Non-obligate predator bacterium burkholderia casidae and uses thereof

Abstract

A novel predator bacterium Burkholderia casidae is disclosed. The invention is directed to the isolation and use of Burkholderia casidae to control microbial diseases of plants. The genetic, biochemical and physiological characteristics of Burkholderia casidae are described. Biocontrol compositions comprising Burkholderia casidae , and antimicrobial compounds and antimicrobial preparations prepared from Burkholderia casidae are also disclosed, as are methods for accomplishing all of the foregoing.

What is claimed is:  
     
         1 . A substantially pure culture or suspension of a bacterium  Burkholderia casidae  or variant thereof, which  Burkholderia casidae  or variant exhibits biocontrol activity against a microorganism, and has 
 a) a 16S rRNA gene comprising a sequence that is at least 97% similar to the sequence of SEQ ID NO:1 as determined by Clustal Analysis; and    b) a cellular fatty acid composition comprising about 16% to about 20% C16:0 fatty acid, about 18% to about 22% C16:1 fatty acid, and about 35% to about 45% C18:1 (11,12) fatty acid;    wherein the microorganism is a bacterium, yeast, filamentous fungi, protozoan or algae.    
     
     
         2 . The substantially pure culture or suspension of  claim 1 , which bacterium or variant thereof has a 16S rRNA gene comprising a sequence identical to that of SEQ ID NO:1; and a cellular fatty acid composition comprising about 18% C16:0 fatty acid, about 21% C16:1 fatty acid and about 39% C18:1 (11,12).  
     
     
         3 . A substantially pure culture or suspension of  Burkholderia casidae  strain 2.2N having the accession number ATCC 55961, or a variant thereof.  
     
     
         4 . The substantially pure culture or suspension of  claim 1 ,  2  or  3 , wherein the culture or suspension comprises at least 80% cysts.  
     
     
         5 . The substantially pure culture or suspension of  claim 1 ,  2  or  3 , wherein the culture or suspension comprises at least 80% cells.  
     
     
         6 . The substantially pure culture or suspension of  claim 1 ,  2  or  3 , wherein the  Burkholderia casidae  or variant thereof has been inactivated.  
     
     
         7 . The substantially pure culture or suspension of  claim 6 , wherein the  Burkholderia casidae  or variant thereof has been inactivated by treating with heat or alcohol.  
     
     
         8 . A cell-free filtrate or cell fraction prepared from the substantially pure culture or suspension of  claim 1 ,  2  or  3 .  
     
     
         9 . A cell-free filtrate or cell fraction prepared from the substantially pure culture or suspension of  claim 6 .  
     
     
         10 . An antimicrobial preparation comprising an alcohol-extract of a cell, cyst, culture, suspension, cell-free filtrate or cell fraction of a bacterium  Burkholderia casidae  or variant thereof, which  Burkholderia casidae  or variant exhibits biocontrol activity against a microorganism, and has 
 a) a 16S rRNA gene comprising a sequence that is at least 97% similar to the sequence of SEQ ID NO:1 as determined by Clustal Analysis; and    b) a cellular fatty acid composition comprising about 16% to about 20% C16:0 fatty acid, about 18% to about 22% C16:1 fatty acid, and about 35% to about 45% C18:1 (11,12) fatty acid;    wherein the microorganism is a bacterium, yeast, filamentous fungi, protozoan or algae.    
     
     
         11 . The antimicrobial preparation of  claim 10 , wherein the bacterium or variant thereof has a 16S rRNA gene comprising a sequence identical to that of SEQ ID NO:1; and a cellular fatty acid composition comprising about 18% C16:0 fatty acid, about 21% C16:1 fatty acid, and about 39% C18:1 (11,12).  
     
     
         12 . The antimicrobial preparation of  claim 11 , wherein the bacterium is  Burkholderia casidae  strain 2.2N having the accession number ATCC 55961, or a variant thereof.  
     
     
         13 . The antimicrobial preparation of  claim 10 ,  11  or  12 , wherein the cell, cyst, culture, suspension, cell-free filtrate or cell fraction of  Burkholderia casidae  or variant has been inactivated of any live bacterium.  
     
