WO2013152353A1

WO2013152353A1 - Biocontrol of nematodes

Info

Publication number: WO2013152353A1
Application number: PCT/US2013/035638
Authority: WO
Inventors: Jonathan Margolis; Reed Nathan Royalty; Varghese THOMAS
Original assignee: Bayer Cropscience Lp
Priority date: 2012-04-06
Filing date: 2013-04-08
Publication date: 2013-10-10

Abstract

This invention presents compositions and methods for controlling plant pathogenic nematodes using synergistic combinations of microorganisms.

Description

BIOCONTROL OF NEMATODES

FIELD OF INVENTION

[0001] The present invention relates to control of plant parasitic nematodes.

BACKGROUND OF INVENTION

[0002] Plant parasitic nematodes cause significant damage to a wide variety of crops, resulting in global crop yield losses estimated to range from 5% to 12% annually. Root damage by nematodes is very common and leads to stunted plants, which have smaller root systems, show symptoms of mineral deficiencies in their leaves and wilt easily. Damage by nematodes also predisposes plants to infection by a wide variety of plant pathogenic fungi and bacteria.

[0003] In order to combat and control nematodes, farmers typically use chemical nematicides. These range from gas and liquid fumigation, such as methyl bromide and chloropicrin, to application of organophosphates and carbamates, such as thionazin and oxamyl. Use of these chemical nematicides has been ongoing for several decades. Despite the effectiveness of the chemical nematicide in controlling target nematodes, there are serious limitations to these methods. One limitation is that chemical nematicides cannot act against nematodes that have already penetrated the root. Another limitation is the danger associated with the production and use of chemical nematicides. Chemical nematicides are highly toxic and can lead to human poisoning and death. As a result, countries have restricted and sometimes banned certain pesticides. Methyl bromide in particular is banned in most countries due to its ozone depleting effects.

[0004] Because of these restrictions and bans, there are a lack of viable nematode solutions. The present invention provides a safe and effective means to replace or lessen the use of chemical pesticides. It is also unique in providing a solution that both inhibits nematode penetration into the plant root and then prevents maturation of those nematodes that manage to overcome this initial barrier. SUMMARY OF THE INVENTION

[0005] The present invention provides methods and compositions for the control of plant parasitic nematodes. The invention provides a method for controlling nematodes comprising applying to a plant, a plant part or a locus of the plant an effective amount of Bacillus subtilis QST713, mutants of Bacillus subtilis QST713 or metabolites of Bacillus subtilis QST713. In some embodiments, the Bacillus subtilis QST713 is applied as a fermentation product that includes the Bacillus subtilis QST713, its metabolites and, optionally, residual fermentation broth.

[0006] In some embodiments, the target nematodes are disease-causing root knot nematodes. In certain instances, the nematodes are from the species Meloidogyne. The present compositions kill eggs of root knot nematodes, decrease root knot nematode plant penetration, and/or inhibit maturation of root knot nematodes that penetrate plants.

[0007] In other embodiments, the target nematodes are cyst nematodes. In certain instances, the nematodes are from the species Heterodera. In other instances, the nematodes are from the species Globodera.

[0008] In some embodiments, the above-described compositions are mixed with at least one other pesticide, such as a fungicide, insecticide, nematicide or herbicide. In one embodiment, the pesticide is a nematicide. In certain embodiments the Bacillus subtilis 713- based nematicide is tank mixed with a commercially available formulated nematicide. In other embodiments, the Bacillus subtilis 713-based composition is mixed with the active ingredient and then formulated, such that the multiple actives form one product.

[0009] The present invention further provides any of the compositions of the present invention further comprising a formulation inert or other formulation ingredient, such as polysaccharides (starches, maltodextrins, methylcelluloses, proteins, such as whey protein, peptides, gums), sugars (lactose, trehalose, sucrose), lipids (lecithin, vegetable oils, mineral oils), salts (sodium chloride, calcium carbonate, sodium citrate), and silicates (clays, amorphous silica, fumed/precipitated silicas, silicate salts). In some embodiments, such as those in which the compositions are applied to soil, the compositions of the present invention comprise a carrier, such as water or a mineral or organic material such as peat that facilitates incorporation of the compositions into the soil. In some embodiments, such as those in which the composition is used for seed treatment or as a root dip, the carrier is a binder or sticker that facilitates adherence of the composition to the seed or root. In another embodiment in which the compositions are used as a seed treatment the formulation ingredient is a colorant. In other compositions, the formulation ingredient is a preservative. [0010] In some embodiments the compositions are applied to plants, plant parts, or loci of the plants, such as soil, prior to planting. In other embodiments that compositions are applied at planting. In still others the compositions are applied after planting.

[0011] In certain embodiments, application of the compositions is preceded by a step comprising identifying that the plant or plant locus for growth needs treatment. In some embodiments, identifying includes determining that the locus for plant growth exceeds the economic threshold for nematode infestation.

[0012] In some embodiments, the present invention encompasses a kit that includes Bacillus subtilis QST713 and instructions for its use as a nematicide. In some embodiments these instructions are a product label. In some embodiments, these instructions are for use of the Bacillus subtilis as a nematicide in combination with a chemical nematicide. In certain instances, the instructions direct the user to use the chemical nematicide at a rate that is lower than the rate recommended on a product label for the chemical nematicide when used as a standalone treatment.

BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 shows the effect of QST713 whole broth treatment on galling of roots infested with root knot nematodes. QST713 whole broth is designated as AQ713 in the figure.

[0014] Figure 2 shows the effect of treatment with the SERENADE^® ASO product at various rates on seedlings infested with root knot nematodes. Specifically, results show extent of root galling and penetration and effects on nematode development.

[0015] Figure 3 represents root knot nematode eggs per plant treated with various batches of AQ713 whole broth as compared to untreated plants (designated as UTC in the figure).

[0016] Figure 4 represents root knot nematode eggs per plant treated with the SERENADE SOIL^® product, either independently or in combination with the INLINE^® product (with active ingredients of 1,3-dichloropropene + chloropicrin) as compared to untreated plants (designated as UTC) and plants treated with the INLINE^® product alone. Various treatments set forth in Table 2 are designated as Tl, T2, T3 and T4 in the figure.

DETAILED DESCRIPTION OF THE INVENTION

[0017] All publications, patents and patent applications, including any drawings and appendices, herein are incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[0018] The following description includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed inventions, or that any publication specifically or implicitly referenced is prior art.

[0019] The SERENADE^® product (EPA Registration No. 69592-12) contains a unique, patented strain of Bacillus subtilis (strain QST713) and many different lipopeptides that work synergistically to destroy disease pathogens and provide superior antimicrobial activity. The SERENADE^® product is used to protect plants such as vegetables, fruit, nut and vine crops against diseases such as Fire Blight, Botrytis, Sour Rot, Rust, Sclerotinia, Powdery Mildew, Bacterial Spot and White Mold. The SERENADE^® products are available as either liquid or dry formulations which can be applied as a foliar and/or soil treatment. Copies of EPA Master Labels for SERENADE^® products, including SERENADE^® ASO, SERENADE^® MAX, and SERENADE SOIL^®, are publicly available through National Pesticide Information Retrieval System's (NPIRS^®) USEPA/OPP Pesticide Product Label System (PPLS).

[0020] SERENADE^® ASO (Aqueous Suspension- Organic) contains 1.34% of dried QST713 as an active ingredient and 98.66% of other ingredients. SERENADE^® ASO is formulated to contain a minimum of 1 x 10⁹ cfu/g of QST713 while the maximum amount of QST713 has been determined to be 3.3 x 10¹⁰ cfu/g. Alternate commercial names for

SERENADE^® ASO include SERENADE BIOFUNGICIDE^®, SERENADE SOIL^® and

SERENADE^® GARDEN DISEASE. For further information, see the U.S. EPA Master Labels for SERENADE^® ASO dated January 4, 2010, and SERENADE SOIL^®, each of which is incorporated by reference herein in its entirety.

[0021] SERENADE^® MAX contains 14.6% of dried QST713 as an active ingredient and 85.4% of other ingredients. SERENADE^® MAX is formulated to contain a minimum of 7.3 x 10⁹ cfu/g of QST713 while the maximum amount of QST713 has been determined to be 7.9 x 10¹⁰ cfu/g. For further information, see the U.S. EPA Master Label for SERENADE^® MAX, which is incorporated by reference herein in its entirety.

[0022] Bacillus subtilis QST713, its mutants, its supernatants, and its lipopeptide metabolites, and methods for their use to control plant pathogens and insects are fully described in U.S. Patent Nos. 6,060,051 ; 6, 103,228; 6,291,426; 6,417, 163; and 6,638,910; each of which is specifically and entirely incorporated by reference herein for everything it teaches. In these U.S. patents, the strain is referred to as AQ713, which is synonymous with QST713. Bacillus subtilis QST713 has been deposited with the NRRL on May 7, 1997, under the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purpose of Patent Procedure under Accession Number B21661. Any references in this specification to QST713 refer to Bacillus subtilis QST713 (aka AQ713) as present in the SERENADE^® products, deposited under NRRL Accession No. B21661, or prepared in bioreactors or shake flasks under conditions that simulate production of the SERENADE^® product.

[0023] The above-referenced patents describe testing of the supernatant of Bacillus subtilis QST713 whole broth against the N2 strain of the nematode Caenorhabditis elegans. Such tests showed that the supernatant lacked nematicidal activity.

[0024] At the time of filing U.S. Patent Application No. 09/074,870 in 1998, which corresponds to the above patents, the QST713 strain was designated as a Bacillus subtilis based on classical, physiological, biochemical and morphological methods. Taxonomy of the Bacillus species has evolved since then, especially in light of advances in genetics and sequencing technologies, such that species designation is based largely on DNA sequence rather than the methods used in 1998. After aligning protein sequences from B. amyloliquefaciens FZB42, B. subtilis 168 and QST713, approximately 95% of proteins found in B. amyloliquefaciens FZB42 are 85% or greater identical to proteins found in QST713; whereas only 35% of proteins in B. subtilis 168 are 85% or greater identical to proteins in QST713. However, even with the greater reliance on genetics, there is still taxonomic ambiguity in the relevant scientific literature and regulatory documents, reflecting the evolving understanding of Bacillus taxonomy over the past 15 years. For example, a pesticidal product based on B. subtilis strain FZB24, which is as closely related to QST713 as is FZB42, is classified in documents of the Environmental Protection Agency as B. subtilis var. amyloliquefaciens. Due to these complexities in nomenclature, this particular Bacillus species is variously designated, depending on the document, as B. subtilis, B. amyloliquefaciens, and B. subtilis var. amyloliquefaciens. Therefore, we have retained the B. subtilis designation of QST713 rather than changing it to B.

amyloliquefaciens, as would be expected currently based solely on sequence comparison and inferred taxonomy.

