WO2015034629A1 - Methods and compositions for control of mite infestations using a newly discovered species of burkholderia - Google Patents

Abstract

Disclosed herein are methods and compositions for controlling mite infestations in plants. The compositions are obtained from cultures of a newly-discovered bacterial isolate, Burkholderia A396. The strain is unique based on its ribosomal RNA sequence, the nucleotide sequences of a number of additional loci, and a variety of biochemical characteristics.

Description

METHODS AND COMPOSITIONS FOR CONTROL OF MITE INFESTATIONS USING A NEWLY DISCOVERED SPECIES OF BURKHOLDERIA

[001] This PCT application claims the benefit under 35 U.S.C 1 19(e) of U.S.

provisional application serial number 61/874,986 filed on September 7, 2013. The content of which is incorporated herein in reference in its entirety.

INCORPORATION of SEQUENCE LISTING

[002] This instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy is named MBI206_0004_ST25.txt and is 11,342 bytes in size.

FIELD

[003] The present disclosure is in the field of biopesticides and pest control; in particular microbial pesticides and the microbial strains that produce them.

BACKGROUND

[004] Natural products are substances produced by microbes, plants, and other organisms. Microbial natural products offer an abundant source of chemical diversity, and there is a long history of utilizing natural products for pharmaceutical purposes. Despite the emphasis on natural products for human therapeutics, where more than 50% are derived from natural products, only 11% of pesticides are derived from natural sources. Nevertheless, natural product pesticides have a potential to play an important role in controlling pests in both conventional and organic farms.

Secondary metabolites produced by microbes (e.g., bacteria, actinomycetes and fungi) provide novel chemical compounds which can be used either alone or in combination with known compounds to effectively control insect pests and to reduce the risk for resistance development.

[005] Increasing environmental concerns and problems caused by synthetic chemicals have stimulated interest in the development of biopesticides for pest management. Application of synthetic chemicals not only can cause environmental hazards but also may affect non-target organisms including bees, humans and other mammals. The use of biopesticides such as fungi, bacteria, baculoviruses, and some botanicals merits attention because they have demonstrated effective pest control with a high degree of safety to non-target organisms and low environmental impact. A number of economically important pests are successfully controlled by biopesticides. For example, Bacillus thuringiensis (Bt) is a bacterial biopesticide that has been used successfully to control lepidopteran, dipteran and coleopteran pests (4, 19, 34, 52), and is popularly applied as foliar or in transgenic plants (58). Chromobacterium subtsugae is another example of a recent product brought to market for insect control (47, 48, 60).

[006] With the development of increasing resistance to chemical pesticides, the spectrum of available pesticides is narrowing. In addition, non-naturally-occurring pesticides can have detrimental environmental effects, as discussed above.

Accordingly, there is a need for new, naturally-occurring pesticides to which plant pathogens have not developed resistance, and which have minimal environmental effects.

[007] Bacterial species of the genus Burkholderia are ubiquitous organisms found in soil, the rhizosphere, insects, fungi and water (14, 56). The Burkholderia genus, β- subdivision of the proteobacteria, comprises more than 60 species that inhabit diverse ecological niches (16). Although they have traditionally been known as plant pathogens (B. cepacia being the first discovered and identified as a pathogen causing disease in onions (7)) several Burkholderia species exhibit beneficial interactions with their plant hosts (9, 12). Other Burkholderia species are able to fix atmospheric nitrogen (8) and can nodulate plant roots (12). Additionally, some Burkholderia species have been found to have potential as biocontrol products against soilborne (6), foliar (30), and postharvest (1 1, 21, 27, 28, 55, 76) plant pathogens, and have been used as bioremediators to treat polluted soil or groundwater (36, 40). Certain

Burkholderia species have been found to secrete a variety of extracellular enzymes with proteolytic, lipolytic and hemolytic activities, as well as toxins, antibiotics and siderophores (71). Such metabolic diversity (71) makes the genus Burkholderia very desirable for biotechnological applications.

[008] Some of the known toxins produced by Burkholderia sp. include toxo flavin (l,6-dimethylpyrimido[5,4-e]-l,2,4-triazine-5,7(lH, 6H)-dione), which possesses antibacterial, antifungal and herbicidal activities (29) and fervenulin (a tautomeric isomer of toxoflavin), which possesses antibacterial and nematicidal activities;

rhizobitoxin ([2-amino-4-(2-amino-3-hydroxypropoxy)-?ra«s-but-3-enoic-acid]) which, among other phytotoxic effects, induces foliar chlorosis due to inhibition of cystathione-p-lyase (53); bongkrekic acid, which inhibits adenine nucleotide translocase as well as cell apoptosis (25); rhizonins A and B, hepatotoxic

cyclopeptides that were first discovered from a fungus Rhizopus sp. but later were shown to be produced by a bacterial endosymbiont of the genus Burkholderia (57); tropolone (2-hydroxy-2,4,6-cycloheptatrien-l-one), a non-benzenoid aromatic compound with both phenolic and acidic moieties and proven antimicrobial, antifungal, and insecticidal properties (49) and rhizoxin, a macrocyclic polyketide, which kills rice seedlings through binding to β-tubulin and inhibiting the normal cell division cycle (31). Rhizoxin also demonstrates broad anti-tumor activity in vitro (65). Tropolone is produced by B. plantarii; this compound has been reported to show insecticidal activity against Tyrophagus putrescentiae (Formosan subterranean termite), Dermatophaguoides farina (mould mite) and Coptotermes formosanus (house dust mite) (49); and repellency against Callosobruchus chinensis (cigarette beetle) (61).

[009] On the other hand, several Burkholderia species are also opportunistic human pathogens (13, 45, 50), the most well-known of which are the species of the

Burkholderia cepacia complex (Bcc), as well as B. gladioli and B. fungorum. B. pseudomallei and B. mallei are the only other known members of the genus

Burkholderia that are primary pathogens to humans and animals, causing melioidosis in humans (13) and glanders in horses (50). The Burkholderia cepacia complex (Bcc) has emerged as an important group of opportunistic pathogens, particularly for patients with suppressed immune systems, and more specifically for cystic fibrosis patients (56). The species of the Bcc are phenotypically nearly identical, making their identification and differentiation very difficult by common biochemical tests. The Bcc is composed of seventeen officially recognized strains (66) that have been isolated both from cystic fibrosis patients and from diverse environmental samples. They are versatile microorganisms with large complex genomes (40), able to metabolize a wide variety of carbon sources (39), and produce diverse secondary metabolites (71). Members of the Bcc complex have highly homologous 16S rR A gene sequences, but only moderate whole-genome DNA-DNA hybridization values (30-60%), contributing to the difficulty of unequivocally identifying and

differentiating them (15, 67).

[010] Extracts from cultures of a newly-discovered Burkholderia isolate (A396) contain compounds that have been shown to possess herbicidal, algicidal, insecticidal, nematicidal and fungicidal activities. See, e.g., WO 201 1/106491. However, whether the A396 isolate corresponds to an existing species, or represents a newly-discovered species of Burkholderia (and, if the latter, the degree of relatedness of Burkholderia A396 to other Burkholderia species) remains unknown. Moreover, the existence of additional pesticidal activities in Burkholderia A396 cultures remains to be determined.

SUMMARY

[Oi l] Disclosed herein is a new species of the genus Burkholderia (Isolate A396), characterized by unique biochemical activities, fatty acid composition, and nucleotide sequences encoding 16S rR A, atpD, gltB, gyrB, recA, lepA, phaC and trpB. This new strain, named Burkholderia rinojensis, when applied to plants, exhibits activity against armyworms (e.g., beet armyworm) and mites (e.g., two-spotted spider mites).

[012] Accordingly, also provided are methods for controlling armyworm and mite infestations in a plant. The methods include application, to a plant, of a culture, whole-cell broth, cell fraction, supernatant, filtrate or extract of Burkholderia A396. Application can be either internal or external, and the compositions can be applied to any part of a plant, the soil or growth medium, or seeds.

[013] Also provided are pesticidal (e.g., miticidal, anti-lepidopteran) compositions comprising a culture, whole-cell broth, cell fraction, supernatant, filtrate or extract of Burkholderia A396. Such compositions can optionally include other insecticides or pesticides, either naturally-occurring or man-made.

[014] In further embodiments, plants comprising one or more of a culture, whole- cell broth, cell fraction, supernatant, filtrate or extract of Burkholderia A396 are provided. The compositions can be present on the exterior of the plant or internally.

[015] Progeny of the aforementioned plants are also provided. In addition, seeds from the aforementioned plants, and from their progeny, are provided.

BRIEF DESCRIPTION OF THE DRAWINGS

[016] Figure 1 shows the Neighbor Joining Tree for Isolate A396 based on 16S rRNA sequences. Branches corresponding to partitions reproduced in less than 50% bootstrap replicates are collapsed. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (2000 replicates) are shown next to the branches. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree.

[017] Figure 2 shows the Neighbor-Joining tree for isolate A396 based on concatenated sequences of seven MLST loci. Branches corresponding to partitions reproduced in less than 50% bootstrap replicates are collapsed. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (2000 replicates) are shown next to the branches. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree.

[018] Figure 3 shows the average (mean) percent mortality for BAW larvae on broccoli leaf discs treated with 3% A396 WCB, at 3 days post-exposure. Positive control (Xentari^®) and negative control (water) results are also shown. Results for 3% A396 WCB and Xentari^® are not statistically different (SAS Analysis, one way ANOVA, P value < 0.0001, LSD, a = 0.05).

[019] Figure 4 shows the average percent mortality of TSSM adults three days after exposure to fava bean leaf discs treated with 6% A396 WCB or 6% heat-treated A396 WCB. Positive control (Avid^®) and negative control (water) results are also shown. Results for 6% A396 WCB, 6% heat-treated WCB and Avid^® are not statistically different (SAS Analysis, one way ANOVA, P value < 0.0001, LSD, a = 0.05).

DETAILED DESCRIPTION

[020] Practice of the present disclosure employs, unless otherwise indicated, standard methods and conventional techniques in the fields of agriculture, plant molecular biology, entomology, cell biology, molecular biology, biochemistry, recombinant DNA and related fields as are within the skill of the art. Such techniques are described in the literature and thereby available to those of skill in the art. See, for example, Alberts, B. et al, "Molecular Biology of the Cell," 5^th edition, Garland Science, New York, NY, 2008; Voet, D. et al "Fundamentals of Biochemistry: Life at the Molecular Level," 3^rd edition, John Wiley & Sons, Hoboken, NJ, 2008;

Sambrook, J. et al, "Molecular Cloning: A Laboratory Manual," 3^rd edition, Cold Spring Harbor Laboratory Press, 2001 ; Ausubel, F. et al, "Current Protocols in Molecular Biology," John Wiley & Sons, New York, 1987 and periodic updates; Glover, DNA Cloning: A Practical Approach, volumes I and II, IRL Press (1985), volume III, IRL Press (1987); Perbal, A Practical Guide to Molecular Cloning, John Wiley & Sons (1984); Rigby (ed.), The series "Genetic Engineering" (Academic Press); Setlow & Hollaender (eds.), The series "Genetic Engineering: Principles and Methods," Plenum Press; Gait (ed.), Oligonucleotide Synthesis: A Practical Approach, IRL Press (1984, 1985); Eckstein (ed.) Oligonucleotides and Analogues: A Practical Approach, IRL Press (1991); Hames & Higgins, Nucleic Acid

Hybridization: A Practical Approach, IRL Press (1985); Hames & Higgins, Transcription and Translation: A Practical Approach, IRL Press (1984); B.

