Bacillus subtilis strain NCD-2 is an excellent biocontrol agent for plant soil-borne diseases,and the lipopeptide fengycin is one of the active antifungal compounds in strain NCD-2.The regulator Pho P and its sensor kinase Pho R compose a two-component system in B.subtilis.In this study,the pho R-and pho P-knockout mutants were constructed by in-frame deletion and the role of Pho R/Pho P on the production of fengycin was determined.Inactivation of pho R or pho P in B.subtilis decreased its inhibition ability against Botrytis cinerea growth in vitro compared to the strain NCD-2 wild type.The lipopeptides were extracted from strain NCD-2 wild type and its mutant strains by hydrochloric acid precipitate,and the lipopeptides from pho R-null mutant or pho P-null mutant almost lost the inhibition ability against B.cinerea growth compared to the lipopeptides from strain NCD-2 wild type.Fast protein liquid chromatography(FPLC) analysis of the lipopeptides showed that inactivation of pho R or pho P genes reduced the production of fengycin by strain NCD-2.The fengycin production abilities were compared for bacteria under low-phosphate medium(LPM) and high-phosphate medium(HPM),respectively.Results indicated that the regulation of fengycin production by the Pho R/Pho P two-component system occurred in LPM but not in HPM.Reverse transcriptional-PCR confirmed that the fengycin synthetase gene fen C was positively regulated by pho P when cultured in LPM.All of these characteristics could be partially restored by complementation of intact pho R or pho P gene in the mutant.These data indicated that the Pho R/Pho P two-component system greatly regulated fengycin production and antifungal ability in B.subtilis NCD-2 mainly under low-phosphate conditions.
