NZ621971B2 - Chromobacterium formulations, compostions, metabolites and their uses - Google Patents

Abstract

Disclosed is a pesticide formulation comprising a whole cell broth, filtrate, supernatant, or extract of a strain of Chromobacterium species containing a the metabolites violacein, deoxyviolacein, or chromamide A. The formulation is used in methods for modulating pest infestations. Also disclosed is the addition of a stabilizing agent such as a benzoic acid salt or lignosulfonate salt to the presticide formulation to give an improved shelf life due to maintenance of physical uniformity and longer insecticide activity after use due to higher resistance to degradation when exposed to sunlight. the addition of a stabilizing agent such as a benzoic acid salt or lignosulfonate salt to the presticide formulation to give an improved shelf life due to maintenance of physical uniformity and longer insecticide activity after use due to higher resistance to degradation when exposed to sunlight.

Description

CHROMOBACTERIUM FORMULATIONS, TIONS, METABOLITES AND THEIR USES TECHNICAL FIELD Provided is the use of or compositions or formulations comprising Chromobacterium species, filtrate, supernatant, extract, pesticidally active compound or metabolite derived therefrom as an acaricide and insecticide, particularly against infestation of one or more pests belonging to the Acarina, Scarabeidae, Drosophilidae, TriozidaeAphidae, Muscidae, Anthomyiidae or Tenebrionidae families. Further ed are biological pesticide (also referred to as biopesticide) formulations, particularly those comprising Chromobacterium species, filtrate, supernatant, extract, metabolites or pesticidally active nds derived therefrom, s for producing them and methods of use as modulating pest infestation. More specifically, provided are stabilized biological pesticides having improved shelf life due to maintenance of physical uniformity and longer insecticide activity after use due to higher resistance to degradation when d to sunlight.

BACKGROUND Natural ts are substances produced by microbes, plants, and other organisms.

Microbial natural products offer an abundant source of chemical diversity, and there is a long y of utilizing l 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 ial to play an important role in controlling pests in both conventional and organic farms.

Secondary metabolites produced by microbes (bacteria, actinomycetes and fungi) provide novel chemical compounds which can be used either alone or in ation with known compounds to effectively l insect pests and to reduce the risk for resistance development. There are several well-known examples of microbial natural products that are successful as agricultural icides (Thompson et al., 2000; Arena et al., 1995; Krieg et al. 1983).

The development of a microbial pesticide starts with the isolation of a microbe in a pure culture. It then proceeds with efficacy and spectrum screening using in vitro, in vivo or pilot scale trials in a ouse and in the field. At the same time, active compounds ed by the microbe are isolated and identified. For the cialization of a microbial pesticide, the microbe has to be economically produced by fermentation at an industrial scale and formulated with patible and approved additives to increase efficacy and to maximize the ease of application as well as storage stability under field conditions. 2012/061503 Chromobacterium In 2000, Dr. Martin and her coworkers at USDA isolated a -pigmented bacteria (PRAA4-l) from a forest soil in Maryland (Martin et al., 2007a). In the initial screening, they found this bacteria to be toxic to Colorado potato beetle and other insect pests n et al., 2007b). This motile, Gram-negative, bacteria was identified as a new species of Chromobacteria, Chromobacterium substsugae sp. nov n et al., 2007c). It is a facultatively aerobic, motile, Gram-negative betaproteobacterium with polar flagella. Colonies formed at 2-3 days on an L-agar plate at 25°C are initially cream colored, gradually turning light to dark violet during the following 24 hours. Colonies of l grow well on peptone based media with an optimum at 25°C, pH 6.5-8.0, and with 0-1.5 % (w/v) NaCl (Martin et al., 2007a).

Since the finding of C. substugae by Martin and her ers, at least three new species of Chromobacteria have been isolated, and characterized; Young et al. (2008) isolated a novel Chromobacterium species, C. aquaticum, from spring water samples in , and Kampfer et al. (2009) isolated two species, C. piscinae and C. pseudoviolaceum, from nmental samples collected in Malaysia.

Of all known species of Chromobacteria, C. violaceum, a gram-negative saprphyte from soil and water. Published information on secondary metabolites produced by Chromobacteria is based on s on C. violaceum only (see, for example, Duran and Menck (2001) for a comprehensive review of the pharmacological and industrial perspectives of C. violaceum). It is normally considered nonpathogenic to humans, but as an opportunistic pathogen, it has occasionally been the causative agent for septicemia and fatal infections in humans and animals. C. violaceum is known to e a purple pigment, violacein, which is a bisindole molecule generated by a fusion of two L- tryptophan les in the presence of oxygen (Hoshino et al., 1987; Ryan and Drennan; 2009).

Violacein biosynthesis is regulated by quorum-sensing, a common mechanism regulating various other secondary metabolism pathways in egative bacteria an et al., 1997).

Other known metabolites of C. violaceum summarized by Duran and Menck (2001) include hydrogen cyanide, ferrioxamine E, B-lactamic glycopeptides 04 and SQ28,546, antibiotics such as aerocyanidin, aerocavin, 3 ydroxy-indoxazene, and monobactam SB-26.180, and an antitumoral depsipeptide FR901228. According to the review article by Duran and Menck (2001), C. violaceum also produces unusual sugar compounds such as extracellular polysaccharides and lipopolysaccharides.

US patent application publication no. US20120100236 also ses compounds obtainable or derived from Chromobacterium species, more particularly, Chromobacterium substugae.

Mites and Acaricides Tetranychus urticae (Two spotted spider mite) is a member of the Tetranychidae family.

Spider mites are perhaps the most important mite pests of ornamentals. They also cause 2012/061503 considerable damage in more than 180 species of greenhouse and field crops. These mites are also among the most difficult arthropod pests to l and resistance to chemicals can develop y (Stamps and Osborne 2009, Osborne, Ehler and s, 1999).

Acaricides are compounds that kill mites (miticides) and ticks (ixodicides). This class of pesticides is large and includes antibiotics, carbamates, formamidine acaricides, pyrethroids, mite growth regulators, and organophosphate acaricides. Besides chemical pesticides, diatomaceous earth and fatty acids can be used to control mites. They typically work through disruption of the cuticle, which dries out the mite. In addition, some essential oils such as peppermint oil, are used to control mites. In spite of the great variety of known acaricide nds, mites remain a serious problem in agriculture because of the damage they cause to the crops. They can produce several generations during one season, which facilitates rapid development of resistance to the acaricide products used.

Hence, new pesticide products with new target sites and novel modes of action are ally needed.

House Flies Musca ica (House flies) are members of the family Muscidae. This family is considered an economic problem domestically and worldwide. Other members of the Muscidae family include face fly, stable fly, and horn fly. They are considered a nuisance and are vectors of human and animal es. Their habits of walking and g on garbage and excrement and on the humans and food make them ideal agents for the transfer of disease organisms. This species can also be a pest to animals and transmit disease through open wounds.

Plant Feeding Flies — Spotted Wing Drosophila The spotted wing Drosophila, Drosophila suzukii is a recent invader to the fruit and vegetable growing areas in the United States. It is far more destructive than a well known related species Drosophila melanogaster and other Drosophila because D. suzukii can feed on and damage in-tact fruits and vegetables, while other Drosophila only feed on decaying plant material.

Root Maggots Root maggots of the family Anthomyidae feed on the roots of l different plants.

Cabbage Root Maggots affect cabbage, cauliflower, broccoli, and Brussels sprouts. (This group of vegetables is also known as ‘cole crops’). Different types of root maggots also occur that affect carrots, onions, and other vegetable crops. Because cole crops are cool-season vegetables, Cabbage Root Maggots are much more prominent in rn zones of the US. They are difficult to control, because they hatch and feed underneath the soil, so you may only know they are there when you notice stunted growth or g foliage.

Green Peach Aphids Myzus persicae, (green peach aphids) are members of the Aphididae family (see US20110054022). As evident by its common name, green peach aphids are pests of a wide range of fruits, vegetables and ornamental plants and have a worldwide presence. These insects are particularly harmful since they not only cause direct damage by feeding on phloem sap but are also potential s for the plum pox virus, the causal agent for Sharka disease, which causes fruit deformation and oration. As a result, infected trees must be uprooted. Attempts have been made to control these pests with various ides. However, resistance is often developed.

Potato Psyllid Bactericera cockerelli, (potato psyllid) is a member offlthe Triozidae family and is a causative agent of zebra chip disease via infestation of gram negative ia. Although it is native to North America, it has been found in New Zealand as well (www.biosecurity.govt.nz/files/pests/potato-tomato-psyllid/psyillid-factsheet.pdf). The potato psyllid generally breeds in solanaceous hosts (such as tomatoes and potatoes). However, they have been found in other plants as well such as capsicum, chilli, eggplant, kumara, poroporo, tamarillo and thornapple.

Litter Beetles obius diaperinus is a serious pest in the poultry industry and is a member of the Tenebrionidae family. The El strain PS86Bl reportedly has activity against Alphitobius (US Pat.

No. 665 to Hickle et al.). Br tenebrionis may have activity against larvae of this beetle as well (US Pat. No. 5,244,660). Litter beetles and a few other coleopteran species act as vectors for protozoan, bacterial, and viral es of chickens and turkeys resulting in significant ic loss. Litter beetles act as a significant reservoir for pathogenic Salmonella species including the more pathogenic varieties, such as S. enterica serotype enteritidis. The problem is that poultry contaminated with pathogenic sms like Salmonella threaten human health. These beetles inhabit the litter, wood, Styrofoam, fiberglass, and polystyrene insulation panels of chicken houses.

Larvae and adult beetles thrive both on bird droppings and on grains used as chicken feed. These large beetle populations and their e habitats within chicken houses make it more difficult to eradicate the Salmonella they carry. In the midst of a heavy litter beetle infestation, or prior to establishing new n populations neither frequent changes in the litter nor dusting with multiple chemical insecticides is a completely effective l for this pest.

Grubs and Scarabs Grubs, such as white grubs (Cyclocephala lurida), Southern Masked , (Rhizotrogus majalz's) se beetle larvae, (Papillia japom'ca) black vine weevil larvae (Otiorhynchus sulcatus), oriental beetle larvae (Anomala orientalz's), members of the aeidae family, have been found to infest turf and pasture grasses. Adult scarabs have been found to infest ornamental plants, and numerous crops around the world. Various pesticides have been tried and include chemical pesticides, nematodes (see, for example, US Patent No. 7,641,573) and Bacillus thuringiensis (see US Patent No. 5,185,158), peromones, and natural repellents such as catnip and chives.

Polyhydroxyalkanoates (PHAs) Bio-plastic is d as a form of plastic sized from renewable resources such as plant starch and microbial species. Some of the biodegradable plastic als under development include polyhydroxyalkanoate (PHA), polylactide, aliphatic polyesters, polysaccharides, and the copolymers and/or blends of these. PHAs in particular include several polymeric esters such polyhydroxybutyrates, droxybutyrate co-hydroxyvalerates (PHBV), polyhydroxybutyrate co- hydroxyhexanoate (PHBHx) and polyhydroxybutyrate roxyoctonoate (PHBO). Poly3- hydroxybutyric acid (PHB) is the most common natural ial PHA. Polyhydroxyalkanoates are 100% biodegradable polymers. Since they have similar properties to various synthetic thermoplastic like polypropylene, PHAs can be used in their place. They are also totally degraded to water and carbon dioxide under c conditions and to methane under anaerobic conditions by micro-organisms in soil, lake water, sewages and sea water. Depending on the number of carbon atom in the chain, PHAs have been divided into two groups: short-chain length (SCL) which consists of 3-5 carbon atoms, and medium-chain length (MCL) which consists of 6-14 carbon atoms (Khanna S, Srivastava AK. 2005). These differences are mainly due to the substrate specificity of the PHA synthases that can accept 3HAs of a certain range of carbon length. The other nown PHA is the copolymer, poly (3-hydroxybutyrate-c0hydroxyvalerate) c0-3HV), which S LC comprise of four- and five-carbon ric units. The tion of these monomeric units can vary, and this affects the physical properties of the polymer, i.e. less brittle with increasing proportion of 3HV unit.

In some microbial species, accumulation of PHA occurs during the presence of excess carbon and a limitation of nitrogen sources (Verlinden et al., 2007). PHAs produced in response to stressful conditions serve as energy storage molecules to be utilized when common energy sources are absent (Solaiman and Ashby, 2005). The plastic polymers accumulate intracellularly as light refracting amorphous storage granules in these organisms (Mukhopadhyay et al., 2005). PHB is synthesized from acetyl-CoA using three tic steps (Krans et al., 1997). From a biotechnological point of view, the ability of bioplastics to be biodegradable makes them a ble substitute for petrochemical-based plastic, an environmental pollutant (Lee, 1996). Increased WO 62977 production of bioplastics can significantly reduce carbon dioxide ons, curtail plastic waste generation and decrease consumption of fossil fuels.

PHAs can be obtained from the following three methods: biosynthesis by microorganisms, ynthesis by transgenic plants, and in vitro biosynthesis using appropriate enzymes (see, for example, US Patnet No. 7,455,999, WO99l43 13). In most bacteria, cells synthesize PHA under growth-limiting substrates other than carbon source such as nitrogen, phosphorus or oxygen.

Accumulated PHA serves as both carbon and energy source during starvation. PHA also serves as a sink for reducing power and could therefore be regarded as a redox regulator within the cell. PHAs are also useful as stereo regular compounds which can serve as chiral precursors for the al synthetic of optically active nds. Such compounds are particularly used as biodegradable carriers for long-term dosage of drugs, medicines, hormones, insecticides and herbicides (Reddy 2003). They are also used as osteosynthetic materials in the stimulation of bone growth owing to their piezoelectric properties, in bone plates, al sutures and blood vessel replacements (Schaefer et al., 2000). Furthermore, there have been disclosures of method of copolymer production by microbiological process using various bacteria e.g. Alcalz'genes eutrophus NCIMB 40124 (EP. 0431883A2) and US Patent No. 7,455,999. EP No. 2236089Al discloses uses of these polymers in multizone ts for orthopedic repair devices and soft tissue fixation devices. WO 9l/009l7Al discloses method for controlling and modifying novel polyester biopolymer by manipulation of the cs and enzymology of synthesis of polyhydroxybutyrate (PHB) and polyhydroxyalkanoate (PHA) polyesters at the molecular level in prokaryotic and eukaryotic cells, especially plants. Al discloses methods and uses as compostable packing materials. WO2008/11054ldiscloses the method of stabilization of polyhydroxybutyrates against l degradation.

Lignin Lignin is a pal constituent of the woody ure of higher plants. Processed lignin is obtained as a by-product of wood pulping reactions. Lignin products include, for example, lignin sulphonates, alkali lignins, and oxylignins which may be obtained from sulphite, te, and alkali waste liquors (Snook, 1982, Handbook for Pulp & Paper Technologists, TAPPI, Atlanta).

Lignin has been found to have a variety of commercial uses. For example, alkali soluble lignin has been used as a dispersing agent. US. Patent No. 3,726,850 discloses the use of an alkali soluble, ozone-treated lignin product, which is essentially free of organically bound sulfur, as a dispersing agent for clays, dyestuffs, pesticides, carbon black and other materials. US. Patent No. 4,666,522 discloses the use of lignosulphonate products for ing emulsions of waxes, oils, fats, ts, and mixtures thereof. Lignin e, has been reported to be useful for applications such as acting as a binder in water-based ng ink compositions. (See, e.g., US. Patent No. 051). US. Patent No. 183 discloses the use of lignin sulphonate products for dispersing luble substances. Furthermore, there have been disclosures of binding of lignin-pesticide complexes (see, for example, US. Patent No. 3,813,236, US. Patent No. 3,929,453, reissued as Re.

