Chromobacterium bioactive compositions and metabolites

Abstract

Provided are bioactive compounds and metabolites derived from Chromobacterium species culture responsible for controlling pests, compositions containing these compounds, methods for obtaining these compounds and methods of using these compounds and compositions for controlling pests.

Claims

1. A method for inhibiting infestation of Aedes aegypti or Aedes albopictus in a location where inhibition is desired comprising applying an effective amount of a fermented whole cell broth collected from Chromobacterium subtsugae sp. nov. (NRRL B-30655); and at least one of a carrier, diluent or adjuvant, to inhibit said infestation at said location.

2. The method according to claim 1, wherein said Aedes aegypti or Aedes albopictus is inhibited by increasing the mortality of said Aedes aegypti or Aedes albopictus.

3. The method according to claim 1, wherein the mortality of Aedes aegypti or Aedes albopictus is increased and wherein there is a mortality of Aedes aegypti or Aedes albopictus of at least about 50% at said location.

4. The method according to claim 1, which further comprises applying another natural or artificial insecticidal substance.

5. The method of claim 4, wherein said another natural or artificial insecticidal substance is applied at said location in rotation.

6. The method according to claim 1, wherein the infestation of Aedes aegypti or Aedes albopictus is inhibited by decreasing the rate of hatching of eggs.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) FIG. 1 is a schematic representation of purification scheme for obtaining the compounds of the invention from culture broth.

(2) FIG. 2 depicts the ESI-LCMS chromatogram for chromamide A (1).

(3) FIG. 3 depicts the HRMS data for chromamide A (1).

(4) FIG. 4 depicts .sup.1H NMR for chromamide A (1) in DMSO-d.sub.6 at 600 MHz.

(5) FIG. 5 depicts .sup.13C NMR for chromamide A (1) in DMSO-d.sub.6 at 600 MHz.

(6) FIG. 6 depicts the HPLC chromatogram for compound B (MW 874).

(7) FIG. 7 depicts chemical structures for chromamide A (1) violacein (2) and deoxyviolacein (3).

(8) FIG. 8 Percentage of mobile nematodes after treatment with filter sterilized C. substugae broth (1undiluted; 0.1diluted 10-fold) after 24 hours.

(9) FIG. 9 Percentage of mobile nematodes after treatment with filter sterilized C. substugae broth (1undiluted; 0.1diluted 10-fold) after 48 hours.

DETAILED DESCRIPTION OF THE INVENTION

(10) While the compositions 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.

(11) 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.

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

(13) 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.

(14) As defined herein, derived from means directly isolated or obtained from a particular source or alternatively having identifying characteristics of a substance or organism isolated or obtained from a particular source. In the event that the source is an organism, derived from means that it may be isolated or obtained from the organism itself or medium used to culture or grow said organism.

(15) As defined herein, 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.

(16) The term supernatant refers to the liquid remaining when cells grown in broth or are harvested in another liquid from an agar plate and are removed by centrifugation, filtration, sedimentation, or other means well known in the art.

(17) As defined herein, filtrate refers to liquid from a whole broth culture that has passed through a membrane.

(18) As defined herein, 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.

(19) As defined herein, 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. As defined herein, an isolated compound is essentially free of other compounds or substances, e.g., at least about 20% pure, preferably at least about 40% pure, more preferably about 60% pure, even more preferably about 80% pure, most preferably about 90% pure, and even most preferably about 95% pure, as determined by analytical methods, including but not limited to chromatographic methods, electrophoretic methods.

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

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

(22) The term pest infestation as defined herein, is the presence of a pest in an amount that causes a harmful effect including a disease or infection in a host population or emergence of an undesired weed in a growth system.

(23) 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 nematocides, insecticides, plant fungicides, plant bactericides, and plant viricides.

(24) As used herein, the term 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.

(25) As used herein, 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(O)H, acyl, oxyacyl, carboxyl, sulfonyl, sulfonamide, sulfuryl, and the like.

(26) As used herein, 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, and substituted alkenyl refers to alkenyl groups further bearing one or more substituents as set forth above.

(27) As used herein, 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, and substituted alkynyl refers to alkynyl groups further bearing one or more substituents as set forth above.

(28) As used herein, 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.

(29) As used herein, 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.

(30) As used herein, 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.

(31) As used herein, 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.