     
         14 . The antimicrobial preparation of  claim 10 ,  11  or  12 , wherein the alcohol-extract is prepared according to a method comprising: 
 a) boiling an alcoholic mixture comprising the cell, culture, suspension, cell-free filtrate or cell fraction and an alcohol;  
 b) clarifying the boiled mixture;  
 c) mixing the boiled mixture with magnesium silicate;  
 d) collecting the magnesium silicate;  
 e) washing the magnesium silicate with water; and  
 f) eluting antifungal compounds from the magnesium silicate with an alcoholic solution, thereby producing the alcohol-extract.  
 
     
     
         15 . A biocontrol composition comprising the substantially pure culture or suspension of  claim 1 ,  2  or  3 .  
     
     
         16 . A biocontrol composition comprising the substantially pure culture or suspension of  claim 4 .  
     
     
         17 . A biocontrol composition comprising the substantially pure culture or suspension of  claim 5 .  
     
     
         18 . A biocontrol composition comprising the substantially pure culture or suspension of  claim 6 .  
     
     
         19 . A biocontrol composition comprising the substantially pure culture or suspension of  claim 7 .  
     
     
         20 . A biocontrol composition comprising sprayed-dried or freeze-dried cells of the substantially pure culture or suspension of  claim 1 ,  2  or  3 .  
     
     
         21 . A biocontrol composition comprising sprayed-dried or freeze-dried cells of the substantially pure culture or suspension of  claim 4 .  
     
     
         22 . A biocontrol composition comprising sprayed-dried or freeze-dried cells of the substantially pure culture or suspension of  claim 5 .  
     
     
         23 . A biocontrol composition comprising sprayed-dried or freeze-dried cells of the substantially pure culture or suspension of  claim 6 .  
     
     
         24 . A biocontrol composition comprising sprayed-dried or freeze-dried cells of the substantially pure culture or suspension of  claim 7 .  
     
     
         25 . A biocontrol composition comprising the cell-free filtrate or cell fraction of  claim 8 .  
     
     
         26 . A biocontrol composition comprising the cell-free filtrate or cell fraction of  claim 9 .  
     
     
         27 . A biocontrol composition comprising the antimicrobial preparation of  claim 10 .  
     
     
         28 . A biocontrol composition comprising the antimicrobial preparation of  claim 11 .  
     
     
         29 . A biocontrol composition comprising the antimicrobial preparation of  claim 12 .  
     
     
         30 . A biocontrol composition comprising the antimicrobial preparation of  claim 13 .  
     
     
         31 . A biocontrol composition comprising the antimicrobial preparation of  claim 14 .  
     
     
         32 . A method for treating or preventing a disease of a plant, comprising applying an effective amount of the biocontrol composition of  claim 15  to the plant.  
     
     
         33 . A method for treating or preventing a disease of a plant, comprising applying an effective amount of the biocontrol composition of  claim 18  to the plant.  
     
     
         34 . A method for treating or preventing a disease of a plant, comprising applying an effective amount of the biocontrol composition of  claim 20  to the plant.  
     
     
         35 . A method for treating or preventing a disease of a plant, comprising applying an effective amount of the biocontrol composition of  claim 23  to the plant.  
     
     
         36 . A method for treating or preventing a disease of a plant, comprising applying an effective amount of the biocontrol composition of  claim 25  to the plant.  
     
     
         37 . A method for treating or preventing a disease of a plant, comprising applying an effective amount of the biocontrol composition of  claim 26  to the plant.  
     
     
         38 . A method for treating or preventing a disease of a plant, comprising applying an effective amount of the biocontrol composition of  claim 27  to the plant.  
     
     
         39 . A method for treating or preventing a disease of a plant, comprising applying an effective amount of the biocontrol composition of  claim 28  to the plant.  
     
     
         40 . A method for treating or preventing a disease of a plant, comprising applying an effective amount of the biocontrol composition of  claim 29  to the plant.  
     
     
         41 . A method for treating or preventing a disease of a plant, comprising applying an effective amount of the biocontrol composition of  claim 30  to the plant.  
     
     
         42 . A method for treating or preventing a disease of a plant, comprising applying an effective amount of the biocontrol composition of  claim 31  to the plant.  
     
