Screening And Identification Of Antagonistic Baeteria Against Botrytis Cinerea And Study Of Its Active Substances

A strain Loq18 was screened from soil sample of Longquan, which could strongly antagonize to Botrytis cinerea from Strawberry,by dilution method.Loq18 was identified as Bacillus subtilis through morphological, physiological, biochemical and molecular identification. Virulence equation of the anfungal activity of fermentation product of Loq18 to Botrytis cinerea from strawberry was drawn, we obtained its minimum inhibitory concentration is 20mg/mL. The crude protein with anfungal activity obtained by ammonium sulfate precipitation from sterilization broth, and the remaining part have none. In order to determine the physical and chemical properties of the antifungal substances produced by Bacillus subtilis strain Loq18, the effects of temperature, pH values, ultraviolet, organic solvents, and protease were studied. The results showed that this crude antifungal substances were thermal stable, about 98% of the initial activity remained after heating at 121℃for 20 min; they were not sensitive to acidity-alkalinity, in the pH range of 4~11were stable; the antifungal activity was almost not be destroyed when they were under UV irradiation for10h; when they were treated by ether and acetone, their inhibition activity remained above 90% of control; they were not sensitive to protease.The Sephadex G-75 column chromatography, DEAE Sepharose Fast Flow ion exchange chromatography and Sephadex G-25 column chromatography were used to purify as LP18.Throughing MALDI-TOF-TOF-MS, biuret reaction and ninhydrin reaction, amino acid composition analysis, LP18 was identified. The results show that protein LP18 is a multisubunit protein and the subunit molecular weight of 4283.7 Da, which contains Leu, Glu, Asp and other 14 kinds of amino acids, with peptide bond andα-amino acids, no lipid and phenyl structure.Studying the antagonistic activity of proteins LP18 to a variety of pathogens, show that the protein LP18 have strong antagonistic activity to Magnaporthe grisea, Curvularia lunata and other strains of pathogenic fungi.So it has the potential applications to prevent and control a variety of plant fungal diseases.Microscopic observation of Botrytis cinerea which was affected by LP18 showed that the antagonistic mechanism may be dissolving the cell wall and changing the cell membrane permeability lead to leakage of protoplasm, so that the mycelium breaks or deformities, and inhibits spore germination.