Isolation, Identification, Characterization and Toxicity Evaluation of Indigenous Strains and Preparation of Indigenous Bacillus thuringiensis-based Biopesticide against Chickpea Pod-borer, Helicoverpa armigera

Abstract

Bacillus thuringiensis Berliner is being used successfully as biological control agent throughout the world as a substitute of hazardous chemical insecticide in agriculture and forestry for the elimination of pests, and in human health sector for the elimination of disease vectors. In Pakistan, being an agricultural country, commercial scale production and application of biological insecticide is essential. The main objective of this study was to explore potential B. thuringiensis isolates from local environments and to produce effective and low cost biopesticides by a simple and effective process (shake flask technique/fermentation) for the control of chickpea pod-borer, Helicoverpa armigera Hübner of lepidoptera group. To achieve this objective 150 soil samples collected from different regions of Pakistan were screened and eighty one B. thuringiensis isolates were obtained from 33 (22%) soil samples, identified as B. thuringiensis by using phase contrast microscope and standard tests. These B. thuringiensis isolates contained crystal of different shapes but majority contained typical bipyramidal with cuboidal or irregular crystal. Polymerase chain reaction results indicated that 85.19% isolates was positive for cry1 gene (Lepidoptera specific) showing that cry1 gene occur frequently in our B. thuringiensis isolates. The SDS-PAGE results indicated that variations exist in the protein profile of spore-crystal of B. thuringiensis isolates but the protein profile of the majority was similar to reference standard strain. Results of preliminary screening bioassay at 500 μg toxin/mL diet indicated that toxic B. thuringiensis isolates and reference strain caused 96.55-100% whereas non-toxic caused - 7.33-45.33% mortality against 1 st instar larvae of H. armigera. Non-toxic B. thuringiensis isolates did not contain typical bipyramidal crystal. These results indicated that correlation exist between crystal morphology and toxicity to H. armigera. Bioassay results of toxic B. thuringiensis isolates indicated that LC 50 and potency of the most toxic B. thuringiensis isolate, PA-Sb-46.3 were 4.54 μg/mL, 1177515 IU/mg and relative potency 73.6. Relative potency showed that it was 73.6 times toxic than reference strain. B. thuringiensis var. kurstaki (HD-I-S-1980) viiiThe biopesticide was prepared from locally available low cost ingredients: dried beef blood, molasses and mineral salts (ZnCl 2, MgCl 2, MnCl 2, CaCl 2 and FeCl 3 ) which were used as medium for the laboratory scale production of B. thuringiensis biopesticide by shake flask technique. Indigenous B. thuringiensis isolate PA-Sb-46.3 which produced two crystals: bi-pyramidal and cuboidal was found 73.6 times toxic against H. armigera than reference strain B. thuringiensis var. kurstaki (HD-I-S-1980) was used. Medium was fermented for 72 hours at 30 ± 2 o C and 160 rpm. After 72 h fermented medium showed 95-99 % sporulation, with spore yield of 3.97 X 10 9 spore/mL and LC 50 value to 1 st instar larvae of H. armigera was 0.53 μg/mL diet. Preservatives and diluents used in the biopesticide were found to be effective when stored it at room temperature over a period of 30 months. The three years field results of biopesticide with exotic and chemical insecticides indicated that biopesticide was effective against H. armigera. These observations suggested that the biopesticide produced was effective and highly economical for the industrial scale production to manage H. armigera in Pakistan.