Isolation, Identification, Fermentation Of Antagonistic Strain RS-25 Against Verticillium Dahliae And Purification Of Antifungal Polypeptide

Cotton Verticillium wilt is one of the most dangerous fascicular disease caused by Verticillium dahliae Kleb.Biological control which people pay more attention now is a highly effective measure for management of cotton Verticillium wilt.The antagonistic strain RS-25 which produces antifungal protein against Verticillium dahliae was isolated from the cotton rhizosphere soils.The crude extracts of antifungal protein can be obtained from the cell free supernatant of fermentation liqour of strain RS-25 by ammonium sulphate precipitation.The antifungal protein showed antibiosis activity to phytopathogenic fungi,such as Verticillium dahliae,Rhizoctonia solani, Fusarium oxysporum,Fusarium moniliforme,Aureobasidium pullulans and so on.In the experiment,the antagonistic strain RS-25 was identified by physiological and biochemical characteristics as well as the sequence of 16S rDNA.Physiological and biochemical characteristics were very similar to Bacillus amyloliquefaciens.By sequencing 16S rDNA we found that it was showed 99.62%homology to AB325583 Bacillus amyloliquefaciens.Based on the phenotypic characteristics,physiological and biochemical characteristics as well as analysis of the 16S rDNA sequence,the strain RS-25 was identified as Bacillus amyloliquefaciens.In order to increase the quantity of antifungal protein from B.amyloliquefaciens RS-25,the strain was cultured under different conditions.The carbon source,nitrogen source and inorganic salts of the fermentation cultrue medium were screened.The results showed that the proper components were sucrose,tryptone and K₂HPO₄,CaCl₂ and MgSO₄.7H₂O.By orthogonal design,the optimal proportions of fermentation medium were sucrose 2.0%,tryptone 1.0%,KH₂PO₄ 0.20%,CaCl₂.2H₂O 0.005%,MgSO₄.7H₂O 0.005%.Several technical and engineering parameters during the shake flask fermentation of antifungal proteins were studied by means of the orthogonal experiment design.The optimum shaking flask fermentation condition were proper inoculation 4%,and at age of 10 hours,shaking flask volume is at 30 mL/250 mL,at 180 r·min^-1for 48 h under 28℃.Under the conditions,the most quantity of antifungal protein was obtained.After optimization,diameter of inhibition zone increased by 25.4%.The B.amyloliquefaciens strain RS-25 were cultured in fermentation medium for 48 h at 28℃.The supematant was collected by centrifugation at 8000 r·min^-1for 15 min at 4℃. The crude extract of antifungal protein was obtained from the cell free supematant of fermentation liqour of strain RS-25 by ammonium sulphate precipitation.A antifungal polypeptide A2 purified by DEAE Sepharose Fast Flow chromatography and reversion phase chromatography,molecular weight of it was estimated to be 9000 daltons by SDS-PAGE.Assays for the antagonistic activity of antifungal polypeptide A2 were done on agar plates.The antifungal polypeptide A2 was found to be thermostable,resistant to pepsin,trypsin,and proteinase K.Its active pH range was wide,from 4.0 to 10.0,the antifungal activity was best when pH was 6.0 to 8.0.The antifungal polypeptide A2 was demonstrated as a glycoprotein that contained 1.81%.N-terminal amino acid residues analysis showed 15 amino acid sequence: H₂N-Ala-Ser-Gly-Gly-Thr-Val-Gly-Ile-Tyr-Gly-Ala-Asn-Met-Arg-Ser.Using this amino acid sequence as a target,the similarity of polypeptide A2 was searched with BlastP program on Internet.It was showed 100%homology to 42～56 positional amino-acid residue sequence of protein from B.amyloliquefaciens FZB42.