Isolation, Identification Of Antagonistic Strain 12-34 Against Cotton Verticillium Wilt And Purification Of Antifungal Protein

Cotton is one of the most important economic crop in our country.However, Verticillium wilt of cotton,which is one of the most dangerous fascicular disease caused by Verticillium dahliae Kleb,is the maior diseases which seriously affect the yield and quality of cotton in our country.Using biological control has became the new methord of management of cotton Verticillium wilt.The antagonistic strain 12-34 which produces antifungal protein against Verticillium dahliae was isolated from the cotton rhizosphere soils.The strain has a wide antifungal spectrum.In the experiment,the antagonistic strain 12-34 was identified by physiological and biochemical characteristics as well as the sequence of 16S rDNA.Physiological and biochemical characteristics were very similar to Bacillus velezensis.Based on the phenotypic characteristics,physiological and biochemical characteristics as well as analysis of the 16S rDNA sequence,the strain 12-34 was identified as Bacillus velezensis.We cultured 12-34 strain under different conditions and studied the antagonistic activities of the solid ferment productions.The assay showed that the best medium composition contained 5%maizena,3%beef extraction,0.10%CaCl₂ and 0.05%KCl.The optimum shaking flask fermentation condition were proper inoculation 10%,shaking flask volume is at 50mL/250mL,at 200 r·min^-1 for 48 h under 37℃.With ammonium sulphate fractional precipitation,a antifungal protein purified by DEAE Sepharose Fast Flow chromatography and CM Sepharose Fast Flow chromatography,molecular weight of it was estimated to be 35000 daltons by SDS-PAGE.Through tests of stabilization of the rough protein done by protein enzyme and by high temperature,the proteins was found to be thermostable,resistant to protein enzyme. The antifungal activity was best when pH was 6.0 to 8.0 and stable under alkaline conditions.The research may use for reference on the mechanism of antifungal proteins of the antifungal mirobe in the microsclerotiniasphere of cotton field and on the wilt resistant gene engineering of cotton.