Isolation, Antibiosis, Physical And Chemical Features Of Antifungal Protein From Rice Endophytic Antagonistic Bacterium Bacillus Subtilis

Bacillus subtilis G87 was an endophytic antagonistic bacterium isolated from rice roots. after the culture filtrate was precipitated by 60% amonium sulfate and dialysed, the crude antifungal protein could be obtained. The protein content was determined to be 3.52 mg/ml in the culture filtrate by ultraviolet spectrophotometry. Through Sephadex G-50 column chromatography of the crude protein, two absorption peaks occurred and peakⅠh ad strong antifungal activity. Through SDS-PAGE ,peakⅠc ontained two components with the molecular weight of 35.946 kD and 59.954 kD repectively according to Coomassie brilliant blue R250 staining, or seven components whose molecular weight ranged from 25.046 kD to 82.799 kD according to silver staining. Because these protein components were not further purified and their antifungal activity was not measured, the components and molecular weight of the pure antifungal proteins could not be in detail determined.The crude protein could obviously inhibit the mycelial growth, conidial germination and germ-tube growth of rice pathogenic fungi such as Gibberella fujikutoi and Magnaporthe grisea. Generally speaking, the inhibition of the protein on the mycelial growth of M. grisea was stronger than that of G. fujikuroi. At the concentration of 1.20 mg/ml, the inhibition rate of the protein on the mycelial growth of M. grisea ranged from 60% to 80%. But, the inhibition of the protein on the conidial germination and germ-tube growth of G. fujikuroi were stronger than those of M. grisea. At the concentration of 2.40 mg/ml, the inhibition rates of the protein on the conidial germination and germ-tube growth were over 60%. In addition, the crude protein could damage the hyphal shape of two pathogenic fungi significantly, leading to the hyphal cell shrink and malformation, the cytoplasm concentration and outflow, the cell wall disruption and the hyphal cell collapse. In a word, the damage of the crude protein to the hyphae of M. grisea was more serious than those of G. fujikuro.Characters of the antifungal protein was preliminarily studied. The protein was a kind of glycoprotein that contained 3.33% saccharide, but no lipid, withoutα-amino acid and phenyl structure. It was not very clear about the composition, structure and the antifungal activity of the components. In addition, the isoelectric point of the antifungal protein was 5.90. The protein was not stable at high tempreture (>60℃) and in alkline condition (pH>8), but not sensitive to ultraviolet radiation, chloroform, trypsin, proteinase K and pepsin.