments, the effective amount for soil application is about 1-25 oz of the active ingredient per 1000 ft row. In yet another embodiment, the effective amount is about 2-20 oz, or about 3-15oz, or about 4-10 oz, or about 5-8 oz, of the active ingredient per 1000 ft row.
The compositions, substantially pure culture, whole cell broth, supernatant, filtrate extract or compounds set forth above may modulate plant parasitic nematode infestation by a number of mechanisms. These include but are not limited to exterminating the plant parasitic nematodes at various stages of the life cycle (eggs, larvae or adult), affecting the motility of the plant parasitic nematodes, and/or reducing the number and/or viability of eggs that are laid by the parasitic nematodes.
The said formulated product can be used alone or sequentially with one or more other components as set forth above, such as growth promoting agents in a tank mix or in a program (sequential application called rotation) with predetermined order and application interval during the growing season.
Contact of the plants with the compositions set forth above may be carried out directly or by allowing the compositions to act on their surroundings, habitat or storage space by, for example, immersion, spraying, evaporation, fogging, scattering, painting on, injecting or soil amendment. In the case that the composition is applied to a seed, the composition may be applied to the seed as one or more coats prior to planting the seed using one or more coats using methods known in the art.
The compositions comprising a Chromobacterium sp., Burkholderia sp., and/or Flavobacterium sp. may be applied by root dip at transplanting, specifically by treating a fruit or vegetable with the composition by dipping roots of the fruit or vegetable in a suspension of said composition (about 0.25 to about 1.5% and more particularly about 0.5% to about 1.0% volume by volume) prior to transplanting the fruit or vegetable into the soil.
Alternatively, the composition may be applied by drip or other irrigation system. Specifically, the composition comprising a Chromobacterium sp., Burkholderia sp., and/or Flavobacterium sp. may be injected into a drip irrigation system. In a particular embodiment, the composition may be applied at a rate of about 11 to about 4 quarts per acre.
In yet another embodiment, the composition may be added as an in-furrow application. Specifically, the composition may be added as an in-furrow spray at planting using nozzles calibrated to deliver a total output of 2-6 gallons/acre, or at outputs of up to 20 gallons per acre. Nozzles are placed in the furrow opener on the planter so that the pesticide application and seed drop into the furrow are simultaneous. The mixtures set forth above and, where appropriate, a solid or liquid adjuvant are prepared in known manner. For example, the mixtures may be prepared by homogeneously mixing and/or grinding the active ingredients with extenders such as solvents, solid carriers and, where appropriate, surface-active compounds (surfactants). The compositions may also contain further ingredients such as stabilizers, viscosity regulators, binders, adjuvants as well as fertilizers or other active ingredients in order to obtain special effects.
EXAMPLE
The composition and methods set forth above will be further illustrated in the following, non-limiting Examples. The examples are illustrative of various embodiments only and do not limit the claimed invention regarding the materials, conditions, weight ratios, process parameters and the like recited herein.
A seed treatment trial was conducted to determine the effect of whole cell broths from Flavobacterium sp. H492 (MBI-302), Chromobacterium subtsugae sp. nov. (MBI-203), Burkholderia A396 (MBI-206), and enriched whole cell broth from Flavobacterium sp. H492 (MBI-302) on Heterodera glycines (soybean cyst nematode, SCN) infestation of soybean plants.
