Isolation And Identification Of Bacillus Strains From Farmland Soil In Chengdu Plain And Biological Characteristics And Insecticidal Activity Of Three Bacillus Thuringiensis Isolates

In this paper,98 Bacillus strains were screened by sodium acetate-anti-biotic method from 60 farmland soil samples fetched in Chengdu plain, and the genetic diversity and phylogeny was determined by ERIC-PCR, ARDRA and 16S rDNA sequence analysis. From these strains, three Bt strains was screened and identified by cry gene amplification, and the parasporal crystal was observed by staining. Furthermore, we detected the insecticidal activity of three Bt strains. The main results were as follows:(1) Ninety-eight Bacillus strains were isolated from the farmland soil in Chengdu Plain, ERIC-PCR analysis divided these 98 strains into 6 genotypic groups at the level of 84% similarity. ARDRA analysis divided these strains into 7 groups at the level of 85% similarity, which showed the great diversity was existed among these strains.(2) 16S rDNA sequence of representative strains were analyzed and the phylogeny was constructed, the results suggested that 15 representative strains were belonged to genus of Bacillus and Paenibacillus. The 16S rDNA sequence similarity was 99.8% between strain S11 and B. altitudinis 41KF2bT (AJ831842); and 99.4% between strain S65 and B. firmus IAM 12464T (XD16268); and 100% between strains S33, S60, S69 and B. aryabhattai B8W22T(EF114313). Strains S8, S21, S96 had 99.2%,99.0%,99.0% similarity with B. thuringiensis IAM 12077T(D 16281), respectively, which showed that these three strains were Bt strain, the Bt isolation proportion was 3%.(3) The morphological observation of three Bt strains indicated that these Bt strains were short rod, and could form several shapes of parasporal crystal such as bipyramidal, square and spherical. The cry gene-types of these three Bt strains were identified by PCR amplification using universal primer K5un2 and K3un2, and one 1500bp size band was obtained, the results showed that 3 Bt isolates harbored cryl genes.(4) The prescreening on bioassay of three Bt strains showed that strain S21 was highly toxic against lepidoptera(silkworm) and coleopteron(flour weevil). LC50 was 0.81×108cfu/mL to silkworm, and was 0.89x108cfu/mL to flour weevil, which was lower than 30% concentration of stock solution, and the toxicity to silkworm were much higher than to flour weevil.