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.

What is claimed is: 
     
         1 . A compound that (a) has pesticidal activity; (b) has a molecular weight of about 840-900 as determined by Liquid Chromatography/Mass Spectroscopy (LC/MS) and (c) has an High Pressure Liquid Chromatography (HPLC) retention time of about 7-12 minutes on a reversed phase C-18 HPLC column using a water:acetonitrile (CH 3 CN) gradient solvent system (0-20 min; 90-0% aqueous CH 3 CN, 20-24 min; 100% CH 3 CN, 24-27 min; 0-90% aqueous CH 3 CN, 27-30 min; 90% aqueous CH 3 CN) at 0.5 mL/min flow rate and UV detection of 210 nm and (d) is optionally obtainable from a  Chromobacterium  species. 
     
     
         2 . The compound according to  claim 1 , wherein said compound has an HPLC retention time of about 9 minutes on said reversed phase C-18 reversed phase HPLC column. 
     
     
         3 . The compound according to  claim 1 , wherein said compound has 43 carbons, seven methyl, ten methylene carbons, twelve methines, 6 olefinic methines, eight quaternary carbons as determined by  13 C NMR. 
     
     
         4 . The compound according to  claim 1 - 3 , wherein said compound (i) has a molecular weight of about 840-890 as determined by Liquid Chromatography/Mass Spectroscopy (LC/MS); (ii) has  1 H NMR values of about δ 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 (iii) has  13 C NMR values of about δ 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. 
     
     
         5 . The compound according to  claim 4 , wherein said compound has at least one of: (a) an HPLC retention time of about 9.08 minutes on said C-18 reversed phase HPLC column and (b) a molecular weight of about 860 as determined by Liquid Chromatograph/Mass Spectroscopy (LC/MS). 
     
     
         6 . The compound according to  claim 1 , wherein the compound is selected from the group consisting of
 (A) a compound having the structure ##STR001##   
       
         
           
           
               
               
           
         
       
       or a pesticidally acceptable salt or steriosomers thereof, wherein R is —H, lower chain alkyl containing 1, 2, 3, 4, 5, 6, 7, 8 or 9 alkyl moieties, aryl or arylalkyl moiety, substituted lower alkyl; n is 0, 1, 2, 3, 4, 5, 6, 7, 8 or 9; X is O, NH, NR or S; R1, R2, R3, R4, R5, R6, R7, R8, R9, R10 are each independently H, are the same or different and independently an amino acid side-chain moiety or an amino acid side-chain derivative, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, substituted heterocyclic, cycloalkyl, substituted cycloalkyl, alkoxy, substituted alkoxy, thioalkyl, substituted thioalkyl, hydroxy, halogen, amino, amido, carboxyl, —C(O)H, acyl, oxyacyl, carbamate, sulfonyl, sulfonamide, or sulfuryl;
 (B) a compound having the structure ##STR001a## 
 
       
         
           
           
               
               
           
         
       
       wherein R is —H, lower chain alkyl containing 1, 2, 3, 4, 5, 6, 7, 8 or 9 alkyl moieties, aryl or arylalkyl moiety, substituted lower alkyl; X is O, NH, NR or S; R2a, R2b are independently selected from the group consisting of —H, alkyl, lower-alkyl, substituted alkyl and substituted lower-alkyl; R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11  are each independently H, are the same or different and independently an amino acid side-chain moiety or an amino acid side-chain derivative, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, substituted heterocyclic, cycloalkyl, substituted cycloalkyl, alkoxy, substituted alkoxy, thioalkyl, substituted thioalkyl, hydroxy, halogen, amino, amido, carboxyl, —C(O)H, acyl, oxyacyl, carbamate, sulfonyl, sulfonamide, or sulfuryl;
 (C) a compound having the structure ##STR001b## 
 
       
         
           
           
               
               
           
         
       
       wherein R is —H, lower chain alkyl, aryl or aryl alkyl moiety, substituted lower alkyl; X is O, NH, NR or S; n is 0, 1, 2, 3, 4, 5, 6, 7, 8 or 9; R2a, R2b are independently selected from the group consisting of —H, alkyl, lower-alkyl, substituted alkyl and substituted lower-alkyl; R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11  are each independently H, are the same or different and independently an amino acid side-chain moiety or an amino acid side-chain derivative, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, substituted heterocyclic, cycloalkyl, substituted cycloalkyl, alkoxy, substituted alkoxy, thioalkyl, substituted thioalkyl, hydroxy, halogen, amino, amido, carboxyl, —C(O)H, acyl, oxyacyl, carbamate, sulfonyl, sulfonamide, or sulfuryl;
 (D) a compound having the structure ##STR001c## 
 
