Classifications

• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
  • A01N43/00—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds
  • A01N43/90—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having two or more relevant hetero rings, condensed among themselves or with a common carbocyclic ring system
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
  • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
  • A01N63/20—Bacteria; Substances produced thereby or obtained therefrom
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
  • A61K35/66—Microorganisms or materials therefrom
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
  • A61K35/66—Microorganisms or materials therefrom
  • A61K35/74—Bacteria
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
  • A61K38/12—Cyclic peptides, e.g. bacitracins; Polymyxins; Gramicidins S, C; Tyrocidins A, B or C
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
  • C07K5/02—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link
  • C07K5/0205—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link containing the structure -NH-(X)3-C(=0)-, e.g. statine or derivatives thereof
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
  • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
  • C07K5/08—Tripeptides
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
  • C07K7/04—Linear peptides containing only normal peptide links
  • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
  • C07K7/64—Cyclic peptides containing only normal peptide links
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
  • C12P17/18—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing at least two hetero rings condensed among themselves or condensed with a common carbocyclic ring system, e.g. rifamycin
  • C12P17/188—Heterocyclic compound containing in the condensed system at least one hetero ring having nitrogen atoms and oxygen atoms as the only ring heteroatoms
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P21/00—Preparation of peptides or proteins
  • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
  • A61K2236/10—Preparation or pretreatment of starting material
  • A61K2236/11—Preparation or pretreatment of starting material involving culturing conditions, e.g. cultivation in the dark or under defined water stress
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
  • A61K2236/30—Extraction of the material
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
  • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
  • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

• Natural products are substances produced by microbes, plants, and other organisms. Microbial natural products offer an abundant source of chemical diversity, and there is a long history of utilizing natural products for pharmaceutical purposes. Despite the emphasis on natural products for human therapeutics, where more than 50% are derived from natural products, only 11% of pesticides are derived from natural sources. Nevertheless, natural product pesticides have a potential to play an important role in controlling pests in both conventional and organic farms. Secondary metabolites produced by microbes (bacteria, actinomycetes and fungi) provide novel chemical compounds which can be used either alone or in combination with known compounds to effectively control insect pests and to reduce the risk for resistance development. There are several well- known examples of microbial natural products that are successful as agricultural insecticides (Thompson et al., 2000; Arena et al., 1995; Krieg et al. 1983).
• 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 greenhouse and in the field. At the same time, active compounds produced by the microbe are isolated and identified.
• the microbe has to be economically produced by fermentation at an industrial scale and formulated with biocompatible and approved additives to increase efficacy and to maximize the ease of application as well as storage stability under field conditions.
• Beta-Proteobacterium strain Chromobacterium subtsugae
• Diabrotica virgifera adult and larval Southern Corn Rootworm (Diabrotica virgifera), adult and larval Southern Corn Rootworm (Diabrotica virgifera), adult and larval Southern Corn Rootworm (Diabrotica virgifera), adult and larval Southern Corn Rootworm (Diabrotica virgifera), adult and larval Southern Corn Rootworm (Diabrotica virgifera), adult and larval Southern Corn Rootworm (Diabrotica virgifera), adult and larval Southern Corn Rootworm (Diabrotica).
• Chromobacterium violaceum is studied the most, and published information on secondary metabolites produced by Chromobacteria is based on studies on Chromobacterium violaceum only.
• Duran and Menck (2001) have published a comprehensive review of the pharmacological and industrial perspectives of Chromobacterium violaceum, a Gram-negative saprophyte from soil and water. 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.
• Chromobacterium violaceum is known to produce a purple pigment, violacein, which is a bisindole molecule generated by a fusion of two L-tryptophan molecules 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 Gram-negative bacteria (McClean et al., 1997).
• Menck include hydrogen cyanide, ferrioxamine E, B-lactamic glycopeptides SQ28,504 and SQ28,546, antibiotics such as aerocyanidin, aerocavin, 3 ,6-dihydroxy- indoxazene, and monobactam SB-26.180, and an antitumoral depsipeptide FR901228.
• Chromobacterium violaceum also produces unusual sugar compounds such as extracellular polysaccharides and lipopolysaccharides .
• Nematodes are non- segmented, bilaterally symmetric, worm-like invertebrates that possess a body cavity and complete digestive system but lack respiratory and circulatory systems. Their body wall is composed of a multilayer cuticle, a hypodermis with four longitudinal cords, and internal musculature (Chitwood, 2003). Their body contents are mostly occupied by digestive and reproductive systems. Most nematodes are free-living but a smaller number of species are ubiquitous parasites of animals or plants.
• Root-knot nematodes (Meloidogyne spp.) parasitize a wide range of annual and perennial crops, impacting both quality and quantity of marketable yields. Nematodes in this genus are considered the most economically important plant parasitic nematodes (Whitehead, 1998). Annual crop losses caused by plant-parasitic nematodes have been estimated to exceed US $100 billion (Koenning et al. 1999), with more than half caused by the genus Meloidogyne. The inoculum in this strain comes from eggs that under favorable conditions hatch to release infective second stage larvae (J2s), which migrate in the soil towards a host plant root.
• J2s infective second stage larvae
• Infection occurs through root tip penetration, after which the larvae move to vascular tissue where the nematode becomes sedentary, feeding directly from plant cells.
• the plant responds by producing giant cells that form galls (root knots).
• root knots giant cells that form galls
• root-knot nematodes The most efficient means for controlling root-knot nematodes is via nematicides that inhibit either egg hatching, juvenile mobility and/or plant infectivity.
