Methods and compositions for insecticidal mixtures with synergistic effects

Abstract

The present disclosure provides an insecticidal composition having a synergistic effect, comprising: one or more chromamide compounds; and one or more Scott proteins disposed in the carrier.

Description

[0001] priority

[0002] This application claims priority to U.S. Provisional Application No. 63 / 336,021, filed on April 28, 2022. Technical Field

[0003] The present disclosure relates generally to the field of bioactive compositions and metabolites derived from Chromobacterium for controlling pests, and methods of their use in controlling pests.

[0004] Federally Sponsored Research Statement

[0005] none.

[0006] Incorporated by reference into the material archived on the CD

[0007] none. Background Art

[0008] Without limiting the scope of the present disclosure, its background is described in conjunction with synergistic bioactive compositions and metabolites derived from Chromobacterium for controlling pests, and methods of their use for controlling pests.

[0009] Natural products are substances produced by microorganisms, plants and other organisms. Microbial natural products provide a rich source of chemical diversity, and there is a long history of using natural products for pharmaceutical purposes. Although emphasis is placed on natural products for human treatment, more than 50% of which come from natural products, only 11% of pesticides come from natural sources. However, natural product pesticides may play an important role in controlling pests on traditional and organic farms. The secondary metabolites produced by microorganisms (bacteria, actinomycetes and fungi) provide new compounds that can be used alone or in combination with known compounds to effectively control insect pests and reduce the risk of resistance development. There are several well-known examples of microbial natural products that have been successfully used as agricultural insecticides (Thompson et al., 2000; Arena et al., 1995; Krieg et al., 1983).

[0010] The development of microbial insecticides begins with the isolation of microorganisms in pure culture. They are then screened for efficacy and spectrum using in vitro and in vivo assays or pilot-scale trials in greenhouses or fields. Simultaneously, the active compounds produced by the microorganisms are isolated and identified. For commercialization of microbial insecticides, the microorganisms must be economically produced by fermentation on an industrial scale and formulated with biocompatible and approved additives to enhance efficacy and maximize ease of use and storage stability under field conditions.

[0011] In 2000, Dr. Martin and her colleagues at the USDA isolated a purple-pigmented bacterium (PRAA4-1) from forest soil in Maryland (Martin et al., 2007a). In an initial screen, they found it to be toxic to Colorado potato beetles and other insect pests (Martin et al., 2007b). This motile, Gram-negative bacterium was identified as a new species of Chromobacteria: Chromobacterium substsugae sp. nov (Martin et al., 2007c). It is a facultatively aerobic, motile, Gram-negative betaproteobacterium with polar flagella. Colonies formed on L-agar plates at 25°C within 2-3 days are initially cream-colored, gradually changing from light purple to dark purple over the next 24 hours. Colonies of PRAA4-1 grow well on peptone-based media with an optimum temperature of 25°C, a pH of 6.5-8.0, and 0-1.5% (w / v) NaCl (Martin et al., 2007a).

[0012] Since the discovery of C. substugae by Martin and her colleagues, at least three new species of Chromobacterium have been isolated and characterized; Young et al. (2008) isolated a new species of Chromobacterium, C. aquaticum, from spring water samples in Taiwan, and Kampfer et al. (2009) isolated two species, C. piscinae and C. pseudoviolaceum, from environmental samples collected in Malaysia.

[0013] Of all known species of Chromobacterium, C. violaceum is a Gram-negative species and a common inhabitant of soil and water in tropical and subtropical regions. Published information on secondary metabolites produced by Chromobacterium is based solely on studies of C. violaceum (see, for example, Duràn and Menck (2001) for a comprehensive review of the pharmacology and industrial prospects of C. violaceum). It is generally considered nonpathogenic to humans, but as an opportunistic pathogen, it occasionally becomes a causative agent of sepsis and fatal infections in humans and animals. C. violaceum is known to produce the purple pigment violacein, a bisindole molecule produced by the fusion of two L-tryptophan molecules in the presence of oxygen (Hoshino et al., 1987; Ryan and Drennan, 2009). Violacin biosynthesis is regulated by quorum-sensing, a common mechanism regulating various other secondary metabolic pathways in Gram-negative bacteria (McClean et al., 1997).

[0014] Other known metabolites of C. violaceum, as summarized by Duràn and Menck (2001), include hydrogen cyanide, ferrioxamine E, the beta-lactam glycopeptides SQ28,504 and SQ28,546, antibiotics (e.g., aerocyanidin, aerocavin, 3,6-dihydroxy-indoxazene, and the monobactam SB-26.180), and the antitumor depsipeptide FR901228. According to the review article by Duràn and Menck (2001), C. violaceum also produces unusual sugar compounds, such as exopolysaccharides and lipopolysaccharides.

[0015] US Patent No. 8,715,754, which is incorporated herein by reference in its entirety, also discloses compounds obtainable or derived from Chromobacterium species, more particularly Chromobacterium hemlockii.

[0016] U.S. Patent No. 9,259,007, which is incorporated herein by reference in its entirety, discloses a stable biopesticide comprising a Chromobacterium species, a filtrate, a supernatant, an extract, or an insecticidal active substance derived therefrom having insecticidal activity, which has an improved shelf life due to maintenance of physical homogeneity and a longer insecticidal activity after use due to a higher resistance to degradation when exposed to sunlight. Summary of the Invention

[0017] The present disclosure provides a synergistic insecticidal composition comprising: (a) one or more chromamide compounds; (b) violacein; and (c) one or more proteins selected from the group consisting of Scott 1, Scott 2, and Scott 3 in a carrier. According to one aspect, the one or more chromamide compounds of the composition include chromamides having MW 860, MW 874A, MW 874B, and MW 876. In another configuration, the chromamide compounds include one or more selected from the group consisting of chromamide A having MW 860 (molecular weight 860), chromamide having MW 874A (molecular weight 874), chromamide having MW 874B (molecular weight 874), and chromamide having MW 876 (molecular weight 876).

[0018] The present disclosure provides a synergistic insecticide composition comprising: one or more chromamide compounds; and one or more Scott proteins in a carrier.

[0019] The present disclosure also provides a method for regulating an infestation of pests, comprising: applying an amount of a compound effective for regulating the infestation to the location where regulation is desired, the compound comprising one or more chromamide compounds; and one or more Scott proteins placed in a carrier. The one or more chromamide compounds include one or more chromamides having a molecular weight of 860, a molecular weight of 874A, a molecular weight of 874B, and a molecular weight of 876. The composition further comprises one or more additional insecticides. The pests are selected from the group consisting of Acari, Muscidae, Drosophilidae, Anthomyidae, Aphididae, TrioZidae, Tenebrionidae, and Scarabaeidae.