Buchanan, W. Gruissem & R. Jones (eds.) "Biochemistry and Molecular Biology of Plants," Wiley (2002) and the series "Methods in Enzymology," Academic Press, San Diego, CA. The disclosures of all of the foregoing references are incorporated by reference in their entireties for the purpose of describing methods and compositions in the relevant arts.

[021] Where a range of values is provided, it is understood that included therein is 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. 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.

[022] 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 disclosure pertains. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the disclosed methods and compositions, exemplary methods and materials are now described.

[023] It must be noted that as used herein and in the appended claims, the singular forms "a," and "the" include plural references unless the context clearly dictates otherwise.

Pesticidal activity of Burkholderia A396

[024] A novel bacterial isolate, A396, is disclosed herein. Molecular and biochemical characterization of this isolate revealed it to be a novel species of Burkholderia, named Burkholderia A396 or Burkholderia rinojensis sp. nov.

Although the 16S rRNA sequence of the novel isolate was most closely related to those of B. glumae and B. plantarii; DNA-DNA hybridization experiments revealed a low degree of genetic similarity with these two species, as well as low similarity to other members of the genus Burkholderia, such as B. multivorans and B. cenocepacia. Screening by multi-locus sequence typing indicated that the novel isolate lacked markers associated with members of the B. cepacia complex (Bcc).

[025] The biochemical profile of isolate A396 was unique amongst known species of the genus Burkholderia. B. rinojensis is a Gram-negative, non-sporulating, oxidase-positive, catalase-positive, and urease-positive organism. The cells form small straight rods that grow as cream colored shiny colonies on potato dextrose agar. Colonies turn into a light shade of pink after 48 hours of incubation. The cells are sensitive to cloxacillin, minocycline, nalidixic acid, oxacillin, novobiocin, sulfadiazine, tylosin, oleandomycin, sulfisoxazole; and resistant to lincomycin, vancomycin, troleandomycin, oxytetracycline, polymyxin B and penicillin G. The strain grows well between pH 5 and 9.5, and is able to grow at pH 4.5 in the presence of L-methionine. When evaluated in the Biolog format, Isolate A396 is negative for assimilation of L-arabinose, cellobiose, lactose, maltose, raffinose, D-xylose, dulcitol, citrate and phenylacetate; and is able to assimilate D-glucose, D-mannitol and caprate. It can utilize urea and γ-amino-butyric acid for growth, and can tolerate up to 1% NaCl, but does not grow at NaCl concentrations of 2% or higher.

[026] No clinical isolates of Burkholderia displayed any significant similarity to B. rinojensis, by either molecular or biochemical criteria.

[027] Fermentation supernatants and whole-cell broth of Burkholderia A396 exhibited activity against a number of agricultural pests, including the chewing pest Spodoptera exigua (beet armyworm) and the sucking pest Tetranychus urticae (two- spotted spider mite). Liquid cultures of Burkholderia A396 were toxic upon both ingestion and contact. For example, in diet-overlay assays l%-4% dilutions of A396 whole-cell broth (WCB) resulted in 97-100% mortality of beet armyworm four days after exposure; and 75+22% mortality was observed three days after exposure of beet armyworms to A396 WCB in a leaf disc treatment assay. In contact bioassays, exposure of beet armyworms to A396 WCB resulted in 60+1 1.5% larval mortality; with treated larvae exhibiting discoloration, stunting, liquefied frass and failure to molt.

[028] When two-spotted spider mites were exposed to leaf discs treated with A396 WCB, up to 93% mortality was observed three days after exposure. [029] Pesticidal activity was observed in heat-treated WCB and in cell-free supernatants, suggesting that the activity could be due to a heat-stable enzyme, secreted compound and/or a secondary metabolite.

[030] B. rinojensis also exhibits phytotoxicity and herbicidal activity.

[031] "Burkholderia rinojensis " "Burkholderia A396 " "Isolate A396" and "A396" are used interchangeably to refer to the novel strain of Burkholderia disclosed and characterized herein.

Formulations and Pesticidal Compositions

[032] The present disclosure provides, inter alia, pesticidal (e.g., miticidal and anti- lepidopteran) compositions and formulations comprising a culture, whole-cell broth, cell fraction, supernatant, filtrate or extract of Burkholderia A396.

[033] A "pest" is an organism (procaryotic, eucaryotic or Archael) that increases mortality and/or slows, stunts or otherwise alters the growth of a plant. Pests include, but are not limited to, mites, nematodes, insects (e.g., moths), fungi, bacteria, and viruses.

[034] A "pesticide," as defined herein, is a chemical substance or a substance derived from a biological product that increases mortality and/or inhibits the growth rate of one or more plant pests. Pesticides include but are not limited to miticides, nematocides, insecticides (e.g., anti-lepidopteran agents), herbicides, plant fungicides, plant bactericides, and plant viricides.

[035] A "biological pesticide" as defined herein is a microorganism with pesticidal properties, or a compound produced by such a microorganism.

[036] A "pesticidal composition" is a formulation comprising a pesticide and optionally one or more additional components. Additional components include, but are not limited to, solvents (e.g., amyl acetate, carbon tetrachloride, ethylene dichloride; kerosene, xylene, pine oil, and others listed in EPA list 4a and 4b etc.), carriers, (e.g., organic flour, Walnut shell flour, wood bark), pulverized mineral (sulfur, diatomite, tripolite, lime, gypsum talc, pyrophyllite), clay (attapulgite bentonites, kaolins, volcanic ash, and others listed in EPA list 4a and 4b), stabilizers, emulsifiers (e.g., alkaline soaps, organic amines, sulfates of long chain alcohols and materials such as alginates, carbohydrates, gums, lipids and proteins, and others listed in EPA list 4a and 4b), surfactants (e.g., those listed in EPA list 4a and 4b), antioxidants, sun screens, a second pesticide, either chemical or biological (e.g., insecticide, nematicide, miticide, algaecide, fungicide, bactericide), an herbicide and/or an antibiotic.

[037] A "carrier" as defined herein is an inert, organic or inorganic material, with which the active ingredient is mixed or formulated to facilitate its application to a plant or other object to be treated, or to facilitate its storage, transport and/or handling.

[038] Pesticidal compositions as disclosed herein are useful for modulating pest infestation in a plant. The term "modulate" as defined herein means to alter the amount of pest infestation, or to alter the rate of spread of pest infestation. Generally, such alterations constitute a lowering of the degree and/or rate and/or spread of the infestation.

[039] The term "pest infestation" as defined herein, 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 weed in a growth system. Exemplary plant pests include, but are not limited to, mites (e.g., Tetranychus urticae (Two-spotted spider mite)), moths and their larvae (e.g., Spodoptera exigua (beet armyworm)), fruit flies (e.g., Drosophila suzukii, Drosophila melanogaster), house flies (e.g., Musca domestica), arachnids (e.g., Acari spp.), root maggots (Anthomyidae spp., e.g.

Cabbage Root Maggots), aphids (e.g., Myzus persicae (green peach aphid)), Triozidae spp. (e.g., potato psyllid (Bactericera cockerelli)), beetles (Tenebrionidae spp., e.g., litter beetles (Alphitobius diaperinus)), grubs (e.g., white grub (Cyclocephala lurida), Southern Masked Chafer (Rhizotrogus majalis), Japanese beetle (Popillia japonica) larvae, black vine weevil (Otiorhyncus sulcatus) larvae, Oriental beetle (Anomala orientalis) larvae, scarabs (e.g., Sca -abaeidae spp.), nematodes (e.g., Root-knot nematode (Meloidogyne spp.)), fungi, bacteria, and various plant viruses, for example, Tobacco mosaic virus, Tomato spotted wilt virus, Tomato yellow leaf curl virus, Cucumber mosaic virus, Potato virus Y, Cauliflower mosaic virus, African cassava mosaic virus, Plum pox virus, Brome mosaic virus, Potato virus X, Citrus tristeza virus, Barley yellow dwarf virus, Potato leaf roll virus and Tomato bushy stunt virus.

[040] Pesticidal compositions, as disclosed herein, can be used either for prophylactic or modulatory purposes. When provided prophylactically, the compositions(s) are provided in advance of any symptoms of infestation. The prophylactic administration of the composition(s) serves to prevent, attenuate, or decrease the rate of onset of any subsequent infection or infestation. When provided for modulatory purposes, the composition(s) are provided at (or shortly after) the onset of an indication of infection or infestation. Modulatory administration of the compound(s) serves to attenuate the pathological symptoms of the infection or infestation and to increase the rate of recovery.

[041 ] Additional methods can be employed to control the duration of action of a pesticidal composition. Controlled-release can be achieved through the use of polymers to complex or absorb one or more of the components of the composition. The controlled delivery may be exercised by selecting appropriate macromolecules (for example polyesters, polyamino acids, polyvinyl, pyrrolidone,

ethylenevinylacetate, methylcellulose, carboxymethylcellulose, or protamine, sulfate) and the concentration of macromolecules as well as the methods of incorporation in order to control release. Another possible method to control the duration of action by controlled release preparations is to incorporate compositions as disclosed herein into particles of a polymeric material such as polyesters, polyamino acids, hydrogels, poly(lactic acid) or ethylene vinylacetate copolymers. Alternatively, instead of incorporating these compositions into polymeric particles, the compositions can be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatine- microcapsules and poly(methylmethacylate) microcapsules, respectively, or in colloidal delivery systems, for example, liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules or in macroemulsions. Such techniques are known in the art.

[042] Pesticidal compositions as disclosed herein, (e.g., pesticidal toxins) are produced by culture of Burkholderia A396 in lab scale, pilot scale and/or

manufacturing scale fermentations. Toxins can be produced by fermentation procedures known in the art and formulated directly, or after extraction and purification of the toxin from a fermentation broth. The formulation can include live cells and/or non-viable cells.

[043] The pesticidal compositions disclosed herein 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), microencapsulates, water dispersed granules, flowables (FL), microemulsions (ME), nano-emulsions (NE), etc. In any of the formulations described herein, the percentage of the active ingredient is within a range of 0.01% to 99.99%. Detailed descriptions of pesticide formulations are found, for example, in the Kirk-Othmer Encyclopedia of Chemical Technology; Knowles, A. (2005) New Developments in Crop Protection Product Formulation, Agrow Reports, London, UK; van Valkenburg, W, ed. (1973) Pesticide Formulation, Marcel Dekker, New York, USA; and Knowles, D.A., ed. (1998) Chemistry and Technology of Agrochemical Formulations, Kluwer Academic Publishers, Dordrecht, the Netherlands.

