| The Bacillus subtilis B68and antifungal proteins of the B68showed a high inhibitory activity against Colletotrichum musa, Fusarium oxysporum sp.4, Curvularia sp., Phomopsis mangiferae Ahmad, Colletotrichum gloeosporioiles, Colletotrichum gossypii Southw, Colletotrichum capsici, Colletotrichum gloeosporioides, Colletotrichum gloeosporioides, Colletotrichum gloeosporioides, Colletotrichum gloeosporioides, Fusarium lateritium, Pestalotiopsis psisii, Colletotrichum gloeosporioides, and antibacterial effects of extracted crude protein was better. The active protein showed the strongest antifungal activity against Colletotrichum musa. The lipopeptide biotics which extracts with methanol from hydrochloric acid precipitate also showed a high antimicrobial activity.The filtrated cultured liquid Bacillus subtilis B68was precipitated with ammonium sulfate at w=10%, w=20%, w=30%, w=40%, w=50%, w=60%, w=70%, w=80%, w=90%, w=100%, which in order to draw the antifungal proteins. The results-showed that all groups crude proteins had inhibitory activity against Colletotrichum musa, and precipitated antifungal proteins by w=80%(NH4)2SO4had the strongest inhibitory activity. The w=80%is the best salting ammonium sulfate saturation. The hyphae of Colletotrichum musa which were treated with the antimicrobial substances were made into temporary slide. Placed under an optical microscope and observed. Crude proteins and lipopeptide antifungal substances can cause central and top of plant pathogen hyphae swelling, distorting or deforming, eventually leading mycelium lysis, thus inhibiting pathogen growth and reproduction.The antimicrobial proteins extremely resistant to high temperatures, the property is very stable. Even crude proteins were treated for35min at120℃, but the antimicrobial activity was almost unchanged. It kept relatively stable under the condition of alkaline, neutral and acidic pH value, it had high antifungal activity in broad range of pH value. The antimicrobial proteins had the strongest inhibitory activity activity against Colletotrichum musa when pH value was7, and activity decreased slightly under strong alkaline or strong acidic alkaline pH. The crude proteins were respectively filtrated by ultra filter with tubes of lOku,30ku,50ku, both filtration and retention part of ultrafiltration membrane were activity. The inhibitory activity of the retention parts was stronger than the filtration. The studies showed that the active liquid contains not only the large molecular weight protein, but also the small molecular weight protein, and the majority of active protein is large molecular protein. Crude proteins of the B68were purified by anion exchange chromatography. Three peaks were obtained while the crude proteins eluted through the DEAE Sepharose Fast Flow column, only the fraction P2had antifungal activity against Colletotrichum musa. After the fraction P2were eluted through gel chromatography of the Sephadex G-100, two peaks were obtained, only the fraction P4had antifungal activity against Colletotrichum musa. The fraction P2showed only one protein band in the SDS-PAGE. The results showed the molecular weight of proteins were about38.4ku.The active proteins were identified by NanoLC-ESI-MS/MS. The analysis of the antifungal proteins showed that it might be a kind of hypothetical protein gi254239066, account for93.7%of the total protein content. The molecular weight of hypothetical protein gi254239066was38.060ku, between this kind of protein and the binding protein from pseudomonas aeruginasa C3719had a significant correlation. The other proteins content between0.5%and1.8%, between the protein and oxalate decarboxylase from Bacillus subtilis had a significant correlation. In addition, between the protein and fibrinolytic enzyme also had a significant correlation. |
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