     
         43 . The method according to  claim 32 , wherein the microorganism is a species of a genus selected from the group consisting of Alternaria, Aspergillus, Botrytis, Cercospora, Cercosporidium, Geotrichum, Mycosphaerella, Mucor, Penicillium, Phoma, Phytophthora, Plasmopora, Pseudopeziza, Puccinia, Pythium, Rhizoctonia, Rhizopus, Saccharomyces, Septoria, Sporothrix, Stemphylium, Trichophyton, Verticillium, Erwinia, Pseudomonas and Xanthomonas.  
     
     
         44 . The method according to  claim 33 , wherein the microorganism is a species of a genus selected from the group consisting of Alternaria, Aspergillus, Botrytis, Cercospora, Cercosporidium, Geotrichum, Mycosphaerella, Mucor, Penicillium, Phoma, Phytophthora, Plasmopora, Pseudopeziza, Puccinia, Pythium, Rhizoctonia, Rhizopus, Saccharomyces, Septoria, Sporothrix, Stemphylium, Trichophyton, Verticillium, Erwinia, Pseudomonas and Xanthomonas.  
     
     
         45 . The method according to  claim 34 , wherein the microorganism is a species of a genus selected from the group consisting of Alternaria, Aspergillus, Botrytis, Cercospora, Cercosporidium, Geotrichum, Mycosphaerella, Mucor, Penicillium, Phoma, Phytophthora, Plasmopora, Pseudopeziza, Puccinia, Pythium, Rhizoctonia, Rhizopus, Saccharomyces, Septoria, Sporothrix, Stemphylium, Trichophyton, Verticillium, Erwinia, Pseudomonas and Xanthomonas.  
     
     
         46 . The method according to  claim 35 , wherein the microorganism is a species of a genus selected from the group consisting of Alternaria, Aspergillus, Botrytis, Cercospora, Cercosporidium, Geotrichum, Mycosphaerella, Mucor, Penicillium, Phoma, Phytophthora, Plasmopora, Pseudopeziza, Puccinia, Pythium, Rhizoctonia, Rhizopus, Saccharomyces, Septoria, Sporothrix, Stemphylium, Trichophyton, Verticillium, Erwinia, Pseudomonas and Xanthomonas.  
     
     
         47 . The method according to  claim 36 , wherein the microorganism is a species of a genus selected from the group consisting of Alternaria, Aspergillus, Botrytis, Cercospora, Cercosporidium, Geotrichum, Mycosphaerella, Mucor, Penicillium, Phoma, Phytophthora, Plasmopora, Pseudopeziza, Puccinia, Pythium, Rhizoctonia, Rhizopus, Saccharomyces, Septoria, Sporothrix, Stemphylium, Trichophyton, Verticillium, Erwinia, Pseudomonas and Xanthomonas.  
     
     
         48 . The method according to  claim 37 , wherein the microorganism is a species of a genus selected from the group consisting of Alternaria, Aspergillus, Botrytis, Cercospora, Cercosporidium, Geotrichum, Mycosphaerella, Mucor, Penicillium, Phoma, Phytophthora, Plasmopora, Pseudopeziza, Puccinia, Pythium, Rhizoctonia, Rhizopus, Saccharomyces, Septoria, Sporothrix, stemphylium, Trichophyton, Verticillium, Erwinia, Pseudomonas and Xanthomonas.  
     
     
         49 . The method according to  claim 38 , wherein the microorganism is a species of a genus selected from the group consisting of Alternaria, Aspergillus, Botrytis, Cercospora, Cercosporidium, Geotrichum, Mycosphaerella, Mucor, Penicillium, Phoma, Phytophthora, Plasmopora, Pseudopeziza, Puccinia, Pythium, Rhizoctonia, Rhizopus, Saccharomyces, Septoria, Sporothrix, Stemphylium, Trichophyton, Verticillium, Erwinia, Pseudomonas and Xanthomonas.  
     
     
         50 . The method according to  claim 39 , wherein the microorganism is a species of a genus selected from the group consisting of Alternaria, Aspergillus, Botrytis, Cercospora, Cercosporidium, Geotrichum, Mycosphaerella, Mucor, Penicillium, Phoma, Phytophthora, Plasmopora, Pseudopeziza, Puccinia, Pythium, Rhizoctonia, Rhizopus, Saccharomyces, Septoria, Sporothrix, Stemphylium, Trichophyton, Verticillium, Erwinia, Pseudomonas and Xanthomonas.  
     
