Screening Of An Antagonistic Strain Against Phytopathogen And Study On The Antagonistic Substance

Plant diseases caused by plant pathogens are partially responsible for significant losses or decrease the quality of agricultural products. Biological methods using antagonistic microorganisms or their active metabolites to control plant disease have been extensively studied in recent years. In this study, a strain of Bacillus sp. Y5-18 isolated from the filed soil in HZAU showed broad spectrum for antagonism to phytopathgenic fungi and the taxonomic of Y5-18 was identified. The gene related to antimicrobial substances synthesis of strain Y5-18 was detected and its fermentation conditions were optimized. The antifungal compound produced by strain Y5-18 was isolated and identified and the antagonistic mechanism was executed. The results were as follows:1.77 strains were isolated from filed soil and strain Y5-18 of them showing a broad antifungal activity against tested phytopathogens in vitro was obtained through dual culture method. The strain Y5-18 was identified as Bacillus amyloliquefaciens based on morphological observations, physiological and biochemical characteristics analysis,16S rDNA and tetB-yyaOlyyaR sequences analysis.2.PCR detection of lipopeptides synthesis related genes in strain Y5-18 by three specific primers revealed the presence of HuD, sfp and fenB genes, coding for Iturin, Surfactin and Fengcin, respectively.3.Separating and purifying the crude lipopeptides from the culture filtrate of strain Y5-18 according to lipopeptides extracting method and One-factor experimental design was used to optimize the fermentation conditions of strain Y5-18 based on the production of crude extract. The optimized crude lipopeptide production was from the initial approximately 2800 mg/L to about 3600 mg/L, with an increase of 28.6%.4. Both the culture filtrate and the crude lipopeptides extracted from the Bacillus-free culture broth showed strong growth inhibition activity against the plant diseases phytopathogens in vitro. The antifungal substance in the culture filtrate was relatively stable to temperature and pH. More than 60% of the antifungal activities of the culture filtrate against the growth of Rhizoctonia solani, Fusarium oxysporiumf.sp. cucumerinum and Sclerotinia sclerotiorum still remained after treatment at 100℃ for 30 min. Meanwhile, the antifungal activity of the culture filtrate against the three phytopathogens kept almost unchanged(>80%)when the culture filtrate was exposed to a pH ranging from 2 to 8. At the same time the antifungal activity of the culture filtrate was resistant to proteinase K and organic solvents. All of these are well known characteristics associated with the lipopeptide compounds.5. HPLC analysis showed that the crude lipopeptides mainly included five compounds. Only one fraction showed antifungal activity. The compound was isolated and identified as Iturin A2 based on the molecular weight and peptide ring sequence by using UHPLC-ESI-Q-MS/MS analytical techniques.6. Microscopic observation showed that, after the treatment of Iturin A2 to phytopathogens, protoplasm were condensed, cell wall were perforated, and mycelia were distorted, fractured, irregularly curled and exhibited growth deformation.