No. 29,238, US. Patent No. 4,381,194, US Patent Application Pub. No. 20110015237, US Patent Application Pub. No. 2010136132, US Patent Application Pub. No. 20100278890, US Patent Application Pub. No. 20080113920, US Patent Application Pub. No. 7130, US Patent No. 7,867,507, WO2003/005816, US Patent No. 5,994,266).

Sodium Benzoate Sodium benzoate has been used in various ations as an anti-microbial in food preparations. For example, US Patent No. 6,599,514 discloses synergistic antifungal compositions sing an antifungal agent and a food ve, which produces a synergistic effect on the l antifungal activity of the antifungal composition. Food additives disclosed in US Patent No. 6,599,514 included sorbic acid and sorbates, benzoic acid and benzoates, hydroxy-benzoates, sulphur dioxide and sulphites, biphenyl and tives, nitrites, nitrates, lactic acid, lactates, citric acid and citrates, tartaric acid and tartrates, orthophosphoric acid and hosphates, malates, adipic acid, succinic acid, 1,4-heptonolactone, nic acid, triammoniun citrate, ammonium ferric citrate, calcium disodium EDTA, glycerol, di-, tri- and polyphosphates, fatty acids (E470), mono- and diglycerides of fatty acids (E4 71), esters of mono- and erides of fatty acids, carbonates, gluconates, ne (E92S), sodium hexametaphosphate, butylated hydroxyanisole (BHA), butylated hydroxy toluene (BHT)(E321), l hydroquinone (THBQ), propyl gallate, m heptonate, calcium phytate, diethyl ether, EDTA, disodium ogen EDTA, ethyl acetate, glycerol mono-, di- and tates, glycine, oxystearin, propan-1,2-diol and propan01 and sodium heptonate.

Sodium benzoate has also been used in pesticide formulations. For example, US Patent 4,668,507 by SC Johnson teaches use of sodium benzoate in pesticides contained in rized steel aerosol delivery systems where main mode of stabilization is corrosion inhibition. US Patent No. 5,620,678 discloses an insecticide formulation that include sodium benzoate as corrosion inhibitor. US Patent No. 4,731,379 teaches insecticidal compositions that contain sodium benzoate when used as an animal shampoo to kill fleas. In this patent the use of sodium benzoate is not shown to increase effectiveness of the insecticide or stabilize the product but rather, is thought to assist in healing of wounds of the treated animal. US Patent No. 5,017,620 teaches insecticidal itions that contain sodium benzoate and other known preservatives when used as an anti-microbial to stabilize the product while in storage. US Patent No. 6,841,572 discloses an aqueous solution for treating live plants, crops, trees, pre-harvest fruits, vegetables, leaves, stems, roots and flowers having a pH of between 4.0 and 6.5 and consisting essentially of fungicidally and/or bactericidally In one , the present invention provides a method for modulating infestation of a pest, wherein said pest is selected from an Acari, Muscidae, Drosophilidae, Anthomyidae, Aphididae, Triozidae, Tenebrionidae, and Scarabaiedae, in a location where tion is desired comprising applying an amount of a composition obtained from Chromobacterium subtsugae Nov strain (NRRL B- 30655), effective for ting said infestation.

Also provided herein is a method for modulating infestation of arachnid (Acari or Acarina) AH26(10752692_1):CCG 0752692_1):CCG ae, Aphididae, Triozidae, Tenebrionidae or Scarabaiedae family comprising as active components: (a) a supernatant, filtrate and/or extract of Chromobacterium sp. and/or one or more metabolite(s) from said supernatant, filtrate and/ or extract of Chromobacterium sp. and (b) another pesticidal substance, particularly, an acaracide and/or insecticide which may be effective against one or more insect pests belonging to the Acari, Anthomyidae, Drosophilidae, Muscidae, Aphididae, Triozidae, Tenebrionidae or Scarabaiedae family wherein (a) and (b) may optionally be present in synergistic amounts. The pesticidal substance may be (a) derived from a microorganism; (b) a natural product and/or (c) a chemical pesticide and in particular a chemical insecticide.

In one embodiment, the metabolite may be a compound that (a) has pesticidal activity; (b) has a lar weight of about 840-900 as determined by Liquid Chromatography/Mass Spectroscopy (LC/MS) and (c) has an High Pressure Liquid Chromatography (HPLC) retention time of about 7-12 minutes on a reversed phase C-18 HPLC column using a water:acetonitrile (CH3CN) nt solvent system (0-20 min; 90-0 % aqueous CH3CN, 20-24 min; 100% CH3CN, 24-27 min; 0-90 % aqueous CH3CN, 27-30 min; 90% aqueous CH3CN) at 0.5 mL/min flow rate and UV detection of 210 nm and (d) is optionally obtainable from a Chromobacterium species. The compound in one embodiment may be a peptide.

In a ular embodiment, the compound has 43 carbons, seven , ten methylene carbons, twelve methines, 6 olefinic es, and eight nary carbons as determined by 13C NMR. In more particular embodiments, the compound encompasses compounds “A", “B", “C", “D", depicted as ##STR001#. ##STR001a##, ##STR001b##, ##STR001c## respectively.

In one specific embodiment, the compound “A": (a) is able from a Chromobacterium species; (b) is toxic to a pest; (c) has a lar weight of about 840-890 and more particularly, 860 as determined by Liquid Chromatography/Mass Spectroscopy (LC/MS); (d) has 1H NMR values of 6 8.89, 8.44, 8.24, 8.23, 7.96, 7.63, 6.66, 5.42, 5.36, 5.31, 5.10, 4.13, 4.07, 4.05, 3.96, 3.95, 3.88, 3.77, 3.73, 3.51, 3.44, 3.17, 2.40, 227,211,208, 2.03, 2.01, 1.97, 1.95, 1.90, 1.81, 1.68, 1.63, 1.57, 1.53, 1.48, 1.43, 1.35, 1.24, 1.07, 1.02, 0.96, 0.89, 0.88, 0.87, 0.80 and has 13C NMR values ofo 173.62,172.92,172.25,172.17,171.66,171.28,170.45,132.13,130.04,129.98,129.69,129.69, 125.48, 98.05, 70.11, 69.75, 68.30, 68.25, 64.34, 60.94, 54.54, 52.82, 49.72, 48.57, 45.68, 40.38, 39.90, 38.18, 36.60, 31.98, 31.62, 31.58, 29.53, 28.83, 27.78, 24.41, 23.06, 22.09, 20.56, 19.31, 18.78, 17.66, 15 .80 (e) has an High re Liquid Chromatography (HPLC) ion time of about 7-12 minutes, more ically about 9 minutes and even more specifically about 9.08 min on a reversed phase C-18 HPLC (Phenomenex, Luna 5” C18(2) 100 A, 100 x 4.60 mm) column using a water:acetonitrile (CH3CN) with a gradient solvent system (0-20 min; 90-0% aqueous CH3CN, 20- 24 min; 100% CH3CN, 24-27 min; 0-90 % aqueous CH3CN, 27-30 min; 90% aqueous CH3CN) at 0.5 mL/min flow rate and UV detection of 210 nm.

WO 62977 In another specific embodiment, the compound “B" has the following characteristics: (a) is obtainable from a Chromobacterium species; (b) is toxic to a pest; (c) has a molecular weight of about 850-900 and more particularly, 874 as determined by Liquid tography/Mass Spectroscopy (LC/MS); (d) has an High Pressure Liquid Chromatography (HPLC) retention time of about 7-12 minutes, more specifically about 9 minutes and even more specifically about 9.54 min on a reversed phase C-18 HPLC meneX, Luna 5” C18(2) 100 A, 100 X 4.60 mm) column using a water:acetonitrile ) with a gradient solvent system (0-20 min; 90-0% aqueous CH3CN, 20- 24 min; 100% CH3CN, 24-27 min; 0-90% aqueous CH3CN, 27-30 min; 90% aqueous CH3CN) at 0.5 mL/min flow rate and UV detection of 210 nm.

The metabolite may also be a nd including but not limited to: (A) a compound having the structure ##STR001## ##STR001## or a pesticidally acceptable salt or stereoisomers f, wherein R is —H, lower chain alkyl containing 1, 2, 3, 4, 5 , 6, 7, 8 or 9 alkyl es, aryl or arylalkyl moiety, substituted lower alkyl; X is O, NH, NR or S; n is 0, l, 2, 3, 4,5, 6, 7, 8 or 9; R1, R2, R3, R4, R5, R6, R7, R8, R9, R10, R11 are each independently H, are the same or different and independently an amino acid side-chain moiety or an amino acid side-chain derivative, alkyl, substituted alkyl, l, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, substituted heterocyclic, cycloalkyl, substituted cycloalkyl, alkoxy, substituted alkoxy, thioalkyl, tuted thioalkyl, hydroxy, halogen, amino, amido, carboxyl, -C(O)H, acyl, oxyacyl, carbamate, sulfonyl, sulfonamide, or sulfuryl; (B) a compound having the structure ##STR001a## ## STRO01 all!!! wherein R is —H, lower chain alkyl containing 1, 2, 3, 4, 5 , 6, 7, 8 or 9 alkyl moieties, aryl or arylalkyl moiety, substituted lower alkyl; X is O, NH, NR or S; R2,, R2}, are independently selected from the group consisting of —H, alkyl, lower-alkyl, substituted alkyl and tuted lower-alkyl; R1, R2, R3, R4, R5, R6, R7, R8, R9, R10, R11 are each independently H, are the same or different and independently an amino acid side-chain moiety or an amino acid side-chain tive, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, substituted heterocyclic, cycloalkyl, substituted cycloalkyl, alkoxy, substituted alkoxy, kyl, substituted thioalkyl, hydroxy, halogen, amino, amido, carboxyl, -C(O)H, acyl, oxyacyl, carbamate, sulfonyl, amide, or sulfuryl.

(C) a nd having the structure ##STR001b## ##STR00 1 b## wherein R is —H, lower chain alkyl containing 1, 2, 3, 4, 5, 6, 7, 8 or 9 alkyl moieties, aryl or aryl alkyl moiety, tuted lower alkyl; X is O, NH, NR or S; n is 0, l, 2, 3, 4, 5, 6, 7, 8 or 9; R2,, R2}, are independently ed from the group consisting of —H, alkyl, lower-alkyl, substituted alkyl and substituted lower-alkyl; R1, R2, R3, R4, R5, R6, R7, R8, R9, R10, R11 are each independently H, are the same or different and ndently an amino acid side-chain moiety or an amino acid side-chain derivative, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, tuted heterocyclic, cycloalkyl, tuted cycloalkyl, alkoxy, substituted alkoxy, thioalkyl, substituted thioalkyl, y, halogen, amino, amido, carboxyl, -C(O)H, acyl, oxyacyl, carbamate, sulfonyl, sulfonamide, or sulfuryl.

(D) a compound having the structure ##STROOlc## ##STROO 1 c## n R is —H, lower chain alkyl, aryl or aryl alkyl moiety, substituted lower alkyl containing 1,2, 3,4,5, 6,7, 8 or 9 alkyl moieties; X is O, NH, NR or S; n is 0, 1,2, 3,4,5, 6,7, 8 or 9; R2a, R2b are independently selected from the group consisting of —H, alkyl, lower-alkyl, substituted alkyl and substituted lower-alkyl; R1, R2, R3, R4, R5, R6, R7, R8, R9, R10, R11 are each independently H, are the same or different and independently an amino acid side-chain moiety or an amino acid side-chain derivative, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, substituted heterocyclic, cycloalkyl, tuted cycloalkyl, alkoxy, substituted alkoxy, thioalkyl, substituted thioalkyl, y, halogen, amino, amido, carboxyl, -C(O)H, acyl, oxyacyl, carbamate, sulfonyl, sulfonamide, or sulfuryl.

In a more particular ment, the metabolite is chromamide A (l).

Chromamide A (1) In a particular embodiment, the metabolite is compound “C", has the following characteristics: (a) is obtainable from a bacterium species; (b) is toxic to one or more pests; (c) has a lar weight of about 325-360 and more particularly, about 343 as determined by Liquid tography/Mass Spectroscopy (LC/MS); (d) has an High Pressure Liquid Chromatography (HPLC) retention time of about 8-14 minutes, more specifically about 10 minutes and even more ically about 10.88 min on a reversed phase C-18 HPLC (PhenomeneX, Luna 5” C18(2) 100 A, 100 X 4.60 mm) column using a water:acetonitrile (CH3CN) with a gradient t system (0-20 min; 90-0 % aqueous CH3CN, 20-24 min; 100% CH3CN, 24-27 min; 0 - 90 % aqueous CH3CN, 27-30 min; 90% aqueous CH3CN) at 0.5 mL/min flow rate and UV detection of 210 nm. In a particular embodiment, compound “C" may be violacein (2), a known compound isolated r from Chromobacterium eum.

In another embodiment, another metabolite used in the compositions and methods set forth above, is the compound “D", has the following characteristics: (a) is obtainable from a bacterium species; (b) is toxic to a pest; (c) has a molecular weight of about 315-350 and more ularly, about 327 as determined by Liquid Chromatography/Mass Spectroscopy (LC/MS); (d) has an High Pressure Liquid Chromatography (HPLC) retention time of about 10-15 minutes, more specifically about 12 s and even more specifically about 12.69 min on a reversed phase C-18 HPLC (PhenomeneX, Luna 5” C18(2) 100 A, 100 X 4.60 mm) column using a water:acetonitrile (CH3CN) with a gradient solvent system (0-20 min; 90-0% aqueous CH3CN, 20- 24 min; 100% CH3CN, 24-27 min; 0 - 90 % s CH3CN, 27-30 min; 90% aqueous CH3CN) at 0.5 mL/min flow rate and UV detection of 210 nm. In a particular embodiment, compound “D" may be characterized as deoxyviolacein (3), a known compound ed earlier from Chromobacterium violaceum.

In another specific embodiment, the compound may have the following ure wherein R is —H, lower chain alkyl containing 1, 2, 3, 4, 5 , 6, 7, 8 or 9 alkyl moieties, aryl or aryl alkyl moiety, substituted lower alkyl, halogens; R1, R2, R3, R4, R5, R6, R7, R8, R9 are each independently H, are the same or different, alkyl, substituted alkyl, alkenyl, substituted l, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, substituted heterocyclic, cycloalkyl, substituted cycloalkyl, alkoxy, substituted alkoxy, thioalkyl, tuted thioalkyl, hydroxy, n, amino, amido, carboxyl, -C(O)H, acyl, oxyacyl, carbamate, sulfonyl, sulfonamide, or sulfuryl.

Further provided is a method for (l) ting pest (e .g., nematode, insect, soil-borne bacteria) infestation in a plant comprising applying to the plant and/or seeds thereof and/or substrate used for growing said plant or a method for ting soil borne bacteria in soil comprising applying to the plant, seeds, and/or substrate an amount of (I) a compound that (a) has pesticidal and/or antimicrobial activity; (b) has a molecular weight of about 950 - 1450 as determined by LTQ Orbitrap XL hybrid Fourier Transform Mass Spectrometer.

WO 62977 (c) has 1H NMR 6 values of 5.22 (sext, 1H), 2.62 (dd, 1H), 2.53 (dd, 1H), and 1.31 (d, 3H) (d) has 13C NMR 6 values of 169.2, 67.6, 40.9, and 19.8. (d) comprises the structure —(-O-CHCH3-CH2-CO-)n-, where n=6-50 (e) is able from a Chromobacterium species and (II) optionally another pesticidal substance effective to modulate infestation in said plant.