[0025] Compositions of the present invention can be obtained by culturing Bacillus subtilis QST713 according to methods well known in the art, including by using the media and other methods described in U.S. Patent No. 6,060,051. Conventional large-scale microbial culture processes include submerged fermentation, solid state fermentation, or liquid surface culture. Towards the end of fermentation, as nutrients are depleted, Bacillus subtilis cells begin the transition from growth phase to sporulation phase, such that the final product of fermentation is largely spores, metabolites and residual fermentation medium. Sporulation is part of the natural life cycle of Bacillus subtilis and is generally initiated by the cell in response to nutrient limitation. Fermentation is configured to obtain high levels of colony forming units of Bacillus subtilis and to promote sporulation. The bacterial cells, spores and metabolites in culture media resulting from fermentation may be used directly or concentrated by conventional industrial methods, such as centrifugation, tangential-flow filtration, depth filtration, and evaporation. Fermentation broth and broth concentrate are both referred to herein as "fermentation products." Compositions of the present invention include fermentation products. In some embodiments, the concentrated fermentation broth is washed, for example, via a diafiltration process, to remove residual fermentation broth and metabolites.

[0026] The fermentation broth or broth concentrate can be dried with or without the addition of carriers using conventional drying processes or methods such as spray drying, freeze drying, tray drying, fluidized-bed drying, drum drying, or evaporation.

[0027] The resulting dry products may be further processed, such as by milling or granulation, to achieve a specific particle size or physical format. Carriers, described below, may also be added post-drying.

[0028] Cell-free preparations of fermentation broth of the novel variants and strains of Bacillus of the present invention can be obtained by any means known in the art, such as extraction, centrifugation and/or filtration of fermentation broth. Those of skill in the art will appreciate that so-called cell-free preparations may not be devoid of cells but rather are largely cell-free or essentially cell-free, depending on the technique used (e.g., speed of centrifugation) to remove the cells. The resulting cell-free preparation may be dried and/or formulated with components that aid in its application to plants or to plant growth media. Concentration methods and drying techniques described above for fermentation broth are also applicable to cell-free preparations.

[0029] Metabolites of Bacillus subtilis can be obtained according to the methods set forth in U.S. Patent No. 6,060,051. The term "metabolites" as used herein may refer to semi- pure and pure or essentially pure metabolites, or to metabolites that have not been separated from Bacillus subtilis. In some embodiments, after a cell-free preparation is made by centrifugation of fermentation broth, the metabolites may be purified by size exclusion filtration such as the Sephadex resins including LH-20, G10, and G15 and G25 that group metabolites into different fractions based on molecular weight cut-off, such as molecular weight of less than about 2000 daltons, less than about 1500 daltons, less than about 1000 daltons and so on, as the lipopeptides are between 800 daltons and 1600 daltons.

[0030] Concentration methods and drying techniques described above for formulation of fermentation broth are also applicable to metabolites. [0031] Compositions of the present invention may include formulation inerts added to compositions comprising cells, cell-free preparations or metabolites to improve, efficacy, stability, and usability and/or to facilitate processing, packaging and end-use application. Such formulation inerts and ingredients may include carriers, stabilization agents, nutrients, or physical property modifying agents, which may be added individually or in combination. In some embodiments, the carriers may include liquid materials such as water, oil, and other organic or inorganic solvents and solid materials such as minerals, polymers, or polymer complexes derived biologically or by chemical synthesis. In some embodiments, the carrier is a binder or adhesive that facilitates adherence of the composition to a plant part, such as a seed or root. See, for example, Taylor, A.G., et al., "Concepts and Technologies of Selected Seed Treatments", Annu. Rev. Phytopathol. 28: 321-339 (1990). The stabilization agents may include anti-caking agents, anti-oxidation agents, desiccants, protectants or preservatives. The nutrients may include carbon, nitrogen, and phosphors sources such as sugars, polysaccharides, oil, proteins, amino acids, fatty acids and phosphates. The physical property modifiers may include bulking agents, wetting agents, thickeners, pH modifiers, rheology modifiers, dispersants, adjuvants, surfactants, antifreeze agents or colorants. In some embodiments, the composition comprising cells, cell-free preparation or metabolites produced by fermentation can be used directly with or without water as the diluent without any other formulation preparation. In some embodiments, the formulation inerts are added after concentrating fermentation broth and during and/or after drying.

[0032] Compositions of the present invention may include carriers, which are inert formulation ingredients added to compositions comprising a lipopeptide-containing fermentation product, cell-free preparations of lipopeptides or purified, semi-purified or crude extracts of lipopeptides to improve recovery, efficacy, or physical properties and/or to aid in packaging and administration. Such carriers may be added individually or in combination.

[0033] The compositions of the present invention may be mixed with other chemical and non-chemical additives, adjuvants and/or treatments, wherein such treatments include but are not limited to chemical and non-chemical fungicides, insecticides, miticides, nematicides, fertilizers, nutrients, minerals, auxins, growth stimulants and the like.

