Study On Antifungal Substance From Marine Bacillus Subtilis Dm03

In this study, fourteen strains of bacteria which have more effective and stable antifungal activities against plant pathogenic fungi were screened from two handreds strains of marine bacteria. Amoung them, DM03 strain showed higher inhibitory activity and was selected to be studied further. The following work was focused on the characteristics of its taxonomy, the determination of its antigonistic spectra, the isolation, purification and characterization of the antifungal substances secreted by DM03 strain. The results were summarized as follows:1. Rhizoctonia solani, Fusarium oxysporium f. sp. Vasinfectum and Sclerotinia sclerotiorum were selected as indicator strains, two hundreds strains of marine bacteria from the Third Institute of Oceanography State Oceanic Administration were tested on their antifungal activities, and fourteen strains were screened because of their obvious and stable inhibitory action on plant pathogenic fungi. And then, DM03 strain which owned higher and more stable antifungal activity among them was selected to be investigated further. Preparatory experiment suggested that the fermentation broth of DM03 strain had excellent inhibitory effect on the indicator stains, while the supernatant of its fermentation broth showed little inhibitory effect. Fortunately, the cell-free supernatant of the fermentation broth of DM03 strain cultured in PDA medium revealed high activity of inhibiting the growth of the fungi.2. The inhibition mechanism of DM03 strain toward plant pathogenic fungi was likely nutrients competition combined with the production of antifungal substances, which was proved by the plate inhibition experiment between the fermentation broth of DM03 strain and plant pathogenic fungi.3. The characteristics of the antifungal substances secreted by DM03 strain were investigated, the results indicated that the antifungal substances were resistant to heat, UV light, acid and alkali. After being heated at 100℃for 10 min or irradiated by UV light for 30 min, the substances could still inhibit the growth of the indicator strain, and when pH value was varied from 3 to 11, the inhibitory activity of the substance varied insignificantly. The substances were precipitated using saturated ammonium sulfate solution, redissolved in Tris-HCl buffer, and then filtered using a 100 kDa ultrafiltration cube, the results showed that the active components of the substance was trapped by the ultrafitration cube. After that, the active components was determined using native-PAGE, two protein straps were captured in the PAGE gel. Then the two straps were cut out from the gel plate, hydrolyzed by trypsin and treated as the preparation for MALDI-TOF/MS analysis. Protein mass fingerprinting analysis of the MS spectra suggested that one of the active components had no matched in the NCBInr protein database, which indicated that DM03 strain was likely to produce a novel antifungal protein which had never been reported by others.4. DM03 strain could not produce antifungal substances in the MB medium which was more suitable for its growth, while the active substance could be secreted when DM03 strain grew in PDA or PDB media, this suggested that the production of the active substance may depend on the growth conditions of DM03 strain.