Compound (I) may have the structure: R1 Y x 0H Wherein X, is independently -0, -NR, or -S, wherein R is H or C1-C10alkyl; Y, is independently -O, —S; n = 6-50; R1, R2 are each independently H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, l, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted aryl, heterocyclic, substituted heterocyclic, cycloalkyl, substituted cycloalkyl, alkoxy, substituted alkoxy, thioalkyl, substituted thioalkyl, hydroxy, halogen, amino, amido, carboxyl, -C(O)H, acyl, oxyacyl, carbamate, sulfonyl, sulfonamide, or sulfuryl.

In particular, the compound (I) has the structure: CH3 0 To 0H L n wherein n = 10-25.

In a most specific embodiment, (I) is an alpha-butyric acid.

Further provided is a method for obtaining the nds set forth above. The method ses culturing a strain of a Chromobacterium sp. in a whole cell broth under conditions ient to produce the nd and isolating the compound produced from the whole cell broth.

In a related aspect, disclosed are methods for stabilizing biological pesticide compositions against physical separation and loss of activity due to exposure to ht by applying an amount of a stabilizing agent to said biological pesticide composition effective to stabilize the biological pesticide composition against physical separation and loss of activity due to exposure to ht.

Also provided are compositions sing such agents. In a particular embodiment, the pesticidal composition comprises bacterium sp. filtrate, supernatant, extract or pesticidally active substance derived therefrom which may be present in the amount of at least about 0.5% and particularly between about 0.5 wt% based on dry cell weight to about 30 wt.%. The stabilizing agent may in a particular embodiment be a benzoic acid salt and/or lignin salt, particularly lignin sulfonate salt, ing but not d to sodium, potassium, m, magnesium, ammonium, and combinations thereof and may present in the amount of at least about 2.5 wt% and may preferably be present in the amount of about 5%-15%.

BRIEF PTION OF THE FIGURES Figure l is a schematic representation of purification scheme for obtaining chromamide A (1) violacein (2) and deoxyviolacein (3).from culture broth.

Figure 2 depicts chemical structures for chromamide A (1) violacein (2) and deoxyviolacein (3).

Figure 3 shows results from MBI-203 TSSM Leaf disk assays. CFD ents Cells Freeze Dried, which were then reconstituted in water to ent a l/2x, 1x and 2.5x dose of the original Whole Cell Broth.

Figure 4 shows the mean number of eggs laid by potato psyllid females exposed to MBI- 203 treated pepper leaf.

Figure 5 shows the mean number of eggs oviposited by psyllid females 5 days post exposure to MBI-203 treated and untreated pepper leaf discs.

Figure 6 shows the mean number of Green Peach Aphid (GPA) nymphs and adults on treated pepper leaf discs 24 hours post exposure of adults (P < 0.0001, LSD, 0! = 0.05). Means of the same letter not statistically different from each other. dH2O — negative control, 203 10% v/v — positive l.

Figure 7 shows the mean number of Green Peach Aphid progeny s) on MBI-203 treated pepper leaf discs 3 days post exposure of adults (P < 0.0001, LSD, 0! = 0.05). Means of the same letter not statistically different from each other. dHZO — negative control, Avid 10% v/v — positive control.

Figure 8 shows the mean number of Green Peach Aphid progeny (nymphs) on MBI-203 and DF2 (MBI-203 formulated product) treated pepper leaf discs 3 days post exposure of adults (P < 0.0001, LSD, 0! = 0.05). Means of the same letter not statistically different from each other. dHZO — negative control, Avid 10% v/v — positive control.

Figure 9 is a schematic entation of purification scheme for violacein (2) and oligo-(B- hydroxybutyric acid (4).from e broth.

Figure 10 depicts the results of nematocidal bioassays of various ons and of compound oligo-hydroxybutyric acid (1).

Figures 11A and 11B show the mean number of green peach aphid, Myzus persicae (nymphs and adults) on treated pepper leaf discs 24 hours post exposure. Means of the same letter are not significantly different from each other (LSD, P < 0.0001, or = 0.05). 203 Me — MBI-203 crude t in ol t, Me — Methanol (blank treatment), 203, Ace — MBI-203 crude extract in acetone solvent, Ace — Acetone (blank ent), dHZO — negative control, Avid 10% - positive control.

Figures 12A and 12B show the mean number of green peach aphid, Myzus persicae (nymphs and adults) on treated pepper leaf discs 24 hours post exposure. Means of the same letter are not statistically different from each other. Ace — acetone only (blank treatment, DCM - EA - , , Wash - — ve control, 203 10% - MBI-203 std (positive control).

, MeOH -. dHZO Figure 13 shows the mean number of Green Peach Aphid progeny (nymphs) on treated pepper leaf discs 3 days post exposure of adults (P < 0.0018, LSD, or = 0.05). Means of the same letter not statistically different from each other. VL1 — violacein at 0.5 yg/mL, VL2 — violacein at 1.0 yg/mL, Acetone — blank treatment, dHZO — negative l, Avid 10% v/v — positive control.

Figure 14 shows the mean number of Green Peach Aphid (GPA) nymphs and adults on d pepper leaf discs 24 hours post exposure of adults (P < 0.0001 or = 0.05). Means of the , LSD, same letter not statistically different from each other. dHZO — negative control, 203 10% v/v — positive control.

Figure 15 shows the mean number of Green Peach Aphid (GPA) nymphs and adults on treated pepper leaf discs 24 hours post re of adults (P < 0.0001, LSD, or = 0.05). Means of the same letter not tically different from each other. dHZO — negative control, 203 10% v/v — positive control.

Figure 16 shows MBI-203 @ 3% presun vs postsun mortality, zeroed to water.

Figure 17 shows MBI-203: Cabbage Aphid plant spray EP/EP+ NaBenz.

Figure 18 shows MBI-203 EP +/- Na Benz, Cabbage Aphids plant spray, corrected.

Figure 19 shows MBI-203: EP + CaCO3/NaBenz PRE/POST SUN, normalized to negative control.

Figure 20 shows MBI-203 EP SDP post sun, 3 and 6 day assays.

Figure 21 shows MBI-203 EP w/ Na Benz + lignin in sun, assay day 4, corrected.

Figure 22 shows MBI-203 EP w/ Na Benz + lignin in sun, percent activity loss, assay day 4.

Figure 23 shows MBI-203: EP + lignin + sun, Broccoli CL assay day 3.

DETAILED DESCRIPTION OF THE RED EMBODIMENTS While the itions and methods heretofore are susceptible to various modifications and alternative forms, exemplary embodiments will herein be described in detail. It should be understood, however, that there is no intent to limit the invention to the particular forms disclosed, but on the contrary, the intention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the invention as defined by the appended claims.

Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is included therein. Smaller ranges are also included. The upper and lower limits of these smaller ranges are also included therein, subject to any specifically excluded limit in the stated range.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. gh any methods and als similar or equivalent to those described herein can also be used in the ce or testing of the t invention, the preferred s and materials are now described.

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

As defined herein, “derived from" means directly isolated or ed from a particular source or atively having identifying characteristics of a substance or organism isolated or obtained from a particular source.

A “carrier" as defined herein is an inert, organic or inorganic material, with which the active ient is mixed or formulated to tate its application to plant or other object to be treated, or its storage, transport and/or handling.

The term “modulate" as defined herein is used to mean to alter the amount of pest infestation or rate of spread of pest infestation.

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 .

A “pesticide" as defined herein, is a substance derived from a biological product or chemical substance that increase mortality or inhibit the growth rate of plant pests and includes but is not limited to cides, insecticides, herbicides, plant fungicides, plant bactericides, and plant viricides.

A “biological pesticide" as defined herein is a microorganism with pesticidal properties.

METHODS OF PRODUCTION As noted above, the biological pesticide may se or be derived from an organism having the identifying characteristics of a Chromobacterium species, more ularly, from an organism having the identifying characteristics of a strain of Chromobacterium substugae, more particularly from a strain of Chromobacterium substugae sp. nov. which may have the fying characteristics of NRRL B-30655, or alternatively from any other microorganism. The methods comprise cultivating these organisms and ing the compounds and/or compositions of the present invention by isolating these compounds from the culture of these organisms.

In particular, the organisms are cultivated in nutrient medium using methods known in the art. The organisms may be cultivated by shake flask cultivation, small scale or large scale fermentation (including but not limited to continuous, batch, tch, or solid state fermentations) in laboratory or industrial fermentors performed in suitable medium and under conditions allowing cell growth. The cultivation may take place in suitable nutrient medium comprising carbon and nitrogen sources and inorganic salts, using procedures known in the art. Suitable media are ble may be available from commercial sources or prepared according to published compositions.

After cultivation, the compounds and/or compositions of the present invention may be extracted from the e broth. The extract may be fractionated by chromatography.

Compositions The substances set forth above used in the compositions and methods disclosed herein can be formulated in any manner. Non-limiting formulation examples include but are not limited to fiable concentrates (EC), Wettable powders (WP), soluble liquids (SL), Aerosols, Ultra-low volume concentrate solutions (ULV), Soluble powders (SP), Microencapsulation, Water sed Granules, les (FL), Microemulsions (ME), Nano-emulsions (NE), etc. In any formulation described herein, t of the active ingredient is within a range of 0.01% to 99.99%.

The itions may be in the form of a liquid, gel or solid. Liquid compositions comprise pesticidal compounds derived from a Chromobacterium , e.g. a strain having the identifying characteristics of Chromobacterium substugae sp. Nov and more particularly, having the fying characteristics of NRRL B-30655 (see US Patent No. 607).

A solid ition can be prepared by suspending a solid carrier in a solution of pesticidal compounds and drying the suspension under mild conditions, such as evaporation at room temperature or vacuum evaporation at 65°C or lower.

A ition may comprise gel-encapsulated compounds d from the Chromobacterium strain. Such gel-encapsulated als can be prepared by mixing a gel- forming agent (e.g., gelatin, cellulose, or lignin) with a culture or suspension of live or inactivated Chromobacterium, or a cell-free filtrate or cell on of a Chromobacterium culture or suspension, or a spray- or freeze-dried culture, cell, or cell fraction or in a solution of pesticidal compounds used in the method of the invention; and inducing gel formation of the agent.

The composition may additionally se a surfactant to be used for the purpose of emulsification, dispersion, wetting, spreading, integration, disintegration control, stabilization of active ingredients, and improvement of fluidity or rust inhibition. In a particular embodiment, the surfactant is a non-phytotoxic non-ionic surfactant which ably belongs to EPA Inerts List 4B.

In another particular embodiment, the nonionic surfactant is polyoxyethylene (20) urate. The concentration of surfactants may range between 01-35% of the total formulation, preferred range is -25%. The choice of sing and emulsifying agents, such as non-ionic, anionic, amphoteric and cationic dispersing and emulsifying agents, and the amount employed is determined by the nature of the composition and the ability of the agent to facilitate the dispersion of the compositions of the present invention.

The composition as set forth above also comprises a stabilizing agent, which stabilizes a biological pesticide composition against physical separation and loss of activity due to exposure to sunlight. This stabilizing agent may be a benzoic acid salt or lignin sulfonate salt.

The composition set forth above may be ed with another microorganism and/or pesticide (e.g, nematocide, fungicide, icide, antibiotic or anti-microbial agent). The microorganism may include but is not limited to an agent d from Bacillus sp., Pseudomonas sp., acillus sp., Lecanicillium sp., non-Ampelomyces sp., zyma sp., Streptomyces sp, Burkholderia sp, Trichoderma sp, Gliocladium sp. Alternatively, the agent may be a natural oil or oil-product having fungicidal and/or insecticidal activity (e .g., paraffinic oil, tea tree oil, lemongrass oil, clove oil, cinnamon oil, citrus oil, rosemary oil). Furthermore, the ide may be a single site anti-fungal agent which may include but is not limited to benzimidazole, a demethylation inhibitor (DMI) (e .g ., imidazole, piperazine, pyrimidine, triazole), morpholine, hydroxypyrimidine, opyrimidine, phosphorothiolate, quinone outside inhibitor, quinoline, dicarboximide, carboximide, phenylamide, opyrimidine, phenylpyrrole, aromatic hydrocarbon, cinnamic acid, hydroxyanilide, antibiotic, polyoxin, acylamine, imide, benzenoid (xylylalanine), a demethylation inhibitor selected from the group consisting of imidazole, piperazine, pyrimidine and triazole (e.g.,bitertanol, myclobutanil, penconazole, propiconazole, triadimefon, bromuconazole, cyproconazole, diniconazole, fenbuconazole, hexaconazole, tebuconazole, tetraconazole), myclobutanil, and a quinone outside inhibitor (e .g., strobilurin). The strobilurin may include but is not limited to azoxystrobin, im-methoyl or ystrobin. In yet another particular embodiment, the anti-fungal agent is a quinone, e.g., yfen (5 ,7-dichloroquinolyl 4- fluorophenyl ether). The anti-fungal agent may also be derived from a Reynoutria extract.

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

The composition may as noted above, further comprise an insecticide. The insecticide may include but is not limited to avermectin, Bt, neem oil, spinosads, lderdia sp. as set forth in US Patent Appln. Pub. No. 2011-0207604, entomopathogenic fungi such a ria bassiana and chemical insecticides including but not limited to organochlorine compounds, organophosphorous compounds, carbamates, pyrethroids, and neonicotinoids.

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

Uses The compositions, cultures and supernatants and idal compounds set forth above may be used as pesticides. In particular, the compounds or compositions as set forth above may be used as insecticides, bactericides (against soil-borne bacteria) and nematocides. Specifically, nematodes that may be controlled using the method set forth above include but are not limited to parasitic des such as not, cyst, and lesion des, including but not limited to Meloidogyne sp. Tylenchorhynchus sp, Hoplolaimus sp., Helicotylenchus sp., Pratylenchus sp., Heterodera sp., Globodera, sp., Trichodorus sp. Paratrichodorus sp., Xiphinema sp., and Criconema sp.; particularly Meloidogyne incognita (root knot nematodes), as well as Globodera rostochiensis and globodera pailida (potato cyst nematodes); Heterodera glycines (soybean cyst nematode); Heterodera schachtii (beet cyst nematode); and Heterodera avenae (cereal cyst nematode).

As noted above, the active ingredient(s) and compositions set forth above may also be applied to locations containing Acari (arachnids), such as mites, including but not limited to, Panonychus sp. such as Panonychus citrz' (citrus red mite), and Panonychus ulmz' (red spider mite), Tetranychus sp. such as Tetranychus kanzawi (Kanzawa spider mite), Tetranychus urticae (2 spotted spider mite), Tetranychus pacificus (Pacific spider mite), Tetranychus turkestanii berry mite) and Tetranychus cinnabarinus (Carmine spider mite), Oligonychus sp. such as Oligonychus panicae (avacado brown mite), ychus perseae (persea mite), Oligonychus pratensis (Banks grass mite) and Oligonychus cofleae, Aculus sp. such as Aculus comatus (Peach silver mite), Aculus fockem' (plum rust mite) and Aculus rsz'ci (tomato russet mite), Eotetranychus sp. such as Eotetranychus wilametti, anychus yumensis (yuma spider mite) and anychus sexmaculatis (6-spotted mite), Bryobia rubrioculus (brown mite), Epitrimerus pyri (pear rust mite), tus pyri (Pear leaf r mite), Acalitis essigi (red berry mite), Polyphagotarsonemus latus (Broad mite), Eriophyes m' (citrus bud mite), Brevipalpus lewz'sz' (citrus flat mite), Phylocoptruta oleivora (citrus rust mite), Petrobia s (Brown wheat mite), Oxyenus maxwelli (olive mite), Rhizoglyphus spp., agus spp., Diptacus gigantorhyncus (bigheaded plum mite) and Penthaleaa major (winter grain mite), Avocado red mite, Flat mite, black and red Mango spider mite, Papaya leaf edgeroller mite, Texas citrus mite, European red mite, Grape erineum mite (blister mite), c spider mite, Willamette spider mite; Pink citrus rust mite. Such locations may include but are not limited to crops that are infested with such mites or other arachnids (e .g., aphenids). Such locations may include but are not limited to crops that are infested with such mites or other arachnids (e.g., aphenids).

