Biocontrol activity of the endophytic Bacillus amyloliquefaciens strain CEIZ-11 against Pythium aphanidermatum and purification of its bioactive compounds

• B. Amyloliquefaciens CEIZ-11 harbor genes involved in lipopeptide biosynthesis.
• Crude lipopeptide exhibited strong and broad antifungal activity.
• LC-MS analysis permits the detection of three families of lipopeptides.
• CEIZ-11 provided protection of tomato plants against damping-off.

In a previous study, Bacillus amyloliquefaciens strain CEIZ-11 has been isolated from a compost extract and showed a broad spectrum of antifungal activity against various plant pathogenic fungi. The potential of Bacillus genus to antagonize phytopathogens is due to the production of some metabolites, mainly cyclic lipopeptides. In the present study, polymerase chain reaction was used to screen CEIZ-11 for genes involved in biosynthesis of lipopeptides. Amplicons of expected sizes were detected as ituC and ituD, srfp and fenD involved in iturin, surfactin and fengycin synthesis respectively. Lipopeptides, extracted by acid precipitation of culture filtrate, exhibited strong antifungal activity with MIC values ranging from 9.8 to 156 μg/mL and an excellent stability to high temperature (20 min at 121 °C) and a large range of pH, 2–11. The crude lipopeptides was subjected to reverse phase high-performance liquid chromatography (HPLC). Five major fractions were detected. Each fraction was further purified by revered phase C18 analytical column. Identification of individual fractions was done by liquid chromatography coupled with mass spectroscopy. Three families of lipopeptides were detected including three homologues of iturin A (A2, A3-5 and A6), one fengycin A having fatty acyl chain length of C16 and one surfactin (C12). To evaluate the potential application of B. amyloliquefaciens strain CEIZ-11 to control damping-off, tomato was used as model. The pot experiments results showed that CEIZ-11 could be a promising agent in biocontrol of damping-off.

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