Lipopeptide Surfactin Produced by Bacillus amyloliquefaciens KPS46 is Required for Biocontrol Efficacy Against Xanthomonas axonopodis pv. glycines

Abstract

The biological control of root and foliar diseases of soybeans caused by fungi and bacteria (e.g. Xanthomonas axonopodis pv. glycines, Xag) using the plant growth promoting rhizobacterium Bacillus amyloliquefaciens KPS46 has been previously reported. Disease suppression is thought to be
due, in part, to the production of secondary metabolites. While a wide variety of these active compounds has been identified, their mode of action and mechanism of disease suppression on soybeans are not fully understood. The study used HPLC to identify secondary metabolites produced by B. amyloliquefaciens KPS46 and tested these compounds for biological control activity against soybean bacterial pustule disease caused by Xag. HPLC analyses indicated that a lipopeptide surfactin was present in KPS46 cell-free culture extracts, with maximum yields of ~550 ± 20.3 mg L^-1. Exogenous application of KPS46-produced surfactin to soybean plants directly that inhibited Xag, reduced disease severity and enhanced plant growth. UV mutagenesis of KPS46 and PCR assays were carried out to assess the role of surfactin production on the biocontrol activity. An independently-generated srfAA mutant of KPS46, strain M6, was unable to produce lipopeptide surfactin. The M6 mutant also was severely affected in its ability to produce extracellular enzymes, including endoglucanase, cellulase, and protease; and had reduced motility on the surface of agar compared to the wild-type strain KPS46. In contrast, the M6 mutant had enhanced production of α-amylase, and faster growth rate in nutrient
broth, than did the parental strain. Soybean plant assays using the srfAA mutant and wild-type biocontrol agents against bacterial pustule disease indicated that the mutant strain M6 had significantly less effect on disease reduction compared to the wild-type parental strain. Results of this study suggest that the ability of B. amyloliquefaciens KPS46 to reduce bacterial pustule severity on soybeans is associated with the production of a lipopeptide surfactin encoded by srfAA, and that mutations in this locus also effect extracellular enzyme production.