Phytopathogenic insects controlled by the method set forth above include but are not d to non-Calicidae larvae insects from the order (a) ptera, for e, Aclerz's spp., Adoxophyes spp., Aegeria spp., Agrotis spp., Alabama argillaceae, Amylois spp., Anticarsia gemmatalz's, Archips spp., Argyrotaem'a spp., Autographa spp., Busseola fusca, Cadra cautella, Carposina nipponensis, Chilo spp., Choristoneara spp., Clysz'a ambigaella, locrocis spp., Cnephasia spp., Cochylis spp., Coleophora spp., olomia binotalis, Cryptophlebz'a leacotreta, Cydia spp., Diatraea spp., Diparopsis castanea, Earias spp., Ephestia spp., Eucosma spp., Eapoecilia ambigaella, Eaproctis spp., Eaxoa spp., Grapholita spp., Hedya nabiferana, Heliothz's spp., Hellala andalis, Hyphantria canea, ria lycopersicella, Leacoptera scitella, Lithocollethis spp., Lobesia botrana, Lymantria spp., Lyonetia spp., Malacosoma spp., Mamestra brassicae, Manduca sexta, Operophtera spp., Ostrim'a nubilalz's, Pammene spp., Pandemis spp., Panolis flammea, Pectinophora gossypiella, Phthorimaea opercalella, Pieris rapae, Pieris spp., Platella xylostella, Prays spp., phaga spp., Sesamia spp., Sparganothis spp., tera spp., Synanthedon spp., topoea spp., Tortrix spp., Trichoplasia m' and Yponomeata spp.; (b) Coleoptera, for example, es spp., Alphitobias sp., Anomola spp., e.g., Anomala alis; Anthonomas spp., Atomaria linearis, Chaetocnema tibialis, olites spp., Curculio spp., Cyclocephala spp., e.g., Cyclocephala lurida, Dermestes spp., Diabrotz'ca spp., Epilaclma spp., Eremnas spp., Leptinotarsa decemlz'neata, Lissorhoptras spp., Melolontha spp., Orycaephilus spp., Otiorhynchas spp., Otiorhynchas sulcatus, Phlyctinus spp., Papillia spp., e.g., la japonica, Psylliodes spp., Rhizopertha spp-, eg., rogas majalz's, Sitophilas spp., Sitotroga spp., Tenebrio spp., Triboliam spp. and Trogoderma spp.; (c) 0rth0ptera,f0r example, Blatta spp., Blattella spp., Gryllotalpa spp., Leacophaea maderae, Locusta spp., Periplaneta spp. and Schistocerca spp.; (d) Isoptera, for example, Reticalitermes spp.; (e) Psocoptera, for example, Liposcelis spp.; (f) Anoplara, for example, Haematopz'nus spp., Linognathus spp., Pedicalus spp., Pemphigus spp. and Phylloxera spp.; (g) Mallophaga, for e, Damalinea spp. and Trichodectes spp.; (h) Thysanoptera, for example, Frankliniella spp., Hercinotnrips spp., Taem'othrips spp., Thrips palmi, Thrips tabaci and Scirtothrips aarantii; (i) Heteroptera,f0r example, Cimex spp., Distantiella theobroma, Dysdercas spp., Euchistus spp., Eurygaster spp., Leptocorisa spp., Nezara spp., Piesma spp., Rhodm'as spp., Sahlbergella singularis, Scotinophara spp. and Tm'atoma spp.; (j) Homoptera, for example, Alearothrixusfloccosas, Aleyrodes brassz'cae, Aom'diella spp., Aphididae, Aphis spp., Aspidiotus spp., Bactericera spp., a tabaci, Ceroplaster spp., mphalas 'am, Chrysomphalas dictyospermi, Coccas idam, Empoasca spp., Eriosoma larigeram, oneara spp., Gascardia spp., phax spp., Lecanium corni, Lepidosaphes spp., Macrosiphas spp., Myzas spp., Nephotettix spp., Nilaparvata spp., Paratoria spp., Pemphigus spp., Planococcus spp., Pseudaulacaspis spp., Pseudococcus spp., Psylla spp., Pulvinaria aethiopica, Quadraspidiotus spp., Rhopalosiplmm spp., Saissetia spp., Scaphoideus spp., Schizaphis spp., Sitobion spp., Trialeurodes vaporariorum, Triozidae spp., Trioza erytreae and Unaspis citri; (k) Hymenoptera, for example, Acromyrmex, Alta spp., Cephus spp., Diprion spp., Dipriom'dae, Gilpim'a polytoma, Hoplocampa spp., Lasz'us spp., Monomorium m's, Neodiprion spp., Solenopsz's spp. and Vespa spp.; (l) Diptera, for e, Aedes spp., Antherigona soccata, Bibio hortulanus, hora erythrocephala, Ceratitis spp., Chrysomyia spp., Cuterebra spp., Dacus spp., Delia spp., Delia radicum, Drosophila spp., e.g., hila suzukii; Fannia spp., Gastrophilus spp., Glossina spp., Hypoderma spp., osca spp., Liriomyza spp., a spp., Melanagromyza spp., Musca spp., 0estrus spp., Orseolia spp., Oscinella frit, Pegomyia hyoscyami, Phorbia spp., Rhagoletis pomonella, Sciara spp., Stomoxys spp., Tabanus spp., Tannia spp. and Tipula spp.; (m) aptera, for example, Ceratophyllus spp. and Xenopsylla cheopis ; (n) from the order Thysanura, for example, Lepz'sma rina; (0) Hemiptera, for example, Bactericera sp., e.g., Bactericera cockerelli, .

The active ingredients may be applied to locations containing aeidae pests. These include but are not limited to soil, grass and various ornamental plants, trees and vegetables.

The active ingredient(s) and compositions set forth above may also be applied to locations containing the a Muscz'dae pest. These include but are not limited to indoor environments, garbage, animals, fences, s, barns, milking parlors, farrowing pens etc. containing animals (cattle, pigs, sheep, horses etc.) The active ingredient(s) and compositions set forth above may r be applied to locations containing the active ingredient(s) and compositions containing a Tenebrionidae pest.

These include but are not limited to grains, poultry and poultry dwellings enclosures (fences, corrals, barns, g parlors, farrowing pens etc.) ning animals (cattle, pigs, sheep, horses etc.) EXAMPLES The composition and methods set forth above will be further illustrated in the following, non-limiting Examples. The examples are illustrative of various embodiments only and do not limit the claimed invention regarding the materials, conditions, weight ratios, s parameters and the like recited herein.

Example 1: Extraction of Chromamide, Deoxyviolacein and Violacein from Chromobacterium substugae The following procedure is used for the purification of nds extracted from the culture of Chromobacterium substugae: The culture broth derived from the 10-L fermentation C. substugoe in L-broth is extracted with Amberlite XAD-7 resin (Asolkar et al., 2006) by shaking the cell suspension with resin at 225 rpm for two hours at room temperature. The resin and cell mass are collected by filtration h cheesecloth and washed with DI water to remove salts. The resin, cell mass, and cheesecloth are then soaked for 2 h in acetone/methanol (50/50) after which the acetone/methanol is filtered and dried under vacuum using rotary evaporator to give the crude extract. The crude extract is then fractionated by using ex LH 20 size exclusion chromatography (CHZClz/CH3OH; 50/50) to give 7 fractions e 1). These fractions are then concentrated to dryness using rotary ator and the resulting dry es are screened for biological activity using a feeding assay with Cabbage looper (Trichoplusia m) or Beet armyworm ptero exiguo). The active fractions are then subjected to ed phase HPLC (Spectra System P4000 (Thermo Scientific) to give pure compounds, which are then screened in above mentioned bioassays to locate/identify the active compounds. To confirm the identity of the compound, additional spectroscopic data such as LC/MS and NMR is recorded.

Chromamide A (1) and compound B were obtained from Fraction 1 and 2 respectively, whereas violacein (2) & deoxyviolacein (3) were purified from Fraction 5 obtained from Sephadex LH 20 chromatography.

Purification ofCompounds Purification of chromamide A (1) was performed by using HPLC C-18 column (Phenomenex, Luna 10u C18(2) 100 A, 250 x 10), water:acetonitrile gradient solvent system (0-10 min, 80-75 % aqueous CH3CN; 10-45 min, 75-60 % aqueous CH3CN; 45-55 min, 60-50 % aqueous CH3CN; 55-65 min, 50-100 % aqueous CH3CN; 65-70 min, 100 % CH3CN; 55-70 min, 0—80 % aqueous CH3CN) at 2.5 mL/min flow rate and UV detection of 210 nm. The active compound chromamide A (1), has retention time 23 .19 min.

Purification of invention compound B was performed by using HPLC C-18 column (Phenomenex, Luna 10u C18 (2) 100 A, 250 x 10), acetonitrile gradient solvent system (0-10 min, 80-75% aqueous CH3CN; 10-45 min, 75-60 % aqueous CH3CN; 45-55 min, 60 - 50 % aqueous CH3CN; 55-65 min, % aqueous CH3CN; 65-70 min, 100% CH3CN; 55-70 min, 0—80 % aqueous CH3CN) at 2.5 mL/min flow rate and UV ion of 210 nm, the active compound B, retention time 26.39 min.

Purification of violacein (2) and deoxyviolacein (3) was performed by using HPLC C-18 column (Phenomenex, Luna 10u C18(2) 100 A, 250 x 10), water:acetonitrile gradient t system (0-10 min, 70-60 % s CH3CN; 10-40 min, 60 - 20% aqueous CH3CN; 40-60 min, 20 - 0 % aqueous CH3CN; 60-65 min, 100 % CH3CN; 65-75 min, 0—70 % aqueous CH3CN) at 2.5 mL/min flow rate and UV detection of 210 nm, the active compounds violacein (2), had a ion time 7.86 min and deoxyviolacein (3) retention time 12.45 min.

Mass Spectroscopy Analysis of Compounds Mass spectroscopy analysis of active peaks is performed on a Thermo an LCQ Deca XP Plus electrospray (ESI) instrument using both ve and ve ionization modes in a full scan mode (m/z 100-1500 Da) on a LCQ DECA XPPlus Mass Spectrometer (Thermo Electron Corp., San Jose, CA). Thermo high performance liquid chromatography (HPLC) instrument equipped with Finnigan Surveyor PDA plus detector, autosampler plus, MS pump and a 4.6 mm X 100 mm Luna C18 5p 100A column menex). The solvent system ted of water (solvent A) and acetonitrile (solvent B). The mobile phase begins at 10% solvent B and is linearly increased to 100% solvent B over 20 min and then kept for 4 min, and finally returned to 10% solvent B over 3 min and kept for 3 min. The flow rate is 0.5 mL/min. The injection volume was 10 pL and the samples are kept at room temperature in an auto sampler. The compounds are analyzed by LC-MS ing the LC and reversed phase tography. Mass spectroscopy analysis of the present compounds is performed under the following ions: The flow rate of the nitrogen gas was fixed at 30 and 15 arb for the sheath and aux/sweep gas flow rate, tively. ospray ionization was performed with a spray voltage set at 5000 V and a capillary voltage at 35.0 V. The capillary temperature was set at 400°C. The data was analyzed on Xcalibur software. The chromamide A (1) has a molecular mass of 860 in positive ionization mode. The LC-MS chromatogram for another active nd B suggests a molecular mass of 874 in positive ionization mode. Violacein (2) and deoxyviolacein (3) had the molecular masses of 313 and 327 tively in positive ionization mode.

NMR Spectroscopy Analysis of nds NMR-NMR spectra were measured on a Bruker 600 MHz gradient field spectrometer. The reference is set on the internal standard tetramethylsilane (TMS, 0.00 ppm). The amino acid analyses were carried out on Hitachi 8800 amino acid analyzer.

For structure elucidation, the purified chromamide A with molecular weight 860 is r analyzed using a 600 MHz NMR instrument, and has 1H NMR 6 values at 8.89, 8.44, 8.24, 8.23, 7.96,7.63, 6.66,5.42,5.36,5.31,5.10,4.13, 407,405, 3.96, 3.95, 3.88, 3.77, 3.73, 3.51, 3.44, 3.17, 2.40, 2.27, 2.11, 2.08, 2.03, 2.01, 1.97, 1.95, 1.90, 1.81, 1.68, 1.63, 1.57, 1.53, 1.48, 1.43, 1.35, 1.24, 1.07, 1.02, 0.96, 0.89, 0.88, 0.87, 0.80 (see and has 13C NMR values of 173.62, 172.92, 172.25, 172.17, 171.66, 171.28, 170.45, 132.13, 130.04, 129.98, 129.69, 129.69, 125.48, 98.05, 70.11, 69.75, 68.30, 68.25, 64.34, 60.94, 54.54, 52.82, 49.72, 48.57, 45.68, 40.38, 39.90, 38.18, 36.60, 31.98, 31.62, 31.58, 29.53, 28.83, 27.78, 24.41, 23.06, 22.09, 20.56, 19.31, 18.78, 17.66, .80. The chromamide A was isolated as a white solid, which analyzed for the molecular formula C43H68N6O12 (13 degrees of unsaturation), by ESI high-resolution mass spectrometry (obsd M+ m/z 861.5376, calcd M+ m/z 861.5343). The 1H NMR spectral data of chromamide A in DMSO-d6 exhibited 68 proton signals, in which nine protons [6H: 8.89, 8.44, 8.23, 8.22, 7.96, 7.64, 6.65, 5.10, 4.13], were assigned as either NH or OH due to lack of carbon ation in a heteronuclear correlation NMR (HMQC) analysis. The 13C NMR spectrum, showed seven yl signals [65 173.62, 172.92, , 1.72.17, 171.66, 171.28, 170.45] and in the 1H NMR spectrum, six characteristic a-amino protons signals [65 4.07, 4.06, 3.96, 3.95, 3.88, 3.72] were observed which demonstrate that chromamide A is a peptide.

Interpretation of 2D NMR data led to the assignment of three amino acid units of the six, one leucine (Leu), one valine (Val) and one glutamine (Gln). The presence of these amino acids were confirmed by results of amino acid analysis, which also showed the presence of the above three amino acids. Further analysis of DEPT and 2D NMR spectral data (COSY, HSQC and HMBC) established the presence three sub-structures I, II and III as showed below.