The study included six experimental seed treatments, an untreated control, and a standard (positive) control. Treatments of the seeds included the following:
1. Untreated control
2. Standard—AVICTA® Complete Soybean (Syngenta) 4.17 FS applied at 0.15 mg active ingredient per seed
3. Flavobacterium sp. H492 (MBI-302) @1 gram active ingredient per 100 grams seed
4. Flavobacterium sp. H492 (MBI-302) “enriched” @1 gram active ingredient per 100 grams seed
5. Chromobacterium subtsugae sp. nov. (MBI-203) @ 1 gram active ingredient per 100 grams seed
6. Burkholderia A396 (MBI-206) @0.5 gram active ingredient per 100 grams seed
7. Burkholderia A396 (MBI-206) @1.0 gram active ingredient per 100 grams seed
8. Chromobacterium subtsugae sp. nov. (MBI-203) in-furrow 5 oz per 1000 ft row in a minimum of 20 gallons per acre (GPA).
The term “enriched” (as used in treatment 4 above) refers to a MBI-302 whole-cell broth in which most of the water has been extracted to yield a concentrated cell paste.
Seed Treatment
Soybean seeds (94Y82) were prepared by weighing out the desired amount of seeds per treatment. Coating material, for application to seeds, consisted essentially of a sticking component (e.g. methyl cellulose) and whole cell broth from each of Flavobacterium sp. H492 (MBI-302), Burkholderia A396 (MBI-206), and Chromobacterium subtsugae sp. nov (MBI-203), enriched whole-cell broth from Flavobacterium sp. H492 (MBI-302) or AVICTA® Complete Soybean (Syngenta) (positive control). Negative control seeds were untreated. The amount of coating material was 2% of the weight of the seed. The coating material was applied to the seeds in a seed coater and the seeds were dried overnight.
Nematodes
Heterodera glycines were reared on Hutchinson soybeans for 60 days in a greenhouse, and inoculated into soil just prior to planting.
Planting and Plot Size
The trial was conducted in outdoor microplots (pots approximately 1 foot in diameter) having a volume of 19,000 cm3, arranged in a randomized complete block design with 5 replicate plots for each of the eight treatments. Nematode-free field soil (Wickham fine sandy loam, 70-16-18 S-S-C, 1.0% OM, pH=6.5) was used. One week before planting, lime and fertilizer (OSMOCOAT® 10-10-10 Slow Release) (The Scotts Company) were added to the soil in each microplot and incorporated with a hand trowel. Lime was added to raise the pH of the soil to 7, which is optimal for soybean growth.
Soybean seeds, untreated or treated as described above, were planted in the pots in linear rows, at a density of ten seeds per foot. Immediately prior to planting, 500 cm3 of H. glycines-inoculated soil, containing at least 100 cysts, was mixed into each row. Plots were watered twice a day.
Data recording
Seeds were planted on May 1, 2013. Fifty-seven days after planting (Jul. 30, 2013), plant height (distance from soil surface to plant top) was determined in three plants selected randomly from each plot. From these measurements, an average (mean) height was calculated. Mean plant heights for each of the eight treatments are shown in the third column of Table 1.
The number of cysts associated with each plant was also determined at fifty-seven days after planting. For measurement of cyst number, four core samples of 2.5×12 cm were obtained from each pot. The four samples were combined and homogenized, and 150 cm3 of the homogenate was removed. Cysts were extracted from the 150 cm3 sample by gravity screening on an 80 mesh sieve and counted. Results, expressed as mean cyst number, are shown in the fourth column of Table 1.
Seed production per plot was measured 127 days after planting (i.e., Oct. 8, 2013). All plants in a plot were cut down and placed in paper bags for three days for drying. Dried plants from each plot were hand-threshed, the seeds were collected and adjusted to 13% moisture content, then weighed. Results, expressed as mean seed weight per plot, are shown in the fifth column of Table 1.