(32) As used herein, cycloalkyl refers to ring-containing alkyl groups containing in the range of about 3 up to 8 carbon atoms, and substituted cycloalkyl refers to cycloalkyl groups further bearing one or more substituents as set forth above.

(33) As used herein, 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.

(34) Methods of Production

(35) As noted above, 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.

(36) 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, 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.

(37) After cultivation, a supernatant, filtrate and/or extract of or derived from Chromobacterium sp. may be used in formulating a pesticidal composition.

(38) Alternatively, after cultivation, the compounds and/or metabolites may be extracted from the culture broth.

(39) 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.

(40) Compositions

(41) 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 U.S. Pat. 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 U.S. Pat. No. 7,244,607), or the supernatant, filtrate and/or extract or one or more metabolites or isolated compounds derived from a strain of a Chromobacterium sp. or combinations of the foregoing which in particular have nematocidal activity.

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

(43) The compositions may be in the form of a liquid, gel or solid.

(44) 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.

(45) A composition may comprise gel-encapsulated active ingredient(s). Such gel-encapsulated materials can be prepared by mixing a gel-forming agent (e.g., gelatin, cellulose, or lignin) with a culture or suspension of live or inactivated 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.

(46) The composition may additionally comprise a surfactant to be used for the purpose of emulsification, dispersion, wetting, spreading, integration, disintegration control, stabilization of active ingredients, and improvement of fluidity or rust inhibition. In a particular embodiment, the surfactant is a non-phytotoxic non-ionic surfactant which preferably belongs to EPA List 4B. In another particular embodiment, the nonionic surfactant is polyoxyethylene (20) monolaurate. The concentration of surfactants may range between 0.1-35% of the total formulation, preferred range is 5-25%. The choice of dispersing and emulsifying agents, such as non-ionic, anionic, amphoteric and cationic dispersing and emulsifying agents, and the amount employed is determined by the nature of the composition and the ability of the agent to facilitate the dispersion of the compositions of the present invention. The composition set forth above may 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. 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, pyrethrum). Furthermore, 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, bromuconazole, cyproconazole, diniconazole, fenbuconazole, hexaconazole, tebuconazole, tetraconazole), myclobutanil, an anthranilic diamide (e.g., chlorantranilipole) and a quinone outside inhibitor (e.g., strobilurin). The strobilurin may include but is not limited to azoxystrobin, kresoxim-methoyl or trifloxystrobin. In yet another particular embodiment, the anti-fungal agent is a quinone, e.g., quinoxyfen (5,7-dichloro-4-quinolyl 4-fluorophenyl ether). The anti-fungal agent may also be derived from a Reynoutria extract.

(47) 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.

(48) 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.

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

(50) The 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, offshoots and seeds.

(51) 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. In the case that the composition is applied to a seed, the composition may be applied to the seed as one or more coats prior to planting the seed using one or more coats using methods known in the art.

(52) Uses

(53) The compositions, cultures, supernatants, metabolites and pesticidal compounds set forth above may be used as pesticides. In particular, 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.

(54) Specifically, 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. 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).