I? ”f" 0 [H H,N o N 0 CH3 33 1 0 38 21 m H o CH CH3 H3C%/N NJKsl/‘K 14 o 0 CH3 ' \ \ \ N, MN (‘6 H0 . 0 H (I) , 27 H W 0 The connections of the three sub-structures in 1 were accomplished by routine HMBC NMR analysis using correlations between the OL-amino proton and/or the secondary amide proton and the carbonyl carbon nces and chemical shift consideration. The linkage of C-9 from sub-structure I to C-10 from sub-structure II was established by HMBC ations from CH3-40 [6H: 1.00] and the OL-amino proton of alanine [6H: 3.42] to the C-10 carbon [65 70.11]. This was further confirmed by the three bond HMBC correlation from hydroxyl at [65 5.10] to C-9 at [65 49.78]. The methylene at [65 3.50] from sub-structure 111 showed a three bond HMBC correlation to C-19 [65 68.31] which connected the sub-structure I and II. The quaternary carbon at C-3 [65 98.09] was connected to C-21[65 64.40] through a weak correlation from H-21 [6H: 3.95] er with their chemical shift values to form a one ring system. Lastly, the ring closure linkage was secured by a three-bond HMBC correlation from H3-36 [6H: 1.43] to C-1 [65 172.17], which d the planar structure of chromamide A (1) to be assigned.

The compound B with a molecular weight 874 exhibited r NMR and UV data suggesting that this compound B also belongs to the class of peptide.

The ure for violacein (2) and deoxyviolacein (3) was assigned by comparison of the data of these compounds with those published in the literature. The structures of chromamide A, violacein and deoxyviolacein are shown in Figure 2.

Example 2: Amino acids Analysis of chromamide A mide A (0.05 mg) was hydrolyzed by using liquid phase hydrolysis (6N HCL, 1% , 110°C, 24hr, in vacuum). After cooling, the reaction mixture was dried and the hydrolyzed product was dissolved in Norleu dilution buffer to 1.0 mL volume. A 50m of the sample was loaded onto the ion-exchange column for analysis.

For rds and calibration, an amino acid standards solution for protein hydrolysate on the Na-based i 8800 (Sigma, ) is used to determine response factors, and thus ate the Hitachi 8800 analyzer for all of the amino acids. Each injection contains NorLeucine as an al standard to allow correction of the results for variations in sample volume and Chromatography variables. System utilizes Pickering Na buffers, Pierce Sequanal grade HCl (hydrolysis), a Transgenomic change column and an optimized method developed by Molecular ure Facility (MSF), UC Davis, and the dual amino acid present in the sample are reported. The amino acids present in the sample (Chromamide A) were found to be Glx (Glutamine/Glutamic acid), leu ne) and Val (Valine).

Example 3: Effect of Chromobacterium substugae (MBI-203) against Two Spotted Spider Mites-Bean Plants Freeze dried MBI-203 was mixed with distilled water to varying concentrations of whole cell broth cell equivalent. Uninfested bean plants, Phaseolus vulgaris, were sprayed with MBI-203.

Leaf disks were then taken from sprayed plants and placed in a petri dish as a food source for Two- Spotted Spider Mites, Tetranychus urticae. 10 mites were placed in each dish and incubated at 75°F, 12:12 (L:D). Evaluations of live and dead mites were recorded 1, 3, and 7 days after infestation. 1x CFD is freeze dried material reconstituted to whole cell broth cell concentrations (0.0103 g freeze dried/mL dHZO). The results are shown in Figure 3.

Example 4: Effect of Chromobacterium substugae (MBI-203) against Two d Spider Mites-Marigolds Marigolds, Tagetes erecta, were infested with Two-Spotted Spider Mites, Tetranychus urticae. Formulated product (MBI-203) or Chenopodium ambrosioides (marketed as REQUIEM® by AgraQuest, Inc., Davis, CA) was applied to infested plant and kept in a greenhouse with temperature ranges approximately 72-85°F. To sample, a 6 cm2 leaf surface was harvested and number of live and dead nymphs and immature counted. The results are shown in Table 1. 2012/061503 Table 1: Comparison of 3 with REQUIEM® Day 0 Day 3 Day 5 Day 7 Day 14 Treatment Stage Live Live Live Live Live Example 5: Effect of Chromobacterium substugae (MBI-203) ing against Two Spotted Spider Mites (TSSM)-French Beans French beans infested with TSSM or abamectin-resistant TSSM were sprayed with 0.5%, 1%, 2%, and 4% v/v dilutions of formulated 3 at approximately 100 gal/acre. Mortality was assessed 9 days after application. The results are shown in Table 2.

Table 2: Effect of MBI-203 on TSSM infestation in French Beans ————___ —______ ———____ ———E____ Example 6: Screening MBI-203 against Two d Spider Mites in strawberry The efficacy of five traditional compounds and MBI-203 ingredients were evaluated for TSSM control on field strawberry transplants at the Florida Gulf Coast Research and Education Center. Seedlings were transplanted in the field (day 0). Each 12.5 -ft. plot ted of 20 plants.

Plots were infested four times from day 55 through day 71 with 10 to 20 motile TSSM per plant.

Seventeen treatments of various rates and schedules of application of miticides, some ed with an adjuvant, and a non-treated Check were replicated four times in a RCB design. Treatments were applied using a hand-held sprayer with a spray wand outfitted with a nozzle containing a 45 - degree core and a number four disc. The sprayer was pressurized by C02, to 40 psi, and calibrated to deliver 100 gal per acre. Samples were ted weekly from day 90 before first spray through 2 wks after the last application of treatments (day 154) . Samples consisted of ten randomly selected leaflets per plot and were collected from the middle one-third stratum of the . Motile and egg TSSM were brushed from the leaflets onto rotating sticky discs and d. No phytotoxicity was observed. Results are shown in Tables 3 and 4.

E 0-0 E 0 E 0-0 o-E 0E 0E 000 0.000 0.000 0.0 0000 0000. 0.00 0.000 0.0: 0.000 0.000 0.000 0v E 0-0 00 E 0 0-0 0E 0E 0-0 0: 0.000 0.00 0.000 0.0 0.0 00.00 0.00 0000.0v 00600000080000 0.00 0E0 0.000 0.000 8: E 00 00 0E 00 0-0 0E o-E 0-0 000 0.000 0.0 0.00 0.0 00.00 0000. 00002 0.000 0.00 0.000 0.00 0.00 0v 3.00:8 0E0 00:00 E 26 §000 0-0 0E 00 00 0E 0E 00 Eww-H 0.00 00.0 0.00 0.0 0.0 0E0 0.0 0.00 0E0 0.00 00.: 0000. 03 .50 E 0 000 0-0 0E 00 0-0 0E 0-E 0-0 0.00 0.0 00.0 6030200 0000. 00:363mi 0E0 0.00 0E0 0.0 0.0 0.00 0.00 0.0 0v S0028 E 0 0-0 0E 0 0-0 0E 0E 0E E 0: 0.: 0.0 0.0 E: 95E 0.0 0.: 0.0 0.00 0.00 0.00 E0603 000008 0E0 0000.0v 63656-0..«2335 E E o E E E E E E 0: 08002 00 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 00.0 00.0 E 0E0 600000000 E E E E E E E E 0 63000000 {000008 0.0 0.0 0.0 0.0 0.0 00.0 000 0.0 0.0 0.0 0.0 00.0 0E .00 0E0 mm E E E E E E 0:000: 95 E 0000000360 - GM - 00 0.0 E 0.0 00.0 00.0 6:0 0.0 0.0 0.0 0.0 0.0 MNRNV 0E0 6000000000 6o>_.$6:0.:0_fio:o E E E E E E E E E 00 0.0 0.0 0.0 0.0 0.0 0.0 0.0 00 0.0 00.0 00.0 0E0 60000000000000-0800 00000800060 0306000602000 000E00EE .8 .8 .8 .8 .00 .00 .00 .00 00:00-00: .00 .8 E0 125560.: 050 E0 - 00 00:00-00: 0 00 00 00 M<>OZ< 0 0 0 606588 00 Amodwnc 00.05 -0090 + 0 0003 AEEQEOU 95 E 0000 00050520 Eon-000.0 0E qu 00 E58000 010000002 9000000 0008009000 000660000 2 60008200 002330 000800900 n00002000000m 000300900 00000 + E 0000009000 5 Um AEano 0.:- .E0E Um E E .E0E E E o0Esw<v Esofiomv 000006000005 0E 000 .0500 00080520 00080520 5 000000660 600000000000: .m 00.0 0E + + 03 60000 03000 mam—4H- 000200300 000002000000 0070 SEEEEZ @052 RENE-000m ©3002-0003 00050520 055008 00003008000 2Ea0§000§0 @0308 00030000000 @5300 000-0002 000-0002 0003 00 0.20 00050 00000-00006 "+0 EEO < A®mUDQZH Mmmmmoo .60 0 E 0 0 E E EE EE E-E EE E2 E m; E SE 3% 0me ofim WEN ESE qmmm 2? 58.9 EEE mEE BEE E E 9; EE E EE EU EE E 0 -E 3% 2 08 03 2% 3% 2% 3 89 EEE 0me 58.9 EEEEQGEwEm EE EE EE E E EE 0E Eo m2 3% 2 QEN Ms 3N EE E09 58.9 0E 0.8m 2% at: EEE 332 :8 E EE E EE E EE EE E 3 oqu NEE 2M: 3 3 Ea mom 9}: 26 Ewwb EEE 3: 32 ma WE Soodv E3 ENE E EE 3 E EE QE EE EE 0E .5“ evade—\zwwrfi 2 w EEE 32 new ow: 2 2% 0: mEm 0.8 NE 58.9 603028 3563?: 0 E E-E E HO EE EE EE EE 3 E 55:00 E: E E: boa—=52 0mm 2 Q 2m 2 0E 0% Q: Soodv E SEE ESE» .2038 E EE EE E E 0 E 0E E SE 88.0 2E0: .m 6o>_.56:m=8_w6_65 E: 02 0.2 Ms E9 om 02 Q: new 2; E EEE E EE EE E E-E E EU E-E EE EE 633% m2 SE 638%: {803.8 08 WEN 3 w: 2 as 0.2 ES EEE 0.: 58.0 0E d JEE>E6E E BEES 95 E E E E Em 0.2 0.: 3 E 2 E E Amm 65E EEE w: 3 - - Ema MNRNV N33 623% 6o>€o6$b£fio=o E E E E E E E E E om E EE mm EE ms no cm 3 om mod E23 EEE 6oEEowEEEbéo: EEEEEEESE EoEoEEm-Eo6EoEm EEEEEEEE .8 .8 .8 .8 088-com E E E E 12556.9: BNM .E .8 EEE EEE E NE mm m -- E M<>OZ< E E E m oE Amodwmv 63588 EEG/E + Hog 203 AbEmEoU EE 95 EE GEE qu E AEEEEEE m6 EEEEEEEEE EEQ EEEEEEEm EEE @569: EEEEEEEEE 30:6oa EEEEEEz EEEEEEEEE C+xvew2 620205 26 ,mEoEzmoom E3830 Engage + E .E “Sam AEano E E “om E 2? .E E E E dmfld EEE mLoEEmE moEoE6E 3E5w<V Esofiomv E2629: E imam @869: @869: 6oEEow£Eb .v + + E3 Ema 96E mam—<8 \Hfivafiwvwrfl EEEEEEEEEEE Eoz EEEEEEEEZ @052 EEEEEEEE @mbE<mo< @569: Emmmmoo EEEEEESEEm EEEEEEEESEEE @EfimoE EEEEEEEEEEEE ESE/4% 83m: 83m: Boa +“ 3 :5 EEE>i EEO E3£fi6 < E®m0DQZH MmmmEOU E E E .EEE Example 7: Effect of Chromobacterium substugae (MBI-203) Against House flies Test substance is screened for direct contact efficacy against houseflies s). There are three treatment groups for each compound: 1.5%, 3% and 6% tration plus an untreated control. Each group contains five replicates with approximately 10 s each. Arthropods will be treated with a hand pump sprayer until “full ge" is reached, in 16 oz. drink cups with a beverage lid. At four hours, a cotton ball with 10% sucrose solution is provided for the flies by inserting it into the perforation in the lid. Data is taken at 5, 15 45 60 minutes and 2, 4, and 24 , 30, , hours or until endpoint. Knockdown and mortality were determined by counting the relative number of prone insects. Moribund insects were not included in mortality totals. s are shown below in Table 5.

Example 8: Effect of Chromobacterium substugae (MBI-203) t Litter Beetles Three treatment groups were tested for each compound: 1.5%, 3% and 6% concentration plus an untreated control. Each group contained five replicates with approximately 10 insects each.

Arthropods were treated with a hand pump sprayer until “full coverage" was reached, in 8 or 16 oz deli squat cups with perforated lids with filter paper on the bottom to absorb any excess al.

Knockdown and mortality were observed at 5, 15, 30, 45, 60 minutes and 2, 4, 24, 48, and 72 hours after treatment. own and mortality were determined by counting the relative number of prone insects. Moribund insects were not included in mortality totals. Results are shown below in Table 6. 00 $0.00 $0.00 $0.000 E 00 $0.00 $0.00 $0.00 $0.000 $000 $0.00 $0.00 $0.00 $0.0m $0.00 $0.00 \AHSNHHOE .E 00 $0.00 $0.00 $0000 00 $0.00 $0.00 $0.00 $0.00 $0000 $0.00 $0.00 $0.00 $0.00 $0.00 $0.00 E $0.00 $0.00 $000 00 00:00:02 00 $0.00 $0.00 $0.00 $0.00 $0.00 $0.00 $0.00 $0.00 $0.00 $0.00 $0.00 E0 $0.00 $0.00 $000 E0 $30 $0.00 $0.00 $0.00 $0.000 $0.00 $0.00 $0.00 $0.00 $0.00 $0.00 E0 $0.00 $0.00 $000 E0 $0.00 $0.00 $0.00 $0.00 $0.000 $0.00 $0.00 $0.00 $0.00 $0.00 $0.00 0: $0.00 $0.00 $000 0: $0.00 $0.00 $0.00 $0.00 $0.000 $0.00 $0.00 $0.00 $0.00 $0.00 $0.00 OEM ea 00 $0.00 $0.00 $0.00 :Bowxoosm ea 00 $0.00 $0.00 $0.00 $0.00 $0.00 $0.00 $0.00 $0.00 $0.00 $0.00 $0.0m 00 $0.00 $0.00 $0.00 ea 00 $000 $0.00 $0.00 $0.00 $0.00 $0.00 $0.00 $0.00 $0.00 $0.00 $0.00 ea $0.00 $0.00 $0.00 ea $0. 2 2 $0 $0.00 $0.00 $0.00 $0.00 $0.00 $0.0 $0.0 $0.0 $0.00 $0.00 ea ea 0 $0.00 $000 $000 0 $0.00 $000 $0.00 $000 $000 $0.0 $0.0 $0.0 $0.0 $0.0 $000 0 $0 $0 $0 $0 $0 $0 $0 $0 00m $0.0 $0 $0 20m $0.0 $0.0 $0.0 05 008m 0052 0080. m 3:0: $000 QMMWW 0 505 - 000000000 $0230.00 .0005 :mflWE£§§w€ 0300 0300 Example 9: Effect of MEI-203 on egg laying capacity of potato psyllids, Bactericera cockerelli Methods The egg laying capacity of potato psyllid females exposed to MEI-203 treated pepper leaves were determined. Pepper leaves were excised at the petiole and treated with MEI-203 by dipping for 1 minute. Treatments in the experiment were as follows: MEI-203 at 10% v/v in dHZO, dHZO as negative control and Avid at 10% v/v as positive control. Treated leaves were held in plastic petri dishes with the rims welled off, lined with craft foam with the center diameter cut out to expose treated leaf.

Four, l-day old s were placed in the center of the dish where the treated leaf was exposed (cut out portion of the craft foam), covered with the petri plate cover, and the setup was secured with 2 binder clips. The female adults were allowed to lay eggs and egg count was done 3 to days post exposure.

