Purification And Characterization Of Extracellular Antifungal Proteins From Bio-control Bacterium Of Sclerotinia Sclerotiorum Strain EM7

Sclerotinia rot of colza is a worldwide disease caused by Sclerotinia sclerotiorum, andhad an bad influence of economy and agricultural production. Since no effectivedisease-resistant variety was found at present, the prevention against Sclerotinia rot of colzarelies mainly on chemical control. However, the massive use of chemical pesticide couldcause environmental contamination, unbalance of ecological equilibrium, pesticide residue,induced plant resistance, etc. A lot of application of biological control on crop disease controlhas been reported, but less on Sclerotinia rot of colza.The study isolated a strain of endophytic Bacillus subtilis Em7with inhibition functionagainst sclerotinia rot of colza from the middle part of wheat. After morphology, physiology,biochemistry properties analysis and sequence analysis of16S rDNA, the strain was found tobelong to Bacillus subtilis. Also, bacteriostat test and indoor control effect experimentdemonstrated the development and application value of the strain. On that basis, the studyisolated antifungal protein with inhibitory effect from metabolin out of the cell of bacterialstrain, and probed in its properties. Furthermore, the study isolated and purified the protein togain pure protein with antibiosis function to Sclerotinia rot of colza, and provided theoreticalbasis of further study. The main results of the study are as follows.1. According to bacteriostat test of protein after fractional precipitation of Em7fermentationliquor, the protein fraction with fungistasis mainly existed in precipitation of0-30%and30%-60%ammonium sulfate.2. According to bacteriostat assay of antifungal protein against Sclerotinia rot of colza andpolyacrylamide gel electrophoresis, the protein using30%-60%saturation of ammoniumsulfate, with larger number of antifungal protein and higher antifungal activity,was the targetprotein in further study.3. According to the bacteriostat test against Sclerotinia rot of colza of crude antifungal proteinwith different treatment, it was demonstrated that the crude antifungal protein after freezed-drying could be preserved at least3years. Also, crude antifungal protein has its bestantibiosis activity at pH6.0. Moreover, the crude protein was stable to protease, ultravioletrays, heat and some kinds of organic reagent.4. The antifungal protein P5-2was isolated and purified from Em7fermentation liquor byammonium sulfate precipitation, hydrophobic chromatography, gel filtration chromatography,PAGE and recycling by cutting gel and electroelution. The molecular weight was36.4kDusing SDS-PAGE.5. Antimicrobial proteinP5-2and Bacillus amyloliquefaciens FZB42strains of exogenousprotein BglS are similar. The theory molecular weight of protein27586.15Da.