     
         51 . The method according to  claim 40 , wherein the microorganism is a species of a genus selected from the group consisting of Alternaria, Aspergillus, Botrytis, Cercospora, Cercosporidium, Geotrichum, Mycosphaerella, Mucor, Penicillium, Phoma, Phytophthora, Plasmopora, Pseudopeziza, Puccinia, Pythium, Rhizoctonia, Rhizopus, Saccharomyces, Septoria, Sporothrix, Stemphylium, Trichophyton, Verticillium, Erwinia, Pseudomonas and Xanthomonas.  
     
     
         52 . The method according to  claim 41 , wherein the microorganism is a species of a genus selected from the group consisting of Alternaria, Aspergillus, Botrytis, Cercospora, Cercosporidium, Geotrichum, Mycosphaerella, Mucor, Penicillium, Phoma, Phytophthora, Plasmopora, Pseudopeziza, Puccinia, Pythium, Rhizoctonia, Rhizopus, Saccharomyces, Septoria, Sporothrix, stemphylium, Trichophyton, Verticillium, Erwinia, Pseudomonas and Xanthomonas.  
     
     
         53 . The method according to  claim 42 , wherein the microorganism is a species of a genus selected from the group consisting of Alternaria, Aspergillus, Botrytis, Cercospora, Cercosporidium, Geotrichum, Mycosphaerella, Mucor, Penicillium, Phoma, Phytophthora, Plasmopora, Pseudopeziza, Puccinia, Pythium, Rhizoctonia, Rhizopus, Saccharomyces, Septoria, Sporothrix, Stemphylium, Trichophyton, Verticillium, Erwinia, Pseudomonas and Xanthomonas.  
     
     
         54 . The method according to  claim 32 , wherein the plant is selected from the group consisting of safflower, cotton, flax, oats, canola, poinsettia, chrysanthemum, corn, soybean, wheat, rice, alfalfa, sorghum, peanut, tobacco, tomato, pepper, cucumber, lettuce, green bean, lima beans, peas, cantaloupe, musk melon, citrus fruit, grape, banana, geranium, azalea, rose, tulip, petunia, orchid, carnation, pine, yew and spruce.  
     
     
         55 . The method according to  claim 33 , wherein the plant is selected from the group consisting of safflower, cotton, flax, oats, canola, poinsettia, chrysanthemum, corn, soybean, wheat, rice, alfalfa, sorghum, peanut, tobacco, tomato, pepper, cucumber, lettuce, green bean, lima beans, peas, cantaloupe, musk melon, citrus fruit, grape, banana, geranium, azalea, rose, tulip, petunia, orchid, carnation, pine, yew and spruce.  
     
     
         56 . The method according to  claim 34 , wherein the plant is selected from the group consisting of safflower, cotton, flax, oats, canola, poinsettia, chrysanthemum, corn, soybean, wheat, rice, alfalfa, sorghum, peanut, tobacco, tomato, pepper, cucumber, lettuce, green bean, lima beans, peas, cantaloupe, musk melon, citrus fruit, grape, banana, geranium, azalea, rose, tulip, petunia, orchid, carnation, pine, yew and spruce.  
     
     
         57 . The method according to  claim 35 , wherein the plant is selected from the group consisting of safflower, cotton, flax, oats, canola, poinsettia, chrysanthemum, corn, soybean, wheat, rice, alfalfa, sorghum, peanut, tobacco, tomato, pepper, cucumber, lettuce, green bean, lima beans, peas, cantaloupe, musk melon, citrus fruit, grape, banana, geranium, azalea, rose, tulip, petunia, orchid, carnation, pine, yew and spruce.  
     
     
         58 . The method according to  claim 36 , wherein the plant is selected from the group consisting of safflower, cotton, flax, oats, canola, poinsettia, chrysanthemum, corn, soybean, wheat, rice, alfalfa, sorghum, peanut, tobacco, tomato, pepper, cucumber, lettuce, green bean, lima beans, peas, cantaloupe, musk melon, citrus fruit, grape, banana, geranium, azalea, rose, tulip, petunia, orchid, carnation, pine, yew and spruce.  
     
     
         59 . The method according to  claim 37 , wherein the plant is selected from the group consisting of safflower, cotton, flax, oats, canola, poinsettia, chrysanthemum, corn, soybean, wheat, rice, alfalfa, sorghum, peanut, tobacco, tomato, pepper, cucumber, lettuce, green bean, lima beans, peas, cantaloupe, musk melon, citrus fruit, grape, banana, geranium, azalea, rose, tulip, petunia, orchid, carnation, pine, yew and spruce.  
     