A follow up leaf disc bioassay on MEI-203 treated and untreated leaves was conducted to verify the effect of MEI-203 on the egg laying capacity of potato psyllid s. Treatment of MEI-203 at 3% v/v in water and an untreated control, dHZO only ive control) were used in the assay. Pepper leaf discs (from 3-4 week old pepper ) were cut in s using a 23 mm cookie cutter, selecting a flat portion of the leaf to make sure the disc can be evenly laid flat on the agar plate after treatment with the compound. The bottom of the plates was covered with 30 yL of 1% agar solution, just enough to cover the bottom of the plate to prop leaf discs and maintain humidity.

The agar was allowed to solidify by cooling at room temperature. Treatment of leaf disc was performed by pouring the treatment solution into a glass petri dish. With the solution in the dish, leaf discs were treated by soaking, swirling the dish gently to completely soak and coat the leaf discs. Treatment was performed for 1 minute and treated leaf discs were then allowed to dry for 10- 15 s in fume hood or until the solution had completely dried off. In each solidified agar plate, 20-30 yL dHZO was pipeted onto the agar. Each treated leaf disc was laid individually onto the agar plate, placing the leaf disc abaxial side down on wetted agar, pressed down gently to completely flatten the disc into the agar. In each plate with treated leaf disc (treatment), 4 female psyllids were uced. The petri plates with gravid females were then covered with the petri plate cover, poked with tiny holes for aeration and to prevent condensation. The dishes were sealed with parafilm and kept at room temperature. The number of eggs laid was counted daily, starting a day post uction of s. The experiment was done in 3 replications, repeated two times.

Results A significant reduction of eggs by females exposed to MEI-203 ent was observed. A slight delay in egg oviposition was apparent on females in MEI-203 treated leaf discs. Psyllid 2012/061503 females exposed to 3 treated leaf discs d ovipositing eggs 3 days post exposure (Figure 4). Eggs laid by females peaked on day 7 and declined at day 10. At day 10, mean egg count decreased as eggs laid by females started hatching. The females exposed to the positive control treatment (Avid 10% v/v) were all dead in day 3 with no eggs deposited on the avid treated leaf discs.

Verification leaf disc bioassays confirmed a consistent result with a significant reduction of eggs oviposited by females on MBI-203 treated leaf disc at 3% v/v (Figure 5). A 65% egg reduction was exhibited by females exposed to MBI-203 treated leaf discs compared to females d to untreated leaf discs (dHZO only). These bioassay results indicate that MBI-203 affects the physiology of the psyllid females ing their egg laying capacity.

Example 10: Effect of MEI-203 0n Grubs and Scarabs Control of white grubs 0n asses Insecticides were evaluated for l of white grubs (Southern Masked , Cyclocephala lurida Bland) on a Kentucky bluegrass (Poa pratensis L.) and perennial ryegrass rough m perenne L.) at the North Bend golf course in North Bend, Nebraska. Insecticides were applied to 5 x 5 ft plots arranged in a randomized te block (RCB) design with 5 replications. Liquid products were applied using a C02 sprayer at 40 psi and applying 174 gpa finished spray. Within 24 h following application, all treatments were irrigated with 0.25 in of water. Formulations were evaluated 24 days and 48 days after treatment (DAT) by ng from each plot three, 8-inch diameter turf-soil cores (1.05 ft2 total area) to a depth of 3 inches and ng the number of surviving and moribund grubs. Plots were periodically assessed for phytotoxicity. The s are shown in Tables 7 and 8.

There appears to be a correlation between application rate and percent control for the MBI- 203 DFl treatments. All treatments, except MBI-203 DFl (2 fl oz/100 ft2), outperformed trichlorfon 6% (marketed as DYLOX® 420 SL (6.9 fl.oz/1000 ftz) by Bayer CropScience, Inc., an industry standard insecticide for white grub control). Interestingly, moribund individuals were found in all treatments of MBI-203 AFl and MBI-203 DFl. These numbers (in parenthesis) were not used in the statistical analysis but were included for comparative purposes. No phytotoxicity was observed.

AFl is an aqueous flowable and DFl is a le powder formulation of Chromobacterium substugae.

Table 7: Efficacy MBI-203 in controlling White Grubs 24 days after Treatment (24 DAT) Treatment/ Rate No. WG/1.05 ft2 Mean WG i SE % formulation fl W1000 fiz 1.05 ft2 Control ___-“___. ___—mu ___-___” —m_nnnn-—— ___—mun ___—unn— Table 8 Efficacy MBI-203 in controlling White Grubs 24 days after Treatment (43 DAT) Treatment/ Rate No. WG/1.05 ft2 Mean WG % ——-n-_-___ ___—m“ ___—““1- ___—m— ___-um— Means followed by the same letter are not significantly different 5, LSD=4.4).

Feeding Study of the Effect 0fMBI-203 on Other R00t Feeding Scarabs Unsterilized Groton soil, 4.25 pH, 14% organic matter was infested with the scarab, oriental beetle larvae (Anomala orientalis) and rated on the number of larvae that died. The soil was dosed with aqueous flowable formulation of MBI-203 and the t mortality computed: Table 9: Effect of MBI-203 on Oriental Beetle Larvae Trial 1 Trial 2 % mortality 7 DAT (days after treatment) 1.5 ml product/5 g soil (30% soil mixture) 100 100 1 ml product/5 g soil (20% soil e) 100 100 .5 ml product + .5 ml de-ionized water/5 g soil 87 100 .25 ml product + .75 ml de-ionized 5 g soil 93 100 .1 ml product + .9 ml de-ionized water/5 g soil 40 13 .05 ml product + .95 ml de-ionized water/5 g soil 27 27 Untreated Control (30% moisture) 0 7 Untreated Control (20% moisture) 0 0 A similar trial was set up with the , Rhizotrogus majalis, ean chafer) grubs using 1.5 ml and 1 ml product in 5 g soil. 100/100 larvae were killed at 7 DAT and none were killed in the Control.

WO 62977 Trials were also conducted with black vine weevil larvae Otiorhynchus sulcatus (Curculionidae) with potting media (no food source), carrots and Taxus roots. Results are shown in Tables 10,11,12 and 13.

Table 10: Effect of MBI-203 with Potting Soil —% mortality 7 DAT % mortality 14 DAT .9 ml product/3 g media (30% soil moisture)“—- -6 ml product/3 g media (20% soil moisture)“—- Control (30% re) “—- Control (20% moisture) “—- Table 11: Effect of MBI-203 with Carrots Dipped in Product —% mortality 3 DAT Carrot sliee dipped in product Carrot slice dipped in de-ionized water (control)“ Table 12: Effect of 3 with Taxus Roots —% mortality 3 DAT Torus roots dipped in product and dried .— Taxus roots dipped in de-ionized water and dried“ Table 13: Effect of MBI-203 with Carrot Slice (Dried) —% mortality 3 DAT Carrot slice dipped in product and dried “ Carrot slice dipped in de-ionized water and dried (control)n It s that MBI-203 is very active against root feeding scarab beetles and weevil, particularly when fed a treated food source.

Example 11: Effect of MBI-203 t Cabbage Root Maggot This study was conducted to determine the efficacy of MBI-203 formulations for control of Cabbage Maggots (Delia radicum) on broccoli plants in a caged greenhouse study. Experimental treatments of MBI-203 DF-l (a wettable powder formulation of MBI-203) at rates of 2 0 ft2 and 8 02/ 1000 ftz; MBI-203 DF-2 (a second wettable powder ation of MBI-203) at rates of 2 02/ 1000 ft2 and 8 02/ 1000 ftz. Experimental treatments were compared to the cial standard, RADIANT® (marketed by DowAgro Sciences and containing spineforam as an active ingredient) at a rate of l lb/gal.

The number of live adult insects was recorded weekly up to 14 days after the third ation (l4DA-C) and the number of live larvae was recorded weekly up to 21 DA-C. Results showed that MBI 203 DF-l had a significantly fewer number of adults emerge by the last evaluation date, and was comparable to RADIANT®. MEI-203 DF-l had a significantly lower number of adults emerge than the UTC by the last evaluation date, and was able in control to Radiant.

Table 14. Maggot Count. Average number of larval Delia radicum insects per treatment, listed by evaluation date.

No. Name Unit Code —-—____ -——————n I_-—____ Table 15. Percent l. Average percent control of larval Delia radicum insects per treatment, listed by evaluation date.

No. Name Unit Code —-—____ -——————n -——————n I_-—____ Example 12: Efficacy of MEI-203 DF1 and 3 DF2 for the control of Drosophila suzukii (Spotted Wing Drosophila (SWD)) on Strawberry in the ouse This study was conducted to determine the cy of 3 DF1 and MEI-203 DF2 for the control of Spotted Winged Drosophila (SWD) on strawberry crops in the greenhouse.

Experimental treatments of MEI-203 DF1 and MEI-203 DF2 were applied at rates of l lb/a and 4 lb/a to replicate plots. Treatments were compared to the commercial standard, Entrust® at a rate of 1.5 oz/a. All treatments were combined with the surfactant SILWET® L77 (Chemtura AgroSolutions, Inc.) at a rate of 0.05% v/v. Each ate plot ning one strawberry plant was caged to prevent the migration of insect populations.

Third generation laboratory reared SWD winged adults were released on each caged strawberry transplant. Adult SWD counts were recorded at before application (pre-count), 4 days after application (DAA), 7 DAA, and 11 DAA. The number of SWD larvae per berry was recorded at 14 DAA, 21 DAA, 28 DAA, and 35 DAA. Statistics were analyzed using ANOVA mean comparison with LSD test and (1:0.05.

Following the first application, MEI-203 treatments showed progressive reduction of d Winged Drosophila adults and a rate response was observed for both DF1 and DF2 products. Although not significantly comparable to ENTRUST®, (Dow AgroBioSciences) containing spinosad as an active ingredient, MBI-203 DF2 at 4 lb/a significantly reduced adult tions by 25% in comparison to the UTC at 7 days after the first application (DAA). By 11 DAA, both DF1 and DF2 at 4 lb/a reduced adult counts by 44%, although not statistically different from the UTC. Both MBI-203 products exhibited significant results for reduction of SWD larvae counts. At all evaluations, DF2 at 4 lb/a significantly reduced the number of larvae per berry by 71%, comparable to ENTRUST®, (Dow AgroBioSciences) ning spinosad as an active ingredient, at 78%; further, by 21 DAA, larvae counts were controlled by all MBI-203 treatments up to 72%, and all were able to ENTRUST®, (Dow AgroBioSciences). A rate response was observed for both MBI-203 DF1 and DF2 with respect to larvae counts.

Results: Table 16. Number of Flies per Tent. Average count of Drosophila suzukii winged adults following release onto plants and applications per treatment, listed by evaluation date.

Trt ent Rate Rate Appl Pre-Count 4 DAA 7 DAA 11 DAA No. Name Unit Code 1 Untreated ABC 25.00 a 23.25 a 16.00 ab 4.00 a 2 MBI-203 DF 1 1 lb/a ABC 25.00 a 21.25 a 14.75 ab 2.75 a Silwet L77 0.05 % v/v ABC 3 MBI-203 DF 1 4 lb/a ABC 25 .00 a 19.75 a 13.50 ab 2.25 a Silwet L77 0.05 % v/v ABC 4 MBI-203 DF 2 1 lb/a ABC 25 .00 a 21.00 a 18.00 a 3.00 a Silwet L77 0.05 % v/v ABC MBI-203 DF 2 4 lb/a ABC 25 .00 a 19.75 a 12.00 b 2.25 a Silwet L77 0.05 % v/v ABC 6 t 1.5 oz/a ABC 25 .00 a 8.25 b 0.00 c 0.00 b Silwet L77 0.05 % v/v ABC Table 17. Percent l. Average percent control of Drosophila suzukii larvae per replicate plot, listed by evaluation date.

Trt ent Rate Rate Appl 14 DAA 21 DAA 28 DAA 35 DAA No. Name Unit Code 1 Untreated ABC 0.00 d 0.00 b 0.00 b 0.00 c 2 MBI-203 DF 1 1 lb/a ABC 41.52 c 54.22 a 42.83 ab 44.44 b Silwet L77 0.05 % v/v ABC 3 MBI-203 DF 1 4 lb/a ABC 54.78 bc 67.85 a 88.11 a 100.00 a Silwet L77 0.05 % v/v ABC 4 MBI-203 DF 2 1 lb/a ABC 52.97 bc 60.58 a 64.33 a 88.89 a Silwet L77 0.05 % v/v ABC MBI-203 DF 2 4 lb/a ABC 71.36 ab 72.16 a 88.11 a 100.00 a Silwet L77 0.05 % v/v ABC 6 Entrust 1.5 oz/a ABC 78.12 a 69.19 a 100.00 a 100.00 a Silwet L77 0.05 % v/v ABC 2012/061503 The findings may be summarized as follows: ° A rate response was observed for MBI-203 DF1 and DF2 for reduction of SWD adults and larvae counts ° MBI-203 DF2 at 4 lb/a significantly reduced adult populations by 25% at 7 DAA ° Throughout the trial, DF2 at 4 lb/a significantly reduced the number of larvae per berry and was comparable to Entrust Although application of both products did not show much effect on adult tion, it icantly reduced the larval population in the next generation.

Example 13: Repellent Effect of MBI-203 t0 Aphids Evaluation of repellent effect of various concentrations MBI-203 for green peach aphids was performed. ically, three treatment trations of MBI-203 in water (1% v/v, 3% v/v and 10% v/v) were evaluated. The MBI-203 10% v/v concentration was used as ve control and dHZO only treatment as negative control. Each treatment solution was added with 0.01% TWEEN Bioassays were performed by treating pepper leaf discs with respective MBI-203 concentrations as mentioned above. Pepper leaf discs (from 3-4 week old pepper plants) were cut in s using a 23mm cookie cutter, selecting a flat portion of the leaf to make sure the leaf disc can be evenly laid flat into the agar plate after treatment with the compound. A 1% agar solution was melted by heating and poured into the 145 mm X 20 mm petri plate, enough to cover the bottom surface of the plate to prop leaf discs and maintain humidity. The agar was allowed to solidify by cooling at room temperature.

Leaf disc treatment was performed by pouring the treatment solution into a glass petri dish.

With the solution in the dish, leaf discs were treated by soaking, swirling the dish gently to completely soak and coat the leaf discs. Treatment of leaf discs by soaking was done for 1 minute.

Treated leaf discs were then d to dry off by taking them out from the solution using forceps and placing them in the fume hood for 10-15 minutes or until the solution had completely dried off in the leaf surface. Once leaf discs are dried, 40 ML water was pipeted onto the agar where leaf disc will be laid. Treated leaf discs were then laid equidistant from each other, onto the wetted surface of the agar, placing each disc abaxial side down. Each disc was pressed down gently to completely flatten into the agar, and 20 3-4 day old GPA adults were uced at the center of the dish using a fine paint brush. Plates were then covered and sealed with parafilm. Petri plate cover was poked with tiny holes for aeration and to prevent condensation.

The test was performed in three replications. Repellency data of adults and nymphs was determined 24 hours after exposure of adults in the plates with treated leaf discs. The number of aphids (adults and nymphs) present in each leaf was counted and data recorded and analyzed.

Results MEI-203 is ent to GPA adults and nymphs with 97 - 99% repellency at different MBI- 203 concentrations (Table 18). Figure 6 shows a statistical difference among treatment concentrations. MEI-203 at 3% and 1% v/v concentrations had a computed mean % ency of 97% and 99% repellency, respectively.

