Antagonistic Mechanisms Of Bacillus Licheniformis To Botrytis Cinerea And Its Antifungal Protein

Bacillus licheniformis W10 was an antagonistic bacterium to Botrytis cinerea and other important plant pathogens. After cultured for 24 hours, the culture and the culture filtrate of the bacterium had same inhibition on the fungus, which suggested that the bacterium could secrete into the culture filtrate antifungal compounds.The culture filtrate had obvious damage to the mycelia and inhibitory effects on the growth of B. cinerea. It was observed that the protoplasm of hyphal cells shrinked and hyphal cells swelled, as well cell wall was destroied, which led to protoplasm leakage from the wound and even lysis of hyphae when B. cinerea was cultured in PS amended with the culture filtrate. The hyphae treated with the culture filtrate for 3h became abnormal. After treated 7 hours, the cell wall was destroied and the hyphal cell died. The hyphal cell lysis occurred soon at the high concentration of the culture filtrate. When the culture filtrate was diluted 10 times, the inhibition rates to the colony growth and the hyphal quantity of B. cinerea were 98.59% and 68.90%, respectively. The culture filtrate was diluted 2 times, the inhibition rates to sporulation, conidial germination and germ-tube growth were 83.64%, 33.71% and 57.69%, respectively. As the dilution of the filtrate was increased, the inhibitory effect of the filtrate could decline and even lose. The effects of the filtrate on the formation and germination of the sclerotia were the same as that on conidia. When diluted 2 times, the culture filtrate showed prominent inhibition to sclerotial formation, with the inhibitory rate of 51.06%. After immersed in the filtrate for 30min and then incubated for 25h , the germination rate of the sclerotia was obviously lower than that of control. But after incubated for 45h, the sclerotia germination rates were 100%, which suggested that the filtrate could delay the sclerotial germination, but not inhibit it.The bacterial metabolites in the culture filtrate could be precipitated by 30%amonium sulfate and the inhibition activity was similar to that of control, while the supernate couldn't inhibit the pathogen growth, which indicated that the antagonistic compound was protein. The crude protein was obtained by the dialysis of the precipitate, there were two bands in SDS-PAGE and IEF-PAGE of the crude protein. Through Sephadex G-75 filtration, the crude protein displayed two absorption peaks at 280nm. Absorption peak I with higher content in the crude protein exhibited strong inhibitory activity to B. cinerea. But absorption peak II with lower content had no inhibition. The refined protein was obtained by the collection of Peak I filtrate, which was further detected by IEF-PAGE and only one protein band appeared. The band was the same as the main protein band in electrophoresis of the crude protein, which indicated that there was only one component in the refined protein. According to ultraviolet spectrophotometry, the concentration of the antifungal protein in the culture filtrate was measured and was 621 μ g/ml.Characters of the antifungal protein was preliminarily studied. The molecular weight was 46,672 dalton and the isoelectric point was 6.71. The antifungal protein was a kind of glycoprotein that contained 6.83% saccharide, and some proline or hydroxyproline, but no lipid. The protein was found to be thermostable at 100℃, and not sensitive to trypsin, proteinase K, ultraviolet radiation and chloroform. The inhibitory activity of the protein was stable in the range of pH 6 to 12, and the strongest at pH 6.