Biological control of peach brown rot (Monilinia spp.) by Bacillus subtilis CPA-8 is based on production of fengycin-like lipopeptides

Bacillus subtilis CPA-8, a strain with demonstrated ability to control Monilinia spp. in peaches, was studied to elucidate its mechanisms of antifungal activity. Growth inhibition assays using cell-free supernatants and butanolic extracts showed strong antifungal activities against Monilinia laxa and Monilinia fructicola. By comparison with the reference B. subtilis strains UMAF6614 and UMAF6639, fengycin, iturin and surfactin lipopeptides were identified by thin layer chromatography in butanolic extracts from cell-free supernatants, indicating that antibiosis could be a major factor involved in the biological control ability of CPA-8. TLC-bioautography analysis confirmed the presence of fengycin, iturin and surfactin lipopeptides but strong antifungal activity could be associated only with fengycin lipopeptides. These results were definitively supported by mutagenesis analysis targeted to suppress fengycin biosynthesis by disruption of the B. subtilis fenB gene. By TLC-bioautography analysis it was possible to identify transformants from CPA-8 with reduced or suppressed antifungal activity, and this phenotype was associated with the lack of fengycin bands. Fruit trials confirmed that fengycin-defective mutants and their cell-free supernatants lost their ability to control peach brown rot disease in comparison with CPA-8 wild type strain or Serenade Max (R), a commercial formulation based on B. subtilis. Furthermore, population dynamics studies determined that CPA-8 fengycin-deficient mutants survived in wounds in peach fruit equally well as the CPA-8 wild type. Taken together our data indicate that fengycin-like lipopeptides play a major role in the biological control potential of B. subtilis CPA-8 against peach brown rot.