Molecular Identification And Purification Of Antifungal Protein Of B21 Against Botrytis Cinerea

An antagonistic bacterial strain B21 was isolated from the leaf, could produce large amount of antimicrobial substance, which inhibited the growth of Botrytis cinerea Pers.ex Fr. strongly. Field test, molecular identification of B21, characteristic analyse and purification of antifungal protein, some results have been showed as follows:The results of field trial indicated that the control efficiency of B21 suspension, 5 fold and 10 fold diluted culture filtrate with water by spraying on the diseased tomato leaves could reach to 67.21%, 63.57% and 51.02% respectively, and all significantly higher than that of the carbendazim(42.26%). The difference of its effectiveness between 5 fole and 10 fold diluted culture filtrate of B21 was significant, while no significant difference was found between 5 fole diluted culture filtrate and suspension of B21. The fruits of tomato plant were treated by spraying the suspension, 5 fold and 10 fold diluted culture filtrate of B21. Results showed the rates of diseased tomato fruits were 7.5%, 9.6% and 13.23% respectively. It was shown that the B21 was very potential and useful on of disease control of tomato in the future.The result of sequence analysis of 16S rDNA showed that strain B21 shared 99.93% homology with published sequence of B. subtilis (accession number: AY 172513). In the phylogenetic tree , both of strain B21 and B. subtilis were on the same branch, which showed that strain B21 was closely related with B. subtilis. Therefore, bacterial strain B21 was identified as B. subtilis.Crude proteins was extracted from precipitation of the cell culture of strain B21 with ammonium sulphate at 80% saturation. The determination of bioactivity of this antagonistic protein showed that its inhibitant activity depressed evidently when treated with 80℃ for 30 minutes, and its inhibitant activity lossed under 100℃ for 30 minutes. All those indicated that the protein could not endure high temperature. The result also showed that the protein was partially sensitive to Trypsin and Proteinase K, and is insensitive to Pronase but sensitive to Chloroform. Its active pH range was wide, from 5.0 to more than 11.0, but partially sensitive to low pH value.Through SDS-PAGE, antagonistic activity tracing, Sephacryl S-200 HR column chromatography and DEAE Fast Flow column chromatography, the antifungal protein was prepared and purified. The purified protein was demonstrated to be one single protein band by SDS-PAGE with a molecular weight of 43KD.