• the development of chemical control for plant-parasitic nematodes is challenging because of both
• halogenated hydrocarbons e.g. ethylene dibromide, methyl bromide
• ethylene dibromide methyl bromide
• microbial plant/nematode pathogens have been reported to be active against plant parasitic nematodes (Guerena, 2006). These biological control agents include the bacteria Bacillus thuringiensis , Burkholderia cepacia, Pasteuria penetrans and P. usgae. Pasteuria Biosciences has launched P. usgae against sting nematodes on turf in the southeastern US. Nematicidal fungi include Trichoderma harzianum, Hirsutella
• rhossiliensis H. minnesotensis , Verticillium chlamydosporum, Arthrobotrys dactyloides, and Paecilomyces lilanicus (marketed as BioAct ® and Melcon ® by Prophyta) .
• Another fungus, Myrothecium verrucaria is available in a commercial formulation, DiTera ® , by Valent Biosciences. This is a killed fungus; hence the activity is due to nematicidal compounds.
• Other commercial bionematicides include Deny ® and Blue Circle ® (B.
• Botanical nematicides such as plant extracts and essential oils, can be used to control nematodes (Kokalis-Burrelle and Rodriguez-Kabana, 2006).
• Chitwood has summarized the options of using plant-derived compounds for nematode control in his recent review article (Chitwood, 2002).
• Siddiqui and Alam (2001) demonstrated that potting soil amended with plant parts from the neem tree (Azadirachta indica) and Chinaberry tree (Melia azadirah) inhibited root-knot nematode development of tomatoes.
• no neem products are currently registered in the US for use against nematodes .
• a new botanical product from Chile (Nema-Q ® ) based on a Quillaja saponaria tree extract containing saponins (bidesmosidic derivatives of quillajie acid substituted with a trisaccharide at C-3 and an oligosaccharide in C-28) has been recently registered as a an organic nematicide through US EPA and listed for organic farming by the Organic
• AUelochemicals are plant-produced compounds that affect the behavior of organisms in the plant's environment. Examples of nematocidal allelochemicals include polythienyls,
• RNA interference has been proposed as a method for controlling nematodes .
• Use of gene silencing via RNAi was first demonstrated on Caenorhabditis elegans and quite recently also for plant parasitic nematodes such as Meloidogyne spp.
• compositions comprising a strain of Chromobacterium sp., particularly a strain of Chromobacterium substugae and more particularly, a strain of Chromobacterium substagae sp. nov. and even more particularly a strain of Chromobacterium substagae sp. nov. having the identifying characteristics of NRRL B-30655 described in US Patent No. 7,244,607.
• a method for modulating nematode infestation in a plant comprising applying to a plant, and/or seeds thereof and/or substrate used for growing said plant an amount of a supernatant, filtrate and/ or extract and/or one or more metabolites from said supernatant, filtrate and/ or extract of a strain of Chromobacterium sp. , particularly a strain of Chromobacterium substugae and more particularly, a strain of Chromobacterium substagae sp. nov. and even more particularly a strain of
• Chromobacterium substagae sp. nov. having the identifying characteristics of NRRL B- 30655 described in US Patent No. 7,244,607 and optionally another nematocidal substance in an amount effective to modulate said nematode infestation.
• a pesticidal combination synergistic to at least one pest comprising as active components: (a) a supernatant, filtrate and/or extract of a strain of Chromobacterium sp., particularly a strain of Chromobacterium substugae and more particularly, a strain of Chromobacterium substagae sp. nov. and even more particularly a strain of Chromobacterium substagae sp. nov. having the identifying characteristics of NRRL B-30655 described in US Patent No.
• Chromobacterium sp. particularly a strain of Chromobacterium substugae and more particularly, a strain of Chromobacterium substagae sp. nov. and even more particularly a strain of Chromobacterium substagae sp. nov. having the identifying characteristics of NRRL B-30655 described in US Patent No. 7,244,607 and (b) another pesticidal substance, wherein (a) and (b) are present in synergistic amounts.
• the pest in a particular embodiment, may be an insect pest, but may also include, but is not limited to, a nematode, plant fungus, plant virus and plant bacteria and weeds. Further, the combination may be a composition.
• 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 nematocide.
• the combination may comprise a supernatant, filtrate and/or extract of a strain of Chromobacterium sp., particularly a strain of Chromobacterium substugae and more particularly, a strain of Chromobacterium substagae sp. nov. and even more particularly a strain of Chromobacterium substagae sp. nov. having the identifying characteristics of NRRL B-30655 described in US Patent No. 7,244,607 and a pesticidal substance derived from a microorganism including but not limited to Bacillus sp. (e.g., Bacillus thuringiensis or Bacillus thuringiensis kur taid) and spinosad.
• the combination may comprise a supernatant, filtrate and/ or extract of a strain of Bacillus sp. (e.g., Bacillus thuringiensis or Bacillus thuringiensis kur taid) and spinosad.
• Chromobacterium sp. particularly a strain of Chromobacterium substugae and more particularly, a strain of Chromobacterium substagae sp. nov. and even more particularly a strain of Chromobacterium substagae sp. nov. having the identifying characteristics of NRRL B-30655 described in US Patent No. 7,244,607 and a pesticidal substance derived from a natural product such as pyrethrum.
• the combination may comprise a supernatant, filtrate and/ or extract of a strain of Chromobacterium sp .
• a strain of Chromobacterium substugae and more particularly, a strain of Chromobacterium substagae sp. nov. and even more particularly a strain of Chromobacterium substagae sp. nov. having the identifying characteristics of NRRL B-30655 described in US Patent No. 7,244,607 and a pesticidal substance which is a chemical pesticide, particularly, an insecticide, where the insecticide includes but is not limited to pyrethrins, spirotetramet and anthranilicdiamide .