(55) 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., Busseola fusca, Cadra cautella, Carposina nipponensis, Chilo spp., Choristoneura spp., Clysia ambiguella, Cnaphalocrocis spp., Cnephasia spp., Cochylis spp., Coleophora spp., Crocidolomia binotalis, Cryptophlebia leucotreta, Cydia spp., Diatraea spp., Diparopsis castanea, Earias spp., Ephestia spp., Eucosma spp., Eupoecilia ambiguella, Euproctis spp., Euxoa spp., Grapholita spp., Hedya nubiferana, Heliothis spp., Hellula undalis, Hyphantria cunea, Keiferia lycopersicella, Leucoptera scitella, Lithocollethis spp., Lobesia botrana, Lymantria spp., Lyonetia spp., Malacosoma spp., Mamestra brassicae, Manduca sexta, Operophtera spp., Ostrinia nubilalis, Pammene spp., Pandemis spp., Panolis flammea, Pectinophora gossypiella, Phthorimaea operculella, Pieris rapae, Pieris spp., Plutella xylostella, Prays spp., Scirpophaga spp., Sesamia spp., Sparganothis spp., Spodoptera spp., Synanthedon spp., Thaumetopoea spp., Tortrix spp., Trichoplusia ni and Yponomeuta spp.; (b) Coleoptera, for example, Agriotes spp., Anthonomus spp., Atomaria linearis, Chaetocnema tibialis, Cosmopolites spp., Curculio spp., Dermestes spp., Diabrotica spp., Epilachna spp., Eremnus spp., Leptinotarsa decemlineata, Lissorhoptrus spp., Melolontha spp., Orycaephilus spp., Otiorhynchus spp., Phlyctinus spp., Popillia spp., Psylliodes spp., Rhizopertha spp., Scarabeidae, Sitophilus spp., Sitotroga spp., Tenebrio spp., Tribolium spp. and Trogoderma spp.; (c) Orthoptera, for example, Blatta spp., Blattella spp., Gryllotalpa spp., Leucophaea maderae, Locusta spp., Periplaneta spp. and Schistocerca spp.; (d) Isoptera, for example, Reticulitermes spp.; (e) Psocoptera, for example, Liposcelis spp.; (f) Anoplura, for example, Haematopinus spp., Linognathus spp., Pediculus spp., Pemphigus spp. and Phylloxera spp.; (g) Mallophaga, for example, Damalinea spp. and Trichodectes spp.; (h) Thysanoptera, for example, Frankliniella spp., Hercinotnrips spp., Taeniothrips spp., Thrips palmi, Thrips tabaci and Scirtothrips aurantii; (i) Heteroptera, for example, Cimex spp., Distantiella theobroma, Dysdercus spp., Euchistus spp., Eurygaster spp., Leptocorisa spp., Nezara spp., Piesma spp., Rhodnius spp., Sahlbergella singularis, Scotinophara spp. and Tniatoma spp.; (j) Homoptera, for example, Aleurothrixus floccosus, Aleyrodes brassicae, Aonidiella spp., Aphididae, Aphis spp., Aspidiotus spp., Bemisia tabaci, Ceroplaster spp., Chrysomphalus conidium, Chrysomphalus dictyospermi, Coccus hesperidum, Empoasca spp., Eriosoma larigerum, Erythroneura spp., Gascardia spp., Laodelphax spp., Lecanium corni, Lepidosaphes spp., Macrosiphus spp., Myzus spp., Nephotettix spp., Nilaparvata spp., Paratoria spp., Pemphigus spp., Planococcus spp., Pseudaulacaspis spp., Pseudococcus spp., Psylla spp., Pulvinaria aethiopica, Quadraspidiotus spp., Rhopalosiphum spp., Saissetia spp., Scaphoideus spp., Schizaphis spp., Sitobion spp., Trialeurodes vaporariorum, Trioza erytreae and Unaspis citri; (k) Hymenoptera, for example, Acromyrmex, Atta spp., Cephus spp., Diprion spp., Diprionidae, Gilpinia polytoma, Hoplocampa spp., Lasius spp., Monomorium pharaonis, Neodiprion spp., Solenopsis spp. and Vespa spp.; (l) Diptera, for example, Aedes spp., Antherigona soccata, Bibio hortulanus, Calliphora erythrocephala, Ceratitis spp., Chrysomyia spp., Culex spp., Cuterebra spp., Dacus spp., Drosophila melanogaster, Fannia spp., Gastrophilus spp., Glossina spp., Hypoderma spp., Hyppobosca spp., Liriomyza spp., Lucilia spp., Melanagromyza spp., Musca spp., Oestrus spp., Orseolia spp., Oscinella frit, Pegomyia hyoscyami, Phorbia spp., Rhagoletis pomonella, Sciara spp., Stomoxys spp., Tabanus spp., Tannia spp. and Tipula spp.; (m) Siphonaptera, for example, Ceratophyllus spp. and Xenopsylla cheopis and (n) from the order Thysanura, for example, Lepisma saccharine. 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. In a particular embodiment, the insect may be a member of the Spodoptera, more particularly, Spodoptera exigua, Myzus persicae, Plutella xylostella or Euschistus sp.

(56) Application of 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.

EXAMPLES

(57) The composition and methods set forth above will be further illustrated in the following, non-limiting Examples. The examples are illustrative of various embodiments only and do not limit the claimed invention regarding the materials, conditions, weight ratios, process parameters and the like recited herein.

Example 1: Extraction of Compounds from Chromobacterium substugae

(58) The following procedure is used for the purification of compounds extracted from the culture of Chromobacterium substugae:

(59) 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.sub.2Cl.sub.2/CH.sub.3OH; 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 armyworm (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.

