Presence of Multiple cry Genes in Bacillus thuringiensis Isolated from Dead Cotton Bollworm Heliothis armigera

Cry genes encoding Cry proteins toxic to Lepidoptera, Coleoptera and Diptera species were studied in thirty seven B. thuringiensis strains isolated from twelve naturally infested Heliothis armigera larvae. To further confirm the isolates, two groups of species-indicative biochemical tests were applied while discriminative biochemical tests being employed to figure out the repetitive strains. A PCR experiment was performed using five sets of universal primers for cry۱, cry۲, cry۳, cry۴, cry۷/۸ genes. All strains reacted appropriately, for B. thuringiensis, to the biochemical tests and while the reactions to the discriminative tests being varied. Based upon the results of the discriminative tests, twenty four non-repetitive strains were selected and employed in the PCR assay. Each of the selected strains presented one cry gene, at least; cry۱ being the most frequently detected one (۹۱.۷%), followed by cry۲ (۸۷.۶%), cry۳ (۵۰%) and cry۴ (۴۲%) but no isolate harbored a coleopteran-active cry۷/۸ gene. All the strains presented combinations of two or more cry genes: ۲۰% presenting cry۱+cry۲, ۱۲.۵% cry۱+cry۳, ۴% cry۲+cry۴, ۲۰% cry۱+cry۲+cry۳, ۲۰% cry۱+cry۲+cry۴, ۴% cry۱+cry۳+cry۴ and ۱۲.۵% carrying all the four cry genes studied and only one strain bearing a single cry gene. The cry۱-cry۲ combination was common in many strains (۷۲.۵%). Genetic characterization of this collection provides an opportunity for selection of strains with improved and multiple insecticidal toxicity.