| Seventy-eight Bacillus strains were gained and purified from thirty-seven soil samples collected in Ruoergai plateau by pour plate method. Using ERIC-PCR,16S rDNA PCR-RFLP and 16S rRNA gene sequencing analysis technique, the genetic diversity and phylogeny of these Bacillus strains were performed. Furthermore, three Bacillus thuringiensis strains were screened from the 78 strains, and the cry gene was amplified, and the biological characteristics and virulence test of was determined. The results showed as follows:The genetic diversity of the 72 bacillus strains were studied by Eric-PCR, the results showed that there were very abundant ERIC-PCR patterns, and genetic diversity existed among the tested bacteria. Based on the dendrogram constructed by Eric-PCR patters, all strains were clustered into 8 genotypes at the 81% similarity.According to the results above,57 Bacillus stains were chosen to do 16S rDNA PCR-RFLP, the results showed that 7 genotypes formed at the boundary of 89% similarity.16S rDNA PCR-RFLP analysis was good in accordance with that of ERIC-PCR analysis. Then,19 representative stains were selected to do 16S rRNA gene sequencing based on the results of ERIC-PCR and16S rDNA PCR-RFLP, and the phylogenic tree was constructed, the results suggested that these bacteria mainly distributed in the 3 phylogenetic branches. They were genus of Bacillus, Brevibacillus and Paneibacillus. Including Bacillus pumilus, Bacillus magaterium, Bacillus thuringiensis.Three strains, i.e. YB12, YB14 and YB19, were identified as Bt of the 78 Bacillus strains, the isolation rate was 3.85%, and the parasporal crystals were diamond and roundness. Cry1 gene was amplified from 3 Bt strains successfully; biological tests showed that the optimal growth temperature was 15-20℃, and the optimal original pH was pH7 and pH8; Insecticidal activity testing showed that the lethality rate of strain YB12,YB14 and YB19 to silkworm was 73.81%,66.67% and 54.76%, respectively. |
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