• a method for synergistically modulating infestation of at least one pest or pest species in a plant comprising applying to a plant and/or seeds thereof and/or substrate for growing said plant the combinations set forth above with an amount of the combination effective to modulate infestation of said pest or pest species.
• isolated compounds obtainable or derived from a strain of Chromobacterium species, more particularly, Chromobacterium substugae and more particularly, a strain of Chromobacterium substagae sp. nov. and even more particularly a strain of Chromobacterium substagae sp. nov. having the identifying characteristics of NRRL B-30655 described in US Patent No. 7,244,607 or alternatively, organisms capable of producing these compounds that can be used to control various pests, and particularly, nematocidal pests.
• the compound may be a compound that (a) has pesticidal activity; (b) has a molecular weight of about 840-900 as determined by Liquid
• the compound in one embodiment may be a peptide.
• the compound has 43 carbons, seven methyl, ten methylene carbons, twelve methines, 6 olefinic methines, and eight quaternary carbons as determined by 13 C NMR.
• the compound "A” (a) is obtainable from a strain of Chromobacterium sp., particularly a strain of Chromobacterium substugae and more particularly, a strain of Chromobacterium substagae sp. nov. and even more particularly a strain of Chromobacterium substagae sp. nov. having the identifying characteristics of NRRL B-30655 described in US Patent No.
• the 13 C NMR spectrum reveals signals for 43 carbons, for seven methyl, ten methylene carbons, twelve methines, 6 olefinic methines, eight quaternary carbons and/or the H NMR spectrum displays characteristics of a typical peptide, illustrating five amide NH signals [ ⁇ ⁇ : 8.89, 8.44, 8.23, 8.22, 7.96] , one amine NH 2 signal [ ⁇ ⁇ : 7.64, 6.65] , six -amino protons [ ⁇ ⁇ : 4.07, 4.06, 3.96, 3.95, 3.88, 3.72] and in the 13 C NMR spectrum, six/seven amide or ester resonances [6 C : 173.62, 172.92, 172.25, 1.72.17, 171.66, 171.28, 170.45].
• the compound "B" has the following
• (a) is obtainable from a strain of Chromobacterium sp., particularly a strain of Chromobacterium substugae and more particularly, a strain of Chromobacterium substagae sp. nov. and even more particularly a strain of Chromobacterium substagae sp. nov. having the identifying characteristics of NRRL B-30655 described in US Patent No.
• (b) is toxic to a pest; (c) has a molecular weight of about 850-900 and more particularly, 874 as determined by Liquid Chromatography 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 (Phenomenex, Luna 5 ⁇ CI 8(2) 100 A, 100 x 4.60 mm) column using a water: acetonitrile (CH 3 CN) with a gradient solvent system (0-20 min; 90-0% aqueous CH 3 CN, 20-24 min; 100% CH 3 CN, 24-27 min; 0-90% aqueous CH 3 CN, 27-30 min; 90% aqueous CH 3 CN) at 0.5 mL/min flow rate and UV detection of 210 nm.
• CH 3 CN acetonitrile
• 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;
• n is 0, 1, 2, 3, 4, 5, 6, 7, 8 or 9;
• R l 5 R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R n 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, substituted cycloalkyl, alkoxy,
• 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;
• R2a, R2b are independently selected from the group consisting of -H, alkyl, lower-alkyl, substituted alkyl and substituted lower-alkyl;
• R l5 R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R n 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,
• 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;
• 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;
• R 1 ; R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R n 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,
• 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;
• R l 5 R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R n 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
• the compound is chromamide A (1).
• Chromamide A may be obtained by (a) culturing a strain of Chromobacterium sp., particularly a strain of Chromobacterium substugae and more particularly, a strain of Chromobacterium substagae sp. nov. and even more particularly a strain of Chromobacterium substagae sp. nov. having the identifying characteristics of NRRL B- 30655 described in US Patent No. 7,244,607 in a culture medium or whole cell broth under conditions sufficient to produce said compound to obtain a Chromobacterium culture and (b) isolating said compound produced from the whole cell broth of (a).
• the compound in step (b) may be isolated by (i) applying the whole cell broth to at least one of an ion exchange column, a size exclusion column or a reversed phase HPLC column to obtain column fractions; (ii) assaying the column fractions for pesticidal activity and (iii) concentrating column fractions of (ii) to obtain isolated compound.
• compositions, particularly pesticidal compositions comprising said compounds as well as other compounds obtainable from a strain of Chromobacterium sp., particularly a strain of Chromobacterium substugae and more particularly, a strain of Chromobacterium substagae sp. nov. and even more particularly a strain of
• Chromobacterium substagae sp. nov. having the identifying characteristics of NRRL B- 30655 described in US Patent No. 7,244,607 with pesticidal activity.
• These other compounds may have the following characteristics: (a) a molecular weight of about 315- 360 as determined by Liquid Chromatography/Mass Spectroscopy (LC/MS); (b) an High Pressure Liquid Chromatography (HPLC) retention time of about 8-15 minutes on a reversed phase C-18 HPLC column using a water: acetonitrile (CH 3 CN) with a gradient solvent system (0-20 min; 90-0 % aqueous CH 3 CN, 20-24 min; 100% CH 3 CN, 24-27 min; 0 - 90 % aqueous CH 3 CN, 27-30 min; 90% aqueous CH 3 CN) at 0.5 mL/min flow rate and UV detection of 210 nm and may be obtained by (A) culturing a a strain of Chromo
• Chromobacterium substagae sp. nov. having the identifying characteristics of NRRL B- 30655 described in US Patent No. 7,244,607 in a culture medium under conditions sufficient to produce said compound to obtain a a strain of Chromobacterium sp., particularly a strain of Chromobacterium substugae and more particularly, a strain of Chromobacterium substagae sp. nov. and even more particularly a strain of
• Chromobacterium substagae sp. nov. having the identifying characteristics of NRRL B- 30655 described in US Patent No. 7,244,607 culture and (B) isolating said compound produced from the whole cell broth of (A).