[0034] Nematicides with which the Bacillus subtilis-b&sed compositions of the present invention may be mixed may be chemical or biological nematicides. The term

"chemical nematicide," as used herein, excludes fumigants, and the term "fumigants" encompasses broad spectrum pesticidal chemicals that are applied to soil pre-planting and that diffuse through the soil (in soil air and/or soil water) and may be applied as gases, such as methyl bromide, volatile liquids, such as chloropicrin, or volatile solids, such as dazomet. [0035] In some embodiments, the chemical or biological nematicide is a commercially available formulated product and is tank mixed with the compositions of the present invention. In other embodiments, the chemical or biological nematicide is mixed with the Bacillus subtilis QST713 -based composition prior to formulation such that the compositions form one formulated product.

[0036] Chemical nematicides used in such mixtures are carbamates, oxime carbamates and organophosphorous nematicides. Carbamate nematicides include benomyl, carbofuran (FURADAN^®), carbosulfan and cloethocarb. Oxime carbamates include alanycarb, aldicarb (TEMIK^® or as part of the AVICTA^® Complete Pak seed treatment from Syngenta), aldoxycarb, (STAND AK^®), oxamyl (V YD ATE^®), thiodicarb (part of the AERIS^® seed-applied system from Bayer CropScience), and tirpate. Organophosphorous nematicides include fensulfothion (DANSANIT^®), ethoprop. (MOCAP^®), diamidafos, fenamiphos, fosthietan, phosphamidon, cadusafos, chlorpyrifos, dichlofenthion, dimethoate, fosthiazate, heterophos, isamidofos, isazofos, phorate, phosphocarb, terbufos, thionazin, triazophos, imicyafos, and mecarphon. Parenthetical names following each compound are representative commercial formulations of each of the above chemicals. Other chemical nematicides useful for such mixtures include spirotetramat (MOVENTO^®), MON37400 nematicide and fipronil.

[0037] Biological nematicides include chitin and urea mixtures, compost extracts and teas (both aerated and nonaerated); compositions comprising the fungus Myrothecium verrucaria and/or metabolites therefrom (commercially available as DITERA^®), compositions comprising the fungus Paecilomyces lilacinus (commercially available as, for example, MELOCON^® or BIO ACT^®); compositions comprising the bacterium Pasteuria including P. usgae (commercially available as, for example, ECONEM^®); compositions comprising bacteria from the Bacillus sp., including Bacillus firmus (including the strain deposited as CNMC 1-1582 with the Collection Nationale de Cultures de Microorganismes, Institute Pasteur, France on May 29, 1995, and commercially available as, for example, VOTIVO^®), Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus pumilus (including the strain deposited with NRRL as B-30087 on January 14, 1999, and its mutants) and Bacillus cereus; and compositions comprising nematicidal Streptomycete sp., such as Streptomyces lydicus (commercially available as ACTINOVATE^®). Biological nematicides also include botanically-based nematicides such as products based on neem plants (including seeds or oil from the plants) or azidirachtin, a secondary metabolite of neem seeds, sesame oil-based products (such as DRAGONFIRE^®), carvacrol, and products based on plant extracts (such as NEMA-Q^®, obtained from the Quillaja saponaria tree of Chile). Biological nematicides also include isolated compounds produced by bacteria, such as the mectins, which are produced by Streptomyces avermentilis, including abamectin (consisting of a combination of abamectin Bla and B ib) and avermectin B2a, and the harpin proteins, originally identified in Erwinia amylovora, including harpinEA and harpin_ap.

[0038] While applicants do not wish to be held to any particular theory, it is thought that Bacillus subtilis QST713 controls nematodes by inhibiting development of nematodes after the nematodes have penetrated the roots and established feeding sites. Other microorganisms used for nematode control have distinct modes of action. For example, the VOTIVO^® product, based on Bacillus firmus CNMC 1-1582, is thought to inhibit penetration of roots by nematodes. Pasteuria usgae, the active ingredient of the ECONEM^® product, attaches to and penetrates the nematode cuticle while the nematode is in its free living stage in soil such that the nematode is incapacitated or killed before it can enter the root. Other microorganisms inhibit nematode ability to establish feeding sites.

[0039] The present invention encompasses compositions comprising a synergistic combination of two or more microorganisms, where each microorganism has a distinct mode of action for controlling nematodes. Such microorganisms include those with the following modes of action: (i) diminishing nematode ability to locate a root; for example, through disruption of nematode movement or chemosensory function; (ii) repelling the nematode, wherein the microorganism produces compounds that are aversive to the nematode; (iii) inhibiting nematode root penetration; (iv) inhibiting nematode establishment of root feeding sites; and (v) inhibiting nematode maturation after root penetration.

[0040] In one embodiment, the composition comprises a synergistic combination of two or more microorganism having the following modes of action: inhibiting nematode root penetration, inhibiting nematode establishment of root feeding sites, and inhibiting nematode maturation after root penetration. In still another embodiment, the two or more microorganisms have the following modes of action: inhibiting nematode root penetration and inhibiting nematode maturation after root penetration. A "synergistic combination" represents a quantity of a combination of two or more microorganisms that is statistically significantly more effective against insects, nematodes and/or phytopathogens than either microorganism alone.