Powder and Dust formulations

[044] These simple formulations usually contain 0.1-25% of the active ingredient. However, higher concentrations of active ingredient can be used depending on the potency and particular application. The pesticide toxin is mixed with a solid carrier, preferably of small particle size. Solid carriers can include: silicate clays (e.g., attapulgite, bentonites, volcanic ash, montmorillionite, kaolin, talc, diatomites, etc.), carbonates (e.g., calcite, dolomite, etc), synthetics (precipitated silica, fumed silica, etc.), ground botanicals (e.g., corn cob grits, rice hulls, coconut shells, etc.), organic flour (e.g., Walnut shell flour, wood bark, etc.) or pulverized mineral (e.g., sulfur, diatomite, tripolite, lime, gypsum talc, pyrophyllite, etc.). Additional inert ingredients that can be used in dust formulations are listed in EPA Inert List 4a

(www.epa.gov/opprd001/inerts/inerts_list4Acas.pdf) for conventional formulations and in EPA Inert List 4b (www.epa.gov/opprd001/inerts/inerts_list4Bname.pdf) for organic formulations. Small particle size can be achieved by mixing the active ingredient with the carrier and pulverizing in a mill. Dusts are defined as having a particle size less than 100 microns; with increased toxicity correlated with smaller particle size. Factors to be taken into consideration in the selection of a dust formulation include its compatibility, fineness, bulk density, flowability, abrasiveness, absorbability, specific gravity and cost. Exemplary dust formulations are provided in Table 1.

Table 1

Formulation Formulation Formulation Formulation Formulation components A 15 C D

Active

0.65 5 10 25 ingredient

Talc

Kaolin or other

49.35 95 75 clay [045] A dust formulation can also be prepared from a dust concentrate (e.g., 40% active ingredient, 5% stabilizer, 20% silica, 35% magnesium carbonate) added at 1- 10% to a 1 : 1 organic filler/talc combination.

[046] The dust formulation can be used as a contact powder (CP) or tracking powder (TP) against crawling insects.

[047] A dust formulation with high flowability can be applied by pneumatic equipment in greenhouses.

Granular and pellet formulations

[048] For manufacture of granular and pellet formulations, pesticidal toxin is applied in liquid form to coarse particles of porous material (e.g., clay, walnut shells, vermiculite, diatomaceous earth, corn cobs, attapulgite, montmorillioinite, kaolin, talc, diatomites, calcite, dolomite, silicas, rice hulls, coconut shells, etc.). The granules or pellets can be water dispersible, and can be formed by extrusion (for pesticidal actives with low water solubility), agglomeration or spray drying. Granules can also be coated or impregnated with a solvent-based solution of the pesticidal toxin. The carrier particles can be selected from those listed in EPA Inert List 4a (www.epa.gov/opprd001/inerts/inerts_list4Acas.pdf) for conventional formulations and from those listed in EPA Inert List 4b

(www.epa.gov/opprd001/inerts/inerts_list4Bname.pdf) for organic formulations. The active ingredient can be absorbed by the carrier material or coated on the surface of the granule. Particle size can vary from 250 to 1250 microns (0.25 mm to 2.38 mm) in diameter. The formulations usually contain 2 to 10 percent concentration of the active ingredient(s). The granules are applied in water or whorls of plant or to soil at a density of 10 kg/ha. Granular formulations of systemic insecticides are used for the control of sucking and soil pests by application to soil. Whorl application is done for the control of borer pests of crops such as sorghum, maize and sugarcane, etc. These types of formulations reduce drift and allow for slower release of the pesticidal composition.

[049] Granular pesticides are most often used to apply chemicals to the soil to control weeds, fire ants, nematodes, and insects living in the soil or for absorption into plants through the roots. Granular formulations are sometimes applied by airplane or helicopter to minimize drift or to penetrate dense vegetation. Once applied, granules release the active ingredient slowly. Some granules require soil moisture to release the active ingredient. Granular formulations also are used to control larval mosquitoes and other aquatic pests. Granules are used in agricultural, structural, ornamental, turf, aquatic, right-of-way, and public health (biting insect) pest control operations.

[050] Application of granular formulations is common in pre-emergence herbicides or as soil insecticides for direct application and incorporation into soil or other solid substrates where plants grow. Granules or pellets can also be applied in-furrow. Granules are commonly used for application to water, such as in flooded rice paddies.

[051] A typical granule formulation includes (%w/w) 1-40% active ingredient, 1-2% stabilizer, 0-10% resin or polymer, 0-5% surfactant, 0-5% binder and is made up to 100% with the carrier material.

Wettable powder formulations

[052] Wettable powder is a powdered formulation which yields a rather stable suspension when diluted with water. It is formulated by blending the pesticidal agent with diluents such as attapulgite (a surface active agent) and auxiliary materials such as sodium salts of sulfo acids. Optionally stickers are added to improve retention on plants and other surfaces. Wettable powders can be prepared by mixing the pesticidal toxin (10-95%) with a solid carrier, plus 1-2% of a surface-active agent to improve suspendibility. The overall composition of the formulation includes the active ingredient in solid form (5.0-75%), an anionic dispersant and an anionic or nonionic wetting agent.

[053] A typical example of a wettable powder formulation includes 10-80% active ingredient, 1-2% wetting agents (e.g., benzene sulphonates, naphthalene sulphonates, aliphatic suplhosuccinates, aliphatic alcohol etoxylates, etc.), 2-5% dispersing agent (e.g., lignosulphonates, naphthalene sulphonate-formaldehyde condensates, etc.), and 0.1-1% antifoaming agent (e.g., isopar M (Exxon/Mobil)), made up to 100% with an inert filler or carrier (e.g., diatomaceous earth, silica, etc.).

Emulsifiable concentrate (EC) formulations

[054] EC formulations are concentrated pesticide formulation containing an organic solvent and a surface-active agent to facilitate emulsification with water. When EC formulations are sprayed on plant parts, the solvent evaporates quickly, leaving a deposit of toxin from which water also evaporates. Exemplary emulsifying agents in insecticide formulations include alkaline soaps, organic amines, sulfates of long chain alcohols and materials such as alginates, carbohydrates, gums, lipids and proteins. Emulsifying agents can be selected from those listed in EPA Inert List 4a (www.epa.gov/opprd001/inerts/inerts_list4Acas.pdf) for conventional formulations and from those listed in EPA Inert List 4b

(www.epa.gov/opprd001/inerts/inerts_list4Bname.pdf) for organic formulations.

Solution formulations

[055] A solution formulation is a concentrated liquid pesticide formulation that can be used directly, or which can be diluted, in the case of a soluble concentrate. Soluble concentrates and solutions are water- or solvent-based mixtures with complete miscibility in water.

[056] A typical example of a solution concentrate formulation includes 20-70% active ingredient, 5-15% wetting agent, 5-10% antifreeze, and is made up to 100% with water or a water-miscible solvent.

[057] Depending on the nature and stability of the pesticidal toxin, a solution formulation can optionally include thickeners, preservatives, antifoam, pH buffers, UV screens, etc.

Aerosol and fumigant formulations

[058] In an insecticidal aerosol, a pesticide is suspended as minute particles having sizes ranging from 0.1 to 50 microns in air as a fog or mist. This can be achieved, e.g., by burning the toxin, or by vaporizing it by heating. Release of a toxin dissolved in a liquefied gas, through a small hole, may cause particles of the toxin to float in air with the rapid evaporation of the released gas.

[059] A chemical compound which is volatile at ambient temperatures and sufficiently toxic is known as a fumigant. Fumigants generally enter an insect via its tracheal system. Fumigants are used for the control of insect pests in storage bins and buildings, and for control of certain insects and nematodes in the soil. Most fumigants are liquids held in cans or tanks and often comprise mixtures of two or more gases. Alternatively, phosphine or hydrogen phosphide gas can be generated in the presence of moisture from a tablet made up of aluminum phosphide and ammonium carbonate. The advantage of using a fumigant is that sites that are not easily accessible to other chemicals can be reached with fumigants, due to the penetration and dispersal of the gas. Commonly used fumigants are ethylene dichloride carbon tetrachloride (EDCT), methyl bromide, aluminum phosphide and hydrocyanic acid. Fertilizer Mixtures

[060] A fertilizer mixture can be manufactured by addition of an insecticidal composition, as disclosed herein, to a chemical fertilizer, or by spreading the composition directly on the fertilizer. Fertilizer mixtures are applied at the regular fertilizing time and provide both plant nutrients and control of soil insects. In an exemplary fertilizer formulation, urea (2% solution) is mixed with an insecticidal composition and sprayed for supply of nitrogen to the plant and for realizing effective pest control.

Poison Baits.

[061] Poison baits consist of a base or carrier material attractive to the pest species and a chemical toxicant in relatively small quantities. The poison baits are used for the control of, e.g., fruit flies, chewing insects, wireworms, white grubs in the soil, household pests, rats in the field and slugs. These formulations are useful for situations in which spray application is difficult. A common base used in dry baits is wheat bran moistened with water and molasses. For the control of fruit sucking moths, fermenting sugar solution or molasses with a toxin is used.

Formulations for seed treatments

[062] Seed treatments include application of a pesticidal composition, optionally in combination with other bioactive, antagonistic or symbiotic agents, to the surface of a seed prior to sowing. The pesticidal toxins, compounds and compositions disclosed herein can be formulated for seed treatments in any of the following modes: dry powder, water slurriable powder, liquid solution, flowable concentrate, flowable emulsion, emulsion, microcapsules, gel, or water-dispersible granules; or can be applied to seeds by spraying on the seed before planting.

[063] In the case of a dry powder, the active ingredient is formulated similarly to a wettable powder, but with the addition of a sticking agent, such as mineral oil, instead of a wetting agent. For example: one kg of purified talc powder (sterilized for 12 h), 15 g calcium carbonate, and 10 g carboxymethyl cellulose are mixed under aseptic conditions following the method described by Nandakumar et al. (2001). Pesticidal compositions, or organisms expressing one or more pesticidal toxins, are mixed in a 1 :2.5 ratio (suspension to dry mix) and the product is shade dried to reduce moisture content to 20-35%.

[064] Compositions for seed treatment can 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. For liquid compositions, the active ingredient can be dissolved in a suitable carrier or solvent.

[065] A composition can 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 composition comprising a pesticidal compositions as disclosed herein, and optionally a second pesticide or herbicide, and inducing gel formation of the agent.

[066] The composition can additionally comprise a surfactant to be used for the purpose of emulsification, dispersion, wetting, spreading, integration, disintegration control, stabilization of active ingredients, improvement of fluidity and/or rust inhibition. In a particular embodiment, the surfactant is a non-phytotoxic, non-ionic surfactant, such as those listed in EPA List 4B. In another particular embodiment, the nonionic surfactant is polyoxyethylene (20) monolaurate. The concentration of surfactant can range between 0.1-35% of the total formulation, e.g., from 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 composition.

Formulations comprising microorganisms

[067] Pesticidal compositions as set forth above can be combined with a microorganism. The microorganism can be a plant growth promoter. Suitable microorganisms include, but are not limited to, Bacillus sp. (e.g., Bacillus firmus, Bacillus thuringiensis, Bacillus pumilus, Bacillus licheniformis, Bacillus

amyloliquefaciens, Bacillus 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), Burkholderia sp., Trichoderma sp., Gliocladium sp., Myrothecium sp., Paecilomyces spp., Sphingobacterium sp., Arthrobotrys sp., Chlorosplenium sp., Neobulgaria sp., Daldinia sp., Aspergillus sp., Chaetomium sp., Lysobacter sp., Lachnum papyraceum, Verticillium suchlasporium, Arthrobotrys oligospora, Verticillium chlamydosporium, Hirsutella rhossiliensis, Pochonia chlamydosporia, Pleurotus ostreatus, Omphalotus olearius, Lampteromyces japonicas, Brevudimonas sp. , Muscodor sp., Photorhabdus sp., and Chromobacterium sp. (e.g., C. subtsugae). Agents obtained or derived from such microorganisms can also be used in combination with the pesticidal

compositions disclosed herein.