• one compound used in said composition set forth above, compound "C” has the following characteristics: (a) is obtainable from a strain of
• Chromobacterium sp. particularly a strain of Chromobacterium substugae and more particularly, a strain of Chromobacterium substagae sp. nov. and even more particularly a strain of Chromobacterium substagae sp. nov. having the identifying characteristics of NPvRL B-30655 described in US Patent No. 7,244,607;
• (b) is toxic to pests;
• (c) has a molecular weight of about 325-360 and more particularly, about 343 as determined by Liquid Chromatography Mass Spectroscopy (LC/MS);
• (d) has an High Pressure Liquid
• HPLC Chromatography retention time of about 8-14 minutes, more specifically about 10 minutes and even more specifically about 10.88 min on a reversed phase C-18 HPLC (Phenomenex, Luna 5 ⁇ CI 8(2) 100 A, 100 x 4.60 mm) column using a water: ace tonitrile (CH 3 CN) with a gradient solvent system (0-20 min; 90-0 % aqueous CH 3 CN, 20-24 min; 100% CH 3 CN, 24-27 min; 0 - 90 % aqueous CH 3 CN, 27-30 min; 90% aqueous CH 3 CN) at 0.5 mL/min flow rate and UV detection of 210 nm.
• compound "C” may be violacein (2) , a known compound isolated earlier from Chromobacterium violaceum.
• another compound used in the composition set forth above, the compound "D" has the following characteristics: (a) is obtainable from a
• Chromobacterium species (b) is toxic to a pest; (c) has a molecular weight of about 315- 350 and more particularly, 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 minutes and even more specifically about 12.69 min on a reversed phase C-18 HPLC (Phenomenex, Luna 5 ⁇
• compound "D” may be characterized as deoxy violacein (3), a known compound isolated earlier from Chromobacterium violaceum.
• compositions may further optionally comprise a second substance, wherein said second substance is a chemical or biological pesticide and/or at least one of a carrier, diluent, surfactant or adjuvant.
• a method of using the compounds e.g., compounds "A”, “B”, “C” and “D"
• compositions set forth above to modulate pest infestation, particularly nematocidal pests in a plant comprising applying to the plant an amount of the compound or compositions and optionally a second chemical or biological pesticide effective to modulate said pest infestation.
• the use of the compounds set forth above for formulating a composition for modulating pest infestation in a plant is
• FIG. 1 is a schematic representation of purification scheme for obtaining the compounds of the invention from culture broth.
• FIG. 2 depicts the ESI-LCMS chromatogram for chromamide A (1).
• FIG.3 depicts the FIRMS data for chromamide A (1).
• FIG. 4 depicts 3 ⁇ 4 NMR for chromamide A (1) in DMSO- , at 600 MHz.
• FIG. 5 depicts 13 C NMR for chromamide A (1) in DMSO- 6 at 600 MHz.
• FIG. 6 depicts the HPLC chromatogram for compound B (MW 874).
• FIG. 7 depicts chemical structures for chromamide A (1) violacein (2) and deoxyviolacein (3).
• FIG. 8 Percentage of mobile nematodes after treatment with filter sterilized C.
• substugae broth (lx - undiluted; O.lx - diluted 10-fold) after 24 hours.
• FIG. 9 Percentage of mobile nematodes after treatment with filter sterilized C.
• substugae broth (lx - undiluted; O.lx - diluted 10-fold) after 48 hours.
• derived from means directly isolated or obtained from a particular source or alternatively having identifying characteristics of a substance or organism isolated or obtained from a particular source.
• source is an organism
• derived from means that it may be isolated or obtained from the organism itself or medium used to culture or grow said organism.
• whole broth culture refers to a liquid culture containing both cells and media. If bacteria are grown on a plate the cells can be harvested in water or other liquid, whole culture.
• filtrate refers to liquid from a whole broth culture that has passed through a membrane.
• extract refers to liquid substance removed from cells by a solvent (water, detergent, buffer) and separated from the cells by centrifugation, filtration or other method .
• metabolite refers to a compound, substance or byproduct of a fermentation of a microorganism, or supernatant, filtrate, or extract obtained from a microorganism that has pesticidal and particularly, insecticidal activity.
• an "isolated compound” is essentially free of other compounds or substances, e.g., at least about 20% pure, preferably at least about 40% pure, more preferably about 60% pure, even more preferably about 80% pure, most preferably about 90% pure, and even most preferably about 95% pure, as determined by analytical methods, including but not limited to chromatographic methods, electrophoretic methods.
• a “carrier” is an inert, organic or inorganic material, with which the active ingredient is mixed or formulated to facilitate its application to plant or other object to be treated, or its storage, transport and/or handling.
• modulate is used to mean to alter the amount of pest infestation or rate of spread of pest infestation.
• pest infestation is the presence of a pest in an amount that causes a harmful effect including a disease or infection in a host population or emergence of an undesired weed in a growth system.
• a "pesticide” is a substance derived from a biological product or chemical substance that increase mortality or inhibit the growth rate of plant pests and includes but is not limited to nematocides, insecticides, plant fungicides, plant bactericides, and plant viricides.