[0020] The present disclosure also provides a method for regulating an infestation of an insect pest, wherein the pest is selected from the group consisting of Acari, Muscidae, Drosophilidae, Anopheles, Aphididae, Psyllidae, Tenebrionidae, and Scarabaeidae, comprising applying to a locus where regulation is desired an amount of a effective for regulating the infestation, the a comprising: (a) one or more chromamide compounds selected from the group consisting of chromamide A (MW 860), chromamide MW 874A, chromamide MW 874B, and chromamide MW 876; and (b) one or more Scott proteins (selected from the group consisting of Scott 1, Scott 2, and Scott 3) in a carrier. The locus where regulation is desired is on a plant, plant seed, or in soil. In one configuration, the pest is Acari, and the Acari are mites. In one configuration, the pests are selected from the group consisting of Tetranychus sp., Musca sp., Myzus sp., Bactericera sp., Cyclo cephala sp., Alphitobius sp., Drosophila sp., Delia sp., Rhizotrogus sp., Popillia sp., Anomalasp., and Otiorhynchus sp. In one configuration, the pests include Musca domesitcas, Drosophila suzukii, Delia radicum, Myzus persicae, Bactericera cockerelli, Alphitobius diaperinus xi, Cyclocephala lurida, Rhizotrogus maialis, Popilla japonica, Otiorhynchus sulcatus, or Anomala Orientalis. In one configuration, the composition comprises a supernatant, a filtrate, or an extract from whole cell broth. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] For a more complete understanding of the features and advantages of the present disclosure, reference is now made to the detailed description of the disclosure and to the accompanying drawings, in which:

[0022] Figure 1 Schematic diagram of the purification scheme used to obtain chromamide, violacein (2), and deoxyviolacein (3) from culture broth.

[0023] Figure 2 The chemical structures of chromamide A (1), violacein (2), and deoxyviolacein (3) are depicted.

[0024] Figure 3 is a plot of the mortality percentages for the sample.

[0025] Figure 4 The activity graph is obtained by varying the concentration of scott1 while keeping chromamide constant, and the graph is obtained by varying the concentration of chromamide while keeping scott1 constant.

[0026] Figure 5 This is a diagram of a cabbage looper bioassay.

[0027] Figure 6 is a graph showing the results of insecticidal bioassays of various purified compounds with and without Sct1.

[0028] Figure 7 is a graph showing the effect of varying the concentrations of MW 860 and Sct1 on insecticidal activity. DETAILED DESCRIPTION

[0029] As defined herein, "derived from" means directly isolated or obtained from a specified source, or alternatively having the identifying characteristics of a substance or organism isolated or obtained from a specified source.

[0030] As defined herein, a "carrier" is an inert, organic or inorganic material with which an active ingredient is mixed or formulated to facilitate application of the active ingredient to a plant or other subject to be treated, or storage, transport and / or handling of the active ingredient.

[0031] As defined herein, the term "modulate" is used to mean changing the amount of pest infestation or the rate of spread of pest infestation.

[0032] As defined herein, the term "pest infestation" refers to the presence of pests in amounts that result in deleterious effects, including disease or infection in a host population or the appearance of unwanted weeds in a growing system.

[0033] As defined herein, a "pesticide" is a substance derived from a biological product or chemical that increases the mortality or inhibits the growth rate of plant pests, including but not limited to nematocides, insecticides, herbicides, fungicides, bactericides, and viricides.

[0034] As defined herein, a "biopesticide" is a microorganism that possesses insecticidal properties.

[0035] As defined herein, "whole broth culture" refers to a liquid culture containing cells and medium. If the bacteria are grown on plates, the cells can be harvested in water or other liquid whole culture.

[0036] The term "supernatant" refers to the liquid remaining when cells are grown in broth or collected in another liquid from an agar plate and removed by centrifugation, filtration, sedimentation, or other methods known in the art.

[0037] As defined herein, "filtrate" refers to the liquid from a whole broth culture that has passed through a membrane.

[0038] As defined herein, "extract" refers to liquid material that has been removed from cells by a solvent (water, detergent, buffer) and separated from the cells by centrifugation, filtration, or other methods.

[0039] As used herein, "MBI-203" refers to any microbial-based insecticide containing Chromobacterium subtsugae and spent fermentation medium as active ingredients.

[0040] Chromamide compounds are compounds having a molecular weight of 860 and its homologues (including but not limited to 874A, 874B and 876) and having a structure described herein, which can be obtained by: (a) culturing a strain of Chromobacterium hemlocki in a culture medium or whole cell broth under conditions sufficient to produce the compound, and (b) isolating the compound produced from the whole cell broth of (a).

[0041] "Scott protein" and "Sctt protein" are defined herein as the nucleotide sequence (SEQ ID 4, 5, 6) or the protein sequence as shown in Seq ID No 1, 2 or 3 and listed as Scott1 (Scott 1, Sct1), Scott2 (Scott 2, Sct2) or Scott3 (Scott 3, Sct3).

[0042]

[0043]

[0044]

[0045]

[0046] The present disclosure relates to synergistic insecticidal mixtures comprising at least one of chromamide A (MW 860), MW 874A, MW 874B, and MW 876 and one or more proteins selected from Scott 1, Scott 2, and Scott 3, as well as compositions and methods of controlling insects thereof. In some configurations, the compositions further comprise one or more additional insecticides. Insecticides include, but are not limited to, pyrethrins, spirotetramet, and organochlorines.

[0047] The present disclosure also relates to a method of synergistically controlling insects by contacting the insects or their food supply with a synergistically effective amount of a) chromamide A MW 860, MW 874A, MW 874B, MW 876, and b) one or more proteins selected from Scott 1, Scott 2, and Scott 3.

[0048] In addition, the present disclosure also relates to a method for preparing a synergistic insecticide composition containing the above compounds.

[0049] Previous in-house studies have shown that the insecticidal activity present in MBI-203 whole cell broth is primarily attributable to synergistic interactions between secondary metabolites (chromamide analogs) and proteins (Scott 1, Scott 2, and / or Scott 3). However, these studies were conducted using mixed chromamides (a cocktail of four analogs) and Sct1.

[0050] The present invention also relates to a method for protecting plants from attack or infestation by insects, which comprises contacting the plants or the soil or water in which the plants are growing with a synergistically effective amount of a combination of the above-mentioned compounds.

[0051] As used herein, the term chromamide A having a molecular weight of 860 (MW 860) has the following structure and includes synthetic compositions.

[0052] As used herein, the term chromamide A having a molecular weight of 874 (MW 874A) has the following structure and includes synthetic compositions.

[0053] As used herein, the term chromamide A having a molecular weight of 874 (MW 874B) has the following structure and includes synthetic compositions.

[0054] As used herein, the term chromamide A having a molecular weight of 876 (MW 876) has the following structure and includes synthetic compositions.

[0055] In one configuration, the metabolite can be a compound having the following characteristics: (a) insecticidal activity; (b) a molecular weight of about 840-900 as determined by liquid chromatography / mass spectrometry (LC / MS), and (c) a high pressure liquid chromatography (HPLC) retention time of about 7-12 minutes on a reverse phase C-18 HPLC column using a water:acetonitrile (CH3CN) gradient solvent system (0-20 min; 90-0% CH3CN in water, 20-24 min; 100% CH3CN, 24-27 min; 0-90% CH3CN in water, 27-30 min; 90% CH3CN in water) at a flow rate of 0.5 mL / min and UV detection at 210 nm, and (d) optionally obtainable from a Chromobacterium species. In one configuration, the compound can be a peptide.

[0056] In a specific configuration, the compound has 43 carbon atoms, as determined by 13C NMR: 7 methyl carbons, 10 methylene carbons, 12 methine carbons, 6 olefinic methine carbons, and 8 quaternary carbons. In a more specific configuration, the compound includes compounds "A," "B," "C," and "D," which are depicted as ##STR001#, ##STR001a##, ##STR001b##, and ##STR001c##, respectively.

