Toxicity evaluation of Aphidicidal crystalliferous toxins from Bacillus thuringiensis strains: a molecular study

Abstract

PurposeAphis gossypii and A. punicae are the most damaging pests. The emergence of large populations has created concern among farmers because this pest complex is considered critical as it has a significant effect on major crops around the globe. The lack of new technologies for the control of A. gossypii and A. punicae is also worrying due to the indiscriminate use of chemical insecticides. Besides, this leads to the rapid development of resistance, which strangles their control in the field. Hence, there is a dire need to find the effective biocontrol agent for the management of Aphis gossypii and A. punicae.MethodsThe present investigation emphasizes the isolation and characterization of Aphidicidal Bacillus thuringiensis from the Andaman and Nicobar Islands, Karnataka, Assam, Arunachal Pradesh, and Sikkim soil samples. Phase contrast and scanning electron microscopy analysis used to characterize and identify the crystal morphology. Molecular profiling of Bt cry genes was determined by PCR using aphidicidal cry gene-specific primers, and molecular cloning and sequencing were carried out. Protein profiling by SDS–PAGE analysis was further studied. Finally, a qualitative bioassay of insecticidal activity was carried out against A. gossypii and A. punicae.ResultsA total of 65 Bacillus-like colonies were screened; 15 putative Aphidicidal B. thuringiensis isolates were identified based on morphological as well as through microbiological studies. Spherical and amorphous crystal inclusion was predominantly present in 34.28% of the Bt isolates. Crystal protein profiling of Bt isolates by SDS–PAGE analysis showed the presence 130, 73, 34, 25, and 13 kDa bands, among which 50−66 kDa bands were present abundantly. The detection of the cry gene of these isolates was done by PCR analysis, which indicated that cry1, cry2A, cry3A, and cry11A were on plasmid DNA. All cry genes were 80–100% homologous when aligned on alignment tool NCBI-BLASTn and BLASTp. All isolates of Bt were tested for their insecticidal activity against aphids. Three of the 15 isolates are Aphidicidal toxin specific by PCR analysis which were observed to be toxic to Aphis gossypii and A. punicae at a concentration of 35 μg/mL. The observed physical changes were induced by B. thuringiensis infection; these strains had been re-isolated from the dead aphids, and the presented results fulfilled Koch’s postulates.ConclusionThe present study brought promising Bt isolates, primarily capable of creating an efficient biocontrol agent for Aphis gossypii and A. punicae and various sucking pests soon.