Diversity of Insecticidal Crystal proteins (ICPs) of indigenous Bacillus thuringiensis strains

Objectives: The purpose of this study was to characterize the indigenous Bacillus thuringiensis (Bt) isolated from the soil samples of central development region of Terai. Methods: A total of 50 soil samples collected from cultivated and barren fields of Terai region. Isolation was carried out using the acetate selection protocol as described by (Russell and Al 1987) with a slight modification. The Nutrient broth (NB) was acetated by using 0.25M sodium acetate which is a selective enrichment method for isolation of Bt. Characterization of the isolate was done by phenotyping methods (microscopy and biochemical). Results: No distinct variation was observed between the isolates of cultivable and uncultivable lands. Bt were categorized into7 different types based on colony morphology. The dominant colony was fried egg type identical with the reference strain, followed by flat white type of colony. The result showed that even though the colony morphology is same but the ICPs (Insecticidal crystal proteins) shapes produced by them vary, rod shapes (53.57%), spherical (10.71%), ovoid (8.3%), amorphous (17.85%), capheaded (9.5%). ICPs morphology reveal the cry1, cry2, cry3, cry4, cry8, cry 9, cry10 and cry11 types of gene may be present in the native isolates. Conclusion: This study represents the first report of several indigenous Bacillus thuringiensis strains with significantly different ICPs producing stains from hot tropical climate.

Interactions between molecular chaperone P20 and Cyt2Ba7 toxin inBacillus thuringiensis

P20 or 20-kilodalton protein is a molecular chaperone protein inBacillus thuringiensis(Bt) which can increase yields and facilitates crystal formation of various insecticidal crystal proteins (ICPs). In previous studies, aB. thuringiensisinsecticidal protein gene,cyt2Ba7, was cloned, expressed but its expression level is very low inB. thuringiensis. In this study, various expression vectors were constructed by incorporatingp20 in forward or reverse direction in the upstream ofcyt2Ba7 and transformed into aB. thuringiensisacrystalliferous strain 4Q7. The result showed that in the presence of P20, the expression of Cyt2Ba7 was significantly increased. Especially whenp20 gene was reversely inserted in the upstream ofcyt2Ba7 gene, the expression of Cyt2Ba7 was increased ∼3.2 times meanwhile more and bigger crystals were observed under electron microscopy. By using purified Cyt2Ba7, P20 protein and P20-specific antiserum, immunoblotting and ligand blot analysis demonstrated a strong binding affinity between P20 and Cyt2Ba7. These results reveal that P20 can promote the crystal formation and enhance the expression of Cyt2Ba7 as a molecular chaperone, which can be a powerful tool to boost the ICPs production inB. thuringiensisand help develop more effective insect control strategies.

Receptors and Lethal Effect of Bacillus thuringiensis Insecticidal Crystal Proteins to the Anticarsia gemmatalis (Lepidoptera, Noctuidae)

Bioassays with insecticidal crystal proteins (ICPs) from Bacillus thuringiensis have demonstrated that Cry1Aa, Cry1Ac, and Cry1Ba are the most active toxins on larvae of the Anticarsia gemmatalis. The toxins Cry1Da and Cry1Ea are less toxic, and toxins Cry2Aa are not active. Binding of these ICPs to midgut sections of the A. gemmatalis larvae was studied using streptavidin-mediated detection. The observed staining patterns showed that Cry1Aa and Cry1Ac bound to the brush border throughout the whole length of the midgut. However, the binding sites of Cry1Ba were not evenly distributed in the midgut microvilli. The in vivo assays against larvae of 2nd instar A. gemmatalis confirmed the results from the in vitro binding studies. These binding data correspond well with the bioassay results, demonstrating a correlation between receptors binding and toxicity of the tested ICPs in this insect.