Screening Of Bacillus Thuringiensis Strains Against Xanthomonas Oryzae Pv. Oryzae And Its Active Compounds

Bacillus thuringiensis is the most successful biological pesticides. But a little interest was shown in antimicrobes of B. thuringiensis. The discovery of antimicrobial active substances produced by the strains of B. thuringiensis is still in the initial stage.Firstly,400 B. thuringiensis strains were screened against Xanthomonas oryzae pv. Oryzae. The strains YBT-2368,YBT-2474 and YBT-2532 were shown to produce antibacterial activity against Xanthomonas oryzae pv. Oryzae. YBT-2368 and YBT-2532 were identified as B.thuringiensis by the method of observing the presence of parasporal crystals.The culture medium and conditions were optimized for the production of the active substance against Xanthomonas oryzae pv. Oryzae using the Taguchi design method. The production of the active substance was increased, significantly.Results showed that the fermentation supernatant and the active substances in crude extract of YBT-2532 possessed a significant antibacterial activity against Xanthomonas oryzae pv. Oryzae by comparison with the control under greenhouse conditions.Physical and biochemistry characteristics of the antimicrobial active substance produced by the B.thuringiensis strain YBT-2532 were determined. It is not sensitive to temperature, protease, pH. After incubation for 1 h at 70 ℃, there is still 75% activity retained. Proteinase k,trypsin and pepsin had no effect on the activity. The pH had effect on the antimicrobial activity. However, the active substance can be stable in the range of pH 2.0-12.0. It can be dissolved in water and methanol, weakly dissolved in ethanol, insoluble in acetone, chloroform and dichloromethane. Purification of active substance was achieved using gel chromatography, ion exchange chromatography, Solid phase extraction and HPLC. The purified active substance was analysed by HPLC-IT-MS, its molecular mass was 796.9 Da.