Partial Characterization And Purification Of Antifungal Protein From Bacillus Subtilis G8 Strain

The antifungal activity, inhibitory mechanisms, greenhouse and field protective efficacy against cucumber sclerotium of antifungal protein of Bacillus subtilis strain G8 was studied. And purification and characteristic of antifungal protein was also studied to lay a foundation for further application.Plate inhibition method was used to ascertain inhibiting spectrum of G8 and disk diffusion method was applied to test the antifungal activity of metabolism products of G8. The result showed that G8 strain and its metabolism products had a broad inhibition spectrum. Both bacteria strain and metabolism products had antifungal activity against Sclerotinia sclerotiorum (Lib.) de Bary, Fusarium oxysporum fsp. Vesinfectum, Ascochyta citrullina Smith, Rhizoctonia solani, Phytophthora parasitica var.nicotianae, Fusarium moniliforme var. intermedium, Xanthomonas campestris pv. Malvacearum, Cladosporium flulvum, Rhizoctonia cerealis, Colletotrichum gossypii Southw, Pythium aphanidermatum, Phytophthora cactorum, Botrytis cinerea, Alternaria solani Soraue.Spraying fermentation liquor on leaves in greenhouse at 200mL/L concentration showed that the protective efficacy was 66.67% and curative efficacy was 60.87%, both of the efficacy not as good as chemical germicide. Soil treatment showed that after inoculating Sclerotinia sclerotiorum (Lib.) de Bary in soil for 36 hours applied the antifungal bacteria in the soil and it could comtrol the Cucumber Sclerotium. And applied the antifungal bacteria before or after the proper time it had no efficacy. So we can say that G8 strain could not be used directly, it was better to study G8's antifungal substance.The crude antifungal protein of Bacillus subtilis strain G8 was obtained by the combination of 40%(NH4)2SO4 fraction. The property and inhibition spectrum of crude antifungal protein was studied. The result showed that the crude antifungal protein of G8 had a abroad inhibition spectrum,and the inhibitory rate on Fusarium moniliforme var. intermedium was range from 60% to 80%, on Sclerotinia sclerotiorum (Lib.) de Bary and Botrytis cinerea was above 80%. The antifungal protein of G8 resistant to acid, adjust the pH value of the antifungal protein to 1.0 for 16 hours the inhibitory rate on Sclerotinia sclerotiorum (Lib.) de Bary was 69.2%. The antimicrobial activity showed that it was stable during heat treatment and was retained 80.8% even after at 100℃for 60 min and 80℃had no activity on this protein's structure. The antifungal protein was also stable after the treatment with organic solvents. And the antimicrobial activity was lasting for a long time at the normal temperature.In order to screen what were the optimal medium and fermentative condition for producing the antifungal protein, the antagonistic abilities to cucumber sclerotium was tested after the antifungal protein was obtained by precipitation of the cell-free culture of G8 with 40% ammonium sulphate. Experiment indicated that the best substrate for Bacillus subtilis strain G8′s growth was LB medium. The fermentation conditions were optimized as follows: pH 7.0, temperature 37℃, 30% inoculums, incubation time 36h and rotational speed 180r/m, bacteria growth mass and antifungal protein reached their maximum. Accumulation of antifungal protein was in direct proportion to the quantities of bacteria in the fermentation conditions.The studies on the action mechanisms of the antifungal protein demonstrated that they could break down the hyphae and leakagen the protoplasm, turn the hyphal cells into bigger sacs and manifold the hyphal forfication. The antifungal protein also can inhibit the Sclerotium of the pathogens from germination. And the studies on the action mechanisms of the the volatile substance of G8 demonstrated that they could make the diameters of mycelium of the pathogen different and turn the hyphal cells into bigger sacs, and the protoplasm browned and concentrated, but did not penetrate outside of cell.Two antagonistic peaks were obtained from the crude extract of G8 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.