[0041] In one embodiment, the microorganisms are selected from those provided above in the description of biological nematicides. In another embodiment, the microorganisms are from the Bacillus sp. In still another embodiment, the microorganisms are selected from the group consisting of Bacillus firmus, Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus cereus, Bacillus megaterium, Bacillus pumilus, Bacillus thuringiensis, Bacillus mycoides, Paenibacillus spp., and Pasteuria spp. In a more particular embodiment, the microorganisms are Bacillus subtilis QST713 and Bacillus firmus CNMC 1-1582. In yet another embodiment, the microorganisms are Bacillus subtilis QST713 or mutants thereof and Bacillus firmus CNMC 1-1582 or mutants thereof. The term "mutant" refers to a genetic variant derived from the parent strain; e.g., Bacillus subtilis QST713 or Bacillus firmus CNMC 1-1582. In one embodiment, the mutant has one or more or all the identifying (functional) characteristics of a parent strain, such as QST713 or CNMC 1-1582. In another embodiment, the mutant or a fermentation product thereof controls nematodes at least as well as the parent strain. Mutants may be genetic variants having a genomic sequence that has greater than about 85%, greater than about 90%, greater than about 95%, greater than about 98%, or greater than about 99% sequence identity to the parent strain. Mutants may be obtained by treating parent strain cells with chemicals or irradiation or by selecting spontaneous mutants from a population of parent strain cells (such as phage resistant or antibiotic resistant mutants) or by other means well known to those practiced in the art.

[0042] The microorganisms (e.g., the Bacillus subtilis QST713 and the Bacillus firmus CNMC 1-1582) are used in a synergistic ratio. The skilled person is able to determine the synergistic ratios for the present invention by routine methods. The skilled person understands that these ratios refer to the ratio within a combined-formulation as well as to the calculative ratio of each component when both components are applied as mono-formulations to a plant to be treated. The skilled person can calculate this ratio by simple mathematics since the volume and the amount of each microorganism component in a mono-formulation is known to the skilled person.

[0043] Compositions of the present invention are useful to control plant parasitic nematodes, such as, for example, root-knot, cyst, lesion and ring nematodes, including

Meloidogyne spp., Heterodera spp., Globodera spp., Pratylenchus spp. and Criconemella sp. Compositions are also useful to control Tylenchulus semipenetrans, Trichodorus spp.,

Longidorus spp., Rotylenchulus spp., Xiphinema spp., Belonolaimus spp. (such as B.

longicaudatus), Criconemoides spp., Tylenchorhynchus spp., Hoplolaimus spp., Rotylenchus spp., Helicotylenchus spp., Radopholus spp. (such as R. citrophilis and /?, similis), Ditylenchus spp. and other plant parasitic nematodes. In some embodiments the targets are cyst nematodes, such as Heterodera glycines (soybean cyst nematodes), Heterodera schachtii (beet cyst nematode), Heterodera avenae (Cereal cyst nematode), Meloidigyne incognita (Cotton (or southern) root knot nematode), Globodera rostochiensis and Globodera pallida (potato cyst nematodes). In other embodiments, the targets are root knot nematodes, such as M. incognita (cotton root knot nematode), M. javanica (Javanese root knot nematode), M. hapla (Northern root knot nematode), and M. arenaria (peanut root knot nematode).

[0044] The term "control," as used herein, means killing, reducing in numbers, and/or reducing growth, feeding or normal physiological development, including, for root knot nematodes, the ability to penetrate roots and to develop within roots. An effective amount is an amount able to noticeably reduce pest growth, feeding, root penetration, maturation in the root, and/or general normal physiological development and symptoms resulting from nematode infection. In some embodiments symptoms and/or nematodes are reduced by at least about 5%, at least about 10%, at least about 20%, at least about, 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90%.

[0045] Compositions of the present invention are used to treat a wide variety of agricultural and/or horticultural crops, including those grown for seed, produce, and landscaping. Representative plants that can be treated using the compositions of the present invention include but are not limited to the following: bulb vegetables; cereal grains; citrus fruits (such as grapefruit, lemon, and orange); cotton and other fiber crops; curcurbits; fruiting vegetables; leafy vegetables (such as celery, head and leaf lettuce, and spinach); legumes; oil seed crops; peanut; pome fruit (such as apple and pear); stone fruits (such as almond, pecan, and walnut); root vegetables; tuber vegetables; corm vegetables; tobacco, strawberry and other berries; cole crops (such as broccoli and cabbage); grape; pineapple; and flowering plants, bedding plants, and ornamentals (such as fern and hosta). Compositions of the present invention are also used to treat perennial plants, including plantation crops such as banana and coffee and those present in forests, parks or landscaping.

[0046] Compositions described herein are applied to a plant, a plant part, such as a seed, root, rhizome, corm, bulb, or tuber, and/or a locus on which the plant or the plant parts grow, such as soil, in order to control plant parasitic nematodes. The compositions of the present invention may be administered as a foliar spray, as a seed/root/tuber/rhizome/bulb/corm treatment and/or as a soil treatment. The seeds/root/tubers/rhizomes/bulbs/corms can be treated before planting, during planting or after planting.

