The Antagonistic Substance Produced By A30 Strain To Rhizoctonia Solani And The Gene Mutagenesis Analysis

Antagonistic strain Bacillus subtilis A30 was isolated from the phyllosphere of rice. It showed strong antagonistic activities to several plant pathogens eg. plant fungal pathogens Rhizoctonia solani, Magnaporthe grisea et al, and plant bacterial pathogen Xanthomonas oryzae pv. oryzae et al in vitro.From the curves of growth and antagonistic activity of strain A30, it is known that antagonistic substance could be detected when the strain grew in mid-exponential period, and reached to maximum when it grew in stable period.From the A30 cell-free culture one antagonistic substance, which was named PI, was purified by 40% saturation ammonium sulphate precipitation, FPLC-Source 15 PHE column and HPLC CLC-ODS column. Its molecular weight was primarily determined as 1476.7Da according to the LC-ESI-MS analysis. PI was thermostable, resistant to proteinase K, trypsin and organic solvents, but partial sensitive to acid pH. Its ninhydrin reaction was negative, but its ninhydrin reaction and biuret reaction became positive after acid hydrolysis. These results showed PI maybe a circle peptide or a peptide with some non-ammonium acid compositions. However, the antagonistic substance from the A30 cell-free culture could be cut-off when using centrifugal filtration with 30KDa cut, the reason maybe that this antagonistic substance was bound with a protein or other high-molecular compound.The A30 strain was transformed by plasmid pTVl-OK harboring transposon Tn917 and 6 transformants, which contained plasmid and showed resistance toerythromycin and kanamycin, were obtained. The cells of one transformant were treated at 43 "C for IShours after it cultured to OD of 0.5. Mutants showed resistance to erythromycin but sensitive to kanamycin were screened out, and they had lost the plamids. No antagonistic activity to Rhizoctonia solani was.detected from these mutants. Besides, Rifampin-resistance mutants were obtained after the induction of A30 strain by serial concentrations of Rifampin sequentially. The Rifampin-resistance mutants showed almost the same antagonistic activity as the wild strain.The hyphae and sclerotia of Rhizoctonia solani which had been dipped into the A30 cell-free culture grew abnormally, i.e. the hyphae branches became more but shorter, thinner and twisted; the sclerotia's germination was partially inhibited. In vitro test with rice leaves, it was found that the rice leaves had smaller or no lesions when the hyphae of Rhizoctonia solani were handled with A30 cell-free culture. The A30 culture showed good control efficiency to rice sheath blight in the field, its control efficiency was similar to that of Jinggangmycin.