Isolation and identification of native Bacillus thuringiensis isolates and production of biological pesticide based on effective strains

The objectives of the present study, including 7 projects, were isolation and identification of effective biocontrol Bacillus thuringiensis (Bt) strains and optimization of mass production and formulation of the selected strains. Seventy native Bt strains isolated from different regions of Iran were studied. Project 1: The identification of the strains was performed based on insecticidal genes and crystal protein profiles of isolates. Characterization included the identification of cry, vip and cyt genes coding protein that are effective against lepidoptera, diptera, and coleoptera. Bt strains containing some of the previously described cry gene composition as well as potentially novel Cry proteins were identified. Results showed that genes coding Lepidoptera, Diptera and Coleoptera active crystal proteins occur in different frequencies in the native isolates. Isolates containing vip3Aa gene were the most abundant (82.6%) followed by the cry2 (56.5%), cry1 (49%) and cry9 (30%). Twenty-two distinct cry1-type profiles were identified from only cry1 harboring isolates when were analyzed with specific primers. Several of them were found to be different from all previously published profiles. For example the new Lepidoptera active cry gene profile "cry1Aa, cry1Ab, cry1Ac, cry1Ad, cry1C, cry1D, cry1E, cry1F, cry1I", the new diptera active cry gene profile "cry2Ab, cry4B, cry10, cry11, cry17, cry19, cyt1, cyt2" and the new coleopteran active cry gene profile "cry8B , cry18 , cry28 , cry 8B , cry7A , cry34 ,35" were identified. Project 2: Morphological characteristics of crystals were analyzed by gram staining and observation with common and phase contrast microscopes. All Bt isolates were further characterized by SDS-PAGE of their crystal protein products. For more characterization of the isolates, plasmids of each isolates isolated and observed on the agarose gels. Microscopic studies showed that about 40% of isolates contained more than one type of crystals. The results showed that 47.17% of isolates produce bipyramidal parasporal inclusions, 30% cuboidal in shape, 28.57% elliptical in shape, 10% spindly in shape, 5.71% spherical in shape, and 27.4% of isolates showed irregular and other forms. The results revealed that the Iranian strains synthesize a protein or group of proteins with a molecular mass between 28 and 140 kDa, and some of them had a further protein of 21-28 kDa. The isolates produced 1-5 different protein bands and about 70% of the isolates showed more than one band. Crystal protein patterns of about 8% of the isolates were comparable with Bt subsp. kurstaki. The plasmid profile analysis detected a great complexity in the content of plasmids, providing 12 different plasmid profiles, with several others being similar, not considered as different. The results showed that isolates contained between 1 and 6 plasmids with estimated molecular masses of 4 to 130 MDa. Eight isolates showed four plasmids with masses of 4.9, 9.6, 30 and 47 MDa resembling the Bt subsp. kurstaki pattern. However, most of the isolates containing Lepidopteran-specific active cry genes showed some identical bands, indicating that they probably harbored plasmids of the same size. Finally, based on morphological and molecular studies, 20 isolates were selected for bioassays on Lepidoptera, 20 isolates were selected for bioassays on dipteran pests and 12 isolates for bioassays on Coleoptera. Project 3: in this study, the isolates were studied for novel insecticidal genes. The native strains showed a wide genetic diversity based on insecticide genes contents, and each isolate contained between 5 to 20 different insecticide genes. Furthermore, some isolates when were assayed for cry1C, cry5, cry6, cry9, cry9d, cry9b, cry18 and cry24 genes, showed different size bands from that were expected. This results showed that these isolates may contain novel cry genes. PCR reactions for these isolates and genes were repeated; PCR products were cloned to TA cloning vector pTZ57R/T, and transformed to E. coli strain DH5α. For analysis of the cloned sequences, two strategies, including restriction enzyme patterns (RFLP patterns) and sequencing were used. The oBtained sequences were aligned with cry genes sequences in NCBI and other data gene banks. Finally, the selected sequences analyzed by ClustalX, Mega4 and Tree view software. Finally, 9 sequences were identified as new cry-type genes which were similar to cry1 and vip genes.