Characterization Of The Antifungal Substances Produced By Bacillus Amyloliquefaciens Strain YN-1

A strain of Bacillus sp. YN-1 isolated from soil in an orchard located in Zhengzhou showed broad spectrum antagonism to phytopathogenic fungi, its fermentation broth remained inhibitory effect on many phytopathogenic fungi after being sterilized at 121℃for 20min and subsequently being stored at 4℃for 6 months. The strain was identified as Bacillus amyloliquefaciens by observation of morphological, physiological and biochemical characteristics, and analysis of the DNA sequences of 16S rDNA and ITS.This study included three aspects: PCR-detection of lipopeptides synthesis related genes in strain YN-1 by four primers designed basing on the known lipopeptide genes ; Separating and purifying strain YN-1 broth directionally according to lipopeptides extracting methord, identifying the extracted substance as well as verification of the extracted substance as the main component of strain YN-1 broth ; Exploring active extract separating parameters by HPLC and quantitative and qualitative determination of lipopeptides produced by strain YN-1 with HPLC-ESI-MS and MALDI-TOF-MS method basis of the known separation experimental parameter. Results showed that three genes including sfp, fenB, ituA or bamA were detected in genome of strain YN-1; The properties of the extract substance ,proved to the main antifugal component of strain YN-1 broth, were inhibitory to many phytopathogenic fungi and insensitive to temperature and proteinase k, which were concordant to lipopeptides; HPLC separating parameters of the extract substance obtained by using gradient elution technique were determined by nice peaks and high resolution image. Nine kinds of lipopeptides C14-IturinA, C15-IturinA, C16-IturinA, C14-FengycinA, C15-FengycinA, C16-FengycinA, C17-FengycinA, C16-FengycinB and C17-FengycinB were identified in the antimicrobial extract by the mass spectrum analysis, in which five substances C16-IturinA, C14-FengycinA, C16-FengycinA, C17-FengycinA and C17-FengycinB were major components. The results provided a solid foundation for applying and revealing the efficiency of biocontrol on phytopathogenic fungi of strain YN-1.