[0057] In one specific configuration, compound "A" has the following characteristics: (a) can be obtained from a species of Chromobacterium; (b) is toxic to pests; (c) has a molecular weight of about 840-890, more particularly 860, as determined by liquid chromatography / mass spectrometry (LC / MS); (d) has 1H NMR δ values ​​are 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, and with 13C NMR δ values ​​are 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, and 28. 83, 27.78, 24.41, 23.06, 22.09, 20.56, 19.31, 18.78, 17.66, 15.80; (e) using a water:acetonitrile (CH3CN) gradient solvent system (0-20 min; 90-0% CH3CN in water, 20-24 min; 100% CH3CN, 24-27 min; 0-90% CH3CN in water, 27-30 min; 90% CH3CN in water) on a reverse phase C-18 HPLC (Phenomenex, Luna 5μC18(2)100A, 100×4.60 mm) column with a high pressure liquid chromatography (HPLC) retention time of about 7-12 minutes, more specifically about 9 minutes, even more specifically about 9.08 minutes, at a flow rate of 0.5 mL / min and UV detection at 210 nm.

[0058] In another specific configuration, compound "B" has the following characteristics: (a) can be obtained from a Chromobacterium species; (b) is toxic to pests; (c) has a molecular weight of about 850-900, more particularly 874, as determined by liquid chromatography / mass spectrometry (LC / MS); (d) is purified by reverse phase C-18 HPLC (Phenomenex, Luna) at a flow rate of 0.5 mL / min and UV detection at 210 nm using a gradient solvent system of water: acetonitrile (CH3CN) (0-20 min; 90-0% CH3CN in water, 20-24 min; 100% CH3CN, 24-27 min; 0-90% CH3CN in water, 27-30 min; 90% CH3CN in water) 5μ C18(2) 100A, 100×4.60 mm) column with a high pressure liquid chromatography (HPLC) retention time of about 7-12 minutes, more specifically about 9 minutes, and even more specifically about 9.54 minutes.

[0059] Metabolites can also be compounds, including but not limited to:

[0060] (A) Compounds with the structure ##STR001##:

[0061]

[0062] or a pesticidally acceptable salt or stereoisomer thereof, wherein R is -H, a lower chain alkyl group containing 1, 2, 3, 4, 5, 6, 7, 8 or 9 alkyl groups. wherein R1, R2, R3, R4, R5, R6, R7, R8, R9, R10, and R11 are each independently H, the same or different, and independently represent an amino acid side chain group or amino acid side chain derivative, an alkyl group, a substituted alkyl group, an alkenyl group, a substituted alkenyl group, an alkynyl group, a substituted alkynyl group, an aryl group, a substituted aryl group, a heteroaryl group, a substituted heteroaryl group, a heterocyclyl group, a substituted heterocyclyl group, a cycloalkyl group, a substituted cycloalkyl group, an alkoxy group, a substituted alkoxy group, a thioalkyl group, a substituted thioalkyl group, a hydroxyl group, a halogen group, an amino group, an amido group, a carboxyl group, —C(O)H, an acyl group, an oxyacyl group, a carbamate group, a sulfonyl group, a sulfonamide group, or a sulfone group;

[0063] (B) Compounds having the structure ##STR001a##:

[0064]

[0065] wherein R is —H, a lower chain alkyl group containing 1, 2, 3, 4, 5, 6, 7, 8 or 9 alkyl groups, an aryl or arylalkyl group, or a substituted lower alkyl group; X is O, NH, NR or S; R2a and R2b are independently selected from the group consisting of —H, alkyl, lower alkyl, substituted alkyl and substituted lower alkyl; R1, R2, R3, R4, R5, R6, R7, R8, R9, R10 and R11 are each independently H, the same or different and independently wherein the first and second groups are amino acid side chain groups or amino acid side chain derivatives, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclyl, substituted heterocyclyl, cycloalkyl, substituted cycloalkyl, alkoxy, substituted alkoxy, thioalkyl, substituted thioalkyl, hydroxy, halogen, amino, amide, carboxyl, —C(O)H, acyl, oxyacyl, carbamate, sulfonyl, sulfonamide, or sulfone.

[0066] (C) Compounds having the structure ##STR001b##:

[0067]

[0068] wherein R is —H, a lower chain alkyl group containing 1, 2, 3, 4, 5, 6, 7, 8 or 9 alkyl groups, an aryl or arylalkyl group, or a substituted lower alkyl group; X is O, NH, NR or S; n is 0, 1, 2, 3, 4, 5, 6, 7, 8 or 9; R2a and R2b are independently selected from the group consisting of —H, alkyl, lower alkyl, substituted alkyl and substituted lower alkyl; R1, R2, R3, R4, R5, R6, R7, R8, R9, R10 and R11 are each independently H, are the same or different and are independently an amino acid side chain group or an amino acid side chain derivative, an alkyl group, a substituted alkyl group, an alkenyl group, a substituted alkenyl group, an alkynyl group, a substituted alkynyl group, an aryl group, a substituted aryl group, a heteroaryl group, a substituted heteroaryl group, a heterocyclyl group, a substituted heterocyclyl group, a cycloalkyl group, a substituted cycloalkyl group, an alkoxy group, a substituted alkoxy group, a thioalkyl group, a substituted thioalkyl group, a hydroxyl group, a halogen group, an amino group, an amide group, a carboxyl group, —C(O)H, an acyl group, an oxyacyl group, a carbamate group, a sulfonyl group, a sulfonamide group, or a sulfone group.

[0069] (D) Compounds having the structure ##STR001c##:

[0070]

[0071] wherein R is —H, lower chain alkyl, aryl or arylalkyl group, substituted lower alkyl containing 1, 2, 3, 4, 5, 6, 7, 8 or 9 alkyl groups; X is O, NH, NR or S; n is 0, 1, 2, 3, 4, 5, 6, 7, 8 or 9; R2a, R2b are independently selected from the group consisting of —H, alkyl, lower alkyl, substituted alkyl and substituted lower alkyl; R1, R2, R3, R4, R5, R6, R7, R8, R9, R10, R11 are each independently H, are the same or different and are independently an amino acid side chain group or an amino acid side chain derivative, an alkyl group, a substituted alkyl group, an alkenyl group, a substituted alkenyl group, an alkynyl group, a substituted alkynyl group, an aryl group, a substituted aryl group, a heteroaryl group, a substituted heteroaryl group, a heterocyclyl group, a substituted heterocyclyl group, a cycloalkyl group, a substituted cycloalkyl group, an alkoxy group, a substituted alkoxy group, a thioalkyl group, a substituted thioalkyl group, a hydroxyl group, a halogen group, an amino group, an amide group, a carboxyl group, —C(O)H, an acyl group, an oxyacyl group, a carbamate group, a sulfonyl group, a sulfonamide group, or a sulfone group.

[0072] In a more specific configuration, the metabolite is chromamide A (1).

[0073]

[0074] In a particular configuration, the metabolite is compound "C" having the following characteristics: (a) being obtainable from a Chromobacterium species; (b) being toxic to one or more pests; (c) having a molecular weight of about 325-360, more particularly about 343, as determined by liquid chromatography / mass spectrometry (LC / MS); and (d) being HPLC-MS / HPLC-MS with a gradient solvent system of water:acetonitrile (CH3CN) (0-20 min; 90-0% CH3CN in water, 20-24 min; 100% CH3CN, 24-27 min; 0-90% CH3CN in water, 27-30 min; 90% CH3CN in water) at a flow rate of 0.5 mL / min and UV detection at 210 nm. 5μ C18 (2) 100A, 100×4.60 mm) column with a high pressure liquid chromatography (HPLC) retention time of about 8-14 minutes, more specifically about 10 minutes, and even more specifically about 10.88 minutes. In a specific configuration, compound "C" can be violacein (2), which is a known compound earlier isolated from Chromobacterium violaceum.