(60) 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.

(61) Purification of Compounds

(62) Purification of chromamide A (1) was performed by using HPLC C-18 column (Phenomenex, Luna 10u C18(2) 100 A, 25010), water:acetonitrile gradient solvent system (0-10 min, 80-75% aqueous CH.sub.3CN; 10-45 min, 75-60% aqueous CH.sub.3CN; 45-55 min, 60-50% aqueous CH.sub.3CN; 55-65 min, 50-100% aqueous CH.sub.3CN; 65-70 min, 100% CH.sub.3CN; 55-70 min, 0-80% aqueous CH.sub.3CN) 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.

(63) Purification of invention compound B was performed by using HPLC C-18 column (Phenomenex, Luna 10u C18 (2) 100 A, 25010), water:acetonitrile gradient solvent system (0-10 min, 80-75% aqueous CH.sub.3CN; 10-45 min, 75-60% aqueous CH.sub.3CN; 45-55 min, 60-50% aqueous CH.sub.3CN; 55-65 min, 50-100% aqueous CH.sub.3CN; 65-70 min, 100% CH.sub.3CN; 55-70 min, 0-80% aqueous CH.sub.3CN) at 2.5 mL/min flow rate and UV detection of 210 nm, the active compound B, retention time 26.39 min (see FIG. 6).

(64) Purification of violacein (2) and deoxyviolacein (3) were performed by using HPLC C-18 column (Phenomenex, Luna 10u C18(2) 100 A, 25010), water:acetonitrile gradient solvent system (0-10 min, 70-60% aqueous CH.sub.3CN; 10-40 min, 60-20% aqueous CH.sub.3CN; 40-60 min, 20-0% aqueous CH.sub.3CN; 60-65 min, 100% CH.sub.3CN; 65-75 min, 0-70% aqueous CH.sub.3CN) at 2.5 mL/min flow rate and UV detection of 210 nm, the active compounds violacein (2), had a retention time 7.86 min and deoxyviolacein (3) retention time 12.45 min.

(65) Mass Spectroscopy Analysis of Compounds

(66) Mass spectroscopy analysis of active peaks is performed on a Thermo Finnigan LCQ Deca XP Plus electrospray (ESI) instrument using both positive and negative ionization modes in a full scan mode (m/z 100-1500 Da) on a LCQ DECA XP.sup.plus Mass Spectrometer (Thermo Electron Corp., San Jose, Calif.). Thermo high performance liquid chromatography (HPLC) instrument equipped with Finnigan Surveyor PDA plus detector, autosampler plus, MS pump and a 4.6 mm100 mm Luna C18 5 g 100 A column (Phenomenex). 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 L 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 313 and 327 respectively in positive ionization mode.

(67) NMR Spectroscopy Analysis of Compounds

(68) 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.

(69) For structure elucidation, the purified chromamide A with molecular weight 860 is further analyzed using a 600 MHz NMR instrument, and has .sup.1H 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. 4) and has .sup.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, 15.80 (see FIG. 5). The chromamide A was isolated as a white solid, which analyzed for the molecular formula C.sub.43H.sub.68N.sub.6O.sub.12 (13 degrees of unsaturation), by ESI high-resolution mass spectrometry (obsd M.sup.+m/z 861.5376, calcd M+m/z 861.5343) (FIG. 3). The .sup.1H NMR spectral data of chromamide A in DMSO-d.sub.6 exhibited 68 proton signals, in which nine protons [.sub.H: 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 correlation in a heteronuclear correlation NMR (HMQC) analysis. The .sup.13C NMR spectrum, showed seven carbonyl signals [.sub.C: 173.62, 172.92, 172.25, 1.72.17, 171.66, 171.28, 170.45] and in the .sup.1H NMR spectrum, six characteristic -amino protons signals [.sub.H: 4.07, 4.06, 3.96, 3.95, 3.88, 3.72] were observed which demonstrate that chromamide A is a peptide.

(70) 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.