Table 18. Mean t (%) repellency of Green Peach Aphid (GPA) adults and nymphs on pepper leaf discs treated with different MEI-203 concentrations.

MEI-203ml 3% WV MEI-203ml 1% W “ MEI-203 std 10% v/v Example 14: MEI-203 application reduces aphid y MEI-203 at 3% v/v concentration was tested to determine the effect of the compound on the progeny production of green peach aphid (GPA) . Bioassays were performed by treating pepper leaf discs with MEI-203 at 3% v/v. Pepper leaf discs (from 3-4 week old pepper plants) were cut in circles using a 23 mm cookie cutter, selecting a flat n of the leaf to make sure the disc can be evenly laid flat on the agar plate. A 1% agar solution was melted by heating and 30 yL poured in each petri plate (16mm X 35mm vented, polystyrene petri ), just enough to cover the bottom of the plate to prop leaf discs and maintain humidity. The agar was allowed to solidify by cooling at room temperature. Leaf disc treatment was performed by pouring the treatment solution into a glass petri dish. With the solution in the dish, leaf discs were treated by soaking, gently swirling the dish to completely soak and coat the leaf discs. Treatment of leaf discs by soaking was done for 1 minute. Treated leaf discs were allowed to dry for 10-15 s in fume hood or until the solution had completely dried off the leaf surface. Each treated leaf disc was laid individually onto the agar plate, placing the leaf disc l side down on wetted agar, pressed down gently to completely flatten the disc into the agar. In each plate with treated leaf disc (treatment), 6 GPA adults (3 - 4 day old) were introduced. The plates with adult aphids were then covered with parafilm. Parafilm covers were poked with tiny holes for aeration and to prevent condensation.

Plates were incubated at room temperature. Progeny (early nymphal instars) were counted 3 days after adult exposure to treated leaf discs. The experiment was done in 3 replications, and the whole experiment was repeated 5 times.

Figure 7 shows the MEI-203 significantly affected progeny tion of GPA adults 3 days post exposure to treated leaf discs. The graph in figure five is the result of the five trials ted and reduction in progeny of GPA adults was observed to be more than 50% compared to the negative control (water only treatment) and its performance is comparable to the positive control (10% Avid), showing statistically the same in y production. When the number of y production was tested in comparison with the formulated product DF2, there was no statistical difference between 3 standard and DF2 at 3% v/v concentration (Figure 8). A progeny (nymphs) reduction of more than 90% was exhibited in MEI-203 and DF2 treatments ed from the negative control, which showed to be significantly better than Avid at 10% v/v concentration (positive l).

Example 15: Extraction of Violacein and Oligo-(B-hydroxybutyric acid) from Chromobacterium gae.

The following procedure was used for the purification of compounds extracted from the culture of Chromobacterium substugae: The whole culture broth (WCB) derived from the 20-L fermentation C. substugae in L-broth was extracted by liquid-liquid extraction method using ethyl acetate. The ethyl acetate layer was separated and dried under vacuum using rotary evaporator to give the crude extract. The crude extract was then fractionated by using different solvent such as dichloromethane (DCM), ethyl acetate (EA), methanol (MeOH) and washing with mixture of solvent (WASH). These fractions were then concentrated to dryness using rotary evaporator and the resulting dry residues are ed for biological activity using different pest ts, nematodes). The active fractions were then subjected to Sephadex LH 20 size exclusion chromatography (CH2C12/CH3OH; 50/50) to give 10 fractions (Figure 9). These fractions were then concentrated to dryness using rotary evaporator and the resulting dry residues ions) were screened for biological ty using insect repellency assay with green peach aphids, progeny production of green peach aphid (GPA) and nematicidal bioassay (M. incognito and/0r M. hapla). The active fractions were then ted to reversed phase HPLC (Spectra System P4000 (Thermo ific) to give pure compounds, which were then screened in above mentioned bioassays to locate/identify the active compounds. To confirm the identity of the nd, additional spectroscopic data such as LC/MS and NMR were recorded.

The potent insecticidal repellent compound was isolated from fractions F8, F9 & F10 and was identified as violacein (2). The nematicidal active compound from the main DCM fraction was identified as oligo-(B-hydroxybutyric acid) (4).

Mass Spectroscopy Analysis of Compounds Mass spectroscopy analysis of active peak was performed on a Thermo Finnigan LCQ Deca XP Plus electrospray (ESI) ment using both positive and negative ionization modes in a full scan mode (m/z 100-1500 Da) on a LCQ DECA XPPlus Mass Spectrometer (Thermo Electron Corp., San Jose, CA). Thermo high performance liquid tography (HPLC) instrument equipped with an Surveyor PDA plus detector, autosampler plus, MS pump and a 4.6 mm x 100 mm Luna C18 5p 100A column (Phenomenex). The solvent system consisted of water (solvent A) and acetonitrile (solvent B). The mobile phase begins at 10% solvent B and is ly increased to 100% solvent B over 20 min and then kept for 4 min, and finally returned to 10% solvent B over 3 min and kept for 3 min. The flow rate is 0.5 mL/min. The injection volume was 10 pL and the samples are kept at room temperature in an auto sampler. The compounds are analyzed by LC-MS utilizing the LC and reversed phase chromatography. Mass spectroscopy is of the present compounds is performed under the following conditions: The flow rate of the nitrogen gas was fixed at 30 and 15 arb for the sheath and aux/sweep gas flow rate, respectively. Electrospray ionization was performed with a spray voltage set at 5000 V and a capillary e at 35.0 V. The capillary temperature was set at 400°C. The data was analyzed on Xcalibur software. The analysis of oligo- (B-hydroxybutyric acid) (1) was performed using LTQ ap XL hybrid r Transform Mass Spectrometer at UC Davis Mass ometry facility.

NMR Spectroscopy Analysis of Compounds NMR-NMR spectra were measured on a Bruker 600 MHz gradient field spectrometer. The reference is set on the internal standard tetramethylsilane (TMS, 0.00 ppm).

Purification ofCompounds The dichloromethane (DCM) fraction was triturated with methanol and the white solid Obtained was filtered off to give oligo-(B-hydroxybutyric acid) (1).

Identification ofcompounds Oligo-(fl-hydroxybutyric acid) (I) The 1H NMR spectrum of compound 4 ted signals at 6 5.22 (sext), 2.62 (dd) and 2.53 (dd) with a ve ities of l proton each. In addition to these a methyl signal at 6 1.31 as a doublet was also observed. The 13C NMR spectrum showed only four carbon signals at 6 169.2, 67.6, 40.9 and 19.8. The detail 1D & 2D NMR analysis resulted to the partial structure of —(-O- CH2-CO-)- with a fragment mass of 86. The MALDI-TOF-ESI MS ( of this compound exhibited typical signal pattern as for a mixture of oligomers with molecular masses of [(n x 86) + Na], corresponding the product was a mixture of Oligo-(B-hydroxybutyric acid) 1 with n = 10- 25. This compound has been reported to be isolated from several ia (Singh et al., 2011; Maskey et al., 2002; Hahn et al., 1995). Potency of this compound obtained from DCM fraction was confirmed in an in vitro assay using M. hapla which showed 75% immobility (Figure 10).

In vitro testing offractions andpure compound ofChromobacterium gae.

The fractions and pure compounds were ved in Dimethylsulfoxide (DMSO) and were tested in an in vitro 96-well plastic cell-culture plate bioassay. Around 15-20 nematodes in a 50 ul water solution were exposed to 100 pl of a 4mg/ml of sample for a 24 hour period at 25°C. Once the incubation period was completed, results were recorded based on a visual grading of immobility of the juvenile nematodes (J2’s) in each well treated with samples; each treatment was tested in well repetitions of four. Results were shown in Figure 5, which shows the results oftwo different 96- well plate bioassays of C. substugae fractions and compound 4. Three controls were ed in each trial; 1 positive (1% Avid) & 2 negative (DMSO & water). Both trials (T1) and (T2) were carried out using M. hapla nematodes.

Isolation and identification of compounds responsible for repellency The main fractions such as DCM, EA, MeOH and WASH were tested for repellency activity using the green peach aphid (GPA) bioassay as bed in detail below. The most potent repellency was observed for EA and MEOH fractions. The LCMS is of these fractions showed similar chemical profile, so since the yield of MeOH fraction was higher than EA fraction, the detail chemistry work was carried out using MeOH on. The MeOH on further fractionated using Sephadex LH 20 size exclusion chromatography (CHZClz/CH3OH; 50/50) to give 10 fractions (Figure 9). The most potent activity was observed in ns F9 and F10. Bioassay guided purification of these fractions by combination of HPLC and Sephadex LH 20 gave violacein as the compound responsible for repellent ty. Violacein was also tested in aphid progeny testing.

Example 16: Repellent Effect of 3 Fractions on Green Peach Aphid Choice bioassays on Green Peach Aphid adults (GPA) were done using MBI-203 fractions and pure nd. Fractions and pure compound (violacein) obtained from the extraction of cell paste of C. substugoe (MBI-203) were tested for insect efficacy h repellency bioassays.

Pepper leaf discs (from 3-4 week old pepper plants) were cut in circles using a 23 mm cookie cutter, selecting a flat portion of the leaf to make sure the leaf disc can be evenly laid flat into the agar plate after treatment of the compound. One percent (%) agar in water was prepared. The 1% agar solution was melted by heating and poured into the 145mm X 20mm petri plate, enough to cover the bottom surface of the plate to prop leaf discs and maintain humidity. The agar was allowed to solidify by g at room temperature.

Leaf disc treatment was done by pipetting gently 100 ML of MBI-203 extract unto the underside of the leaf disc. Treated leaf discs were then allowed to dry off by laying the disc flat unto a labeled 12-well plate cover. Once leaf discs are dried, 40uL water was pipetted unto the agar where leaf disc will be laid. Treated leaf discs were then laid equidistant from each other, unto the wetted surface of the agar, placing each disc abaxial side down with the treated surface up. Each disc was pressed down gently to completely flatten into the agar. After laying treated leaf discs, 20 3-4 day old GPA adults were introduced at the center of the dish using a fine paint brush. Plates was then covered and sealed with parafilm. Petri plate covers were poked with tiny holes for on and prevent condensation.

All tests were done in three replications. To select the t for testing initial testing of the crude extract was done using methanol and acetone as a solvent. This test showed that acetone was better as a solvent. Succeeding samples testing including fractions and pure compound (violacein) were carried out using e as a solvent. Data for repellency of adults and nymphs was determined 24 hours post exposure of adults on d leaf discs. The number of aphids (adults and nymphs) was counted and data recorded and analyzed. The percent repellency was computed as: % Repellency = 100-{([N+A]"on treated leaf" )/[N+A]"on petri dish" X100}, where A stands for adults and N stands for nymphs.

Results Repellency tests for MEI-203 fractions started with the crude extracts. Crude extracts in methanol and acetone solvents were tested and analysis of result showed significant differences as shown in Figure 11. Leaf discs treated with crude t of MEI-203 in both methanol and acetone solvents resulted in a statistically significant ence in settling response of green peach aphid nymphs and adults than those of the negative control. Figure 11A and B showed a repellent effect of MEI-203 on the aphids. The methanol solvent however showed a ent effect on aphids (Figure 11A), showing no statistical difference with the MEI-203 extract and the positive control (avid 10%). The solvent acetone showed to be a good solvent to use in ons as it did not exhibit repellency on GPA nymphs and adults, having the mean number of nymphs and adults settling in the leaf disc tically the same with the ve control e 11B).

Fractions of MEI-203 exhibited a strong repellency on GPA nymphs and adults. Statistical difference among treatment means were observed e 12A and B). Fractionated materials EA and MeOH caused 100% repellency on nymphs and adults while Wash material 94% repellency which is not statistically different from the positive control (MEI-203 10% v/v) (Table 19).

Table 19. Mean percent repellency of MEI-203 fractionated samples.

Fractionated Mean % material Repellency DCM 77.2 EA 100 203 10% (+ C) 98.9 Wash 94 MeOH 100 203 10% (+ C) 100 Furthermore, the 10 fractions obtained from MeOH fraction were tested using acetone as a solvent; the s with pure violacein compound (F9, F10) showed high repellency effect on adults and nymphs of green peach aphids (Table 20). Only 2 non-violacein fractions showed high repellency effect (F2 and F3). The fractions F9 and F10 were further purified which gave violacein which had 100% repellency. The data revealed that violacein is the responsible compound in causing repellency to sucking insects. It appears that onated materials F6 — F10 contained violacein with F9 and F10 containing pure violacein compound.

Table 20. Mean percent repellency of ons obtained from fractionation of MeOH fraction (F1 — F10).

Fractionated material Mean % ency F1 81 .5 F2 92 .9 F3 95 .6 F4 76 .8 F5 84 .3 F6 90.1 F7 96 F8 91 .5 F9 100 F10 100 Example 17: Violacein reduces aphid progeny Two concentrations of pure violacein compound in acetone, 0.5 yg/mL and 1.0 yg/mL were used in the test to determine the effect of the compound on the progeny production of green peach aphid (GPA) adults. Bioassays were performed by treating pepper leaf discs with the different violacein concentrations in e. Pepper leaf discs (from 3-4 week old pepper plants) were cut into 23 mm diameter discs, selecting a flat portion of the leaf to make sure the disc can be evenly laid flat onto the agar plate after treatment with the compound. A 1% agar solution was prepared, melted by heating and 30 yL poured into petri dishes (16 mm X 35 mm vented, polystyrene petriplates), just enough to cover the bottom of the plate to support leaf discs and maintain humidity.

The agar was allowed to solidify by cooling at room temperature. Treatment with violacein was done by gently spreading 100 yL of the sample solution onto the leaf disc using a 200 yL an.

Treatments were set up in triplicate. d leaf discs were allowed to dry in the hood for 5-10 s. The positive control was 10% v/v Avid and the negative l was dHZO, acetone was used as the blank. In each solidified agar plate, 20-30 yL dHZO was pipetted onto the agar to maintain humidity. Each treated leaf disc was laid individually onto the agar plate, placing the leaf disc abaXial side down on wetted agar, pressed down gently to completely flatten the disc onto the agar. In each plate with treated leaf disc ment), 6 GPA adults (3 - 4 day old) were introduced.

The plates with adult aphids were then d with lm. Cover parafilm was poked with holes aeration to prevent condensation, and was kept at room temperature. Progeny (early l instars) were counted 3 days after adult exposure to treated leaf discs. The ment was done in 3 replications, and the whole experiment was repeated 2 times.

As shown in Figure 13, violacein at 1.0 M g/mL icantly d y production of adult aphids. A reduction of about 50% was observed compared to the negative control (water only treatment). The positive control (10% Avid) had the least number of progeny as most of the exposed adults died 3 days post exposure. Two trials were med in this experiment, each treatment ated three times. Both trials provided consistent results with violacein significantly affecting progeny.

Example 18: Other Violacein producers display repellency against aphids The repellent effect of other Chromobacterium species on green peach aphids was also evaluated. Chromobacterium species evaluated are: Chromobacterium piscz'nae DSM 23278, C. pseudoviolaceum DSM 23279, C. haemolyticum DSM 19808 and C. aquaticum DSM 19852. Two of the species are violacein producing species (C. piscinae and C. pseudoviol) while the two other species are documented to not produce violacein. Microorganisms were grown on LB broth at 26°C, and 100 rpm for 5 days. At the end of the fermentation, broths were harvested and aliquoted for bioassay. Treatment concentrations at 5% v/v in water were tested on GPA adults. MBI-203 10% v/v concentration was used as positive control and dH2O only treatment as negative control. Each treatment solution was added with 0.01% TWEEN 20.