• alkyl refers to a monovalent straight or branched chain hydrocarbon group having from one to about 12 carbon atoms, including methyl, ethyl, n- propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-hexyl, and the like.
• substituted alkyl refers to alkyl groups further bearing one or more substituents selected from hydroxy, alkoxy, mercapto, cycloalkyl, substituted cycloalkyl, heterocyclic, substituted heterocyclic, aryl, substituted aryl, heteroaryl, substituted heteroaryl, aryloxy, substituted aryloxy, halogen, cyano, nitro, amino, amido,— C(0)H, acyl, oxyacyl, carboxyl, sulfonyl, sulfonamide, sulfuryl, and the like.
• alkenyl refers to straight or branched chain hydrocarbyl groups having one or more carbon-carbon double bonds, and having in the range of about 2 up to 12 carbon atoms
• substituted alkenyl refers to alkenyl groups further bearing one or more substituents as set forth above.
• alkynyl refers to straight or branched chain hydrocarbyl groups having at least one carbon-carbon triple bond, and having in the range of about 2 up to 12 carbon atoms
• substituted alkynyl refers to alkynyl groups further bearing one or more substituents as set forth above.
• aryl refers to aromatic groups having in the range of 6 up to 14 carbon atoms and "substituted aryl” refers to aryl groups further bearing one or more substituents as set forth above.
• heteroaryl refers to aromatic rings containing one or more heteroatoms (e.g., N, O, S, or the like) as part of the ring structure, and having in the range of 3 up to 14 carbon atoms and "substituted heteroaryl” refers toheteroaryl groups further bearing one or more substituents as set forth above.
• heteroatoms e.g., N, O, S, or the like
• alkoxy refers to the moiety— O-alkyl-, wherein alkyl is as defined above, and “substituted alkoxy” refers to alkoxyl groups further bearing one or more substituents as set forth above.
• thioalkyl refers to the moiety— S- alkyl-, wherein alkyl is as defined above, and "substituted thioalkyl” refers to thioalkyl groups further bearing one or more substituents as set forth above.
• cycloalkyl refers to ring-containing alkyl groups containing in the range of about 3 up to 8 carbon atoms
• substituted cycloalkyl refers to cycloalkyl groups further bearing one or more substituents as set forth above.
• heterocyclic refers to cyclic (i.e., ring-containing) groups containing one or more heteroatoms (e.g., N, O, S, or the like) as part of the ring structure, and having in the range of 3 up to 14 carbon atoms and "substituted heterocyclic” refers to heterocyclic groups further bearing one or more substituent's as set forth above.
• heteroatoms e.g., N, O, S, or the like
• compounds or metabolites may be obtained, are obtainable or derived from an organism having the identifying characteristics of a Chromobacterium species, more particularly, 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 identifying characteristics of NRRL B-30655, or alternatively from any other microorganism.
• the methods comprise cultivating these organisms and obtaining the compounds and/or compositions of the present invention by isolating these compounds from the culture of these organisms .
• 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, fed-batch, or solid state fermentations) in laboratory or industrial fermentors performed in suitable medium and under conditions allowing cell growth.
• the cultivation may take place in suitable nutrient medium comprising carbon and nitrogen sources and inorganic salts, using procedures known in the art. Suitable media are available may be available from commercial sources or prepared according to published compositions.
• Chromobacterium sp. may be used in formulating a pesticidal composition.
• the compounds and/or metabolites may be extracted from the culture broth.
• the extract may be fractionated by chromatography. Chromatographic fractions may be assayed for toxic activity against, for example, Cabbage looper (Trichoplusia ni) or Beet armyworm (Spodoptera exigua) using methods known in the art. This process may be repeated one or more times using the same or different chromatographic methods.
• Compositions may comprise whole broth cultures, liquid cultures, or suspensions of a strain from a Chromobacterium sp., e.g. a strain having the identifying characteristics of Chromobacterium substugae sp. Nov and more particularly, having the identifying characteristics of NRRL B-30655 (see US Patent No. 7,244,607), as well as supernatants , filtrates or extracts obtained from a strain of a Chromobacterium sp., e.g. a strain having the identifying characteristics of Chromobacterium substugae sp. Nov and more particularly, having the identifying characteristics of NRRL B-30655 (see US Patent No.
• compositions set forth above can be formulated in any manner.
• Non-limiting formulation examples include, but are not limited to, Emulsifiable concentrates (EC), Wettable powders (WP), soluble liquids (SL), Aerosols, Ultra-low volume concentrate solutions (ULV), Soluble powders (SP), Microencapsulation, Water dispersed Granules, Flowables (FL), Microemulsions (ME), Nano-emulsions (NE), etc.
• percent of the active ingredient is within a range of 0.01% to 99.99%.
• compositions may be in the form of a liquid, gel or solid.
• a solid composition can be prepared by suspending a solid carrier in a solution of active ingredient(s) and drying the suspension under mild conditions , such as evaporation at room temperature or vacuum evaporation at 65°C or lower.
• a composition may comprise gel-encapsulated active ingredient(s).
• gel-encapsulated materials can be prepared by mixing a gel-forming agent (e.g., gelatin, cellulose, or lignin) with a culture or suspension of live or inactivated Chromobacterium, or a cell-free filtrate or cell fraction 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.
• a gel-forming agent e.g., gelatin, cellulose, or lignin
• the composition may additionally comprise a surfactant to be used for the purpose of emulsification, dispersion, wetting, spreading, integration, disintegration control, stabilization of active ingredients, and improvement of fluidity or rust inhibition.
• 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.
• the surfactant is a non-phytotoxic non-ionic surfactant which preferably belongs to EPA List 4B.