[0075] In another configuration, another metabolite used in the above compositions and methods is compound "D" having the following characteristics: (a) being obtainable from a Chromobacterium species; (b) being toxic to pests; (c) having a molecular weight of about 315-350, more particularly about 327, as determined by liquid chromatography / mass spectrometry (LC / MS); and (d) being HPLC-MS / HPLC-MS with a gradient solvent system of water:acetonitrile (CH3CN) (0-20 min; 90-0% CH3CN in water, 20-24 min; 100% CH3CN, 24-27 min; 0-90% CH3CN in water, 27-30 min; 90% CH3CN in water) at a flow rate of 0.5 mL / min and UV detection at 210 nm. 5μ C18 (2) 100A, 100×4.60 mm) column with a high pressure liquid chromatography (HPLC) retention time of about 10-15 minutes, more specifically about 12 minutes, even more specifically about 12.69 minutes. In a specific configuration, compound "D" can be violacein (3), which is a known compound earlier isolated from Chromobacterium violaceum.

[0076] In another specific configuration, the compound may have the following structure:

[0077]

[0078] wherein R is —H, lower chain alkyl containing 1, 2, 3, 4, 5, 6, 7, 8, or 9 alkyl groups, aryl or arylalkyl groups, substituted lower alkyl, halogen; and R1, R2, R3, R4, R5, R6, R7, R8, and R9 are each independently H, the same or different, and are alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclyl, substituted heterocyclyl, cycloalkyl, substituted cycloalkyl, alkoxy, substituted alkoxy, thioalkyl, substituted thioalkyl, hydroxy, halogen, amino, amide, carboxyl, —C(O)H, acyl, oxyacyl, carbamate, sulfonyl, sulfonamide, or sulfone.

[0079] Also provided is a method for (1) regulating an infestation of pests (e.g., nematodes, insects, soil-borne bacteria) in a plant, comprising applying to the plant and / or its seeds and / or a substrate for growing the plant an amount of:

[0080] I) A compound having the following characteristics: (a) having insecticidal and / or antimicrobial activity; (b) having a molecular weight of about 950-1450 as determined by an LTQ Orbitrap XL hybrid Fourier transform mass spectrometer; (c) having 1H NMR δ values ​​of 5.22 (sext, 1H), 2.62 (dd, 1H), 2.53 (dd, 1H) and 1.31 (d, 3H); (d) having 13C NMR δ values ​​of 169.2, 67.6, 40.9 and 19.8; (d) comprising the structure —(—O—CHCH3—CH2—CO—) n —, wherein n=6-50; (e) is obtainable from a Chromobacterium species, and (II) optionally another insecticidal substance effective in regulating infestation in said plant.

[0081] Compound (I) may have the following structure:

[0082]

[0083] wherein X is independently —O, —NR, or —S, wherein R is H or C1-C10 alkyl; Y is independently —O, —S; n=6-50; R1 and R2 are each independently H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclyl, substituted heterocyclyl, cycloalkyl, substituted cycloalkyl, alkoxy, substituted alkoxy, thioalkyl, substituted thioalkyl, hydroxy, halogen, amino, amide, carboxyl, —C(O)H, acyl, oxyacyl, carbamate, sulfonyl, sulfonamide, or sulfone.

[0084] In particular, compound (I) has the following structure:

[0085]

[0086] wherein n = 10 to 25. In the most specific configuration, (I) is α-butyric acid.

[0087] Also provided is a method for obtaining the above compound, which comprises culturing a strain of Chromobacterium sp. in a whole cell broth under conditions sufficient to produce the compound, and isolating the produced compound from the whole cell broth.

[0088] In a related aspect, a method for stabilizing a biopesticide composition against physical separation and loss of activity due to exposure to sunlight is disclosed, the method comprising applying to the biopesticide composition an amount of a stabilizer effective to stabilize the biopesticide composition against physical separation and loss of activity due to exposure to sunlight. Compositions comprising such agents are also provided. In a specific configuration, the insecticide composition comprises a filtrate, supernatant, extract of a Chromobacterium species, or an insecticidal active substance derived therefrom, which can be present in an amount of at least about 0.5%, particularly from about 0.5 wt % to about 30 wt % based on dry cell weight. In a specific configuration, the stabilizer can be a benzoate and / or a lignin salt, particularly a lignin sulfonate (including but not limited to sodium, potassium, calcium, magnesium, ammonium, and combinations thereof), and can be present in an amount of at least about 2.5 wt %, preferably in an amount of about 5%-15%.

[0089] The above-mentioned substances used in the compositions and methods disclosed herein can be formulated in any manner. Non-limiting examples of formulations 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), microcapsules, water-dispersible granules, flowables (FL), microemulsions (ME), nanoemulsions (NE), and the like. In any formulation described herein, the percentage of active ingredient is in the range of 0.01% to 99.99%.

[0090] The composition may be in liquid, gel or solid form.The liquid composition comprises an insecticidal compound derived from a strain of Chromobacterium, such as a strain having the identifying characteristics of Chromobacterium hemlockii, more particularly a strain having the identifying characteristics of NRRL B-30655 (see US Pat. No. 7,244,607).

[0091] Solid compositions can be prepared by suspending a solid carrier in a solution of the pesticidal compound and drying the suspension under mild conditions (eg, evaporation at room temperature or under vacuum at 65°C or below).

[0092] The composition may comprise a gel-encapsulated compound derived from a strain of Chromobacterium. Such gel-encapsulated materials may be prepared by mixing a gel-forming agent (e.g., gelatin, cellulose, or lignin) with a live or inactivated culture or suspension of Chromobacterium, or a cell-free filtrate or cell fraction of a Chromobacterium culture or suspension, or a spray-dried or freeze-dried culture, cells, or cell fraction, or a solution of a pesticidal compound used in the disclosed methods; and inducing gel formation of the agent.

[0093] The composition may also additionally comprise a surfactant for emulsification, dispersion, wetting, spreading, integration, disintegration control, stabilization of the active ingredient and improvement of flowability or inhibition of rust. In a particular configuration, the surfactant is a non-phytotoxic nonionic surfactant, which preferably belongs to EPA Inerts List 4B. In another particular configuration, the nonionic surfactant is polyoxyethylene (20) monolaurate. The concentration of the surfactant may be between 0.1-35% of the total formulation, with a preferred range of 5-25%. The choice of dispersing and emulsifying agents (e.g., nonionic, anionic, amphoteric and cationic dispersants and emulsifiers) and the amount used are determined by the nature of the composition and the ability of the agent to promote dispersion of the disclosed composition.

[0094] The composition as described above further comprises a stabilizer which stabilizes the biopesticide composition against physical separation and loss of activity due to exposure to sunlight. The stabilizer may be a benzoate or a lignin sulfonate.