       
         
           
           
               
               
           
         
       
       wherein R is —H, lower chain alkyl, aryl or aryl alkyl moiety, substituted lower alkyl containing 1, 2, 3, 4, 5, 6, 7, 8 or 9 alkyl moieties; X is O, NH, NR or S; n is 0, 1, 2, 3, 4, 5, 6, 7, 8 or 9; R2a, R2b are independently selected from the group consisting of —H, alkyl, lower-alkyl, substituted alkyl and substituted lower-alkyl; R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11  are each independently H, are the same or different and independently an amino acid side-chain moiety or an amino acid side-chain derivative, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, substituted heterocyclic, cycloalkyl, substituted cycloalkyl, alkoxy, substituted alkoxy, thioalkyl, substituted thioalkyl, hydroxy, halogen, amino, amido, carboxyl, —C(O)H, acyl, oxyacyl, carbamate, sulfonyl, sulfonamide, or sulfuryl. 
     
     
         7 . The compound according to  claim 1 - 2 , wherein said compound has at least one of (a) an HPLC retention time of about 9.54 minutes on said C-18 reversed phase HPLC column and (b) a molecular weight of about 874 as determined by Liquid Chromatograph/Mass Spectroscopy (LC/MS). 
     
     
         8 . A composition comprising (a) the compound of  claims 1 ; (b) a pesticidally acceptable carrier and (c) optionally a second substance, wherein said second substance is a chemical or biological pesticide and/or at least one of a carrier, diluent, surfactant, or adjuvant. 
     
     
         9 . A method for obtaining the compound of  claim 1  comprising:
 (a) culturing a  Chromobacterium  strain in a culture medium under conditions sufficient to produce said compound to obtain a  Chromobacterium  culture; 
 (b) isolating said compound produced in (a) from the whole cell broth of (a). 
 
     
     
         10 . A method for obtaining a compound selected from the group consisting of (i) the compound of  claims 1  and (ii) a compound having the following characteristics: (a) has pesticidal properties; (b) has a molecular weight of about 315-360 as determined by Liquid Chromatography/Mass Spectroscopy (LC/MS) and (c) has an High Pressure Liquid Chromatography (HPLC) retention time of about 8-15 minutes on a reversed phase C-18 HPLC column using a water:acetonitrile (CH 3 CN) with a gradient solvent system (0-20 min; 90-0% aqueous CH 3 CN, 20-24 min; 100% CH 3 CN, 24-27 min; 0-90% aqueous CH 3 CN, 27-30 min; 90% aqueous CH 3 CN) at 0.5 mL/min flow rate and UV detection of 210 nm comprising: (A) culturing a  Chromobacterium substugae  Nov strain in a culture medium under conditions sufficient to produce said compound to obtain a  Chromobacterium sutbsugae  Nov culture; (B) isolating said compound produced in (A) from the whole cell broth of (A). 
     
     
         11 . The method according to  claims 9 - 10 , wherein said compound produced in step (A) is isolated by (a) applying the whole cell broth to at least one of an ion exchange column, a size exclusion column or a reversed phase HPLC column to obtain column fractions; (b) assaying the column fractions for pesticidal activity and (c) concentrating column fractions of (b) to obtain isolated compound. 
     
     
         12 . A method for modulating pest infestation in a plant comprising applying to the plant and/or seeds thereof and/or substrate used for growing said plant an amount of (A) at least one of: (i) the compound of  claims 1 ; (ii) a compound having the following characteristics: (a) has pesticidal properties; (b) has a molecular weight of about 315-360 as determined by Liquid Chromatography/Mass Spectroscopy (LC/MS); (c) has an High Pressure Liquid Chromatography (HPLC) retention time of about 8-15 minutes on a reversed phase C-18 HPLC column using a water:acetonitrile (CH 3 CN) with a gradient solvent system (0-20 min; 90-0% aqueous CH 3 CN, 20-24 min; 100% CH 3 CN, 24-27 min; 0-90% aqueous CH 3 CN, 27-30 min; 90% aqueous CH 3 CN) at 0.5 mL/min flow rate and UV detection of 210 nm and (d) is optionally obtainable from a  Chromobacterium  species and (B) optionally another pesticidal substance effective to modulate said pest infestation. 
     