• the nonionic surfactant is polyoxyethylene (20) monolaurate.
• the concentration of surfactants may range between 0.1-35% of the total formulation, preferred range is 5-25%.
• dispersing and emulsifying agents such as non-ionic, anionic, amphoteric and cationic dispersing and emulsifying agents , and the amount employed is determined by the nature of the composition and the ability of the agent to facilitate the dispersion of the compositions of the present invention.
• the composition set forth above may be combined with another microorganism and/or pesticide (e.g., nematocide, fungicide, insecticide).
• the microorganism may include but is not limited to an agent derived from Bacillus sp., Pseudomonas sp., Brevabacillus sp., Lecanicillium sp., non- Ampelomyces sp., Pseudozyma sp., Streptomyces sp, Burkholderia sp, Trichoderma sp, Gliocladium sp.
• 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, pyrethram).
• fungicidal and/or insecticidal activity e.g., paraffinic oil, tea tree oil, lemongrass oil, clove oil, cinnamon oil, citrus oil, rosemary oil, pyrethram.
• the pesticide may be a single site anti-fungal agent which may include but is not limited to benzimidazole, a demethylation inhibitor (DMI) (e.g., imidazole, piperazine, pyrimidine, triazole), morpholine, hydroxypyrimidine, anilinopyrimidine, phosphorothiolate, quinone outside inhibitor, quinoline, dicarboximide, carboximide, phenylamide, anilinopyrimidine, phenylpyrrole, aromatic hydrocarbon, cinnamic acid, hydroxyanilide, antibiotic, polyoxin, acylamine, phthalimide, benzenoid (xylylalanine) , a demethylation inhibitor selected from the group consisting of imidazole, piperazine, pyrimidine and triazole (e.g.,bitertanol, myclobutanil, penconazole, propiconazole, triadimefon, bromucon
• the strobilurin may include but is not limited to azoxystrobin, kresoxim- methoyl or trifloxystrobin.
• the anti-fungal agent is a quinone, e.g., quinoxyfen (5,7-dichloro-4-quinolyl 4-fluorophenyl ether).
• the anti-fungal agent may also be derived from a Reynoutria extract.
• the fungicide can also be a multi-site non- inorganic, chemical fungicide selected from the group consisting of chloronitrile, quinoxaline, sulphamide, phosphonate, phosphite, dithiocarbamate, chloralkythios, phenylpyridin- amine, cyano-acetamide oxime.
• the composition may as noted above, further comprise an insecticide.
• the insecticide may include but is not limited to avermectin, Bt (e.g., Bacillus thuringiensis var. kurstaki), neem oil, spinosads , Burkholderdia sp. as set forth in WO2011/106491 , entomopathogenic fungi such a Beauveria bassiana and chemical insecticides including but not limited to organochlorine compounds, organophosphorous compounds, carbamates, pyrethroids, pyrethrins and neonicotinoids.
• the composition may further comprise a nematocide.
• This nematocide may include, but is not limited to, avermectin, microbial products such as Biome (Bacillus firmus) , Pasteuria spp and organic products such as saponins.
• compositions may be applied using methods known in the art. Specifically, these compositions may be applied to plants or plant parts .
• Plants are to be understood as meaning in the present context all plants and plant populations such as desired and undesired wild plants or crop plants (including naturally occurring crop plants).
• Crop plants can be plants, which can be obtained by conventional plant breeding and optimization methods or by biotechnological and genetic engineering methods or by combinations of these methods, including the transgenic plants and including the plant cultivars protectable or not protectable by plant breeders' rights.
• Plant parts are to be understood as meaning all parts and organs of plants above and below the ground, such as shoot, leaf, flower and root, examples which may be mentioned being leaves, needles, stalks, stems, flowers, fruit bodies, fruits, seeds, roots, tubers and rhizomes.
• the plant parts also include harvested material, and vegetative and generative propagation material, for example cuttings, tubers, rhizomes, off-shoots and seeds.
• compositions set forth above Treatment of the plants and plant parts with the compositions set forth above may be carried out directly or by allowing the compositions to act on their surroundings , habitat or storage space by, for example, immersion, spraying, evaporation, fogging, scattering, painting on, injecting.
• the composition may be applied to the seed as one or more coats prior to planting the seed using one or more coats using methods known in the art.
• compositions, cultures, supernatants, metabolites and pesticidal compounds set forth above may be used as pesticides.
• the compositions, cultures, supernatants, metabolites and pesticidal compounds as set forth above may be used as insecticides and nematocides, alone or in combination with one or more pesticidal substances set forth above.
• nematodes that may be controlled using the method set forth above include but are not limited to parasitic nematodes such as root-knot, cyst, and lesion nematodes, including but not limited to Meloidogyne sp. Tylenchorhynchus sp, Hoplolaimus sp., Helicotylenchus sp., Pratylenchus sp., Heterodera sp., Globodera, sp., Trichodorus sp.
• parasitic nematodes such as root-knot, cyst, and lesion nematodes, including but not limited to Meloidogyne sp. Tylenchorhynchus sp, Hoplolaimus sp., Helicotylenchus sp., Pratylenchus sp., Heterodera sp., Globodera, sp., Trichodorus sp.
