Isolation and Identification of Native Strains of Bacillus Thuringiensis Producing Protein Toxin with the Ability to Eliminate Greenhouse Pests and Optimize Spore Production

One of the serious competitors in producing better and more agricultural products is insects. Bacillus thuringiensis is a gram-positive and facultative anaerobic endospored bacillus that is of interest as a biological insecticide in agriculture and has many advantages, including a specific effect on pests, no harmful effects on humans, the environment, and non-target insects, low cost, and It is easy to use compared to chemical types. These bacteria are able to produce different types of toxins. In this research, with the aim of isolating native strains of Bacillus thuringiensis with insecticidal effects against greenhouse pests, samples were taken from the soil of different greenhouse areas around Isfahan and the strains were isolated using sodium acetate selective method. Then identification of strains and initial screening was done based on biochemical tests and observation of toxin crystals. Molecular identification of strains was done using PCR with universal primers and determination of ۱۶S rDNA gene sequence, and molecular method and gral-nem and CU specific primers were used to identify genes synthesizing protein crystals against greenhouse pests. Optimization of spore production on selected strains and the standard Bacillus thuringiensis strain (PTCC ۱۴۹۴) using carbon sources (glucose, starch factory effluent, molasses and glycerol) and nitrogen sources (yeast extract, corn steep and ammonium sulfate) and mineral salts (Persian Gulf water, salt aquarium water for saltwater fish) was done. In the end, the biotechnological potential of the mentioned strains was investigated in terms of the production of protease, chitinase, lipase, amylase and lecithinase enzymes. From the ۵۰ investigated soil samples, ۷۰ Bacillus strains were isolated, and ۲۰ strains were selected in screening based on phenotypic characteristics and observation of protein crystals with Coomassie Brilliant Blue staining by phase contrast microscope. Specific genes with insecticidal properties against butterflies, semi-balan, bibalan, gab-balan, balan curtain in strain A with a length of ۱۸۰-۲۰۰ base pairs and specific genes with insecticidal properties against nematodes, balan curtain, in strain ۵۸ with an approximate length of ۴۸۹ -۴۷۴ base pairs were identified respectively. The results of molecular identification showed that A and ۵۸ strains are most similar to Bacillus thuringiensis strain. Optimization in strains A, ۵۸ and ۱۴۹۴ showed that the carbon source of the starch waste of the chips factory, the nitrogen source of yeast extract and the mineral salts of Persian Gulf water were the best sources for all three strains. Also, all three strains showed good hydrolysis activity.

کلیدواژه ها:

Bacillus thuringiensis ، parasporal ، native isolates ، greenhouse pests