Patent Publication Number: US-2020296972-A1

Title: Composition and method for reducing fungal infections in crops

Description:
CROSS REFERENCE TO RELATED APPLICATIONS 
     This application is a continuation of U.S. patent application Ser. No. 15/886,124, filed on Feb. 1, 2018, which is incorporated herein by reference in its entirety. 
    
    
     FIELD OF THE DISCLOSURE 
     This disclosure relates to methods and compositions for reducing or eliminating fungal infections of growing and/or harvested crops using non-pathogenic bacteria. 
     BACKGROUND OF THE DISCLOSURE 
     Members of the species  Bacillus  have been reported to be useful for preventing infections and/or promoting the growth of crops. For example,  Bacillus pumilus  strain QST2808,  Bacillus pumilus  strain GB34,  Bacillus Subtilis  strain QST713,  Bacillus Subtilis  strain GB03, various strains of  Bacillus thuringiensis  and  Bacillus firmus,  and  Bacillus amyloliquefaciens  strain FZB42 have been used in commercially available biocontrol products. 
     While the known natural, non-pathogenic, biological fungicides have achieved some commercial success, there remains a need for safe and highly effective biological fungicides. 
     SUMMARY OF THE DISCLOSURE 
     This disclosure relates to a safe (non-toxic, non-pathogenic) biological fungicide composition containing a protein source inoculated with from 5×10 7  cfu to 5×10 9  cfu of a  Bacillus amyloliquefaciens  strain per gram of the protein source. The combination of a protein source and a biocidal strain of a  Bacillus amyloliquefaciens  has been found to be more effective at reducing and/or inhibiting fungal infections in crops than either component alone, providing an unexpected and synergistic improvement. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1  is a bar chart showing the inhibitory effect of fermentation medium effluent from different bacterial strains on  Fusarium oxysporum  conidia germination. 
         FIG. 2  is a bar chart showing the inhibitory effect of fermentation medium effluent from different bacterial strains on  Fusarium oxysporum  conidia numbers. 
         FIG. 3  is a bar chart showing the inhibitory effect of adding a composition containing a protein source inoculated with a strain of  Bacillus amyloliquefaciens  to a soil in which a plant is grown. 
         FIG. 4  is a bar chart showing that the addition of the  Bacillus amyloliquefaciens  strain alone, without the protein source, to soil in which a plant is grown does not effectively inhibit  Fusarium oxysporum.    
         FIG. 5  is a bar chart showing that soybean flour inoculated with a strain of  Bacillus amyloliquefaciens  is much more effective than soybean flour alone at inhibiting  Fusarium oxysporum  in soil. 
     
    
    