TABLE 1





Treatment
Concentration
Avg. Height
Cyst No.
Seed Wt.







Untreated
—
31.8 (a)
35.9 (ab)
 84.2 (b)


AVICTA ®

34.3 (a)
13.8 (b)
100.8 (ab)


MBI-302
1%
33.9 (a)
44.1 (a)
 91.3 (b)


MBI-302 enr
1%
33.9 (a)
22.4 (ab)
105.1 (ab)


MBI-203
1%
35.6 (a)
16.4 (ab)
111.3 (ab)


MBI-206
0.5%  
34.7 (a)
20.0 (ab)
113.5 (ab)


MBI-206
1%
33.9 (a)
17.6 (ab)
 98.0 (a)


MBI-203 IF
5 oz/103 ft.
36.4 (a)
17.0 (ab)
136.8 (a)



LSD
 3.589
 0.259
 42.72



Std. Dev.
 3.336
 0.241
 39.71



CV
 9.72
17.78
 37.77





Test substances listed in the “Treatment” column included a commercial nematicide (AVICTA ®), whole-cell broths from Flavobacterium sp. H492 (MBI-302), Chromobacterium subtsugae sp. nov. (MBI-203), and Burkholderia A396 (MBI-206), and enriched whole cell broth from Flavobacterium sp. H492 (MBI-302 enr).


Test substances were coated onto seeds (soybean 94Y82), prior to planting, at the concentration (g/100 g seed) shown in the “Conc.” column, except for MBI-203 IF which was applied to soil just prior to planting at a concentration of 5 oz per 1000 ft in a minimum of 20 gallons per acre.


Heights are expressed in cm; seed weights are expressed in grams per plot.


Mean values followed by the same letter do not significantly differ (p = 0.1, Student-Newman-Kuels test)


LSD: Least significant difference at 90% confidence level (p = 0.1)


Std. Dev.: Standard deviation


CV: Coefficient of Variation






Data Analysis
Least significant difference (Fisher's LSD) values were obtained for plant height, cyst number and seed weight values, as shown in Table 1. Values that differ from the control untreated value by more than the LSD indicate that the difference in values has a 90% probability of resulting from the treatment. By this criterion, both seed coating with, and in-furrow application of, MBI-203 reverse the growth inhibition caused by SCN infestation; and both performed better than the commercial nematicide (Table 1, Column 3).
With respect to cyst number, whole-cell broth from MBI-203 and MBI-206, and enriched whole-cell broth from MBI-302 all reduced the number of cysts associated with infested plants (Table 1, Column 4).
Finally, in-furrow application of MBI-203 increased the amount of seed produced by SCN-infested plants (Table 1, Column 5).
These results show that application of MBI-203, MBI-206 and/or MBI-302 to seeds or soil is effective in reducing the number of SCN cysts in the soil, after seeds are planted in SCN-infested soil. They also show that MBI-203 reduces growth inhibition caused by SCN infestation, and increases seed yield in SCN-infested soybean plants. They also show that MBI-302 and MBI-206 treated plots are not different from the commercial nematicide in yields per plot.
This invention may be embodied in other forms or carried out in other ways without departing from the spirit or essential characteristics thereof. The present disclosure is therefore to be considered as in all aspects illustrate and not restrictive, and all changes which come within the meaning and range of equivalency are intended to be embraced therein.
Various references are cited throughout this specification, each of which is incorporated herein by reference in its entirety.

## Claims

1. A method for modulating soybean cyst nematode infestation of a plant comprising contacting the plant, one or more seeds of the plant,and/or soil in which the plant is grown, a composition comprising a bacterial strain of Chromobacterium subtsugae sp. nov. having NRRL Accession No. B-30655, in an amount effective to modulate said soybean cyst nematode infestation.

2. The method of claim 1, wherein the contacting comprises applying the composition to a seed of the plant prior to planting.

3. The method of claim 1, wherein the composition is added to the soil in furrow.

4. The method of claim 1, wherein the method reduces the number of soybean cyst nematodes in the soil or plant, or on the plant, compared to a plant that has not been contacted with the composition.

5. The method of claim 1, wherein the method promotes growth of the plant, compared to a plant that has not been contacted with the composition.

6. The method of claim 1, wherein the method increases the yield of seeds produced by the plant, compared to a plant that has not been contacted with the composition.

7. The method of claim 1, wherein the plant is a soybean plant.

8. The method of claim 1, wherein the composition is a whole cell broth of Chromobacterium the subtsugae sp. nov.