Identification, moleculer typing and determination of biological activities of Bacillus species isolated from soils in Ankara

In this study, 60 Bacillus strain were isolated from different soil samples in Ankara and identified with Analitic Profile Index (API). Bacillus thuringiensis was also identified by 16S rRNA sequence analysis. Total proteins of each isolates were isolated and analysed by sodium dodecyl sulphate gel electrophoresis (SDS-PAGE). The protein banding patterns of the isolates were compared with each other, genetic distances were calculated and dendrogram was established. The genetic distances between isolates cahnged from 0% to 95%. According to the dendrogram isolates were divided into two main clusters. The first cluster contains B. cereus, B. anthracis and B. thuringiensis. The second cluster B. sphaericus, B. subtilis, B. licheniformis, B. laterosporus, B. pumilus, B. megaterium, B. stearothermophilus, B. brevis, B. firmus and B. circulans. Plasmid DNAs of the isolates were isolated and their sizes were calculated. 57 of the isolates contained 1-9 plasmid DNA changing from 1,1 kb and 19,3 kb in size while 3 isolates had no plasmid DNA. A polymerase chain reaction- restriction fragment lenght polymorphism (PCR-RFLP) method for identification of cry genes from B. thuringiensis was established. The results showed that Bacillus thuringiensis FÖ21 contained cry1 (272 bp) and cry2 (1556 bp) genes, while Bacillus thuringiensis FÖ16 and Bacillus thuringiensis FÖ19 contained only cry2 gene. cry2 gene was restricted with DdeI enzyme in order to vii find cry2 types. All three isolates had cry2Aa and cry2Ab genes. The crystal proteins of the isolates were analysed by SDS-PAGE. The results showed that isolates had two crystal proteins (130 kDa and 65 kDa weight). The morphological structure of parasporal crystals of the B. thuringiensis were investigated by Scanning Electron Microscopy (SEM) morphologically. Results showed that all the isolates carried bipyramidal, spherical and cuboidal structures. The toxic effects of the isolates carrying parapsoral crystals were determined by the bioassay using third instars larvae of Ephestia kuehniella (Lepidoptera) and Trogoderma granarium (Coleoptera). The LC50 and LC99 values of the isolates were determined by Probit analyse and mortality were calculated by Abbot?s formula. Mortality of the Bacillus thuringiensis FÖ21, Bacillus thuringiensis FÖ16 and Bacillus thuringiensis FÖ19 against E. kuehniella larvae were 83%, 80%, and 57% respectively. Toxic effect of Bacillus thuringiensis FÖ21, Bacillus thuringiensis FÖ19 were found better than the reference strain tested. In conlusion, the results show that the B. thuringiensis isolates obtained in this study could be used as an effective biological control agent.