[0095] The above composition can be combined with another microorganism and / or pesticide (e.g., nematicide, fungicide, insecticide, antibiotic or antimicrobial). Microorganisms can include, but are not limited to, agents derived from Bacillus sp., Pseudomonas sp., Brevabacillus sp., Lecanicillium sp., non-Ampelomyces sp., Pseudozymas p., Streptomyces sp., Burkholderia sp., Trichoderma sp., Gliocladium sp.). Alternatively, the agent can be a natural oil or oil product (e.g., paraffin oil, tea tree oil, lemongrass oil, clove oil, cinnamon oil, citrus oil, rosemary oil) with fungicidal and / or insecticidal activity. In addition, the insecticide can be a single-site antifungal agent, which can include, but is not limited to, benzimidazoles, demethylation inhibitors (DMIs) (e.g., imidazoles, piperazines, pyrimidines, triazoles), morpholines, hydroxypyrimidines, anilinopyrimidines, thiophosphates, quinone external inhibitors, quinolines, dicarboximides, formimides, phenylamides, anilinopyridines, phenylpyrroles, aromatic hydrocarbons, cinnamic acid, hydroxyanilines, antibiotics, polyoxins, amides, phthalimides, benzene derivatives (xylylalanine), demethylation inhibitors selected from the group consisting of imidazoles, piperazines, pyrimidines, and triazoles (e.g., biphenyltriazole). The invention also includes but is not limited to tertanol, myclobutanil, penconazole, propiconazole, triadimefon, bromuconazole, cyproconazole, diniconazole, fenbuconazole, hexaconazole, tebuconazole, tetraconazole), myclobutanil and quinone external inhibitors (e.g., strobilurin). Strobilurins can include but are not limited to azoxystrobin, kresoxim-methoyl or trifloxystrobin.In another particular configuration, the antifungal agent is a quinone, such as quinoxyfen (5,7-dichloro-4-quinolyl-4-fluorophenyl ether). The antifungal agent may also be derived from Reynoutria extract.

[0096] The fungicide may 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-acetamideoxime.

[0097] As mentioned above, the composition may also include an insecticide. Insecticides may include, but are not limited to, avermectin, Bt, neem oil, spinosads, Burkholderdia sp. as described in U.S. Patent Application Publication No. 2011-0207604, entomopathogenic fungi (e.g., Beauveria bassiana), and chemical insecticides, including but not limited to organochlorine compounds, organophosphorus compounds, carbamates, pyrethroids, and neonicotinoids.

[0098] As mentioned above, the composition may also include a nematicide. Such nematicides may include, but are not limited to, avermectins, microbial products (e.g., Biome (Bacillus firmus, Pasteuria spp.), and organic products (e.g., saponins).

[0099] The above compositions, cultures and supernatants and insecticide compounds can be used as insecticides. In particular, the above compounds or compositions can be used as insecticides, bactericides (against soil-borne bacteria) and nematocides. Specifically, nematodes that can be controlled using the above methods include, but are not limited to, parasitic nematodes such as root-knot nematodes, cyst nematodes, 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.; in particular, Meloidogyne incognita (root-knot nematode), Globodera rostochiensis, and Globodera spp. pailida (potato cyst nematode); Heterodera glycines (soybean cyst nematode); Heterodera schachtii (beet cyst nematode); and Heterodera avenae (cereal cyst nematode).

[0100] As noted above, the active ingredients and compositions described above can also be applied to loci containing Acari (arachnids) (e.g., mites), including, but not limited to, Panonychus sp., such as Panonychus citri (citrus red mite) and Panonychus ulmi (red spider mite), Tetranychus sp., such as Tetranychus kanzawi (Kanzawa spider mite), Tetranychus urticae (2-spotted spider mite), Tetranychus pacificus (Pacific spider mite), Tetranychus turkestanii (Strawberry mite), and Tetranychus cinnabarinus (Carmine spider mite). mite), Oligonychus sp., for example, Oligonychus panicae (avacado brown mite), Oligonychus perseae (persea mite), Oligonychus pratensis (Banks grass mite), and Oligonychus coffeae, Aculus sp., for example, Aculus cornatus (Peach silver mite), Aculus fockeni (Plum rust mite), and Aculus lycopersici (Tomato russet mite), Eotetranychus sp.), for example, Eotetranychus wilametti, Eotetranychus yumensis (yuma spider mite) and Eotetranychus sexmaculatis (6-spotted mite), Bryobia rubrioculus (brown mite), Epitrimerus pyri (pearrust mite), Phytoptus pyri (pear leaf blister mite), Acalitis essigi (redberry mite), Polyphagotarsonemuslatus (broadmite), Eriophyes sheldoni (citrus bud mite), Brevipalpus spp. lewisi (citrus flat mite), Phylocoptruta oleivora (citrus rust mite), Petrobialateens (Brown wheat mite), Oxyenus maxwelli (olive mite), Rhizoglyphus spp., Tyrophagus spp., Diptacus gigantorhyncus (bigheaded plum mite), and Penthalea major (winter grain mite), Avocado red mite, Flat mite, and mite), black and red mango spider mite, papaya leaf roller spider mite, Texas citrus mite, European red mite, grape spider mite (blister mite), Pacific spider mite, Willamette spider mite; pink citrus rust mite. Such locations may include, but are not limited to, crops infested by such mites or other arachnids, such as aphids. Such locations may include, but are not limited to, crops infested by such mites or other arachnids, such as aphids.

[0101] Plant pathogenic insects controlled by the above method include, but are not limited to, non-Culicidae larvae insects from the following orders: (a) Lepidoptera, such as Acleris spp., Adoxophyes spp., Aegeria spp., Agrotis spp., Alabama argillaceae, Amylois spp., Anticarsia gemmatalis, Archips spp., Argyrotaenia spp., Autographa spp., Busseola fusca, Cadra cautella, Carposinanipponensis, Chilo spp.), Choristoneura spp., Clysia ambiguella, Cnaphalocrocis spp., Cnephasia spp., Cochylis spp., Coleophora spp., Crocidolomia abinotalis, 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, Manducasexta, 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) from the order of the Coleoptera, for example, Agriotes spp., Alphitobius sp., Anomola spp., for example, Anomala orientalis, Anthonomus spp., Atomaria linearis, Chaetocnema tibialis), Cosmopolites spp., Curculio spp., Cyclocephala spp., for example, Cyclocephalalurida, Dermestes spp., Diabrotica spp., Epilachna spp., Eremnus spp.), potato beetles (Leptinotarsa ​​decemlineata), Lissorhoptrus spp., Melolontha spp., Orycaephilus spp., Otiorhynchus spp., Otiorhynchus sulcatus, Phlyctinus spp., Popillia spp., for example, Popilla japonica, flea beetles (Psylliodes spp.), Rhizopertha spp., for example, Rhizotrogus majalis, rice weevils (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. 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) from the order of the Homoptera, for example, Aleurothrixus floccosus, Aleyrodes brassicae, Aonidiella spp., Aphididae, Aphis spp., Aspidiotus spp., Bactericera 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.(k) from the order of the Hymenoptera, for example, Acromyrmex, Atta, Cephus, Diprion, Diprionidae, Gilpinia, Pseudocycota ... 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., Cuterebra spp., Dacus spp., Delia spp., Delia radicum, Drosophila spp.), for example, Drosophila suzukii, 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) from the order Siphonaptera, for example, Ceratophyllus spp. and Xenopsylla cheopis; (n) from the order Thysanura, for example, Lepisma saccharina; (o) from the order Hemiptera, for example, Bactericera sp.), such as the potato psyllid (Bactericera cockerelli).

[0102] The active ingredient can be applied to locations where Scarabaeidae pests are present. These include, but are not limited to, soil, grass, and various ornamental plants, trees, and vegetables.

[0103] The active ingredients and compositions described above can also be applied to locations containing Muscidae pests. These include, but are not limited to, indoor environments containing animals (cattle, pigs, sheep, horses, etc.), garbage, animals, pens, corrals, barns, milking parlors, breeding pens, etc.