Formulations comprising second pesticides

[068] Pesticidal compositions as set forth above can be combined with a second pesticide (e.g., nematocide, fungicide, herbicide, insecticide, algaecide, miticide, or bactericide). Such an agent can be a natural oil or oil product having fungicidal, bactericidal, miticidal, nematicidal, acaricidal and/or insecticidal activity (e.g., paraffinic oil, tea tree oil, lemongrass oil, clove oil, cinnamon oil, citrus oil, rosemary oil, pyrethram). Furthermore, the pesticide can 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, an anthranilic diamide (e.g.,

chlorantranilipole) 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 can also be derived from a Reynoutria extract.

[069] The fungicide can also be a multi-site non-inorganic, chemical fungicide selected from the group consisting of chloronitrile, quinoxaline, sulphamide, phosphonate, phosphite, dithiocarbamate, chloralkythios, phenylpyridin-amine, and cyano-acetamide oxime.

[070] The composition can, as noted above, further comprise an insecticide. The insecticide can include but is not limited to an avermectin, Bt (e.g., Bacillus thuringiensis var. kurstaki), neem oil, spinosads, Chromobacteria sp. (e.g., as set forth in US Patent No. 7,244,607), entomopathogenic fungi such a Beauveria bassiana and chemical insecticides including but not limited to organochlorine compounds, organophosphorous compounds, carbamates, pyrethroids, pyrethrins and neonicotinoids.

[071] As noted above, the composition may further comprise a nematocide. This nematocide may include, but is not limited to, avermectin, microbial products such as Biome (Bacillus firmus), Pasteuria spp and organic products such as saponins.

Seed Coating Agents

[072] The compositions disclosed herein can also be used in combination with seed- coating agents. Such seed coating agents include, but are not limited to, ethylene glycol, polyethylene glycol, chitosan, carboxymethyl chitosan, peat moss, resins and waxes or chemical fungicides or bactericides with either single site, multisite or unknown mode of action.

Anti-Phytopathogenic agents

[073] The compositions disclosed herein can also be used in combination with other anti-phytopathogenic agents, such as plant extracts, biopesticides, inorganic crop protectants (such as copper), surfactants (such as rhamnolipids; Gandhi et ah, 2007), natural oils such as paraffin oil and tea tree oil possessing pesticidal properties, chemical fungicides or bactericides with either single site, multisite or unknown modes of action. As defined herein, an "anti-phytopathogenic agent" is an agent that modulates the growth of a plant pathogen, e.g., a pathogen causing soil-borne disease on a plant, or alternatively prevents infection of a plant by a plant pathogen. Plant pathogens include but are not limited to fungi, bacteria, mites, insects, nematodes, actinomycetes and viruses.

[074] An anti-phytopathogenic agent can be a single-site anti-fungal agent which can 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). In a more particular embodiment, the antifungal agent is 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). In a most particular embodiment, the antifungal agent is myclobutanil. In yet another particular embodiment, the antifungal agent is a quinone outside inhibitor (e.g., strobilurin). The strobilurin may include but is not limited to azoxystrobin, kresoxim-methyl 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).

[075] In yet a further embodiment, the fungicide is a multi-site non-inorganic, chemical fungicide selected from the group consisting of chloronitrile, quinoxaline, sulphamide, phosphonate, phosphite, dithiocarbamate, chloralkythios, phenylpyridine- amine, and cyano-acetamide oxime.

[076] In yet a further embodiment, the anti-phytopathogenic agent can be streptomycin, tetracycline, oxytetracycline, copper, or kasugamycin.

Methods for modulating pest infestation

[077] According to the present disclosure, methods for modulating pest infestation in a plant are provided. The methods comprise application to a plant, or to the soil or substrate in which the plant is growing, of a pesticidal composition as disclosed herein. In particular, the compositions as set forth above can be used as, for example, insecticides and/or miticides, alone or in combination with one or more second pesticidal substances.

[078] Phytopathogenic insects that can be controlled using the compositions and methods set forth above include but are not limited to non-Culicidae larvae insects from the order (a) Lepidoptera, for example, Acleris spp., Adoxophyes spp., Aegeria spp., Agrotis spp., Alabama argillaceae, Amylois spp., Anticarsia gemmatalis, Archips spp., Argyrotaenia spp., Autographa spp., Busseola fusca, Cadra cautella, Carposina nipponensis, Chilo spp., Choristoneura spp., Clysia ambiguella,

Cnaphalocrocis spp., Cnephasia spp., Cochylis spp., Coleophora spp., Crocidolomia binotalis, Cryptophlebia leucotreta, Cydia spp., Diatraea spp., Diparopsis castanea, Earias spp., Ephestia spp., Eucosma spp., Eupoecilia ambiguella, Euproctis spp., Euxoa spp., Grapholita spp., Hedya nubiferana, Heliothis spp., Hellula undalis, Hyphantria cunea, Keiferia lycopersicella, Leucoptera scitella, Lithocollethis spp., Lobesia botrana, Lymantria spp., Lyonetia spp., Malacosoma spp., Mamestra brassicae, Manduca sexta, Operophtera spp., Ostrinia nubilalis, Pammene spp., Pandemis spp., Panolis flammea, Pectinophora gossypiella, Phthorimaea operculella, Pieris rapae, Pieris spp., Plutella xylostella, Prays spp., Scirpophaga spp., Sesamia spp., Sparganothis spp., Spodoptera spp., Synanthedon spp., Thaumetopoea spp., Tortrix spp., Trichoplusia ni and Yponomeuta spp.; (b) Coleoptera, for example, Agriotes spp., Anthonomus spp., Atomaria linearis, Chaetocnema tibialis,

Cosmopolites spp., Curculio spp., Dermestes spp., Diabrotica spp., Epilachna spp., Eremnus spp., Leptinotarsa decemlineata, Lissorhoptrus spp., Melolontha spp., Orycaephilus spp., Otiorhynchus spp., Phlyctinus spp., Popillia spp., Psylliodes spp., Rhizopertha spp-, Scarabeidae, Sitophilus spp., Sitotroga spp., Tenebrio spp., Tribolium spp. and Trogoderma spp.; (c) Orthoptera, for example, Blatta spp., Blattella spp., Gryllotalpa spp., Leucophaea maderae, Locusta spp., Periplaneta spp. and Schistocerca spp.; (d) Isoptera, for example, Reticulitermes spp.; (e) Psocoptera, or example, Liposcelis spp.; (f) Anoplura, for example, Haematopinus spp., Linognathus spp., Pediculus spp., Pemphigus spp. and Phylloxera spp.; (g)

Mallophaga, for example, Damalinea spp. and Trichodectes spp.; (h) Thysanoptera, or example, Frankliniella spp., Hercinotnrips spp., Taeniothrips spp., Thrips palmi, Thrips tabaci and Scirtothrips aurantii; (i) Heteroptera, for example, Cimex spp., Distantiella theobroma, Dysdercus spp., Euchistus spp., Eurygaster spp., Leptocorisa spp., Nezara spp., Piesma spp., Rhodnius spp., Sahlbergella singularis, Scotinophara spp. and Tniatoma spp.; (j) Homoptera, for example, Aleurothrixus floccosus, Aleyrodes brassicae, Aonidiella spp., Aphididae, Aphis spp., Aspidiotus spp., Bemisia tabaci, Ceroplaster spp., Chrysomphalus aonidium, Chrysomphalus dictyospermi, Coccus hesperidum, Empoasca spp., Eriosoma larigerum, Erythroneura spp., Gascardia spp., Laodelphax spp., Lecanium corni, Lepidosaphes spp., Macrosiphus spp., Myzus spp., Nephotettix spp., Nilaparvata spp., Paratoria spp., Pemphigus spp., Planococcus spp., Pseudaulacaspis spp., Pseudococcus spp., Psylla spp., Pulvinaria aethiopica, Quadraspidiotus spp., Rhopalosiphum spp., Saissetia spp., Scaphoideus spp., Schizaphis spp., Sitobion spp., Trialeurodes vaporariorum, Trioza erytreae and Unaspis citri; (k) Hymenoptera, for example, Acromyrmex, Atta spp., Cephus spp., Diprion spp., Diprionidae, Gilpinia polytoma, Hoplocampa spp., Lasius spp., Monomorium pharaonis, Neodiprion spp., Solenopsis spp. and Vespa spp.; (I) Diptera, for example, Aedes spp., Antherigona soccata, Bibio hortulanus, Calliphora erythrocephala, Ceratitis spp., Chrysomyia spp., Culex spp., Cuter ebra spp., Dacus spp., Drosophila melanogaster, Fannia spp., Gastrophilus spp., Glossina spp., Hypoderma spp., Hyppobosca spp., Liriomyza spp., Lucilia spp., Melanagromyza spp., Musca spp., Oestrus spp., Orseolia spp., Oscinella frit, Pegomyia hyoscyami, Phorbia spp., Rhagoletis pomonella, Sciara spp., Stomoxys spp., Tabanus spp., Tannia spp. and Tipula spp.; (m) Siphonaptera, for example, Ceratophyllus spp. unci Xenopsylla cheopis and (n) from the order Thysanura, for example, Lepisma saccharina.

[079] Application of an effective pesticidal control amount of a pesticidal composition as disclosed herein is provided. Said pesticidal composition is applied, alone or in combination with another pesticidal substance, in an effective pest control or pesticidal amount. An effective amount is defined as that quantity of pesticidal composition, alone or in combination with another pesticidal substance, that is sufficient to prevent or modulate pest infestation. The effective amount and rate can be affected by pest species present, stage of pest growth, pest population density, and environmental factors such as temperature, wind velocity, rain, time of day and seasonality. The amount that will be within an effective range in a particular instance can be determined by laboratory or field tests.

Methods of application

[080] The pesticidal compositions disclosed herein, when used in methods for modulating pest infestation, can be applied using methods known in the art.

Specifically, these compositions can be applied to plants or plant parts by spraying, dipping, application to the growth substrate (e.g., soil) around the plant, application to the root zone, dipping roots prior to planting, application to plants as a turf or a drench, through irrigation, or as soil granules. 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 obtained by conventional plant breeding and optimization methods, by biotechnological and genetic engineering methods or by combinations of these methods, including transgenic plants and plant cultivars protectable or not protectable by plant breeders' rights. Plant parts are to be understood as meaning 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 plant parts also include harvested material, and vegetative and generative propagation material, for example cuttings, tubers, rhizomes, off-shoots and seeds. [081] Application can be external, (e.g. by spraying, fogging or painting) or internal (e.g., by injection, transfection or the use of a vector). When applied internally, the compositions can be intracellular or extracellular (e.g., present in the vascular system of the plant or in the extracellular space).