[0047] Compositions described herein are also applied to a plant, a plant part, such as a seed, root rhizome, corm, bulb, or tuber, and/or a locus on which the plant or the plant parts grow, such as soil, in order to increase crop yield. In some embodiments, crop yield is increased by at least about 5%, in others by at least about 10%, in still others at least about 15%, and in still others by at least about 20%.

[0048] When used as a seed treatment, the compositions of the present invention are applied at a rate of about 1 x 10² to about 1 x 10⁹ cfu/seed, depending on the size of the seed. In some embodiments, the application rate is 1 x 10⁴ to about 1 x 10⁷ cfu/seed.

[0049] When used as a soil treatment, the compositions of the present invention can be applied as a soil surface drench, shanked-in, injected and/or applied in-furrow or by mixture with irrigation water. The rate of application for drench soil treatments, which may be applied at planting, during or after seeding, or after transplanting and at any stage of plant growth, is about 4 x 10¹¹ to about 8 x 10¹² cfu per acre. In some embodiments, the rate of application is about 1 x 10¹² to about 6 x 10¹² cfu per acre. The rate of application for in-furrow treatments, applied at planting, is about 2.5 x 10¹⁰ to about 5 x 10¹¹ cfu per 1000 row feet. In some embodiments, the rate of application is about 6 x 10¹⁰ to about 4 x 10¹¹ cfu per 1000 row feet. Those of skill in the art will understand how to adjust rates for broadcast treatments (where applications are at a lower rate but made more often) and other less common soil treatments.

[0050] Bacillus subtilis-based compositions of the present invention may be applied independently or in combination with one or more other nematicides, such as chemical and biological nematicides. In some embodiments, Bacillus subtilis QST713 is co-formulated with at least one other nematicide and the co-formulated product is applied to the plant or plant locus. In some other embodiments, the Bacillus subtilis-b&sed compositions are tank mixed with commercially available formulations of the chemical or biological nematicides and applied to plants and plant loci. In other embodiments, the Bacillus subtilis -based compositions of the present invention are applied to plants and/or plant loci immediately before or after the commercially available formulations of the chemical or biological nematicides. In other embodiments, the Bacillus subtilis-b&sed compositions of the present invention are applied to plants and/or plant loci in rotation with the commercially available formulations of the chemical or biological nematicides. In one instance, the Bacillus subtilis-b&sed compositions are applied as a seed treatment or as an in-furrow or drench treatment, as discussed in more detail below. In some instances of the above embodiments, the commercially available formulations of the chemical or biological nematicides are applied at a rate that is less than the rate recommended on the product label for use of such nematicides as stand-alone treatments.

[0051] In other embodiments, the Bacillus -based compositions of the present invention are applied to plants and/or plant loci following application of a fumigant. Fumigants can be applied by shank injection, generally a minimum of 8 inches below the soil surface. Liquid formulations of fumigants can also be applied through surface drip chemigation to move the fumigant to a depth of 8 inches or more below the soil surface. Treated soil beds are covered with a plastic tarp to retain the fumigant in the soil for several days. This is done before planting and allowed to air out prior to planting. The Bacillus-based compositions described herein would be applied after such air-out period either prior to, at the time of, or post-planting. In some instances, the fumigants are applied at a rate that is less than the rate recommended on the product label.

[0052] Chemical and biological nematicides are described above. Fumigant nematicides include halogenated hydrocarbons, such as chloropicrin (CHLOR-O-PIC^®); methyl bromide (METH-O-GAS^®) and combinations thereof (such as BROM-O-GAS^® and TERR-O- GAS^®); 1 ,3-dichloropnopene (TELONE^® II, TELONE^® EC, CURFEW^®) and combinations of 1,3-dichlonopropene with chloropicrin (TELONE^® C-17, TELONE^® C-35, and INLINE^®); methyl iodide (MIDAS^®); methyl isocyanate liberators, such as sodium methyl dithiocarbamate (VAPAM^®, SOILPREP^®, METAM-SODIUM^®); combinations of 1,3 dichloropropoene and methyl isothiocyanate (VORLEX^®); and carbon disulfide liberators, such as sodium

tetrathiocarbonate (ENZONE^®); and dimethyl disulphide or DMDS (PALADINO^®). Example commercial formulations of each of the above fumigants are provided in parentheses after the chemical name(s).

[0053] Compositions of the present invention may also be applied as part of an integrated pest management ("IPM") program. Such programs are described in various publications, especially by university cooperative extensions. Such programs include crop rotation with crops that cannot host the target nematode, cultural and tillage practices, and use of transplants. For example, the Bacillus -based compositions could be applied after a season of growth with mustard or other nematode suppressive crop.

[0054] In some embodiments, application of the compositions of the present invention to plants, plant parts or plant loci is preceded by identification of a locus in need of treatment. Such identification may occur through visual identification of plants that appear chlorotic, stunted, necrotic, or wilted (i.e., that appear to have nutrient deficiencies) typically coupled with knowledge of a history of nematode problems; plant sampling; and/or soil sampling. Plant sampling may occur during the growing season or immediately after final harvest. Plants are removed from soil and their roots examined to determine the nature and extent of the nematode problem within a field. For root knot nematode, root gall severity is determined by measuring the proportion of the root system which is galled. Galls caused by root knot nematodes may be distinguished from nodules of nitrogen-fixing soil bacteria because galls are not easily separated from the root. Root knot nematode soil population levels increase with root gall severity. In some instances, the detection of any level of root galling suggests a root knot nematode problem for planting any susceptible crop, especially in or near the area of sampling. Cyst nematodes may also be identified by plant sampling and scrutiny of roots for cysts.

