| The antifungal activity, fermentation medium,fermentation conditions and inhibitory mechanisms against cucumber sclerotium of antifungal protein of Bacillus pumilus strain BSH-4 was primarily studied.And for further application of this strain,the isolation and characteristic of its antifungal crude protein were also studied.The method of in dual culture was used to ascertain inhibiting spectrum of BSH-4 and disk diffusion method was applied to test the antifungal activity of antifungal protein of BSH-4. The result showed that BSH-4 strain and its antifungal protein had a broad inhibition spectrum. Both bacteria strain and antifungal protein had antifungal activity against Sclerotinia sclerotiorum,Pythium spp.,Mycosphaerella melonis,Fusarium oxysporum,Rhizoctonia solani,Alternaria solani,Phytophthora capsici and Phytophthora parasitica et al.After several transferred,its antifungal activity was not changed.In order to improve the activity of BSH-4 to producing antifungal protein, the fermentation medium and conditions of the strain were studied via single factor test and orthogonal design. The results showed that the optimal fermentation medium was composed of corn starch 2%, yeast extract 2%, CaCl2 0.4%.And the optimal fermentation condition was fermentation period of 48 hours,rotation speed of 180r/min,cultural temperature of 37℃, initial pH value of 6. 0, inoculation quantity of 6 %,and medium volume of 25mL in 50mL flask. Under such conditions, the concentration of the antifungal protein which were produced from BSH-4 was about 16.8μg/mL.To gain physical and chemical properties of the antifungal protein, the effects of heat, pH, UV irradiation, organic solvents, metal ions prolease were studied,and also used different medium to ascertain its species.The result showed that this crude antifungal proteins were thermal stable, about 90% of the initial activity remained after heating at 100°C for 1 h; they were stable in the pH range of 6–9; the antifungal activity was almost not be destroyed when they were under UV irradiation for 10h; they were stable to ether,acetone, ethyl acetate and chloroform,but methanol could reduce above 20% of the initial activity; Ag+,Cu2+ and Zn2+ ions at the concentration of 10mmol/L could reduced their 40% initial activity and they were not sensitive to proteinase. The result also showed this protein was not chitinase, carboxymethyl cellulase andβ-1,3– glucanase.To ascertain the antagonistic mechanisms of this crude antifungal protein to S. sclerotiorum,the inhibition activities on mycelium growth against S. sclerotiorum were determined by mycelium growth rate method in lab. And its effect on this pathogen's biology characteristics, such as mycelium shape, sclerotium formation, sclerotium germination and membrane osmotic potential were also studied. The results showed that its EC50 value was 10.13μg/mL, which was significantly lower than Diethofencarb and Procymidone;After treated with this crude antifungal protein, the mycelia became etiolation and malformation, but had not yet found in the cytoplasm extravasation;This crude antifungal protein could delay the formation of sclerotium, the sclerotium numbers decreased contrasted that of the water control, and the weight reduced slightly;At the concentration of 250μg/mL of this crude antifungal protein,it could not restrain the sclerotium germination completely;It had no significant effect on the membrane osmotic potential.Two antagonistic peaks were obtained from the crude antifungal protein after Sephadex G-100 column chromatography. Condece the higher inhibition activity component and showed two band on SDS-PAGE. Antifungal activity was detected after distilling the divided band with Tris-HCl buffer. Clear inhibition zone was appeared on colonies of S. sclerotiorum of Low Molecular Weight protein.
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