Bioassays were performed by treating pepper leaf discs as previously described. Each treated disc was pressed down gently to completely flatten into the agar. After laying treated leaf discs, 20 3-4 day old GPA adults were introduced at the center of the dish using a fine paint brush.

Plates were then covered and sealed with parafilm. Petri plate covers were poked with tiny holes for on and to prevent condensation.

The test was done in three replications. Repellency data of adults and nymphs was determined 24 hours after exposure of adults into the plates with treated leaf discs. The number of aphids s and nymphs) settled on each leaf disc was d and data recorded and analyzed.

Percent repellency was calculated as follows: % Repellency = Where N: number of nymphs, and A=number of adult aphis Results are shown in Tables 21 and 22 and Figures 14 and 15. Several violacein-producing Chromobacterium species showed repellency to GPA adults and nymphs with tical differences among treatment means. Chromobacterium species producing violacein had mean % repellency of 75% (C. piscinae), 86% (C. pseudoviolaceum ), while non-violacein producers (C. aquaticum and C. haemolyticum) were not statistically different from the untreated control (water). A slight trend of repellency was observed for the olacein ers.

Table 21. Mean percent (%) repellency of Green Peach aphid (GPA) adults and nymphs exposed to pepper leaf discs treated with different Chromobacterium species.

Treatment Mean % Repellency Cpseudow’olaceum _E MEI-203 Std 10% Table 22. Mean t (%) repellency of Green Peach aphid (GPA) adults and nymphs exposed to pepper leaf discs treated with different Chromobacterium species WWW.

Ms... 10%“ Example 19: Comparison of stability of Chromobacterium formulation with and Without Sodium Benzoate Formulation 1 contains Chrombacterium cell concentrated harvest 32 parts, Chrombacterium Fermentation Broth Supernatant 62.5 parts, n-Hexanol 1 part, Sodium Alginate 0.5 parts, Sorbitan Ester Ethoxylate 2 parts, and d-limonene 2 parts. These formulation ingredients are chosen for their onality in ng uniform and stable mixtures and are also preferred due to their g on US EPA list 4. A listing of an ingredient on EPA list 4 deems that it is of minimal concern in terms of effect on the environment and toxicology. Formulation 2 contains acterium Cell concentrated harvest 32 parts, Chrombacterium Fermentation Broth atant 54.5 parts, n-Hexanol 1 part, Sodium Alginate 0.5 parts, Sorbitan Ester Ethoxylate 2 parts, and Sodium Benzoate 10 parts.

Table 23 illustrates results of storage of ations l & 2 over and extended period of time.

Table 23: Storage of Formulations 1 and 2 Formulation Visual Observation: 30 days 120 days One day Formulation 1 Uniform purple liquid Separation of white Non uniform Grey light brown layers on brown liquid with top. d purple white crusting in head color space.

Uniform purple liquid Uniform purple liquid Uniform purple liquid Formulation 2 is more stable than Formulation 1. It appears that water e salts of benzoic acid stabilizes biological pesticide itions against physical separation and loss of activity due to exposure to ht. The benzoate ions provide solvency and electrolyte balancing properties such that the biological matrix remains uniform this results in extended shelf life for the pesticide composition. The benzoate ions also provide ultraviolet radiation absorption for the product once it has been applied to field crops. The UV protection extends the insecticide ty for at least l more days.

Exanple 20: Effect of Sodium Benzoate on Pest Mortality The end product MBI-203 containing Chromobacterium substugae, d-limonene, hexanol, ene glycol, and paraben formulation (MEI-203 EP) combined with calcium carbonate, sodium benzoate, or um oxide were placed into plastic petri dishes and sealed with parafilm.

Plates were placed outside for 7 hours in the sunlight. After exposure to sun, material was brought inside and diluted to 1.5% and 3% v/v concentrations with autoclaved Millipore water. Material was then placed on artificial diet, dried, and fed to neonate Cabbage Looper, Trichoplusia ni. Mortality was scored 3 and 4 days after infestation of diet. The results are shown in Table 24 and Figure 16.

Table 24: Effect of Sodium Benzoate on Pest Mortality Composition Mortality (% loss) EP + CaCO3 ~30% loss EP+ TiO2 ~60% loss ~25% loss EP + Purshade EP + Na benzoate ~10% loss EP only ~30% loss Broccoli Study 4-5 week old n broccoli were sprayed with 3% v/v dilutions of MBI-203 end product (d-limonene formulation) 03 EP) with and without sodium benzoate. Each plant in a 9 in2 pot received 500 uL of treatment. Tween-20 at a final concentration of 0.01% was ed in all samples. Five adult Cabbage Aphids, Brevicoryne brassicae, were placed on each plant. Plants and insects were incubated under growth lights °F, 16h light/8h dark). Alive Aphids were scored 3, 5 , and 7 days after infestation. It appeared that the sodium benzoate formulation provided better control than the end product alone (Figure 17).

Red Cabbage Study % v/v dilutions of MBI-203 end product (d-limonene formulation) 03 EP) with and without sodium benzoate were sprayed onto Red Cabbage at approximately 30 gal/acre treatment rates. A formulation blank (lot 24033) at 10% v/v was also included in the assay.

After plants had dried, they were infested with 10 Cabbage Aphids. The assay was scored on days 3, 6, and 8. Percent control was ined by applying the Henderson-Tilton correction. The results are shown in Figure 18. The end product + sodium benzoate formulation had better control of aphids than the end product alone or the formulation blank alone.

Example 21: Comparison of Sodium Benzoate and Calcium Carbonate ted 3 end product (MEI-203 EP) with various concentrations of sodium benzoate or calcium carbonate were exposed to sunlight for 1 day in sealed plastic petri dishes. Sun- exposed and unexposed al were then diluted to 3% v/v dilutions and applied to artificial diet.

Neonate Cabbage s were exposed to diet and mortality was scored 4 days after infestation of diet. Results are shown in Figure 19. Sodium benzoate at 10% and 15% concentrations appeared to provide the least degradation in activity at the most economical price.

Example 22: Effect of Lignin Sulfonate Undiluted spray dried cells with various additives were poured into a plastic vial and exposed to the environment for 4 days, 40-65°F, sunny with some days of les). Exposed and unexposed samples were diluted to 6% v/v and d onto Packman Broccoli plants. Dried plants were infested with 5 immature Cabbage . Aphids were scored 3 and 6 days after infestation.

Control was determined by applying the Henderson-Tilton correction. Spray dried cells with sodium benzoate had one of the highest kills at day 3 and maintained control as the assay continued to day 6 (see Figure 20).

MBI-203 end product (d-limonene formulation) (MEI-203 EP) was mixed with various concentrations of sodium te and lignin sulfonate. Treatments were diluted to 10% v/v concentrations and pipetted into plastic petri dishes and exposed to sunlight for 4 continuous days. s were then tested for e Looper activity by treating artificial diet. Lignin ate treatments tended to form a crust over on top of artificial diet. End product with only sodium benzoate seemed to have the most ing kill before and after sun exposure (see Figures 21 and 22).

In another study, 10% v/v dilutions of MEI-203 end product (d-limonene formulation) with % sodium benzoate and various concentrations of lignin sulfonate were exposed to sunlight for 4 utive days in sealed plastic petri dishes. After exposure, 4 week old Packman li were sprayed with treatments at imately a 30 gal/acre rate. Three late second instar Cabbage Looper larvae were placed on each treated plant and mortality was scored on days 3 and 4 of the assay. Of the plants in the sun, the sodium benzoate only sample had the highest control (see Figure 23).

Although this invention has been described with reference to specific embodiments, the details thereof are not to be construed as limiting, as it is obvious that one can use various equivalents, changes and modifications and still be within the scope of the t invention.

Various references are cited throughout this specification, each of which is incorporated herein by reference in its entirety.

References Cited Asolkar, R. N., Jensen, P. R., Kauffman, C. A., Fenical, W. 2006. Daryamides A-C, Weakly Cytotoxic Polyketides from a -Derived Actinomycete of the Genus Streptomyces strain CNQ—085 J. Nat. Prod. 69: 1756-1759.

Arena, J. P., K. K. Liu, et al. (1995). "The mechanism of action of avermectins in Caenorhabditis elegans - correlation between activation of ate-sensitive de current, ne binding and biological activity." Journal of tology 81: 286-294.

Byrom, D. (1991). “Copolymer production.” EP 0431883 A2.

Duran, N., G. Z. Justo, et al. (2007). "Minireview. Violacein: ties and biological activities." Biotechnol. Appl. Biochem. 48: 3.

Duran, N. and C. F. M. Menck (2001). "Chromobacterium violaceum: a review of pharmacological and industrial perspectives." Crit. Rev. Microbiol. 27: 201-222.

Furneaux, G. C. (2005). “Compostable packaging al and method.” WO 030482 A1.

Hahn, S. K.; Chang, Y. K.; Lee, S. Y. (1995). Recovery and characterization of poly(3- ybutyric Acid) synthesized in Alcaligenes eutrophus and recombinant Escherichia coli.

Applied & Environ. Microbiol61. 34-39.

Hoshino, T., T. Takano, et al. (1987). "Biosynthesis of violacein: origins of the hydrogen, nitrogen and oxygen atoms in the 2-pyrrolidone nucleus." Agric. Biol. Chem. 51: 2733-2741.

Huismar, G. W.; Peoples, O. P.; Skraly, F. A. (2000). “Transgenic microbial polyhydroxyalkanoate producers.” WO 11188 A1.

Huismar, G. W.; Skraly, F. A.; Martin, D. P., Peoples, O. P. (1999). “Biological systems for manufacture of polyhydroxyalkanoate polymers containing 4-hydroxyacids.” WO 14313 A2.

Hungria, M., S. Astolfi-Filho, et al. (2005). "Genetic characterization of Chromobacterinm isolates from black water environments in the Brazilian Amazon." Lett. Appl. Microbiol. 41: 17-23.

Khanna S, Srivastava AK. (2005). “Recent es in microbial polyhydroxyalkanoates” Process mistry” 40, 607-619.

Krans RG, t KK, Locke TA, Madigan MT (1997). “Polyhydroxyalkanoate production in Rbodobacier capsulaius.‘ Genes, mutants, expression, and physiology.” Appl. Environ. Microbiol. 63, 3003-3009.

Krieg, A., A. M. Huger, et a1. (1983). "Bacillus thuringiensis var. tenebrionis: Ein neuer, gegenuber Larven von Coleopteren wirksamer yp." Z. Angew. Entomol._96: 500-508.

Kampfer, P., H.—J. Busse, et a1. . "Chromobacterium piscinae sp. nov. and bacterium pseudoviolaceum sp. nov., from environmental samples." Int. J. Syst. Evol. Microbiol. 59: 2486- 2490.

Lee, S.Y. . “Bacterial polyhydroxyalkanoates”. Biotechnol. Bioeng. 49, 1-14.

Martin, P. A. W., D. Gundersen-Rindal, et al. (2007a). "Chromobacterium substugae sp. nov., a betaproteobacterium toxic to Colorado potato beetle and other insect pests." Int. J. Syst. Evol.

Microbial. 57: 993-999.

Martin, P.A., A.D.S. Shropshire, et al., (2007b). " Chromobacterium substugae sp. nov for control of insect pests" US. Patent 7244607 B2.

Martin, P.A.W., Hirose, E., and Aldrich, J .R. 20070. " Toxicity of bacterium substugae to southern green stink bug (Heteroptera:Pentatomidae) and corn rootworm (Coleoptera:Chrysomelidae)".

J. Econ. Entomol. 100: 4.

Martin, P.A.W., Blackburn, M., et a1. (2004), "Two New Bacterial Pathogens of Colorado Potato Beetle (Colorado: Chrysomelidae)" , J. Econ. Entomol. 97:774-780 (2004).

Martin, P.A.W., "A Freeze-Dried Diet to Test Pathogens of Colorado potato ", Biological Control 29:109-114 (2004).

Maskey, R. M.; Kock, 1.; Shaaban, M.; Grun-Wollny, 1.; Helmke, E.; Mayer, F.; Wagner-Dobler, 1.; Laatsch, H. (2002) Low molecular weight oligo-B-hydroxybutyric acid and 3-hydrox-N-phenyl- butyramide new products from microorganism. Polymer bulletin. 49, 87-93.

McClean, K. H., M. K. Winson, et a1. (1997). "Quorum sensing and Chromobacterium eum.‘ exploitation of Violacein production and inhibition for the detection of N-acyl homoserine lactones" iology 143: 3703-3711.

Mukhopadhyay M., Patra A., Paul, A. K., (2005). “Production of poly (3-hydroxybutyrate) and poly (3-hydroxybutyrate-cohydroxy-valerate)” by Rhodopsudomonas ris SP5212” J. Micro.

Biotech, 21, 765-769.

Peoples, O. P.; Sinskey, A. J. . “ Method for producing novel polyester biopolymer.” WO 910091 A1.

Reddy, C. S. K.; Ghai, R.; Kalia, R.V.C. (2003). “Polyhydroxyalkanoates: an overview” Bioresource Technology.” 87, 137-146.

Ryan, K. S. and C. L. Drennan (2009). "Divergent pathways in the biosynthesis of bisindole l products." try&Biology 16: 351-364.

Thompson, G. D., R. Dutton, et al. (2000). "Spinosad - a case study: an example from a natural products discovery programme." Pest Management Science 56: 696-702.

Young, C.-C., A. B. Arun, et al. . "Chromobacterium aquaticum sp. nov., isolated from spring water samples." Int. J. Syst. Evol. Microbiol. 58: 877-880.

I/WE

Claims (11)

CLAIM :

1. A method for modulating ation of a pest, wherein said pest is selected from an Acari, Muscidae, Drosophilidae, Anthomyidae, Aphididae, Triozidae, Tenebrionidae, and Scarabaiedae, in a location where tion is desired comprising applying an amount of a composition obtained from Chromobacterium subtsugae Nov strain (NRRL B-30655), effective for modulating said infestation.

2. The method according to claim 1, wherein the on where modulation is desired is on a plant, plant seed or in soil.

3. The method according to claim 1 or claim 2, wherein said pest is an Acari and the Acari is a mite.

4. The method according to claim 3, wherein said mite is a Tetranychus sp..

5. The method ing to claim 1 or claim 2 wherein said pest is selected from a Musca sp., Myzus sp., Bactericera sp., Cyclocephala sp., Alphitobius sp., Drosophila sp., Delia sp., Rhizotrogus sp., Popillia sp., Anomala sp., and Otiorhynchus sp..

6. The method according to claim 1 or claim 2, wherein said pest is selected from a Musca domesitcas, Drosophila suzukii, Delia radicum, Myzus persicae, Bactericera cockerelli, Alphitobius diaperinusxi, Cyclocephala lurida, Rhizotrogus majalis, Popilla japonica, ynchus sulcatus, and Anomala orientalis.

7. The method of claim 4, wherein the ychus sp. is Tetranychus urticae.

8. The method of claim 1 or claim 2, wherein the pest is a larva of a aiedae.

9. The method of any one of claims 1 to 8, n the composition is selected from a whole cell broth, supernatant, filtrate, and extract obtained from Chromobacterium subtsugae Nov strain (NRRL B-30655).

10. The method of claim 9, wherein the composition is a whole cell broth. AH26(10752692_1):CCG

11. The method of claims 9 or 10, n the whole cell broth comprises violacein, deoxyviolacein, or chromamide A. Marrone Bio Innovations, Inc. By the Attorneys for the Applicant SPRUSON & FERGUSON Per: AH26(10752692_1):CCG