[082] Treatment of plants and plant parts with the compositions disclosed herein can be carried out directly or by allowing the compositions to act on a plant's

surroundings, habitat or storage space by, for example, immersion, spraying, evaporation, fogging, scattering, painting on, and/or injecting. In the case in which the composition is applied to a seed, the composition can be applied to the seed as one or more coats, using methods known in the art, prior to planting the seed.

[083] Pesticidal compositions as disclosed herein can also be applied to seeds; e.g., as a seed coating. Different adherents ("stickers") can be used in the manufacture of seed coatings, including, for example, methyl cellulose, alginate, carrageenan and polyvinyl alcohol. The adherent is dissolved in water at a concentration of 1-10% and stored at room temperature before application to the seeds. Seeds are soaked in adherent solution (e.g., 3 ml/100 seeds) for 15 min, scooped out and mixed with organic matter (e.g., 1.5 g/100 seeds) in plastic bags and shaken vigorously. This process can also be automated using a seed coating machine.

[084] For priming seeds with compositions as disclosed herein, seeds are soaked in twice the seed volume of sterile distilled water containing suspensions of pesticidal composition, or talc formulation (dry formulation) (4-10 g kg^"1 of seed, depending on seed size), and incubated at 25 ±2°C for 12-24 h. The suspension is then drained off and the seeds are dried under shade for 30 min and used for sowing. Alternatively, if the pesticidal composition is a culture, whole-cell broth or supernatant, seeds are soaked in the culture, broth or supernatant, or a dilution thereof, for an appropriate period of time (e.g., 15 min, 30 min, 1 hour, 2 hours, 4 hours, 8 hours, 12 hours, 24 hours), then dried.

[085] The compositions can also be used as soil amendments, e.g., in combination with a carrier such as a talc formulation. Formulations for soil amendment can also include clays, emulsifiers, surfactants and stabilizers, as are known in the art. For preparation of talc based formulations, one kg of purified talc powder (sterilized for 12 h), 15 g calcium carbonate, and 10 g carboxymethyl cellulose are mixed under aseptic conditions following the method described by Nandakumar et al. (2001). Suspensions of a pesticidal composition, or an organism that produces a pesticidal composition, are mixed in a 1 :2.5 ratio (suspension to dry mix) and the product is shade-dried to reduce moisture content to 20-35%.

[086] For soil amendment, formulations (e.g., talc formulations) can be applied at densities of between 2.5 - 10 Kg ha^"1 at sowing and/or at different times after emergence, or both, depending on the crop.

[087] The compositions disclosed herein can also be applied to soil using methods known in the art. See, for example, the USDA website at

naldc.nal.usda.gov/download/43874/pdf, accessed February 20, 2013. Such methods include but are not limited to fumigation, drip irrigation or chemigation, broadcast application of granules or sprays, soil incorporation (e.g., application of granules), soil drenching, seed treatment and dressing, and bare root dip.

EXAMPLES

Example 1: Isolation of microorganism

[088] Microbial isolate A396 was isolated from a soil sample. The sample was suspended in sterile water, serially diluted and plated onto agar plates of various compositions. Isolate A396 was recovered from Potato Dextrose Agar (PDA) plates that had been incubated at 25 °C in the dark for approximately one week.

Example 2: Bacterial cultivation and production of test substances

[089] Isolate A396 was deposited with the ARS-NRRL Collection under accession code NRRL B-50319. Isolate A396 was maintained on PDA plates at 25 °C, and cultures were grown in liquid medium (Hy-Soy 15 g/L, NaCl 5 g/L, ΚΗ₂Ρ0₄5 g/L, MgS0₄ x 7H₂0 0.4 g/L, ( H₄)₂S0₄ 2 g/L, glucose 5 g/L, pH 6.8) in 250ml to 2L fermentation flasks at 200 rpm, 25°C for five days. Whole-cell broth (WCB) was obtained from these five-day cultures without further purification and stored at -80°C until use in toxicity assays. For certain experiments, WCB was heat-treated by incubation at 60°C for two hours. For larger volumes, fermentation is performed in SIP (sterilization in place) tanks with dissolved oxygen and pH control.

[090] To obtain cell-free supernatants for testing, cells were removed from cultures by centrifugation and the supernatant was further clarified by filtration through a 0.22 μιη nylon filter. Example 3: 16S rRNA gene sequence

[091] Isolate A396 was grown on PDA plates overnight at 25°C, in the dark. Fresh growth was scraped from the plate using a sterile disposable loop. The collected biomass was suspended in extraction buffer, and DNA was extracted using an UltraClean^® Microbial DNA Isolation Kit (MoBio Laboratories, Inc., Carlsbad,CA). DNA extract was checked for quality and quantity by electropohoresis of a 5 μΐ sample on a 1% agarose gel and comparison to molecular weight standards (Hi-Lo Mass Ladder, Bionexus, Oakland, CA).

[092] The 16S rRNA-encoding portion of the genome of Isolate A396 was amplified by PCR. PCR reactions were assembled as follows: 2 μΐ DNA extract, 5 μΐ PCR buffer, 1 μΐ dNTPs (10 mM each), 1.25 μΐ forward primer (27F, AGA GTT TGA TCM TGG CTC AG; SEQ ID NO: l), 1.25 μΐ reverse primer (1525R, AGA GTT TGA TCC TGG CTC AG; SEQ ID NO:2) and 0.25 μΐ Taq polymerase; and the total reaction volume was adjusted to 50 μΐ using sterile nuclease-free water. The amplification reaction included an initial denaturation step at 95°C for 10 minutes, followed by 30 cycles of 94°C for 30 sec, 57°C for 20sec, and 72°C for 30sec; and a final extension step at 72°C for 10 minutes. The concentration and size of the amplification product was estimated by electrophoresis of a 5 μΐ sample of the completed reaction mixture on a 1% agarose gel; with comparison to molecular weight markers (Hi-Lo Mass Ladder, Bionexus, Oakland, CA). Excess primers, dNTPs and enzyme were removed from the PCR product using an UltraClean^® PCR clean up kit (MoBio Laboratories, Inc., Carlsbad, CA).

[093] The purified PCR product was directly sequenced using primers 27F and 1525R. The 16S sequence obtained is shown below:

TGGAGAGTTTGATCCTGGCTCAGATTGAACGCTGGCGGCATGCCTTACAC

ATGCAAGTCGAACGGCAGCACGGGTGCTTGCACCTGGTGGCGAGTGGCGA

ACGGGTGAGTAATACATCGGAACATGTCCTGTAGTGGGGGATAGCCCGGC

GAAAGCCGGATTAATACCGCATACGATCTACGGATGAAAGCGGGGGATCT

TCGGACCTCGCGCTATAGGGTTGGCCGATGGCTGATTAGCTAGTTGGTGG

GGTAAAGGCCTACCAAGGCGACGATCAGTAGCTGGTCTGAGAGGACGATC

AGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAG

TGGGGAATTTTGGACAATGGGGGAAACCCTGATCCAGCAATGCCGCGTGT

GTGAAGAAGGCCTTCGGGTTGTAAAGCACTTTTGTCCGGAAAGAAATCCT

TTGGGCTAATACCCCGGGGGGATGACGGTACCGGAAGAATAAGCACCGG

CTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGTGCGAGCGTTAATC

GGAATTACTGGGCGTAAAGCGTGCGCAGGCGGTTTGTTAAGACAGATGTG

AAATCCCCGGGCTTAACCTGGGAACTGCATTTGTGACTGGCAAGCTAGAG

TATGGCAGAGGGGGGTAGAATTCCACGTGTAGCAGTGAAATGCGTAGAG ATGTGGAGGAATACCGATGGCGAAGGCAGCCCCCTGGGCCAATACTGACG

CTCATGCACGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTC

CACGCCCTAAACGATGTCAACTAGTTGTTGGGGATTCATTTCCTTAGTAAC

GTAGCTAACGCGTGAAGTTGACCGCCTGGGGAGTACGGTCGCAAGATTAA

AACTCAAAGGAATTGACGGGGACCCGCACAAGCGGTGGATGATGTGGATT

AATTCGATGCAACGCGAAAAACCTTACCTACCCTTGACATGGTCGGAATC

CTGAAGAGATTCGGGAGTGCTCGAAAGAGAACCGATACACAGGTGCTGC

ATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACG

AGCGCAACCCTTGTCCTTAGTTGCTACGCAAGAGCACTCTAAGGAGACTG

CCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAGTCCTCATGGCCC

TTATGGGTAGGGCTTCACACGTCATACAATGGTCGGAACAGAGGGTTGCC

AACCCGCGAGGGGGAGCTAATCCCAGAAAACCGATCGTAGTCCGGATTGC

ACTCTGCAACTCGAGTGCATGAAGCTGGAATCGCTAGTAATCGCGGATCA

GCATGCCGCGGTGAATACGTTCCCGGGTCTTGTACACACCGCCCGTCACA

CCATGGGAGTGGGTTTTACCAGAAGTGGCTAGTCTAACCGCAAGGAGGAC

GGTCACCACGGTAGGATTCATGACTGGGGTGAAGTCGTAACAAGGTAGCC

GTATCGGAAGGTGCGGYTGGATCACCTCCTT (SEQ ID NO:3)

[094] This sequence was imported into MEGA5 software (www.megasoftware.net/), and compared with available type strains of the genus Burkholderia using EZ-Taxon (eztaxon-e.ezbiocloud.net/), a manually curated database of type strains of prokaryotes that provides identification tools using a similarity-based search, and nucleotide BLAST (blast.ncbi.nlm.nih.gov/Blast.cgi). According to the EZ-Taxon results, 25 type strains of Burkholderia had a 16S rRNA gene sequence with a similarity of 97% or greater to that of A396. The closest matches were B. plantarii LMG 9035^T (98.9% pairwise similarity) and B. glumae LMG 2196^T (98.69% pairwise similarity).

[095] The 16S rRNA sequence of Isolate A396 was also very similar to those of several members of the Burkholderia cepacia complex (Bcc), a group of opportunistic human pathogens. When compared to all type strain sequences for Burkholderia, the lowest degree of similarity in 16S sequence was 94.13%. The 16S rRNA gene sequence of Isolate A396 was 95.17% similar to that of Pandorea thiooxidans ATSB16^T. No significant similarities (above 97%) were found to other taxa.

[096] A neighbor joining tree was built, using MEGA5, with the A396 16S rRNA sequence and 16S rRNA sequences for all type strains within the genus Burkholderia that possessed 97% or greater similarity to that of A396. Sequences were aligned by MUSCLE. The bootstrap consensus tree inferred from 2000 replicates is taken to represent the evolutionary history of the taxa analyzed. The evolutionary distances were computed using the Jukes-Cantor method and are in the units of the number of base substitutions per site. The analysis involved 10 nucleotide sequences. Codon positions included were lst+2nd+3rd+Noncoding. All ambiguous positions were removed for each sequence pair. There were a total of 1529 positions in the final dataset. Evolutionary analyses were conducted in MEGA5, and the phylogenetic tree is shown in Figure 1. Estimates of evolutionary divergence between Isolate A396 and the closest matches indicate that there is no definitive species level match to A396.

The closest branches in the tree include B. plantarii LMG 9035^T and B. glumae LMG 2196^T.