[0055] Soil sampling offers a means to determine the number of nematodes and/or nematode eggs infesting a certain volume of soil or roots. Soil sampling may be conducted when a problem is first suspected, at final harvest, or any time prior to planting a new crop, including prior to crop destruction of the previous crop. University cooperative extension programs offer soil sampling services, including the University of Florida, Oregon State University and the University of Nebraska-Lincoln. In addition, such programs provide guidance for how to collect samples. For example, in one method of post-harvest predictive sampling, samples are collected at a soil depth of 6 to 10 inches from 10 to 20 field locations over 5 or 10 acres (depending on value of the crop, with fewer acres sampled for higher value crops) in a regular zigzag pattern. In a method of testing established plants, root and soil samples are removed at a soil depth of 6 to 10 inches from suspect plants that are symptomatic but that are not dead or dying, i.e., decomposing.

[0056] In some embodiments, identification involves determining whether an economic threshold of nematode infestation has been reached; i.e., a point at which expected economic losses without treatment exceed treatment costs. The economic threshold varies depending on the crop, geography, climate, time of planting, soil type, and/or soil temperature. Numerous papers have been published on this topic and guidelines are available from university cooperative extension programs in different areas. See, for example, Robb, J.G., et al., "Factors Affecting the Economic Threshold for Heterodera schachtii Control in Sugar Beet," Economics of Nematode Control January-June 1992; Hafez, Saad L., "Management of Sugar Beet

Nematode," University of Idaho Current Information Series (CIS) 1071 (1998); and UC IPM Pest Management Guidelines: Tomato UC ANR Publication 3470 Nematodes A. Ploeg, Nematology, UC Riverside (January 2008). Determining the economic threshold for a particular crop at a particular time of year is well within the skill set of one of ordinary skill in the art.

[0057] In some embodiments, the soil sampling reveals that the nematode infestation will cause yield that is about 80%, about 90%, or about 95% of normal for uninfested soil.

[0058] In some embodiments, the economic threshold of root knot juveniles per kilogram of soil sample is at least about 250, at least about 300, at least about 500, at least about 750, at least about 1000, at least about 2000, at least about 3000, at least about 4000, at least about 5000, or at least about 6000.

[0059] In some embodiments, the economic threshold of cyst nematode eggs and larvae per 1 cm³ soil is at least about 0.5, at least about 1, at least about 2, at least about 3, at least about 4. According to Hafez (1998), supra, a cyst may be estimated as 500 viable eggs and larvae.

[0060] The following examples are given for purely illustrative and non-limiting purposes of the present invention. EXAMPLES

Example 1

Activity of Bacillus subtilis QST713 against Meloidogyne javanica

[0061] Studies were conducted with cucumber seeds var. Sultan to determine activity of QST713 against Meloidogyne javanica, root knot nematode. 50 mL centrifuge tubes containing 20 g sand and one ungerminated seed were treated with different rates of whole broth of QST713 or of the commercially available SERENADE^® ASO product. The whole broth and SERENADE^® ASO product differ in that the product is much more concentrated in terms of colony forming units (cfu) and metabolites, with the product having at least 1 log greater cfu than the whole broth. In addition, the product is formulated with, among other things, preservatives, such that the product is more acidic than the whole broth. To obtain whole broth cultures of QST713, seed flasks containing Luria Broth (LB) were inoculated with QST713 and grown overnight at 30 °C. The next day, aliquots from each seed flask were inoculated into 200 ml of a soy-based medium in a 1 L shake flask and grown until sporulation. Briefly, the shake flask culture was maintained at a temperature between 30 °C and 32 °C and at a shaker setting of 200 to 220 rpm. After approximately 3 days of incubation, when cell growth and metabolite production had stopped, the culture broth was harvested. The treated seeds were allowed to germinate and grow in the greenhouse. Four to five days after treatment (DAT) each tube was inoculated with 100 second- stage juvenile root knot nematodes. 10 DAT the seedlings were scored for percentage root galling on a 0-4 scale, which is described in Table 1.

[0062] The roots were then stained with acid fuschin to observe nematode penetration and development and observed under a Leica dissecting microscope. For nematode penetration, the total nematode juveniles inside each root were counted. For nematode development, total fat juveniles including late second stage juvenile (J2's) and third stage juvenile (J3's) were counted. Penetration of nematodes into the root and nematode development after penetration were scored as detailed in Table 1. For details on techniques used, see CO. Omwega, et al., "A Nondestructive Technique for Screening Bean Germplasm for Resistance to Meloidogyne incognita," Plant Disease (1988) 72(11): 970-972).

[0063] Table 1. Rating Scheme for Nematode Antagonistic Activity of Bacterial Whole Broths. The galling index was based on the percentage of root galling. The penetration scale was calculated as the mean total number of juvenile nematodes relative to the number of juvenile nematodes in the untreated control (UTC). The development scale reflects the total number of fat juvenile nematodes (late J2 stage/ J3 stage) inside the root. Galling Index Penetration Scale Development Scale

0 None 0 None 0 None

1 1-24% 1 1-10% 1 1-3

2 25-49% 2 11-50% 2 3-10

3 50-74% 3 51-75% 3 11-30

4 >75% 4 76-100% 4 >30

[0064] Figure 1 shows that application of QST713 whole broth decreases root galling. Figure 2 shows that application of various rates of the SERENADE^® ASO product decrease galling, penetration and development compared to the untreated control. Note that because the data is based on the above rating system it is not always possible to observe a dose response.