• Phytopathogenic insects controlled by the method set forth above include but are not limited to non-Culicidae larvae insects from the order (a) Lepidoptera, for example, Acleris spp., Adoxophyes spp., Aegeria spp., Agrotis spp., Alabama argillaceae, Amylois spp., Anticarsia gemmatalis, Archips spp., Argyrotaenia spp., Autographa spp., Busseolafusca, Cadra cautella, Carposina nipponensis, Chilo spp., Choristoneura spp., Clysia ambiguella, Cnaphalocrocis spp., Cnephasia spp., Cochylis spp., Coleophora spp., Crocidolomia binotalis, Cryptophlebia leucotreta, Cydia spp.,
• Coleoptera for example, Agriotes spp., Anthonomus spp., Atomaria linearis, Chaetocnema tibialis, Cosmopolites spp., Curculio spp., Dermestes spp., Diabrotica spp., Epilachna spp., Eremnus spp., Leptinotarsa decemlineata, Lissorhoptrus spp., Melolontha spp.,
• Isoptera for example, Reticuliterm.es spp.
• Psocoptera for example, Liposcelis spp.
• Anoplura for example, Haematopinus spp., Linognathus spp., Pediculus spp., Pemphigus spp. and Phylloxera spp.
• Mallophaga for example, Damalinea spp.
• Thysanoptera for example, Frankliniella spp., Hercinotnrips spp., Taeniothrips spp., Thrips palmi, Thrips tabaci and Scirtothrips aurantii
• Heteroptera or example, Cimex spp., Distantiella theobroma, Dysdercus spp., Euchistus spp.,
• Eurygaster spp. Leptocorisa spp., Nezara spp., Piesma spp., Rhodnius spp., Sahlbergella singularis, Scotinophara spp. and Tniatoma spp.; (j) Homoptera,for example,
• Aleurothrixus floccosus Aleurothrixus floccosus , Aleyrodes brassicae, Aonidiella spp., Aphididae, Aphis spp., Aspidiotus spp., Bemisia tabaci, Ceroplaster spp., Chrysomphalus aonidium,
• Chrysomphalus dictyospermi Coccus hesperidum, Empoasca spp., Eriosoma larigerum, Erythroneura spp., Gascardia spp., Laodelphax spp., Lecanium corni, Lepidosaphes spp., Macrosiphus spp., Myzus spp., Nephotettix spp., Nilaparvata spp., Paratoria spp., Pemphigus spp., Planococcus spp., Pseudaulacaspis spp., Pseudococcus spp., Psylla spp., Pulvinaria aethiopica, Quadraspidiotus spp., Rhopalosiphum spp., Saissetia spp.,
• the active ingredients according to the invention may further be used for controlling crucifer flea beetles (Phyllotreta spp.), root maggots (Delia spp.), cabbage seedpod weevil (Ceutorhynchus spp.) and aphids in oil seed crops such as canola (rape), mustard seed, and hybrids thereof, and also rice and maize.
• the insect may be a member of the Spodoptera, more particularly, Spodoptera exigua, Myzus persicae, Plutella xylostella or Euschistus sp.
• an effective pesticidal control amount of a supernatant, filtrate or extract containing a pesticidally active metabolite, or isolated compound produced by the Chromobacterium sp. or application of combinations of the foregoing is provided.
• the strain or supernatant or filtrate or extract, metabolite and/or compound are applied, alone or in combination with another pesticidal substance, in an effective pest control or pesticidal amount.
• An effective amount is defined as that quantities of microorganism cells, supernatant, filtrate or extract, metabolite and/or compound alone or in combination with another pesticidal substance that is sufficient to modulate pest infestation.
• the effective rate can be affected by pest species present, stage of pest growth, pest population density, and environmental factors such as temperature, wind velocity, rain, time of day and seasonality. The amount that will be within an effective range in a particular instance can be determined by laboratory or field tests.
• composition and methods set forth above will be further illustrated in the following, non-limiting Examples.
• the examples are illustrative of various embodiments only and do not limit the claimed invention regarding the materials, conditions, weight ratios, process parameters and the like recited herein.
• the culture broth derived from the 10-L fermentation C. substugae 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 through 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 Sephadex LH 20 size exclusion chromatography (CH 2 C1 2 /CH 3 0H; 50/50) to give 7 fractions (FIG. 1). These fractions are then concentrated to dryness using rotary evaporator and the resulting dry residues are screened for biological activity using a feeding assay with Cabbage looper (Trichoplusia ni) or Beet army worm (Spodoptera exigua). The active fractions are then subjected to reversed 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 isolated from fractions 1 and 2 respectively, whereas violacein (2) & deoxyviolacein (3) were isolated from fraction 5, all of which were obtained from Sephadex LH 20 chromatography.
• the structures of these compounds are set forth in FIG. 7.
• chromamide A (1) Purification of chromamide A (1) was performed by using HPLC C-18 column (Phenomenex, Luna lOu CI 8(2) 100 A, 250 x 10), water: ace tonitrile gradient solvent system (0-10 min, 80-75 % aqueous CH 3 CN; 10-45 min, 75-60 % aqueous CH 3 CN; 45-55 min, 60-50 % aqueous CH 3 CN; 55-65 min, 50-100 % aqueous CH 3 CN; 65-70 min, 100 % CFLCN; 55-70 min, 0-80 % aqueous CH 3 CN) 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.
• the solvent system consisted 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 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 analysis 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 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 (see FIG. 2).
• the LC-MS chromatogram for another active compound B suggests a molecular mass of 874 in positive ionization mode.
• Violacein (2) and deoxyviolacein (3) had the molecular masses of 313and 327 respectively in positive ionization mode.
• 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.
• the purified chromamide A with molecular weight 860 is further analyzed using a 600 MHz NMR instrument, and has 3 ⁇ 4 NMR ⁇ 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, 4.07, 4.05, 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 FIG.
• the chromamide A was isolated as a white solid, which analyzed for the molecular formula C 4 3H 6 gN 6 0 12 (13 degrees of unsaturation), by ESI high-resolution mass spectrometry (obsd NT mlz 861.5376, calcd M + m/z 861.5343) (FIG. 3).