Example 4: Multi-locus sequence typing

[097] Amplification and sequencing of seven loci (atpD, gltB, gyrB, lepA, phaC, recA and trpB) from Isolate A396 was performed as described by Spilker et al. (63).

The sequences obtained are shown below.

[098] atpD

TGGTCCGCACCATCTGTCTGGGTGCATCCGACGGCCTGCGCCGCGGTCTGGTGGTGAAG A

ACACGCGGAAGCCGATCTCGGTGCCGGTCGGCAAGCCGACCCTCGGCCGGATCATGGA CG

TGCTCGGCCGTCCGATCGACGAAGCGGGCCCGATCGAAAGCGAAACGACGCGTTCGAT CC

ACCAGAAGGCGCCGGCGTTCGACGAACTGTCGCCGTCGACCGAACTGCTCGAAACCGG CA

TCAAGGTCATCGACCTGATCTGCCCGTTCGCGAAGGGCGGCAAGGTCGGTCTGTTCGGT G

GTGCAGGGGTGGGCAAGACCGTCAACATGATGGAGCTCATCAACAACATCGCGAAGG AGC

ACGGCGGTTACTCCGTGTTCGCGGGCGTGGGCGAGCGTACCCGTGAAGGGAACGACTT CT

ACCACGAAATGAAGGACTCGAAC (SEQ ID NO:4)

[099] gltB

CGCGAAGTTCGACGACCTCGTCGGCCGCGCCGATCTGCTCGACACCCGCAAGGGCATCG A

GCACTGGAAGGCGAAAGGCCTCGATTTCGCGCGCGTGTTCTACCAGCCGGAAGGCTGCG A

GGAAGTGGCGCGCCGCCATGTCGAGAGCCAGGAGCACGGCCTCGAACGCGCGCTCGACC A

TACGCTGATCGAGAAGGCGAAGGCCGCGATCGAGAGTGGCGAGCACGTGTCGTTCATCC A

GCCGGTGCGCAACGTGAACCGCACGGTCGGCGCGATGCTGTCCGGCGCGATCGCGAAGC G

GCACGGCCACGACGGTCTCGCCGACGATGCGATCCACATCCAGCTCAAAGGCACGGCGG G

GCAGAGCTTCGGCGCGTTCCTCGCGAAGGGCGTGACGCTC (SEQ ID NO:5) [0100] gyrB

GCTACAAGGTGTCGGGCGGGCTGCATGGGGTCGGCGTGTCGTGCGTGAACGCGCTGTCG A

GCTGGTTGCGGCTGACCGTGCGGCGCAACGGCAAGAAGCACTTCATGGAGTTTCATCGC G

GCGTCGCGCAGAACCGCGTGCTCGAAGAGCAGGACGGCGAGCAGGTGTCGCCGATGCAG C

TCGTCGGGCCGACGGAGAATCGCGGGACTGAAGTGCATTTCATGGCCGATCAGACGATC T

TCGGCACGGTCGAGTTTCACTACGACATTCTCGCGAAGCGGATTCGCGAGCTGTCGTTCT TGAATAACGGCGTGCGGATTCGGTTGACCGACCAGCGCTCGGGCAAGGAAGACGATTTC G

CATTCGCCGGAGGCGTGAAGGGTTTTGTCGAGTACATCAACAAGACCAAGCAGGTGCTG C

ATCCGACGATTTTCCACATCGTCGGCGAGAAGGA (SEQ ID NO:6)

[0101] recA

GCGCTGGCGGCTGCGCTCTCGCAGATCGAGAAGCAGTTCGGCAAGGGCTCGATCATGCG C

CTCGGCGCAGGCGAAGCGGTCGAGGACATCCAGGTGGTGTCCACGGGCTCGCTGGGTCT G

GATATCGCGCTCGGCGTCGGCGGCCTGCCGCGCGGCCGGGTGGTCGAGATCTACGGCCC G

GAATCGTCCGGCAAGACCACGCTGACGCTGCAGGTGATCGCCGAGATGCAGAAGCTCGG C

GGCACGGCGGCGTTCATCGATGCCGAGCACGCGCTCGACGTGCAGTACGCGGGCAAGCT C

GGCGTGAACGTGCAGGAGCTGCTGATCTCGCAGCCGGACACGGGCGAGCAGGCGCTCGA G

ATCGTCGACGCGCTGGTGCGCTCGGGCTCGATT (SEQ ID NO:7) [0102] lepA

GTCGGCGATACGGTCACGCATGCGACCAAGTCGGCGCTCGCGCCGCTGCCGGGCTTCAA G

GAAGTGAAGCCGCAGGTGTTCGCGGGCCTCTATCCGGTCGAGGCGAACCAGTACGACGC G

CTGCGCGAGTCGCTCGAGAAGCTCAAGCTGAACGATGCGTCGCTGCAGTACGAGCCGGA A

GTGTCGCAGGCGCTCGGCTTCGGCTTCCGCTGCGGCTTCCTCGGCCTGCTGCACATGGAG ATCGTGCAGGAACGGCTCGAGCGCGAGTTCGACATGGACCTGATCACGACCGCGCCGAC C

GTCGTCTACGAGGTCGTGAAGAGCGACGGCGCGACGATCACGGTCGAGAATCCGGCGAA G

ATGCCGGAGCTGAGCCGGATCGGCGAGATCCGCGAGC (SEQ ID NO: 8)

[0103] phaC

CGGCGTACGAAGTGGATCGAGGACCAGAAAGCGCTGGGCTTCGATTTCGTGGCATCCGA G

GGCAACGTCGGCGACTGGGACTCCACCAAGCAGGCGTTCGACAAGGTCAAGGCCGAGGT C

GGCGAGGTCGACGTGCTGGTCAACAACGCCGGCATCACGCGCGACGTCGTGTTCCGCAA G ATGACGCACGAGGATTGGACGGCCGTGATCGACACGAACCTCACGAGCCTCTTCAACGT C

ACGAAGCAGGTGATCGACGGCATGGTCGAGCGCGGCTGGGGGCGGATCATCAACATCTC G

TCGGTGAACGGCCAGAAGGGTCAGTTCGGTCAGACCAACTACTCGACCGCGAAGGCCGG C

ATCCACGGCTTCACGATGTCGCTCG (SEQ ID NO:9)

[0104] trpB

TCATCGGCACGGTCGCCGGCCCGCATCCGTATCCGATGATGGTGCGCGACTTCCAGCGCG TGATCGGCGACGAGTGCAAGGTGCAGATGCCCGAGCTGGCCGGCCGTCAGCCGGATGCG G

TGATCGCCTGCGTTGGCGGCGGCTCGAACGCGATGGGCATCTTCTATCCGTACATCGACG ATCGCGACGTGCAGCTGATCGGCGTCGAGGCGGCGGGCGACGGGCTCGACTCGGGCCAT C

ACGCGGCCTCGCTGATCGCCGGCAGCCCGGGCGTGCTGCACGGCAACCGCACCTACCTG C

T (SEQ ID NO: 10)

[0105] These seven sequences were concatenated, and the concatenated sequence

(shown below) was compared to sequences of the same loci from representative

Burkholderia species available from the Bcc MLST database (pubmlst.org/5cc/) to construct a phylogenetic tree. Neighbor-joining trees were built in MEGA version

5.05 software and significance was evaluated by bootstrap analyses.

[0106] The results are shown in Figure 2, which shows a neighbor-joining tree for

Isolate A396 and 56 other Burkholderia species.

[0107] Burkholderia sp. strain A396, concatenated MLST sequence:

TGGTCCGCACCATCTGTCTGGGTGCATCCGACGGCCTGCGCCGCGGTCTGGTGGTGAAGA

ACACGCGGAAGCCGATCTCGGTGCCGGTCGGCAAGCCGACCCTCGGCCGGATCATGGACG

TGCTCGGCCGTCCGATCGACGAAGCGGGCCCGATCGAAAGCGAAACGACGCGTTCGATCC

ACCAGAAGGCGCCGGCGTTCGACGAACTGTCGCCGTCGACCGAACTGCTCGAAACCGGCA

TCAAGGTCATCGACCTGATCTGCCCGTTCGCGAAGGGCGGCAAGGTCGGTCTGTTCGGTG

GTGCAGGGGTGGGCAAGACCGTCAACATGATGGAGCTCATCAACAACATCGCGAAGGAG

C

ACGGCGGTTACTCCGTGTTCGCGGGCGTGGGCGAGCGTACCCGTGAAGGGAACGACTTCT

ACCACGAAATGAAGGACTCGAACCGCGAAGTTCGACGACCTCGTCGGCCGCGCCGATCTG

CTCGACACCCGCAAGGGCATCGAGCACTGGAAGGCGAAAGGCCTCGATTTCGCGCGCGTG

TTCTACCAGCCGGAAGGCTGCGAGGAAGTGGCGCGCCGCCATGTCGAGAGCCAGGAGCA

C

GGCCTCGAACGCGCGCTCGACCATACGCTGATCGAGAAGGCGAAGGCCGCGATCGAGAG T

GGCGAGCACGTGTCGTTCATCCAGCCGGTGCGCAACGTGAACCGCACGGTCGGCGCGATG

CTGTCCGGCGCGATCGCGAAGCGGCACGGCCACGACGGTCTCGCCGACGATGCGATCCAC

ATCCAGCTCAAAGGCACGGCGGGGCAGAGCTTCGGCGCGTTCCTCGCGAAGGGCGTGACG

CTCGCTACAAGGTGTCGGGCGGGCTGCATGGGGTCGGCGTGTCGTGCGTGAACGCGCTGT

CGAGCTGGTTGCGGCTGACCGTGCGGCGCAACGGCAAGAAGCACTTCATGGAGTTTCATC

GCGGCGTCGCGCAGAACCGCGTGCTCGAAGAGCAGGACGGCGAGCAGGTGTCGCCGATG

C

AGCTCGTCGGGCCGACGGAGAATCGCGGGACTGAAGTGCATTTCATGGCCGATCAGACGA TCTTCGGCACGGTCGAGTTTCACTACGACATTCTCGCGAAGCGGATTCGCGAGCTGTCGT