[0104] The active ingredients and compositions described above can also be applied to locations containing the active ingredients and compositions containing Tenebrionidae pests. These include, but are not limited to, grain, livestock, and poultry pens (pens, corrals, barns, milking parlors, mare pens, etc.) containing animals (cattle, pigs, sheep, horses, etc.).

[0105] Example

[0106] The above compositions and methods are further described in the following non-limiting examples. These examples are merely illustrative of various configurations and do not limit the claimed disclosure with respect to the materials, conditions, weight ratios, process parameters, etc. described herein.

[0107] Example 1 includes the use of chromamide A (MW 860), MW 874A, MW 874B, MW 876, violacein, and Scott 1, and their related compositions and methods of controlling insects.

[0108] Example 2 includes chromamide MW 860 and Scott 1 in a carrier, and their related compositions and methods of controlling insects.

[0109] Example 3 includes chromamide MW 874A and Scott 1 in a carrier, and their related compositions and methods of controlling insects.

[0110] Example 4 includes chromamide MW 874B and Scott 1 in a carrier, and their related compositions and methods of controlling insects.

[0111] Example 5 includes chromamide MW 876 and Scott 1 in a carrier, and their related compositions and methods of controlling insects.

[0112] Example 6 includes the use of chromamide A (MW 860), MW 874A, MW 874B, MW 876, violacein, and Scott 2, and their related compositions and methods of controlling insects.

[0113] Example 7 includes chromamide MW 860 and Scott 2 in a carrier, and their related compositions and methods of controlling insects.

[0114] Example 8 includes chromamide MW 874A and Scott 2 in a carrier, and their related compositions and methods of controlling insects.

[0115] Example 9 includes chromamide MW 874B and Scott 2 in a carrier, and their related compositions and methods of controlling insects.

[0116] Example 10 includes chromamide MW 876 and Scott 2 in a carrier, and their related compositions and methods of controlling insects.

[0117] Example 11 includes the use of chromamide A (MW 860), MW 874A, MW 874B, MW 876, violacein, and Scott 3, and their related compositions and methods of controlling insects.

[0118] Example 12 includes chromamide MW 860 and Scott 3 in a carrier, and their related compositions and methods of controlling insects.

[0119] Example 13 includes chromamide MW 874A and Scott 3 in a carrier, and their related compositions and methods of controlling insects.

[0120] Example 14 includes chromamide MW 874B and Scott 3 in a carrier, and their related compositions and methods of controlling insects.

[0121] Example 15 includes chromamide MW 876 and Scott 3 in a carrier, and their related compositions and methods of controlling insects. The following steps are used to purify the compounds extracted from the culture of Chromobacterium hemlockensis disclosed in Example 1:

[0122] A 10-L culture broth derived from fermentation of Chromobacterium hemlockii in L-broth was 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 were collected by filtration through cheesecloth and washed with DI water to remove salts. The resin, cell mass and cheesecloth were then soaked in acetone / methanol (50 / 50) for 2 hours, and the acetone / methanol was then filtered and dried under vacuum using a rotary evaporator to obtain a crude extract. The crude extract was then fractionated using a Sephadex LH20 size exclusion chromatograph (CH2Cl2 / CH3OH; 50 / 50) to obtain 7 fractions ( Figure 1 These fractions were then concentrated to dryness using a rotary evaporator, and the resulting dry residues were screened for biological activity using either Trichoplusia ni or Spodoptera exigua feeding assays. The active fractions were then subjected to reverse-phase HPLC (Spectra System P4000 (Thermo Scientific)) to obtain pure compounds, which were then screened in the aforementioned bioassays to locate / identify the active compounds. Additional spectroscopic data, such as LC / MS and NMR, were recorded to confirm the identity of the compounds.

[0123] Chromamide A (1) and compound B were obtained from fractions 1 and 2, respectively, while violacein (2) and deoxyviolacein (3) were purified from fraction 5, which was obtained from Sephadex LH 20 chromatography.

[0124] Chromamide A(1) was purified by HPLC using a C-18 column (Phenomenex, Luna10u C18(2)100A, 250×10) with a water:acetonitrile gradient solvent system (0-10 min, 80-75% CH3CN in water; 10-45 min, 75-60% CH3CN in water; 45-55 min, 60-50% CH3CN in water; 55-65 min, 50-100% CH3CN in water; 65-70 min, 100% CH3CN; 55-70 min, 0-80% CH3CN in water) at a flow rate of 2.5 mL / min and UV detection at 210 nm. The retention time of the active compound chromamide A(1) was 23.19 minutes.

[0125] Compound B of the present invention was purified by using an HPLC C-18 column (Phenomenex, Luna10u C18(2)100A, 250×10) with a flow rate of 2.5 mL / min and UV detection at 210 nm, and a water:acetonitrile gradient solvent system (0-10 min, 80-75% CH3CN aqueous solution; 10-45 min, 75-60% CH3CN aqueous solution; 45-55 min, 60-50% CH3CN aqueous solution; 55-65 min, 50-100% CH3CN aqueous solution; 65-70 min, 100% CH3CN; 55-70 min, 0-80% CH3CN aqueous solution), and the retention time of active compound B was 26.39 minutes.

[0126] Violabactin (2) and deoxyviolacein (3) were purified by using an HPLC C-18 column (Phenomenex, Luna10u C18 (2) 100A, 250×10) at a flow rate of 2.5 mL / min and UV detection at 210 nm using a water:acetonitrile gradient solvent system (0-10 min, 70-60% CH3CN aqueous solution; 10-40 min, 60-20% CH3CN aqueous solution; 40-60 min, 20-0% CH3CN aqueous solution; 60-65 min, 100% CH3CN; 65-75 min, 0-70% CH3CN aqueous solution). The retention time of the active compound violacein (2) was 7.86 minutes, and the retention time of deoxyviolacein (3) was 12.45 minutes.

[0127] Active peaks were analyzed by mass spectrometry on a Thermo Finnigan LCQ DecaXP Plus electrospray ionization (ESI) instrument using full scan mode (m / z 100-1500 Da) in positive and negative ionization modes on an LCQ DECA XPplus mass spectrometer (Thermo Electron Corporation, San Jose, CA). The Thermo high-performance liquid chromatography (HPLC) instrument was equipped with a Finnigan Surveyor PDA plus detector, an autosampler plus, an MS pump, and a 4.6 mm × 100 mm Luna C18 5μ100A column (Phenomenex). The solvent system consisted of water (solvent A) and acetonitrile (solvent B). The mobile phase started with 10% solvent B, increased linearly to 100% solvent B over 20 minutes, then held for 4 minutes, and finally returned to 10% solvent B over 3 minutes and held for 3 minutes. The flow rate was 0.5 mL / min. The injection volume was 10 μL, and the samples were stored in the autosampler at room temperature. These compounds were analyzed by LC-MS using LC and reverse phase chromatography. Mass spectrometry analysis of the compounds of the present invention was performed under the following conditions: the flow rates of nitrogen were fixed at 30 and 15 arb for the sheath gas and aux / sweep gas, respectively. Electrospray ionization was performed with the spray voltage set to 5000 V and the capillary voltage set to 35.0 V. The capillary temperature was set to 400°C. The data were analyzed on Xcalibur software. In positive ionization mode, the molecular weight of chromamide A (1) was 860. The LC-MS chromatogram showed that the molecular weight of another active compound B was 874 in positive ionization mode. In positive ionization mode, the molecular weights of violacein (2) and deoxyviolacein (3) were 313 and 327, respectively.

