Isolation And Identification Endophytic Bacterium SZG-23from Sour Jujube And Purification Of Its Antifungal Protein

Sour jujube was selected as the materials to isolate endopytic bacteria.76strains of endophytic bacteria were obtained and their antibiotic activities against pathogenic fungi were evaluated in this study. The antagonistic effects showed that8of them expressed antibiotic activity on Alternaria alternata. By measuring antibacterial activity of extracellular from the antagonistic strains, antagonistic substances with higher antibacterial activity were selected especially the substance from SZG-1and SZG-23.The antagonistic effects showed that SZG-23expressed antibiotic activity on Alternaria alternata、Alternaria solani、Bortytis cinerea、 Fusarium oxysporum f.sp.niveum、Fusarium graminearum.In the experiment, the antagonistic strain SZG-23was identified by physiological and biochemical characteristics as well as the sequence of16SrDNA. Physiological and biochemical characteristics were similar to Bacillus amyloliquefaciens. The sequence of16SrDNA has been submitted to GenBank and the accession number is KF483660.Therefore, combining physiological and biochemical reactions and16SrDNA gene sequence analysis, the strain SZG-23was identified as Bacillus amyloliquefaciens.Antagonistic activities of strain SZG-23were evaluated under different culture conditions. The assay showed that the best composition of carbon, nitrogen and inorganic salts are soluble starch、yeast extract and K2HPO4·3H2O. The optimum fermentation condition is pH9,30℃and flask volume is at180r/min. The growth curve of SZG-23was studied,and found that8～18h is the best age of inoculating.Precipitates of the fermentation liquid from strain SZG-23were collected by80%saturation ammonium sulfate and showed significant inhibitory effect on growth of Alternaria solani, while the extraction of the fermentation liquid by organic solvents did not show any antagonistic activity. This showed that the antifungal material must be protein.By the analysis of antibacterial activity and SDS-PAGE showed that the best salting saturation is20%～40%. Observation showed that rough proteins made hyphas swells, fracture, twisted. Through tests of stabilization of the rough proteins done by high temperature、ultraviolet light and different pH,we can see that the rough proteins were unsensitive to ultraviolet light, the proteins was found to be thermostable, the antifungal activity was best when pH was5.0to9.0.With ammonium sulphate fractional precipitation, a antifungal protein purified by DEAE-52chromatography and Sephadex G-100chromatography, molecular weight of it was estimated to be50118daltons by SDS-PAGE.