TCTTGAATAACGGCGTGCGGATTCGGTTGACCGACCAGCGCTCGGGCAAGGAAGACGATT

TCGCATTCGCCGGAGGCGTGAAGGGTTTTGTCGAGTACATCAACAAGACCAAGCAGGTGC

TGCATCCGACGATTTTCCACATCGTCGGCGAGAAGGAGTCGGCGATACGGTCACGCATGC

GACCAAGTCGGCGCTCGCGCCGCTGCCGGGCTTCAAGGAAGTGAAGCCGCAGGTGTTCGC

GGGCCTCTATCCGGTCGAGGCGAACCAGTACGACGCGCTGCGCGAGTCGCTCGAGAAGCT

CAAGCTGAACGATGCGTCGCTGCAGTACGAGCCGGAAGTGTCGCAGGCGCTCGGCTTCGG

CTTCCGCTGCGGCTTCCTCGGCCTGCTGCACATGGAGATCGTGCAGGAACGGCTCGAGCG

CGAGTTCGACATGGACCTGATCACGACCGCGCCGACCGTCGTCTACGAGGTCGTGAAGAG

CGACGGCGCGACGATCACGGTCGAGAATCCGGCGAAGATGCCGGAGCTGAGCCGGATCG

G

CGAGATCCGCGAGCCGGCGTACGAAGTGGATCGAGGACCAGAAAGCGCTGGGCTTCGATT

TCGTGGCATCCGAGGGCAACGTCGGCGACTGGGACTCCACCAAGCAGGCGTTCGACAAGG

TCAAGGCCGAGGTCGGCGAGGTCGACGTGCTGGTCAACAACGCCGGCATCACGCGCGACG

TCGTGTTCCGCAAGATGACGCACGAGGATTGGACGGCCGTGATCGACACGAACCTCACGA

GCCTCTTCAACGTCACGAAGCAGGTGATCGACGGCATGGTCGAGCGCGGCTGGGGGCGGA

TCATCAACATCTCGTCGGTGAACGGCCAGAAGGGTCAGTTCGGTCAGACCAACTACTCGA

CCGCGAAGGCCGGCATCCACGGCTTCACGATGTCGCTCGGCGCTGGCGGCTGCGCTCTCG

CAGATCGAGAAGCAGTTCGGCAAGGGCTCGATCATGCGCCTCGGCGCAGGCGAAGCGGTC

GAGGACATCCAGGTGGTGTCCACGGGCTCGCTGGGTCTGGATATCGCGCTCGGCGTCGGC

GGCCTGCCGCGCGGCCGGGTGGTCGAGATCTACGGCCCGGAATCGTCCGGCAAGACCACG

CTGACGCTGCAGGTGATCGCCGAGATGCAGAAGCTCGGCGGCACGGCGGCGTTCATCGAT

GCCGAGCACGCGCTCGACGTGCAGTACGCGGGCAAGCTCGGCGTGAACGTGCAGGAGCT

G

CTGATCTCGCAGCCGGACACGGGCGAGCAGGCGCTCGAGATCGTCGACGCGCTGGTGCGC

TCGGGCTCGATTTCATCGGCACGGTCGCCGGCCCGCATCCGTATCCGATGATGGTGCGCG

ACTTCCAGCGCGTGATCGGCGACGAGTGCAAGGTGCAGATGCCCGAGCTGGCCGGCCGTC

AGCCGGATGCGGTGATCGCCTGCGTTGGCGGCGGCTCGAACGCGATGGGCATCTTCTATC

CGTACATCGACGATCGCGACGTGCAGCTGATCGGCGTCGAGGCGGCGGGCGACGGGCTCG

ACTCGGGCCATCACGCGGCCTCGCTGATCGCCGGCAGCCCGGGCGTGCTGCACGGCAACC

GCACCTACCTGCT (SEQ ID NO: 11 )

[0108] The sequence type for A396 was submitted to the database and designated ST669. Analysis of the MLST sequences presented above (SEQ ID NOs:4-10) indicated that these seven loci have unique sequences when compared to all available allele types in the MLST database (www.pubmlst.org/bcc/). It is also noteworthy that no amplification of the Isolate A396 recA gene was obtained using 5cospecific primers. Thus, A396 does not share any allele types with any other Bcc or non-5cc isolates in the database. The lack of homology to members of the Bcc group makes it unlikely that Isolate A396 is pathogenic to humans.

Example 5: DNA-DNA Hybridization

[0109] DNA-DNA hybridization (DDH) experiments were performed (DSMZ, Braunschweig, Germany) with DNA from isolate A396 and DNA from type strains that were identified as closely -related according to 16S rRNA phylogenetic results, fatty acid methyl ester (FAME) profile and/or phenotypic characterization; namely, B. multivorans, B. glumae DSM 9512^T (=LMG 2196^T), B. plantarii DSM 9509^T (=LMG 9035^T) and B. cenocepacia DSM 16553^T (=LMG 16656^T). DNA was isolated using a Thermo Spectronic French Pressure Cell (Thermo Spectronic, USA) and was purified by column chromatography on hydroxyapatite as described by Cashion et al. (10). DDH was carried out as described by De Ley et al. (18) with the modifications described by Huss et al. (26) using a model Cary 100 Bio UV/VIS -spectrophotometer equipped with a Peltier Thermostatted multicell changer (Agilent Technologies, Santa Clara, CA) and a temperature controller with in-situ temperature probe (Varian, Palo Alto, CA).

[01 10] The results are shown in Table 2. All DDH experiments yielded low or intermediate DNA -DNA similarities ranging from 1 1.5 to 44.5%. Based on the recommended threshold value of 70% DNA-DNA similarity for the definition of bacterial species (74), isolate A396 is not identical with any of the species tested.

Table 2: Results of hybridization analyses between DNA from Isolate A396 and closely related Burkholderia species

B. cenocepacia B. glumae B. multivorans B. plantarii

% similarity to A396 35.7-44.5 % 1 1.5-20.6 % 37.3-37.4% 33.4-37.4%

Example 6: Antibiotic sensitivity

[01 11] Antibiotic sensitivity of Isolate A396 was tested using antibiotic susceptibility discs (BD Biosciences, Franklin Lakes, NJ) on Muller-Hinton agar inoculated with A396 to confluent growth. Growth inhibition, indicated by a clear halo around the disc, was evaluated after 72 hours incubation at 25°C.

[01 12] These analyses revealed that growth of A396 on Muller-Hinton agar plates was suppressed by kanamycin (30 ug), chloramphenicol (30 ug), ciprofloxacin (5 ug), piperacillin (100 ug), imipenem (10 ug) and sulphamethoxazole/trimethoprin (23.75 ug/25 ug). Growth was resistant to tetracycline (30 ug), erythromycin (15 ug), streptomycin (10 ug), penicillin (10 ug), ampicillin (30 ug), oxytetracycline (30 ug), gentamycin (30 ug) and cefuroxime (30 ug).

[01 13] Additional information related to antibiotic susceptibility was obtained from phenotypic microarray analysis. The antibiotic sensitivity profile (diverse antibiotics distributed across several plates) indicated that A396 is sensitive to cloxacillin, minocycline, nalidixic acid, oxacillin, novobiocin, sulfadiazine, tylosin,

oleandomycin, vancomycin and sulfisoxazole at concentrations ranging from 1 to 4 μg/ml, and resistant to polymyxin B at concentrations of 1-4 μg/ml. Example 7: Additional biochemical characterization

[01 14] Biochemical characterization of Isolate A396 and related Burkholderia species was conducted with GENIII Microbial Identification plates (Biolog, Hayward, CA), used according to the manufacturer's guidelines. The results are shown in Table 3.

The inability of Isolate A396 to metabolize arabinose, cellobiose and citrate

distinguish it from most other Burkholderia species.

Table 3: Biochemical characteristics of

strain A396 and closely related Burkholderia species

A396 B. cenocepacia B. glumae B. multivorans B. plantarii

Assimilation of:

L-Arabinose - + + + +

Cellobiose - + (variable) + +

D-Glucose + + + + +

Lactose - + - +

Maltose +

Raffinose - + (variable)

D-Xylose + + + +

Dulcitol - + + +

D-Mannitol + + + + +

Caprate + + + + ±

Citrate + + + ±

Phenylacetate - + - +

[01 15] In addition, a more extensive characterization of biochemical capabilities was performed using a complete Biolog phenotypic microarray panel. According to

phenotypic microarray results, A396 is capable of using the following substrates as carbon sources for growth: L-proline, D-trehalose, D-mannitol, L-glutamic acid, D- glucose-6-phosphate, a-D-glucose, L-glutamine, D-fructose-6-phosphate, L-malic

acid, pyruvic acid, γ-amino-N-butyric acid, butyric acid, capric acid, caproic acid, 5- keto-D-gluconic acid and dihydroxyacetone.

[01 16] The following compounds can be used by A396 as nitrogen sources for

growth: ammonia, nitrite, nitrate, urea, L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-glutamic acid, L-glutamine, glycine, L-histidine, L-isoleucine, L-lysine, L- phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine,

D-alanine, D-asparagine, D-glutamic acid, D-serine, L-homoserine, L-pyroglutamic acid, ethanolamine, putrescine, agmatine, β-phenylethylamine, N-acetyl-D- glucosamine, adenine, adenosine, cytosine, guanosine, thymine, thymidine, uracil, inosine, xanthine, xanthosine, uric acid, allantoin, parabanic acid, γ-amino-N-butyric acid, ε-amino-N-caproic acid, and a-amino-N-valeric acid. Isolate A396 does not grow at or above 2% NaCl, 3% KC1 or 4% urea. No growth was detected at pH < 4 and >10.

[01 17] Growth was tested at a number of different temperatures and cells were found to grow well at 25, 30 and 37°C, and more slowly at 16°C.

Example 8: Fatty acid analysis of Isolate A396

[01 18] Fatty acid composition was determined by Microbial ID, Inc. (Newark, DE) according to established methods. The most abundant fatty acids in Isolate A396 were Ci_6:0 (24.47%), Ci_7:0 cyclo (8.394%), summed feature 2 (might include 12:0 aldehyde, 16: 1 isol, 14:0 30H and an unknown peak at 10.95; 5.7%), summed feature 3 (might include 16: lco7c, and co6c; 19.65%) and summed feature 8 (might include 18: lco7c, and co6c; 25.16%). The following fatty acids contributed less than 2% each to the total fatty acid composition: 14: lco5c, 17:0, 16: 1 20H, 16:0iso 30H, 16:0 20H, summed feature 5, 18:0, 18: lco7c, l ime, 19:0 and 18: 1 20H.

[01 19] The A396 fatty acid profile was compared to related species and to the MIDI Sherlock database. A similarity index of 0.879 with B. cenocepacia GC subgroup B was observed, and close matches to B. cepacia and B. gladioli were also detected.

Example 9: Toxicity of whole cell broth and cell-free supernatant from Isolate A396 toward Spodoptera exigua (Beet armyworm)

[0120] A colony of Spodoptera exigua (Beet armyworm, BAW) was established from eggs obtained from Bio-Serv (Frenchtown, NJ). Larvae were kept in an incubator at 26°C, with a 12 hour photoperiod, and maintained on an artificial diet containing standard growth nutrients necessary for insect propagation. First and third instar larvae were used in feeding assays and in a contact bioassay. Early second instar larvae were used for leaf disc assays.

[0121] Toxicity was evaluated using whole-cell broth or cell-free supernatant from Isolate A396, both of which were prepared as described in Example 2. Artificial diet overlay assay

[0122] Larval toxicity via feeding was evaluated in artificial diet overlay assays using 96-well microtitre plates (Thermo-Fisher Scientific, Rochester, NY). Dilutions of 4.0, 2.0, 1.0 and 0.5% (v/v) of Isolate A396 whole-cell broth (WCB) or cell-free supernatant (CFS) were prepared in sterile distilled water. Sterile distilled water and Javelin^® WG (a commercial Bt product) were used as the negative and positive controls, respectively. One hundred and fifty microliters of BAW artificial diet was added to each well, followed by 100 iL of test substance, water, or Javelin^®. Forty replicates of each WCB and CFS dilution were assayed (i.e., 40 wells per dilution). The plates were dried in a fume hood at room temperature, and one first instar BAW larva was introduced into each well. Plates were sealed with clear sheets of adhesive mylar and a pinhole was made in the seal over each well for aeration. Plates were then incubated at 26°C with a 12 hour photoperiod. Larval mortality was assessed 3 and 4 days after exposure to the treated diet, and average percent mortality of larvae was determined for each treatment.

