Aphis 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. The 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. A 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. The 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.

中文翻译：

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苏云金芽孢杆菌菌株的蚜虫结晶性毒素的毒性评估：分子研究

蚜虫和A. punicae是危害最大的害虫。大量人口的出现引起了农民的关注，因为这种有害生物复合物被认为是至关重要的，因为它对全球主要农作物都有重大影响。由于不加选择地使用化学杀虫剂，缺乏用于控制棉铃虫和矮牵牛的新技术的问题也令人担忧。此外，这导致了抗性的迅速发展，这扼杀了它们在野外的控制。因此，迫切需要找到有效的生物防治剂来管理棉蚜和pun.a。本研究强调从安达曼和尼科巴群岛，卡纳塔克邦，阿萨姆邦，阿鲁纳恰尔邦和锡金土壤样品中分离和鉴定苏云金芽孢杆菌。相衬和扫描电子显微镜分析用于表征和鉴定晶体形态。Bt cry基因的分子谱分析通过使用蚜虫性cry基因特异性引物的PCR进行测定，并进行了分子克隆和测序。通过SDS-PAGE分析对蛋白质谱进行了进一步研究。最终，对棉球菌和pun。a。进行了杀虫活性的定性生物测定。总共筛选了65个芽孢杆菌样菌落。根据形态学和微生物学研究，鉴定出15种推测的苏云金芽孢杆菌双歧杆菌。球形和非晶态晶体夹杂物主要存在于34.28％的Bt分离物中。通过SDS-PAGE分析对Bt分离物的晶体蛋白谱分析显示，存在130、73、34、25和13 kDa条带，其中大量存在50-66 kDa条带。这些分离物的cry基因的检测通过PCR分析完成，表明cry1，cry2A，cry3A和cry11A在质粒DNA上。当在比对工具NCBI-BLASTn和BLASTp上比对时，所有cry基因都具有80-100％的同源性。测试了所有Bt分离株对蚜虫的杀虫活性。通过PCR分析，在15种分离物中有3种是蚜虫特异性毒素，在35μg/ mL的浓度下观察到它们对棉蚜和短枝曲霉具有毒性。苏云金芽孢杆菌感染可引起观察到的物理变化。这些菌株已从死亡的蚜虫中重新分离出来，并且所提出的结果符合科赫的假设。本研究带来了有希望的Bt分离株，