Characterization of cry genes and insecticidal proteins from indigenous bacillus thuringiensis to develop potential biopesticide

Abstract

Indiscriminate use of chemical pesticides, the cause of serious public health problems and environment pollutions as well as the emergence of pest resistance in many developing countries, could be reduced with Bacillus thuringiensis (Bt) biopesticides for its eco-friendly properties. The work was aimed at controlling vegetable pests by foliar spraying of locally developed potential Bt biopesticides as the facts of immunological adverse effects and increasing insect resistance due to the continuous exposure to the Bt toxins were reported for transgenic crops. Bt isolates were, therefore, isolated from different eco-regions of Bangladesh and characterized based on their phenotype, genetics, proteomics, diversity, toxicity and field efficacy. Upon biochemical typing of 298 indigenous Bt isolates, Bt indiana (17%), kurstaki (16%), thuringiensis (12%) were found to be the most prevalent biotypes in Bangladesh and from the distribution index, an indication of the degree of ubiquity, Bt thuringiensis (0.5) and indiana (0.5) appeared to be more ubiquitous than all other biotypes followed by eleven (0.43), kurstaki, sotto and nine (0.4 for each). Variation in abundance and distribution pattern of 16 different biotypes in Bangladesh was demonstrated in comparison to the other parts of the world. Broad spectrum cytolytic activity was observed to vary with biotypes and 58.36% strains were presumed to insecticidal based on this attribution. The molecular weight based distribution of small plasmids was compared among the biotypes and the maximum diversity was observed among Bt thuringiensis, kurstaki, indiana and israelensis biotypes. The screening for insecticidal genes in the native Bt strains revealed their presence in varied proportion being cry1 to be the most prevalent (30.8%) followed by cry2 (25.5%), cry3 (22.2%) and cry9 (7.2%). Prevalence of cry4, cry8 and cry10 genes were less than 5% and cry11 gene was not found in any strain. The SDS-PAGE analysis of the partially purified Cry proteins revealed the presence of Cry1- type delta endotoxins including Cry1Aa, Cry1Ab, Cry1Af, Cry1B, Cry1C and Cry1M, Cry2, Cry3, Cry8 and Cry9 proteins which were also supported by the cry gene profiles. Genetic diversity among 177 Bt strains were analyzed by RAPD-PCR which revealed 15 genotypes and type 9 and type 11 were found to contain more than 25% of the strains. In combination of RAPD-PCR and cry gene profile analysis, genotype based distribution of cry genes was evaluated and Genotype 1, 6, 9, 11 and 12 were found to contain the most of the cry genes at different extent while cry genes with diversities were most prevalent in genotype 6, 9 and 1. Genetic diversity analyzed thus is highly relevant and significant in discovering novel insecticidal genes in indigenous Bt strains and to deal with the problems of emerging resistance towards Bt toxins. Novel toxicity of indigenous Bt strains was identified at significant level against the larvae of Bactrocera cucurbitae and the efficacy of Bt JSc1 was highly comparable with the reference strain Btk HD-73 with no statistically significant difference. The complete Open Reading Frame (ORF) of cry1A- type gene from Bt strain JSc1 was, therefore, obtained by PCR walking technique and was revealed to be identical to cry1Aa9 and cry1Aa13 genes. Five conserved block of Cry proteins were identified from the deduced amino acid sequence analysis and the 3-D protein structure constructed by homology modeling revealed its 3-domain protein fold. A cost effective medium MSeMC-AS was formulated with defatted soybean meal, molasses, marine water, cystine and ammonium sulfate which demonstrated more than 80% increase in yield during scaling up from shake flask to a 3.0 L bioreactor and the growth kinetics of indigenous Bt strain JSc1 was also revealed faster than the reference Btk HD-73 in this medium. Yield in δ- endotoxin production was enhanced by inhibiting the endogenous protease activity which was demonstrated to be the cause of protein degradation and loss in productivity. From the field trial of Bt biopesticide preparation in cabbage, cauliflower and organic tea farming, no significant differences were observed with the active treatments which indicated high feasibility of the prepared Bt biopesticide in replacing conventional pesticides with no yield loss. These results suggest that the Bt strains of Bangladesh with their unique and diverse properties as well as potentials as revealed in this study, could potentially be utilized not only in controlling the pests in agriculture but also the vectors, protozoa, nematodes etc of public health concern.