Isolation and characterization of native Bacillus thuringiensis isolates from Saudi Arabia

Abstract

A total of 500 soil samples were collected from different localities across the kingdom to isolate native Bacillus thuringiensis (Bt) strains. Sodium acetate- (0.25 M) -selection heat-pasteurization, and 50% ethanol treatment methods were used for Bt isolation. Phenotypic characterization and identification of recovered Bt isolates was accomplished on the basis of morphological characteristics of colonies, parasporal crystal, and spores, as well as hemolytic activity, motility, susceptibility to antibiotics, and carbohydrate utilization (API 50CH system). Analysis of 16S rRNA gene was performed to further confirm Bt species identity and differentiate the spore-forming bacilli. Bioassay tests were conducted on larvae of Aedes caspius and Culex pipiens. Based on results, a total of 64 Bt isolates were recovered and the overall Bt index corresponding to the whole sampling areas was 0.25. Most of Bt isolates showed spherical crystals (56%), while, irregular, bipyramidal, and attached crystal to the spores constituted 14, 10 and 6% respectively. The most common biochemical type (31%) was as that of the reference Bt subsp. kurstaki (Btk). The 16S rRNA gene analysis, using the universal primer, provided an efficient identification of Bt isolates and their discrimination from the closely related Bacillus cereus, as well as from other spore forming bacilli. Bioassay showed that only one Bt isolate exhibited anti-diptera activity similar to Btk strain while most of the native Bt isolates exhibited weak (<30% mortality) or non insecticidal activity. However, results of motility, hemolytic activity, antibiotic-susceptibility patterns, and crystal shape, seem to suggest that many of our Bt isolates may exhibit parasporins activity.

Key words: Bacillus thuringiensis, parasporal crystal, isolation, biochemical type, insecticidal, cry gene, mosquitoes.