Antagonistic Substances Produced By Bacterical Strains To Xanthomonas Oryzae Pv.oryzae

Antagonistic bacterial strains, Bacillus subtilis 5 and Enterobacter cloacae B8 antagonistic bacterial strains, which were isolated from the rhizosphere of rice, showed strongly antagonistic activities to Xanthomonas oryzae pv.oryzae in vitro test. Antagonistic bacterial strain B. subtilis 504 isolated from the rhizosphere of rice showed strongly antagonistic activities to Xanthomonas oryzae pv.oryzae and Rhizoctonia solani in vitro test. The antagonistic substances produced by these three antagonistic bacterial strains were isolated and purified, the partial properties of the antagonistic substances were studied. The mainly results were as follows:1. Bacillus subtilis 5:B. subtilis 5 was screened out from the phyllosphere of rice in Hangzhou, China. It showed strongly antagonistic activity to rice bacterial blight pathogen in vitro test. The antagonistic substances from the cell free culture could be precipitated by (NH4)2SO4 with 75% saturation. The antagonistic activities were thermostable, resistance to organic solvents and trypsin, but partial sensitive to proteinase K. Two antagonistic proteins with 40Kda and 38Kda were isolated and purified by substantially chromatography of FPLC DEAE sephacel column, and FPLC source 15phenyl column. The sequence of the first 20 amino acids of the N-terminal of the 40 Kda antagonistic protein was determined, so was the first 12 amino acids of the N-terminal of the 38 Kda antagonistic protein. One antagonistic substance with 659 Da, which determined by MS, was also isolated and purified by substantially chromatography of FPLC DEAE sephacel column, FPLC source 15phenyl column and HPLC ProRPC 5/10 column.The antagonistic substance from the bacterial cells was isolated and purified partially by FPLC DEAE sephacel column, and FPLC source 15phenyl column after disruption with ultrasonication, and precipitation with (NH4)2SO4. The result from the SDS-PAGE electrophoresis showed the MW of the antagonistic protein was 38Kda.The control efficiency of antagonistic substance extract to rice bacterial blight in field was 6.8%. It was far less than that of Saikuzuo, which was 71.3%.2. Bacillus subtilis 504B, subtilis 504 was screened out from the rhizosphere of rice in Hangzhou, China. It showed strongly antagonistic activity to X. oryzae pv. oryzae, and Rhizoctonia salani in vitro test. The antagonistic substances from the cell free culture could be precipitated by 1.0 mol/L (NH4)2SO4. The antagonistic activities were thermostable, resistance to organic solvents and proteinase K. One antagonistic peptide with MW of 2.5-5Kda was isolated and purified from the cell free culture after the chromatography of FPLC DEAE sephacel column, and HPLC Diol 150 Column, substantially. It showed antagonistic activity to both X. oryzae pv. oryzae and R. solani.3. Enterobacter cloacae B8E. cloacae B8 was screened out from the phyllosphere of rice in Tiantai, Zhejiang, China. It showed strongly antagonistic activity to rice bacterial blight pathogen in vitro test. The antagonistic substance produced by E. cloacae B8 could be extracted from the cultured solid medium by 50mmol/L Tris-HCl (ph7.5) or CH2C12, and could be precipitated by (NH4)2SO4 with 75% saturation. The antagonistic activities were thermostable, resistance to organic solvents and proteinase K, but the antagonistic substance was degraded gradually during the storage at 4癈. The antagonistic substance was purified by silica gel thin layer chromatography and HPLC ProRPC 5/10 column chromatography, the MW of the antagonistic substance was 453 Da obtained from the results of LC-MS analysis.The control efficiency of antagonistic substance extract to rice bacterial blight in field could reach to 61%, which was potential on the application of plant disease control in future after more studies.