[0128] Extraction of violacein and oligo-(1-hydroxybutyric acid) from Chromobacterium hemlockii. The following steps were used to purify compounds extracted from cultures of Chromobacterium hemlockii:

[0129] The whole culture broth (WCB) derived from 20-L fermentation of Chromobacterium hemlockii in L-broth was extracted using ethyl acetate by liquid-liquid extraction. The ethyl acetate layer was separated and dried under vacuum using a rotary evaporator to obtain a crude extract. The crude extract was then graded using different solvents such as dichloromethane (DCM), ethyl acetate (EA), methanol (MeOH) and washed with a mixture of solvents (WASH). The fractions were then concentrated to dryness using a rotary evaporator, and the resulting dried residues were screened for biological activity using different pests (insects, nematodes). The active fractions were then subjected to Sephadex LH 20 size exclusion chromatography (CH2Cl2 / CH3OH; 50 / 50) to obtain 10 fractions. These fractions were then concentrated to dryness using a rotary evaporator and the resulting dry residues (fractions) were screened for biological activity using an insect repellency assay with green peach aphid, progeny production with green peach aphid (GPA), and a nematicidal bioassay (M. incognita and / or M. hapla). The active fractions were then subjected to reverse phase HPLC (Spectra System P4000 (Thermo Scientific)) to obtain pure compounds, which were then screened in the above bioassays to locate / identify the active compounds. Additional spectroscopic data, such as LC / MS and NMR, were recorded to confirm the identity of the compounds.

[0130] To identify the compounds responsible for the insecticidal activity observed in MBI-203 (Chromobacterium hemlockii), a bioassay-guided fractionation / purification was performed. However, it was found that insecticidal activity was lost following the extraction / fractionation process. The individual fractions / compounds had very weak activity compared to the original MBI203 WCB (whole cell broth). However, when these isolated compounds were mixed together, the mixture exhibited similar insecticidal activity.

[0131]

[0132]

[0133] Preliminary results from testing fractions containing chromamide (mixture) and protein (intermediate scott1) showed increased insecticidal activity compared to the individual compounds. Further testing was performed using fractions containing only chromamide (with molecular weights 860, 874A, 874B, and 876) and scott1 (protein), which showed activity comparable to that of MBI-203WCB. Figure 3is a graph of the percent mortality of the samples. These results confirm that the activity between these compounds is the result of a synergistic effect between the two classes of compounds. All compounds were tested at a rate of 2% based on the concentration of each compound present in MBI-203WCB.

[0134]

[0135]

[0136] *=pure sample

[0137] Insecticidal Bioassay: Activity against Trichoplusia ni was tested in a diet overlay bioassay. Appropriate artificial insect diet was dispensed into each well of a standard 96-well plate and allowed to dry. Once the diet solidified, 100 μL of the treatment was pipetted into the appropriate number of wells and allowed to dry. A single first-instar larva was placed into each well of the 96-well plate. Mortality was scored on day 4 after treatment.

[0138] Efficacy is expressed as percentage mortality on day 4 after treatment. The table / graph above shows the results (duplicate) of % mortality.

[0139] Determination of the contribution of the chromamide mixture and SCOTT1 to insecticidal activity. Figure 4 The activity graph is obtained by varying the concentration of scott1 while keeping chromamide constant, and the graph is obtained by varying the concentration of chromamide while keeping scott1 constant.

[0140] To understand the importance and ratio of these two classes of compounds for synergistic effects, in one experiment, the concentration of the chromamide mixture was kept constant and the concentration of SCOTT1 was decreased; in another experiment, the concentration of SCOTT1 was kept constant and the concentration of the chromamide mixture was decreased. The insecticide results showed that there was a significant loss of insecticide activity when the concentration of the chromamide mixture was decreased, indicating that the concentration of the chromamide mixture is very important for synergistic effects.

[0141] Further experiments were conducted using pure chromamide analogs (e.g., MW 860 (Chromamide A), MW 874A, MW 874B, and MW 876) and SCOTT1. The results of these experiments confirmed that chromamide A (MW 860) is essential for synergistic effects with the SCOTT1 protein. Another experiment also confirmed that increasing the concentration of SCOTT1 in combination with chromamide did not increase insecticidal activity. Figure 5This is a diagram of the bioassay for Trichoplusia ni.

[0142] It is contemplated that any configuration discussed in this specification can be implemented with respect to any method, kit, reagent or composition of the present disclosure, and vice versa. In addition, the compositions of the present disclosure can be used to implement the methods of the present disclosure.

[0143] Figure 6 is a graph of the insecticidal bioassay results for various purified compounds with and without Sct1. Previous studies have shown that the insecticidal activity present in MBI-203 whole cell broth is primarily due to a synergistic effect between secondary metabolites (chromamide analogs) and proteins (Sct1). However, these studies were performed using mixed chromamides (a mixture of four analogs) and Sct1. To determine which chromamide analogs were critical for the observed synergistic effects, these individual compounds were purified from MBI-203WCB and tested as mixtures and individual compounds using a chewing bioassay. All compounds were tested at a rate of 3% based on the concentration of each compound present in MBI-203WCB. The "Chroma mixture" contained chromamides MW 860, MW 874A, MW 874B, and MW 876. As expected, the pure chromamide analogs and Sct1 did not show any significant activity when tested alone ( Figure 6 Subsequent testing in two bioassays using cabbage looper (CL) showed that at a 3% dose, MW 860 combined with Sct1 resulted in 92% mortality, while other analogs showed little activity. Therefore, based on these results, MW 860 and Sct1 can be used as markers to predict the insecticidal activity of MBI-203.

[0144] Determination of LC50 values ​​for MW 860 and Sct1. Based on the above test results for purified compounds with and without Sct1, the LC50 values ​​for MW 860, the chromamide mixture, and Sct1 were determined. Individual pure compounds and their mixtures were combined with Sct1 and submitted for LC50 determination. Based on the results of three separate submissions, the LC50 for the MW 860 and Sct1 mixture was found to be between 1.5 and 2.5 μg / mL. The table shows the LC50 results for the purified compounds MW 860 and Sct1.

[0145] Sample ID <![CDATA[LC 50 ]]> Chroma Blend 0 Sct1 0 Chroma mixture + Sct1 1.71 MW 860 0 MW 860+Sct1 2.3 TGAI(D30) 1.25 Positive control (MBI-203-EP) 2.05

[0146] Figure 7Graph showing the effect of varying the concentration of MW 860 and Sct-1 on insecticidal activity. A dose-dependency study of MW 860 and Sct-1 as a mixture was performed. To determine the minimum concentration range of both MW 860 and Sct-1 required for good insecticidal activity, different ratios of the two compounds were prepared and submitted to an insecticidal bioassay. According to the bioassay results, increasing the concentration of MW 860 showed increased insecticidal activity, and a minimum of 3% of MW 860 and Sct-1 was required to achieve approximately 80% control ( Figure 2 ).

[0147] It should be understood that the specific configurations described herein are shown by way of illustration and not as limitations of the present disclosure. The key features of the present disclosure may be used in a variety of configurations without departing from the scope of the present disclosure. Those skilled in the art will recognize, or be able to determine using no more than routine experimentation, many equivalents to the specific steps described herein. Such equivalents are within the scope of the present disclosure and are encompassed by the claims.

