INFECTIONS IN CROPS

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U. S. Application No.

1 5/886, 1 24, filed February 1 , 201 8, which is incorporated herein by reference.

FIELD OF THE DISCLOSURE

[0002] 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

[0003] 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 Substilis 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.

[0004] 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

[0005] This disclosure relates to a safe (non-toxic, non-pathogenic) biological fungicide composition containing a protein source inoculated with from 5x10 ⁷ cfu to 5x10 ⁹ cfu of a Bacillus amyloliquefacias strain per gram of the protein source. The combination of a protein source and a biocidal strain of a Bacillus amyloliquefacies 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 DESCRIPTIONOF THE DRAWINGS

[0006] Figure 1 is a bar chart showing the inhibitory effect of fermentation medium effluent from different bacteria strains on Fusarium oxysporum conidia germination.

[0007] Figure 2 is a bar chart showing the inhibitory effect of fermentation medium effluent from different bacteria strains on Fusarium oxysporum conidia numbers.

[0008] Figure 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.

[0009] Figure 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.

[0010] Figure 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

[0011] 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 to cotton seed meal and soybean flour was very poor regardless of the species of Bacillus and 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 fungicidial bacteria.

[00121 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 Figure 1 , there were exceptions, such as B. pumilus strain BP, which did not appreciably inhibit Fusarium oxysporum. Also, as shown in Figure 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.

[0013] Figure 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.

[0014] Strain OBT 712 is a stock strain sold in most Bacillus- based products sold by

Osprey Biotechnics, Sarasota, Florida, and strain OBT 730 was isolated by Osprey Biotechnics from a soil sample.

[0015] 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 5x10 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 5xl0 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 Figure 3.

[0016] 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 5xl 0 ⁸ cfu strain OBT 712 per gram of soybean flour would deliver 5xl 0 ⁶ cfu strain OBT 712 per gram of soil. Neither this concentration nor 5x10 ⁵ cfu strain OBT 712 per gram of soil appeared to appreciably affect Fusarhm (Figure 4). However, when compared to soy flour alone, soy flour plus strain OBT 712 reduced Fusarium recovery by approximately 80% (Figure 5).

[0017] The inventors have concluded from the above experiments that a soil amendment comprising soy flour inoculated with Bacillus amyloliquefaciem 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.

[0018] 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.

[0019] 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 a seed covering. “Effluents,” refer to filtered liquids obtained from media in which the disclosed bacteria are cultured. [0020] 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.