• the connections of the three sub-structures in 1 were accomplished by routine HMBC NMR analysis using correlations between the a-amino proton and/or the secondary amide proton and the carbonyl carbon resonances and chemical shift consideration.
• the linkage of C-9 from sub-structure I to C-10 from sub-structure II was established by HMBC correlations from CH 3 -40 [ ⁇ ⁇ : 1.00] and the a-amino proton of alanine [ ⁇ ⁇ : 3.42] to the C- 10 carbon [6 C : 70.11]. This was further confirmed by the three bond HMBC correlation from hydroxyl at [ ⁇ ⁇ : 5.10] to C-9 at [6 C : 49.78] .
• the compound B with a molecular weight 874 exhibited similar NMR and UV data suggesting that this compound B also belongs to the class of peptide.
• Chromamide A (0.05 mg) was hydrolyzed by using liquid phase hydrolysis (6N HCL, 1% Phenol, 110°C, 24 hr., 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 50 ⁇ of the sample was loaded onto the ion-exchange column for analysis.
• an amino acid standards solution for protein hydrolysate on the Na-based Hitachi 8800 (Sigma, A-9906) is used to determine response factors, and thus calibrate the Hitachi 8800 analyzer for all of the amino acids.
• Each injection contains NorLeucine as an internal 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 Ion-Exchange column and an optimized method developed by Molecular Structure Facility (MSF), UC Davis, and the individual 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 (leucine) and Val (Valine).
• Toxicity of the compound of interest in fraction 1 (Fl) was confirmed in an in vitro assay using 1 st instar cabbage looper larvae as a test object.
• test insect (1 st instar larvae of cabbage looper) was placed in each well, and the plate was covered with an adhesive seal. The seal was punctured for aeration, and the sealed plate was incubated at 26°C for four days.
• violacein is a potent insecticide with an estimated LC 50 value of 7 * 10 "6 micrograms per well for cabbage looper larvae in an in vitro diet-overlay assay.
• test solution either lx or O.lx filter-sterilized broth
• test solution either lx or O.lx filter-sterilized broth
• 15 nematodes dispensed in 10 ul of DI water were added into each well, plate was closed with a lid, and incubated at 25°C for 24 hours.
• Water and Avid (avermectin) at 20,000x dilution were used as negative and positive controls, respectively. Effect of each compound on nematode mobility was checked after 24 hours by probing each nematode with a needle, and the proportion of immobile nematodes in each treatment was recorded in a notebook using a % scale.
• MBI-203 was tested for its intrinsic activity against the root knot nematode Meloidogyne sp. in two mini drench tests.
• MBI-203 was tested in a greenhouse assay conducted in 45 ml pots. Cucumber seeds cv. Toshka were sown directly into pots filled with a sandy loam soil. Ten days later pots were each treated with 5 ml of a suspension. Hereafter, pots were inoculated with 3000 eggs of M. incognita. Four replicates were prepared for each treatment and rate. The trial was harvested fourteen days after trial application and inoculation. Root galling was assessed according to Zeck's gall index (Zeck, 1971). Specific conditions are set forth below in Table 3. Phytotoxicity was measured as a reduction of growth of the emerged cucumber seedling in comparison to the control.
• Test species Meloidogyne sp. applied at 3000 eggs per mini drench pot
• Test formulation MBI-203 96% liquid formulation
• MBI-203 showed phytotoxicity at the highest concentrations of 50 and 25 ml/L and assessments could not be made at these rates.
• Fosthiazate performed as usual (100% control at 20 ppm).
• Synergy tests were performed by treating artificial diet in 96-well plates and feeding treated diet to neonate larvae. 100 ⁇ ⁇ of treatment were pipetted into multiple wells of each plate. MBI-203 (whole cell broth concentrated to 7.6% dry cell weight) alone, the commercial insecticide alone, and the combination of the 2 were tested using predetermined LC 50 concentrations or fractions thereof. The diet was fan-dried to remove excess moisture. Neonate Beet Armyworm, Spodoptera exigua, or Cabbage Loopers, Trichoplusia ni, were transferred into each well of the multi-well plate. Infested plates were covered with adhesive plate sealer and a single small hole was poked into the sealer over each well to allow for aeration. Plates were stored in an incubator at 26°C, 16h light/8h dark cycle for 3 days. On the third and fourth day after infesting, mortality was scored.
• Chlorantranilipole (marketed as Coragen®, Dupont), Bacillus thuringiensis var. kurstaki (Dipel®, Valent Biosciences), Spinosad (marketed as Entrust®, Dow Agro Sciences), Spirotetramet (marketed as Movento®, Bayer Crop Science) and Pyrethrum pyrethrins (marketed as Pyganic®, Arbico Organics) were tested with MBI-203. As noted above, except where indicated, LC 50 concentrations of MBI-203 and insecticides were used. The results are shown in Table 4. All, but Bt var. kurstaki and 1 instance of LC 50 concentration showed synergism.
• MBI-203 synergy with insecticides against Beet Army Worm (BAW) was tested. Chlorantranilipole (marketed as Coragen®, Dupont), Bacillus thuringiensis var. kurstaki (Dipel®, Valent Biosciences), Spinosad (marketed as Entrust®, Dow Agro Sciences), Spirotetramet (marketed as Movento®, Bayer Crop Science) and Pyrethrum/pyrethrins (marketed as Pyganic®, Arbico Organics) were tested with MBI-203. As noted above, except where indicated, LC 50 concentrations of MBI-203 and insecticides were used. The results are shown in Table 5.

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