Purification, Identification And Inhibitive Mechanism Of Antifungal Protein From Bacillus Amyloliquefaciens Strain HRH317

A lower quantity and quality of crop were caused by the Fungal disease that was the main factor.There was a kind of trend whic the chemical pesticide were replaced gradually by biological control, meanwhile the Bacillus was a kind of bio-control bacteria. In this paper, based on Bacillus amyloliquefaciens HRH317 obtained with the laboratory separation as the main object, the inhibitive activity of antifungal protein produced from metabolites of HRH317 on the wheat Fusarium graminearum were studied. The theoretical basis of strains were provided in biological control applications. The specific research contents include:the the fermentation conditions of antifungal protein were optimized; antifungal protein were separated, purified and identificated; antifungal protein stability were studied; inhibitive mechanism of antifungal protein on Fusarium graminearum were studied; antibacterial protein control effect, soil colonization dynamic and biological safety evaluation were studied.Fermentation condition of strains HRH317 antifungal protein were optimized by the statistical methods the yield of antifungal protein were increased significantly. The best conditions for the production of antifungal protein were:nutrient broth medium(NB),inoculation volume 4%,37℃, and the shaker rotation speed 200r/min was NB, the initial pH value was 7.The inhibitive effect was the best under above the condition combination.The fermentation liquid from Bacillus amyloliquefaciens HRH317 were treated with saturated ammonium sulfate precipitation,then the crude protein extracts were obtained.The crude protin were separated and purified by DEAE Sepharose Fast Flow and Sephadex G-200 column chromatography, then the purified components 2-2 indicated the strongly inhibitive effect on the wheat Fusarium graminearum. The results showed that the molecular weight was about 36 kD by the SDS-PAGE. And the content of antifungal protein was 5.81± 0.04mg/mL. The amino acid composition were detected by HPLC.After SDS-PAGE electrophoresis, the band of antifungal protein were excised and were trypsin enzyme digestion, and then were analyzed by the NanoLC-ESI/MS/MS. And a total of 3 representative peptide sequences were obtained by electrospray ionization tandem mass spectrometry, such as ELISNASDALDK, MTQSPSSLSASVGDR、DSTYSLSSTLTLSK. The antifngal protein were not matched significantly in the network mass spectrometry database. A kind of protein similar to Bacillus secretion of oxalate decarboxylase partially, the results showed that the antifungal protein produced by the strain HRH317 was a novel protein.The results of antifungal protein stability showed that it was stable relatively to heat, acid and alkali, it was not sensitive to proteinase K, trypsin, pepsin.There is still good inhibitive activity dealed with UV irradiation and freeze-thaw cycles. The inhibitive spectrum of antifungal protein were widely, the results suggested that obvious inhibitive effect to 12 kinds of pathogens.Thus it can be seen that there were good development and application prospect in the plant fungal diseases and food antiseptic.The inhibitive mechanism of antifungal protein on the Fusarium graminearum were determined preliminarily by the SEM,TEM and the permeability of cell membrane. Fusarium graminearum hypha and spore germination were inhibited by the antifungal protein, and pathogens organelles were disordered, and cytoplasm were degraded, membrane permeability were increased. The cell wall of pathogens were damged by the antifungal protein was one of the important mechanism, but there was not significant effect to content of the pathogens protein without.The spectrum results of Fusarium graminearum protein SDS-PAGE also proved this point.In this paper, the resulted indicated that the wheat Fusarium graminearum could be controlled markedly by Bacillus amyloliquefaciens HRH317 through pot experiment. disease index of The wheat root dealed with antifungal protein were decreased 62.5% compared with CK, and a good role in promoting growth. After soaking seed and root irrigation treatment, the strain could be colonized in soil. The antifungal activity of antagonistic antibacterial HRH317 recovered from soil were detected and inhibition zone diameter reached 19.6cm. The strain HRH317 was in acute toxicity test. the results showed that there were no pathogenicity to mice, and the strain was proved to have good biological safety.