[0123] The results of this bioassay are shown in Table 4. As demonstrated therein, consumption by 1^st instar BAW larvae of diets treated with different dilutions of A396 WCB and CFS resulted in high mortality. Eighty five percent of the larvae that ingested diet treated with 1.0% A396 WCB died within 3 days. Three-day mortality was 56% after larvae ingested diet treated with 0.5% A396 WCB. Four days after ingestion, the diet treated with A396 WCB at 0.5 and 1.0% resulted in 88.2 and 97.4% mortality, respectively. The negative control exhibited 2% larval mortality. Efficacy of cell- free supernatant was lower than that of WCB. At the maximum dose of 4%, 88.5% mortality was reached by day 4, compared to 100% with WCB. Cell-free supernatants dosed at 2% were half as effective as WCB at the same concentration.

Table 4: Effect of ingestion of WCB and CFS from Isolate A396 on Beet armyworm larvae

Cell-free supernatant Whole-cell broth

Concentration Avg. mortality Avg. mortality Avg. mortality Avg. mortality (vol/vol) on Day 3 (%) on Day 4 (%) on Day 3 (%) on Day 4 (%) 0.5% 1 1.5 20.8 55.6 88.2 1.0% 30.8 37.5 85 97.4

2% 28.6 46.4 90 95

4% 57.7 88.5 90 100

Avg. mortality Avg. mortality Avg. mortality Avg. mortality

Controls on Day 3 (%) on Day 4 (%) on Day 3 (%) on Day 4 (%)

Javelin 98.2 100 100 100 Water 3.6 3.6 1.9 1.9

Leaf disc bioassay

[0124] Toxicity via feeding was also evaluated using treated broccoli leaf discs on 1% water agar in petri plates. Discs were excised from broccoli leaves with a 42mm diameter corer and treated with a 3.0% (vol/vol) solution of A396 WCB, in sterile distilled water. Broccoli discs treated with 3% (vol/vol) Xentari^® (a commercial Bt product) and sterile distilled water were used as the positive and negative controls, respectively. Leaf discs were immersed in each treatment solution for one minute, air dried, and then placed on the agar, abaxial side up. Four newly emerged second instar BAW larvae were introduced into the agar plates containing the treated leaf discs. The agar plates were then covered with Parafilm (punctured with holes for aeration) and kept at room temperature with a 12 hour photoperiod. Treatments were replicated six times. Mortality of larvae was evaluated after 72 hours of exposure to treated leaf discs.

[0125] The results, shown in Figure 3, indicate that, after feeding on A396 WCB- treated leaf discs for 3 days, the mortality of BAW second instar larvae was 75±22 %. This value was not statistically different from the observed mortality of larvae treated with Xentari^®; but was significantly greater that the mortality observed in control (water-treated) larvae. Repeated trials provided consistent results, with A396 WCB providing good control of BAW larvae.

[0126] Experiments were also conducted on BAW first instar larvae with similar results; i.e., exposure of first instar larvae to diets containing dilutions of A396 WCB and CFS resulted in statistically significant high levels of larval mortality. Contact bioassay

[0127] A newly-emerged third instar BAW larva was placed in each of thirty 1.25-oz clear plastic cups (PL1, Solo Cup Company, Highland Park, IL) containing a 1 cm² cube of BAW artificial diet. One microliter of undiluted A396 WCB was applied to the thorax of ten of the larvae using a PB-600 micropipette (Hamilton, Reno, NV). Ten larvae were also treated in the same way with 1 μΐ of heat-treated A396 WCB. To the remaining ten larvae, which served as negative controls, one microliter of sterile water was applied to the thorax.

[0128] The cups containing the treated larvae were covered with Parafilm, punctured for aeration, and incubated at room temperature with a 12 hour photoperiod.

Mortality and morbidity (e.g., stunted growth) were assessed 3 days after treatment, and at the times that negative (water-treated) controls pupated and eclosed. The bioassay was conducted in duplicate.

[0129] The results are shown in Tables 5 and 6. Contact mortality after topical application of A396 WCB to the larval thorax averaged 50% (±20%) at three days after treatment with WCB and up to 90% at three days after application of heat-treated WCB. Of the surviving larvae at three days after treatment with either WCB or heat- treated WCB, an average of 35% exhibited stunted growth. All surviving larvae pupated; however, several did not eclose and others eclosed as stunted individuals and died. Most of the water-treated controls survived, pupated and eclosed normally. Treated larvae also exhibited discoloration and produced liquefied frass.

Table 5: Contact mortality and morbidity in first instar BAW larvae exposed to A396 WCB: Experiment 1*

*Values in all columns are for number of individuals (except for the right-most column), and are presented with respect to all ten starting larvae in each treatment group;

Table 6: Contact mortality and morbidity in first instar BAW larvae exposed to A396 WCB: Experiment 2*

Water 1 0 9 1 0 9 9 10

HT-WCB 4 1 5 7 2 1 1 90

WCB 1 2 7 4 4 2 2 80

*Values in all columns are for number of individuals (except for the right-most column), and are presented with respect to all ten starting larvae in each treatment group;

[0130] In summary, both external contact with, and ingestion of, A396 WCB results in insecticidal activity against Beet armyworm larvae, characterized in part by disruption of molting and cuticle formation. These modes of toxicity are consistent with the action of one or more chitinase enzymes, acting by hydrolysis of the gut lining (e.g., peritrophic membrane) and/or the cuticle of the larva.

Example 10: Toxicity of whole cell broth from Isolate A396 toward

Tetranychus urticae (Two-spotted spider mite)

[0131] A colony of Tetranychus urticae (Two-spotted spider mite, TSSM)) was established from specimens collected in Davis, CA. TSSM was reared on lima beans, Phaseolus lunatus, at 26°C with a 12 hour photoperiod.

[0132] The in vivo efficacy of A396 WCB and heat-treated A396 WCB against TSSM was evaluated in a fava bean leaf disc bioassay. Twelve-well polystyrene plates (Thermo-Fisher Scientific, Rochester, NY) were filled with water-saturated cotton. Fava bean leaf discs were made using a ¾ inch-diameter cork borer.

Solutions of A396 WCB or heat-treated A396 WCB were prepared at 6% (vol/vol) in 0.01% Tween 20. WCB was heat-treated by incubation at 60°C for two hours.

Distilled water and Avid^® 0.15 EC (10% v:v) were used as negative and positive controls, respectively. Leaf discs were immersed in each treatment solution for one minute, then air dried. A treated disc was transferred to each well containing water- saturated cotton. Ten adult TSSM were introduced onto each treated leaf disc, and each treatment was replicated 6 times. Treated TSSM were incubated at room temperature with a 12 hour photoperiod, and mortality of adult mites was evaluated 3 days after their introduction onto the treated leaf discs. Mortality data were analyzed using one-way ANOVA, and significant differences among treatment means were then separated using Fisher's least significant difference at p<0.05 (LSD) (PROC GLM, SAS Institute, 2011).

[0133] The results are shown in Figure 4. A396 WCB at 6% (vol/vol) exhibited significant activity against TSSM adults. Mortality was 93% three days after exposure to treated leaf discs, which was significantly greater than the mortality observed in water-treated mites (paired t-test, P = 0.023) and not significantly different from the mortality obtained with Avid^® treatment. Heat-treated WCB exhibited slightly less miticidal activity than untreated WCB in this experiment; although the difference was not statistically significant. When observed under a microscope, TSSM adults that had died due to A396 treatment were soft, dark in color (i.e., melanized), and disintegrated readily when touched with a paint brush or a pin, indicative of loss of integrity of the exoskeleton.

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Claims

CLAIMS What is claimed is:

1. A method for modulating mite infestation in a plant, the method comprising:

applying to the plant, seeds, or substrate used for growing said plant, an amount of substantially pure culture, whole-cell broth, cell fraction, supernatant, filtrate, or extract obtained from a strain of Burkholderia NRRL B-50319, effective to modulate said mite infestation.

2. A miticidal composition comprising a substantially pure culture, whole-cell broth, cell fraction, supernatant, filtrate, or extract obtained from a strain of Burkholderia, wherein the genome of said strain comprises one or more nucleotide sequences selected from the group consisting of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO: 10.

3. The composition of claim 2, further wherein the strain is sensitive to one or more of the following antibiotics: cloxacillin, minocycline, nalidixic acid, oxacillin, novobiocin, sulfadiazine, tylosin, oleandomycin, and sulfisoxazole at a concentration of 1-4 μg/ml each.

4. The composition of claim 2, further wherein the strain is resistant to polymyxin B at a concentration of 1-4 μg/ml.

5. A composition comprising:

(a) a culture, whole-cell broth, cell fraction, supernatant, filtrate, or extract according to claim 1 ; and

(b) at least one of

(i) a chemical or biological pesticide, and

(ii) at least one of a carrier, diluent, surfactant, or adjuvant.

6. The composition according to claim 5, wherein the chemical or biological pesticide is selected from the group consisting of an herbicide, a bactericide, a nematicide, a fungicide and an insecticide.

7. The composition according to claim 5, wherein the pesticide is selected from the group consisting of Grandevo^® and Regalia^®.

8. The composition according to claim 6, wherein the herbicide is selected from the group consisting of sarmentine and thaxtomin.

9. A method for modulating mite infestation in a plant, the method comprising applying to the plant and/or seeds thereof and/or substrate used for growing said plant an amount of the composition of either of claims 2 or 5 effective to modulate said mite infestation.

10. The method of claim 9, wherein the mite is Tetranychus urticae (two- spotted spider mite).

11. The method of claim 9, wherein the chemical or biological pesticide is selected from the group consisting of an herbicide, a bactericide, a nematicide, a fungicide and an insecticide.

12. The method of claim 9, wherein the pesticide is selected from the group consisting of Grandevo^® and Regalia^®.

13. The method of claim 1 1, wherein the herbicide is selected from the group consisting of sarmentine and thaxtomin.

14. Use of the composition of claim 2, optionally in combination with a second substance, to formulate a miticidal composition, wherein the second substance is selected from the group consisting of one or more of:

(a) a chemical or biological pesticide,

(b) a plant growth-promoting agent,

(c) a carrier,

(d) a diluent,

(e) an adjuvant, (f) a surfactant,

(g) a fertilizer, and

(h) an anti-phytopathogenic agent.

15. The use according to claim 14, wherein the miticidal composition is used for modulating Tetranychus urticae (two-spotted spider mite) infestation in a plant.

16. The use according to claim 14, wherein the pesticide is selected from the group consisting of an herbicide, a bactericide, a nematicide, a fungicide and an insecticide.

17. The use according to claim 14, wherein the pesticide is selected from the group consisting of Grandevo^® and Regalia^®.

18. The use according to claim 16, wherein the herbicide is selected from the group consisting of sarmentine and thaxtomin.

19. A plant comprising the composition of either of claims 2 or 5.

20. A seed comprising the composition of either of claims 2 or 5.