     
         60 . The method according to  claim 38 , wherein the plant is selected from the group consisting of safflower, cotton, flax, oats, canola, poinsettia, chrysanthemum, corn, soybean, wheat, rice, alfalfa, sorghum, peanut, tobacco, tomato, pepper, cucumber, lettuce, green bean, lima beans, peas, cantaloupe, musk melon, citrus fruit, grape, banana, geranium, azalea, rose, tulip, petunia, orchid, carnation, pine, yew and spruce.  
     
     
         61 . The method according to  claim 39 , wherein the plant is selected from the group consisting of safflower, cotton, flax, oats, canola, poinsettia, chrysanthemum, corn, soybean, wheat, rice, alfalfa, sorghum, peanut, tobacco, tomato, pepper, cucumber, lettuce, green bean, lima beans, peas, cantaloupe, musk melon, citrus fruit, grape, banana, geranium, azalea, rose, tulip, petunia, orchid, carnation, pine, yew and spruce.  
     
     
         62 . The method according to  claim 40 , wherein the plant is selected from the group consisting of safflower, cotton, flax, oats, canola, poinsettia, chrysanthemum, corn, soybean, wheat, rice, alfalfa, sorghum, peanut, tobacco, tomato, pepper, cucumber, lettuce, green bean, lima beans, peas, cantaloupe, musk melon, citrus fruit, grape, banana, geranium, azalea, rose, tulip, petunia, orchid, carnation, pine, yew and spruce.  
     
     
         63 . The method according to  claim 41 , wherein the plant is selected from the group consisting of safflower, cotton, flax, oats, canola, poinsettia, chrysanthemum, corn, soybean, wheat, rice, alfalfa, sorghum, peanut, tobacco, tomato, pepper, cucumber, lettuce, green bean, lima beans, peas, cantaloupe, musk melon, citrus fruit, grape, banana, geranium, azalea, rose, tulip, petunia, orchid, carnation, pine, yew and spruce.  
     
     
         64 . The method according to  claim 42 , wherein the plant is selected from the group consisting of safflower, cotton, flax, oats, canola, poinsettia, chrysanthemum, corn, soybean, wheat, rice, alfalfa, sorghum, peanut, tobacco, tomato, pepper, cucumber, lettuce, green bean, lima beans, peas, cantaloupe, musk melon, citrus fruit, grape, banana, geranium, azalea, rose, tulip, petunia, orchid, carnation, pine, yew and spruce.  
     
     
         65 . A method for producing an antimicrobial preparation from  Burkholderia casidae  or variant thereof, comprising: 
 a) boiling an alcoholic mixture comprising a cell, culture, suspension, cell-free filtrate or cell fraction of  Burkholderia casidae  or variant thereof, and an alcohol;    b) clarifying the boiled mixture;    c) mixing the boiled mixture with magnesium silicate;    d) collecting the magnesium silicate;    e) washing the magnesium silicate with water; and    f) eluting antifungal compounds from the magnesium silicate with an alcoholic solution, thereby producing the antimicrobial preparation.    
     
     
         66 . A method for isolating a Burkholderia casidae or variant thereof, which comprises 
 (a) determining the 16S rRNA sequence and the cellular fatty acid composition of a predator bacterium which exhibits biocontrol activity against a microorganism; and    (b) identifying a bacterium having (i) a 16S rRNA gene comprising a sequence that is at least 97% similar to the sequence of SEQ ID NO:1 as determined by Clustal Analysis, and (ii) a cellular fatty acid composition comprising about 16% to about 20% C16:0 fatty acid, about 18% to about 22% C16:1 fatty acid, and about 35% to about 45% C 18:1 (11,12) fatty acid;    thereby isolating the  Burkholderia casidae  or variant thereof.    
     
     
         67 . The method of  claim 66 , wherein the bacterium has (i) a 16S rRNA gene comprising a sequence that is identical to the sequence of SEQ ID NO:1, and (ii) a cellular fatty acid composition comprising about 18% C16:0 fatty acid, about 21% C16:1 fatty acid and about 39% C18:1 (11,12).