     
         13 . The method according to  claims 9 - 12 , wherein said compound used in said method has at least one of: (a) a molecular weight of about 343 as determined by Liquid Chromatography/Mass Spectroscopy (LC/MS) and (b) an HPLC retention of about 10 minutes on said C-18 reversed phase HPLC column. 
     
     
         14 . The method according to  claims 9 - 12 , wherein said compound used in said method has at least one of: (a) a molecular weight of about 327 as determined by Liquid Chromatography/Mass Spectroscopy (LC/MS) and (b) an HPLC retention of about 12 minutes on said C-18 reversed phase HPLC column. 
     
     
         15 . The method according to  claims 9 - 14 , wherein said compound is selected from the group consisting of chromamide A, violacein and deoxyviolacein. 
     
     
         16 . A method for modulating nematode infestation in a plant comprising applying to the plant and/or seeds thereof and/or substrate used for growing said plant an amount of a supernatant, filtrate and/ or extract and/or one or more metabolites of said supernatant, filtrate and/or extract of a strain of  Chromobacterium  sp. and optionally another nematocidal substance in an amount effective to modulate said nematode infestation. 
     
     
         17 . A pesticidal combination synergistic to at least one pest comprising as active components: (a) a supernatant, filtrate and/ or extract of Chromobacterium sp. and (b) another pesticidal substance, wherein (a) and (b) are present in synergistic amounts. 
     
     
         18 . The combination according to  claim 17 , wherein said pest is an insect or nematode pest. 
     
     
         19 . The combination according to  claim 17 , wherein said pesticidal substance is (a) derived from a microorganism; (b) a natural product or (b) a chemical compound. 
     
     
         20 . The combination according to  claim 17 , wherein said pesticidal substance is derived from a microorganism and is selected from the group consisting of  Bacillus  sp. and spinosad. 
     
     
         21 . The combination according to  claim 17 , wherein said pesticidal substance is microorganism is  Bacillus thuringiensis.    
     
     
         22 . The combination according to  claim 17 , wherein said microorganism is  Bacillus thuringiensis kurstaki.  23 The combination according to  claim 17 , wherein said pesticidal substance is a chemical insecticide selected from the group consisting of pyrethrins, spirotetramet and organochlorines. 
     
     
         24 . The combination according to  claim 17 , wherein said pesticidal substance is a natural product and said natural product is pyrethram. 
     
     
         25 . The combination according to  claim 17 , wherein said combination is a composition. 
     
     
         26 . A method for synergistically modulating infestation of at least one pest strain in a plant comprising applying to the plant and/or seeds thereof and/or substrate used for growing said plant an amount of a combination of  claim 17  effective to synergistically modulate infestation of at least one pest in a plant. 
     
     
         27 . A composition comprising (A) a compound having the following characteristics: (a) has pesticidal properties; (b) has a molecular weight of about 315-360 as determined by Liquid Chromatography/Mass Spectroscopy (LC/MS); (c) has an High Pressure Liquid Chromatography (HPLC) retention time of about 8-15 minutes on a reversed phase C-18 HPLC column using a water:acetonitrile (CH 3 CN) with a gradient solvent system (0-20 min; 90-0% aqueous CH 3 CN, 20-24 min; 100% CH 3 CN, 24-27 min; 0-90% aqueous CH 3 CN, 27-30 min; 90% aqueous CH 3 CN) at 0.5 mL/min flow rate and UV detection of 210 nm comprising and (d) is optionally obtainable from a  Chromobacterium  species and (B) at least one of (i) a second substance, wherein said second substance is a chemical or biological pesticide and (ii) at least one of a carrier, diluent, surfactant, or adjuvant, wherein said composition is optionally a pesticidal composition. 
     
     
         28 . Use of a compound selected from the group consisting of (i) the compound of  claims 1 - 7 ; (ii) a compound having the following characteristics: (a) has pesticidal properties; (b) has a molecular weight of about 315-360 as determined by Liquid Chromatography/Mass Spectroscopy (LC/MS); (c) has an High Pressure Liquid Chromatography (HPLC) retention time of about 8-15 minutes on a reversed phase C-18 HPLC column using a water:acetonitrile (CH 3 CN) with a gradient solvent system (0-20 min; 90-0% aqueous CH 3 CN, 20-24 min; 100% CH 3 CN, 24-27 min; 0-90% aqueous CH 3 CN, 27-30 min; 90% aqueous CH 3 CN) at 0.5 mL/min flow rate and UV detection of 210 nm comprising and (d) is optionally obtainable from a  Chromobacterium  species for formulating a composition for modulating pest infestation in a plant.