Example 2

Efficacy of AQ713 for control of Root- Knot Nematodes Eggs in Tomatoes.

[0065] Another experiment was conducted with tomato seeds to test efficacy of QST713 against root knot nematode eggs. AQ713-Batch 1 was a whole broth culture prepared as described in Example 1. AQ713-Batch2 and AQ713-Batch3 were prepared in a bioreactor. Briefly, a vial of stock culture was thawed and transferred to a sterilized flask of Difco Nutrient Broth. The flask culture was then incubated on a rotary shaker at a temperature between 28 °C and 32 °C at a rotation speed of 200 to 220 rpm to promote cell growth and obtain high cell density and then added to 12 L of a soy-based growth medium in a 20 L bioreactor. The bioreactor was set at a temperature setting between 30 °C and 32 °C, at an agitation setting of 500 to 1000 rpm, to a pH buffered between 6 and 8, and to an aeration between 0.5 and 1.0 VVM. After approximately 3 days of incubation, when cell growth and metabolite production had stopped, the culture broth was harvested. Three-week old tomato plants were treated with QST713 by drench. Pots were then kept in a greenhouse for ten days before being inoculated with 5000 root-knot nematode ("RKN") eggs per pot. Plants were harvested forty-two days after nematode inoculation. Eggs were collected from the roots of the tomato plants using a 1 % NaOCl solution as detailed in Hussey RS, Barker KR, "A Comparison of Methods of Collecting Inocula of Meloidogyne spp., Including a New Technique," Plant Disease Reporter, 1973;

57: 1025-1028. AQ713 decreased the number of root knot nematode eggs observed per plant. Data represents direct counts of eggs rather than a scoring system. Results as compared to an untreated sample (UTC) are shown in Figure 3. Example 3

Efficacy of SERENADE SOIL^® product against various nematodes in strawberry

[0066] "Buried bag" studies were conducted to determine the effectiveness of the SERENADE SOIL^® product against various types of nematodes in a strawberry field. Buried bag studies are commonly used to evaluate the effectiveness of soil treatments, especially fumigants. A soil sample containing a known concentration of nematodes was placed in nylon mesh bags and buried at a depth of around six to eight inches in the strawberry plant bed. Various treatments were applied as shown in Table 2. The INLINE^® product was used as a positive control and in combination with the SERENADE SOIL^® product.

Table 2

Trt. No. Treatment Name Rate Appl Code Appl Dates

1 Untreated Check

2 SERENADE SOIL^® 4qt/ac BCD 11/24/11, 01/27/11, 04/01/11

3 INLINE^® followed 20gal/ac A 11/03/10

by SERENADE SOIL^®4qt/ac BCD 11/24/10, 01/27/11, 04/01/11

4 INLINE^® 20gal/ac A 11/03/10

[0067] Bags were collected on December 8, 2010, after treatment B, and the nematodes (adults and larvae) were counted. Results are shown below.

[0068] Means followed by same letter to do not significantly differ at P = 0.05 (Student- Newman-Kewls). [0069] A few weeks after the last application shown above, some plants were removed from the plots and roots analyzed for root knot nematodes, as described in Example 2. Figure 4 shows that all treatments reduced root knot nematode eggs/root.

Claims

CLAIMS We claim:

1. A method for controlling plant pathogenic nematodes comprising applying to a plant or to a locus for plant growth two or more isolated microorganisms, wherein the microorganisms have distinct modes of action.

2. The method of Claim 1 wherein the modes of action are selected from the group consisting of (i) diminishing nematode ability to locate a root; (ii) repellency; (iii) inhibiting nematode root penetration; (iv) inhibiting nematode establishment of root feeding sites; and (v) inhibiting nematode maturation after root penetration.

3. The method of Claim 1 wherein the modes of action are selected from the group consisting of (i) inhibiting nematode root penetration; (ii) inhibiting nematode establishment of root feeding sites; and (iii) inhibiting nematode maturation after root penetration.

4. The method of Claim 2 wherein the microorganisms are Bacillus spp.

5. The method of Claim 2 wherein the microorganisms are selected from the group consisting of Bacillus firmus, Bacillus subtilis, Bacillus amyloliquefaciens, and Pasteuria spp.

6. The method of Claim 3 wherein the microorganisms are Bacillus subtilis QST713 and Bacillus firmus CNMC 1-1582.

7. The method of Claim 1 wherein the microorganisms are applied prior to planting, at planting or after planting.

8. The method of Claim 1 wherein the microorganisms are applied to a seed.

9. A composition comprising a combination of two or more isolated

microorganisms capable of nematode control, wherein the microorganisms have distinct modes of action, in a synergistically effective amount.

10. The composition of Claim 9 wherein the microorganisms capable of nematode control are bacterial strains.

11. The composition of Claim 10 wherein the bacterial strains are Bacillus subtilis QST713 and/or a mutant thereof and Bacillus firmus CNMC 1-1582 and/or a mutant thereof.