[0148] All publications and patent applications mentioned in the specification are indicative of the level of skill of those skilled in the art to which the present disclosure pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[0149] In the claims and / or the specification, when used in conjunction with the term "comprising," the term "a" or "an" can mean "one," but it is also consistent with the meaning of "one or more," "at least one," and "one or more than one." The term "or" as used in the claims is used to mean "and / or" unless expressly indicated to refer only to alternatives or the alternatives are mutually exclusive, although this disclosure supports a definition referring only to alternatives and "and / or." Throughout this application, the term "about" is used to indicate that a value includes the inherent variation of error for the device, the method being employed to determine the value, or the variation that exists between the subjects of study.

[0150] As used in this specification and claims, the words "comprising" (and any form of comprising, such as "comprise" and "comprises"), "having" (and any form of having, such as "have" and "has"), "including" (and any form of including, such as "includes" and "include"), or "containing" (and any form of containing, such as "contains," "contain," etc.) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0151] As used herein, the term "or combinations thereof" refers to all permutations and combinations of the listed items preceding the term. For example, "A, B, C, or combinations thereof" is intended to include at least one of the following: A, B, C, AB, AC, BC, or ABC, and if order is important in a particular context, then also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB. Continuing with this example, combinations containing repetitions of one or more items or terms are expressly included, such as BB, AAA, MB, BBC, AAABCCCC, CBBAAA, CABABB, etc. Those skilled in the art will understand that, unless otherwise apparent from the context, there is generally no limit on the number of items or terms in any combination.

[0152] Although the preparation and use of various configurations of the present disclosure are discussed in detail below, it should be understood that the present disclosure provides many applicable inventive concepts that can be embodied in a variety of specific contexts. The specific configurations discussed herein are merely illustrative of specific ways to prepare and use the present disclosure and do not limit the scope of the present disclosure.

[0153] To facilitate understanding of the present disclosure, a number of terms are defined below. The terms defined herein have meanings that are commonly understood by persons of ordinary skill in the art to which the present disclosure relates. Terms such as "a," "an," and "the" are not intended to refer to only a single entity, but rather to encompass general categories that can be illustrated using specific examples. The terms herein are used to describe specific configurations of the present disclosure, but their use does not limit the present disclosure unless otherwise outlined in the claims.

[0154] When a numerical range is provided, it is understood that unless the context clearly dictates otherwise, the upper and lower limits of that range and every intervening value (to the tenth of the unit of the lower limit) between any other stated value or intervening value in that range are included therein. Smaller ranges are also included. The upper and lower limits of such smaller ranges are also included therein, subject to any specifically excluded limits in the stated range.

[0155] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to the methods and materials described herein can also be used in the practice or testing of the present disclosure, the present application describes preferred methods and materials.

[0156] It must be noted that as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0157] In accordance with the present disclosure, all compositions and / or methods disclosed herein and claimed for protection can be prepared and performed without undue experimentation. Although the compositions and methods of the present disclosure have been described according to preferred configurations, it will be apparent to those skilled in the art that the compositions and / or methods described herein and the steps or sequence of steps in the methods can be modified without departing from the concept, spirit and scope of the present disclosure. It will be apparent to those skilled in the art that all such similar alternatives and modifications are considered to be within the spirit, scope and concept of the present disclosure as defined by the appended claims.

[0158] Citations

[0159] Asolkar, RN, Jensen, PR, Kauffman, CA, Fenical, W. 2006. Daryamides AC, Weakly Cytotoxic Polyketides from a Marine-Derived Actinomycete of the GenusStreptomyces strain CNQ-085J.Nat.Prod.69:1756-1759.

[0160] Martin, PAW, D. Gundersen-Rindal, et al. (2007a). "Chromobacteriumsubstugae sp.nov., a betaproteobacterium toxic to Colorado potato beetle and other insect pests." Int.J.Syst.Evol.Microbiol.57:993-999.

[0161] Martin,P.A.,A.D.S.Shropshire,et al.,(2007b).“Chromobacteriumsubstugae sp.nov for control of insect pests”U.S.Pat.No.7,244,607B2.

[0162] Martin,P.A.W.,Hirose,E.,and Aldrich,J.R.2007c.“Toxicity ofChromobacterium substugae to southern green stink bug(Heteroptera:Pentatomidae)and corn rootworm(Coleoptera:Chrysomelidae)”.J.Econ.Entomol.100:680-684.

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Claims

1. A synergistic insecticidal composition comprising: (a) one or more chromamide compounds; and (b) one or more Scott proteins, The synergistic insecticidal composition is placed in a carrier.

2. The synergistic insecticidal composition of claim 1, wherein the one or more chromamide compounds include one or more chromamide compounds with a molecular weight of 860, a molecular weight of 874A, a molecular weight of 874B, and a molecular weight of 876.

3. The synergistic insecticidal composition according to claim 1, wherein the one or more Scott proteins are selected from Scott 1 SEQ.ID.NO.1, Scott 2 SEQ.ID.NO.2 and Scott 3 SEQ.ID.NO.

3.

4. The synergistic insecticidal composition according to claim 1, further comprising an insecticide.

5. The synergistic insecticidal composition of claim 1, wherein the one or more chromamide compounds are synthetic compounds.

6. The synergistic insecticidal composition of claim 1, wherein the composition comprises a supernatant, a filtrate or an extract from whole cell broth.

7. A synergistic insecticidal composition comprising a compound having the following structure, or a salt or stereoisomer thereof, wherein R is —H, a lower chain alkyl group containing 1, 2, 3, 4, 5, 6, 7, 8 or 9 alkyl groups, an aryl or arylalkyl group, or a substituted lower alkyl group; X is O, NH, NR or S; n is 0, 1, 2, 3, 4, 5, 6, 7, 8 or 9; R1, R2, R3, R4, R5, R6, R7, R8, R9, R10, R11 are the same or different and are independently —H, an amino acid side chain group, an amino acid side chain derivative The invention also includes but is not limited to compounds, such as alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic compound, substituted heterocyclic compound, cycloalkyl, substituted cycloalkyl, alkoxy, substituted alkoxy, thioalkyl, substituted thioalkyl, hydroxy, halogen, amino, amide, carboxyl, -C(O)H, acyl, oxyacyl, carbamate, sulfonyl, sulfonamide and / or sulfone.

8. A method for regulating pest infestation comprising the steps of: An amount of a compound effective for regulating the infestation is applied to the locus where regulation is desired, the compound comprising one or more chromamide compounds; and one or more Scott proteins in a carrier.

9. The method of claim 8, wherein the one or more chromamide compounds comprise: One or more of chromamides of molecular weight 860, molecular weight 874A, molecular weight 874B and molecular weight 876.

10. The method according to claim 8, wherein the pest is selected from the group consisting of Acari, Muscidae, Drosophilidae, Anthomyidae, Aphididae, Psyllidae, Tenebrionidae and Scarabaeidae.

11. The method of claim 8, wherein the pest is Acari, which are mites.

12. The method of claim 11, wherein the mite is spider mite.

13. The method of claim 12, wherein the mite is Tetranychus urticae.

14. The method according to claim 8, wherein: The one or more Scott proteins are selected from Scott 1 SEQ.ID.NO.1, Scott 2 SEQ.ID.NO.2 and Scott 3 SEQ.ID.NO.

3.

15. The composition according to claim 8, wherein The composition further comprises one or more additional insecticides.

16. The method according to claim 8, wherein: The location where regulation is desired is on the plant, plant seeds or in the soil.

17. The method according to claim 8, wherein: The pests are selected from flies, aphids, psyllids, rhinoceros beetles, black beetles, fruit flies, seed flies, root beetles, arc beetles, beetles, aphids, flies and ear beak weevils.

18. The method according to claim 8, wherein: The pests are larvae of the family Scarabaeidae.

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