     DETAILED DESCRIPTION OF THE DISCLOSED EMBODIMENTS 
     Based on experiments in which various  Bacillus  species were screened for their ability to promote growth of basil plants treated with a protein source selected from cotton seed meal, soybean flour and hydrolyzed feather meal, it was determined that the growth response of cotton seed meal and soybean flour was very poor regardless of the species of  Bacillus  and the amount of  Bacillus  used. It was also determined that the growth response to hydrolyzed feather meal was good regardless of the amount or species of  Bacillus  used. This suggested to the inventors that the soybean flour and cotton seed meal was being consumed by bacteria and incorporated in the mass of the bacteria. The bacteria responsible for metabolizing the protein sources could be native bacteria, added bacteria (the protein source inoculant), or a combination of both inoculant and native bacteria. This suggested to the inventors that certain protein sources, such as soybean flour and cotton seed meal, could be used to supplement the growth of a biocontrol organism, such as a fungicidal bacteria. 
     Based on separate experiments in which various  Bacillus  strains were screened for their ability to inhibit germination of conidia in water and their ability to lyse conidia in water, it was determined that the effluent from tryptic soy broth (TSB) media used for cultivating the various  Bacillus  strains tested typically contained secreted substances capable of inhibiting germination of conidia from  Fusarium oxysporum.  However, as shown in  FIG. 1 , there were exceptions, such as  B. pumilus  strain BP, which did not appreciably inhibit  Fusarium oxysporum.  Also, as shown in  FIG. 1 ,  B. amyloliquefaciens  strains OBT 730 (deposited as ATCC Accession No. PTA-124660), and OBT 712 (deposited as ATCC Accession No. PTA-122189) were particularly effective at inhibiting  Fusarium  conidia germination. 
       FIG. 2  shows that the ability of effluents from strains OBT 730 and OBT 712 to lyse  Fusarium oxysporum  conidia were particularly good, with over 70% of the conidia disappearing. Among the remaining conidia, percent germination was much lower for the effluents from strains OBT 730 and OBT 712 than observed when conidia were treated with effluent from other cultures. 
     Strain OBT 712 is a stock strain sold in most  Bacillus -based products sold by Osprey Biotechnics, Sarasota, Fla., and strain OBT 730 was isolated by Osprey Biotechnics from a soil sample. 
     Strain OBT 712 was selected for further analysis. Specifically, experiments were conducted to determine the ability of strain OBT 712 to reduce  Fusarium  conidia concentrations in soil, both with and without soybean flour. Soybean flour was inoculated with 5×10 8  cfu  B. amyloliquefaciens  strain OBT 712 per gram of soybean flour. The inoculated soybean flour was added to soil in amounts of zero, 0.33%, 1% and 5%, and  Fusarium oxysporum  conidia was added at 5×10 5  conidia per gram of soil. The addition of  B. amyloliquefaciens -inoculated soybean flour very substantially reduced  Fusarium  that could be recovered from the soil, as shown in  FIG. 3 . 
     The inventors conducted several other studies to determine whether the OBT 712 strain could reduce  Fusarium  counts in the absence of soy flour and whether the effect on  Fusarium  was due to soy flour alone or if the bacteria was required. One percent soy flour inoculated with 5×10 8  cfu strain OBT 712 per gram of soybean flour would deliver 5×10 6  cfu strain OBT 712 per gram of soil. Neither this concentration nor 5×10 5  cfu strain OBT 712 per gram of soil appeared to appreciably affect  Fusarium  ( FIG. 4 ). However, when compared to soy flour alone, soy flour plus strain OBT 712 reduced  Fusarium  recovery by approximately 80% ( FIG. 5 ). 
     The inventors have concluded from the above experiments that a soil amendment comprising soy flour inoculated with  Bacillus amyloliquefaciens  can be used to reduce  Fusarium  infections in crops. Soy flour appears to have an advantage over other nitrogen sources tested. The amount of soy required to observe a beneficial effect on Fusarium recovery is much lower than other organic nitrogen supplies. At 0.5% soy flour, the nitrogen content of the potting soil is approximately 375 ppm. Typically one would want a nitrogen concentration in the 100-200 ppm range, but given that  B. amyloliquefaciens  is growing on the substrate and assimilating nitrogen it is conceivable that free nitrogen would be much lower than 200 ppm and that detrimental effects to the plant would not be observed. 
     Alternatively, fermentation broth in itself could make a suitable anti-fungal product. One could use routine experimentation to determine the percentage of the material that could be used as a fungicide. The secreted products that are likely responsible for the anti-fungal activity of the  B. amyloliquefaciens  strains are lipopeptides. Examples being iturin, surfactin, and fengycin. Genes encoding for enzymes required for the synthesis of these compounds are present in the strain OBT 712 genome. These peptides have been shown to be haemolytic and as a result have not been commercialized. 
     The compositions (inoculated protein sources and effluents from cultures) disclosed herein can be used to inhibit fungal infections in plants by applying the compositions to the plants, to seeds from which the plants are grown, or to soil in which the plants are grown. Soil concentrations generally refer to concentrations within soil that the roots of the plant contact. Application to seeds refers to incorporating the disclosed compositions in seed covering. “Effluents,” refer to filtered liquids obtained from media in which the disclosed bacteria are cultured. 
     The described embodiments are preferred and/or illustrated, but are not limiting. Various modifications are considered within the purview and scope of the appended claims.