(71) ##STR00006##

(72) The connections of the three sub-structures in 1 were accomplished by routine HMBC NMR analysis using correlations between the -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.sub.3-40 [.sub.H: 1.00] and the -amino proton of alanine [.sub.H: 3.42] to the C-10 carbon [.sub.C: 70.11]. This was further confirmed by the three bond HMBC correlation from hydroxyl at [.sub.H: 5.10] to C-9 at [.sub.C: 49.78]. The methylene at [.sub.H: 3.50] from sub-structure III showed a three bond HMBC correlation to C-19 [.sub.C: 68.31] which connected the sub-structure I and II. The quaternary carbon at C-3 [.sub.C: 98.09] was connected to C-21 [.sub.C: 64.40] through a weak correlation from H-21 [.sub.H: 3.95] together 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 H.sub.3-36 [.sub.H: 1.43] to C-1 [.sub.C: 172.17], which allowed the planar structure of chromamide A (1) to be assigned.

(73) 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.

(74) The structure 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 FIG. 7.

Example 2: Amino Acids Analysis of Chromamide A

(75) 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 l of the sample was loaded onto the ion-exchange column for analysis.

(76) For standards and calibration, 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).

Example 3: Confirmation of Toxicity on Cabbage Looper (Trichoplusia ni)

(77) Toxicity of the compound of interest in fraction 1 (F1) was confirmed in an in vitro assay using 1.sup.st instar cabbage looper larvae as a test object.

(78) Two hundred microliters of commercial cabbage looper diet was distributed in each well of a 96-well microplate. After the diet had solidified, 100 uL of solution containing 50 uL of extract (corresponding to four individual peaks found in fraction 1; H1-H4), 350 uL EtOH and 600 uL sterile DI water was pipetted in each well, after which the plate was dried using a hand-held fan. The amount of extract in each well was 10 micrograms. Each treatment was replicated eight times, and a mixture of pure ethanol and water was used as a negative control.

(79) One test insect (1.sup.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.

(80) The results presented in Table 1 below show good activity (>60% mortality) with a compound in peak H1. This particular peak corresponds with the chromamide A (1) (FIG. 1).

(81) TABLE-US-00001 TABLE 1 Cabbage Looper Mortality (%) at 10 ug/well F1 H1 66.7 F1 H2 11.1 F1 H3 33.3 F1 H4 11.1

Example 4: Determination of LC50 for Violacein for Cabbage Looper (Trichoplusia ni)

(82) The 96-well plate assay system described in the previous example was used to determine the concentration of pure violacein needed to kill 50% of the 1.sup.st instar cabbage looper larvae. The mortality values recorded after 4 days of incubation at 26 C. are presented in Table 2 below. Based on the data, violacein is a potent insecticide with an estimated LC.sub.50 value of 7*10.sup.6 micrograms per well for cabbage looper larvae in an in vitro diet-overlay assay.

(83) TABLE-US-00002 TABLE 2 Effect of Violacein on Cabbage Looper Mortality Violacein % mortality ug/well Day 4 10 100 1 100 0.1 100 0.01 100 0.001 100 0.0001 100 0.00001 71.4 0.000001 14.2 1E07 0

Example 5: Nematicidal Activity of Chromobacterium substugae (MBI-203) Broth on Juvenile Root-Knot Nematodes

(84) To assess the effect of filter-sterilized C. substugae on the motility (and subsequent recovery) of juvenile (J2) root-knot nematodes (Meloidogyne incognita VW6), the following test was conducted on 24-well plastic cell-culture plates:

(85) A 300-ul aliquot of each test solution (either 1 or 0.1 filter-sterilized broth) was added into appropriate wells after which, fifteen 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 20,000 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. To assess the recovery of mobility in each treatment, a volume of 200 ul was removed from each well, and the remaining solution in each well was diluted by adding 2 mL of DI water. Plates were again incubated for 24 hours as described above, after which the second mobility evaluation (48-hour) was performed.

(86) The results presented in FIGS. 8 and 9 show that the undiluted filter-sterilized broth can immobilize the free-living juvenile root-knot nematodes. This effect lasts at least for 48-hours, which suggests that C. substugae broth has nematicidal activity.

Example 6: Effect of Chromobacterium Substugae (MBI-203) Broth on Galling of Cucumber Roots

(87) MBI-203 was tested for its intrinsic activity against the root knot nematode Meloidogyne sp. in two mini drench tests.

(88) Materials and Methods

(89) Specifically 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 4.

(90) Phytotoxicity was measured as a reduction of growth of the emerged cucumber seedling in comparison to the control.

(91) TABLE-US-00003 TABLE 4 Test species MBI-203 Fosthiazate (Standard, EC 150) Meloidogyne sp. applied at 3000 eggs per mini drench pot (in 2 ml) Test plant Cucumis sativus (cucumber cv. Toschka) Test formulation MBI-203 = 96% liquid formulation Test concentrations Mini-drench test #1: 100, 50 ml/L for MBI-203 Mini-drench test #2: 50, 25, 12.5, 6, 3, 1.5 ml/L Test application Drench application
Results
Mini Drench Test No. 1

(92) The activity of the treatments was very high and a reduction of almost 100% was observed when applied at a concentration of 50 ml/L (MBI-203). Minor phytotoxicity was observed for MBI-203. Fosthiazate performed as usual (100% control at 20 ppm).

(93) Mini Drench Test No. 2

(94) MBI-203 showed phytotoxicity at the highest concentrations of 50 and 25 ml/L and assessments could not be made at these rates.

(95) At a concentration of 12.5 ml/L nematode control was over 95% which decreased to 33% at 3 ml/L. At a rate of 1.5 ml/L no activity was recorded.

(96) Fosthiazate performed as usual (100% control at 20 ppm).

Example 8: Synergistic Studies with Chromobacterium substugae (MBI-203) Broth

(97) Synergy tests were performed by treating artificial diet in 96-well plates and feeding treated diet to neonate larvae. 100 uL 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.sub.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., 16 h light/8 h dark cycle for 3 days. On the third and fourth day after infesting, mortality was scored.

(98) The determination of a synergistic, antagonistic, or additive interaction was determined using the methods from (Colby 1967). Due to variation in bioassays, it was determined that ratios between 0 and 0.9 would be considered antagonistic, 0.9-1.1 ratios would be additive, and ratios above 1.1 would be considered synergistic relationships.

(99) MBI-203 synergy with insecticides against Cabbage Loopers 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.sub.50 concentrations of MBI-203 and insecticides were used. The results are shown in Table 5. All, but Bt var. kurstaki and 1 instance of LC.sub.50 concentration showed synergism.

(100) TABLE-US-00004 TABLE 5 MBI-203 + Insecticide: Effect on cabbage loopers Calcu- MBI-203 Product lated Actual alone alone Combo Combo Defined Product Kill % Kill % Kill % Kill % Ratio relation Chlor- 21 3 23.4 33.3 1.42 syn antranilipole Bt var. 61.7 89.6 96 100 1.04 add kurstaki Spinosad 41.5 54.3 72.99 100.00 1.37 syn Spirotetramet 87.9 23.8 86.34 89.87 1.04 add Spirotetramet 90.6 41.5 91.90 94.94 1.03 add (0.5 LC.sub.50); MBI-203 (0.3 LC.sub.50) Pyrethrum 19.7 2.8 21.93 55.37 2.53 syn

(101) 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.sub.50 concentrations of MBI-203 and insecticides were used. The results are shown in Table 6. MBI-203 and Chlorantranilipole interacted additively while Bacillus thuringiensis var. kurstaki and Spinosad showed synergistic control of BAW with MBI-203. Pyrethrum combinations with MBI-203 were antagonistic. Spirotetramet and MBI203 combinations were primarily antagonistic against Beet Armyworm.

(102) TABLE-US-00005 TABLE 6 MBI-203 + Insecticide: Effect on Beet Armyworm Calcu- MBI-203 Product lated Actual alone alone Combo Combo Defined Product Kill % Kill % Kill % Kill % Ratio relation. Chlor- 11.6 9.1 19.69 19.9 1.01 add antranilipole Bt var. 24.5 19.8 39.4 68.3 1.73 syn kurstaki Spinosad 23.8 68.7 83.33 100 1.2 syn Spirotetramet 0 21.6 36.10 27.60 0.76 antag Spirotetramet 0 42.9 38.55 41.67 1.08 add (0.53 LC.sub.50); MBI-203 (0.7 LC.sub.50) Spirotetramet 21.4 53.3 60.57 53.70 0.89 antag Spirotetramet 10 77.5 78.22 41.23 0.53 antag (1.4 LC.sub.50); MBI-203 (1.2 LC.sub.50) Pyrethram 14.4 74.5 78.17 12.16 0.16 antag Pyrethram 70.7 11.1 73.97 27.78 0.38 antag

(103) 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 present invention.

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

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