Tupe Santosh G, Pathan Ejaj K, Deshpande Mukund V
Biochemical Sciences Division, CSIR-National Chemical Laboratory.
Biochemical Sciences Division, CSIR-National Chemical Laboratory;
J Vis Exp. 2017 Jul 30(125):55272. doi: 10.3791/55272.
A major concern when developing commercial mycoinsecticides is the kill speed compared to that of chemical insecticides. Therefore, isolation and screening for the selection of a fast-acting, highly virulent entomopathogenic fungus are important steps. Entomopathogenic fungi, such as Metarhizium, Beauveria, and Nomurea, which act by contact, are better suited than Bacillus thuringiensis or nucleopolyhedrosis virus (NPV), which must be ingested by the insect pest. In the present work, we isolated 68 Metarhizium strains from infected insects using a soil dilution and bait method. The isolates were identified by the amplification and sequencing of the ITS1-5.8S-ITS2 and 26S rDNA region. The most virulent strain of Metarhizium anisopliae was selected based on the median lethal concentration (LC50) and time (LT50) obtained in insect bioassays against III-instar larvae of Helicoverpa armigera. The mass production of spores by the selected strain was carried out with solid-state fermentation (SSF) using rice as a substrate for 14 days. Spores were extracted from the sporulated biomass using 0.1% tween-80, and different formulations of the spores were prepared. Field trials of the formulations for the control of an H. armigera infestation in pigeon peas were carried out by randomized block design. The infestation control levels obtained with oil and aqueous formulations (78.0% and 70.9%, respectively) were better than the 63.4% obtained with chemical pesticide.
开发商业性杀真菌昆虫剂时的一个主要问题是与化学杀虫剂相比的杀灭速度。因此,分离和筛选速效、高毒力的昆虫病原真菌是重要步骤。通过接触起作用的昆虫病原真菌,如绿僵菌、白僵菌和野村菌,比苏云金芽孢杆菌或核型多角体病毒(NPV)更适用,因为后者必须被害虫摄取。在本研究中,我们使用土壤稀释和诱饵法从受感染昆虫中分离出68株绿僵菌菌株。通过对ITS1-5.8S-ITS2和26S rDNA区域进行扩增和测序来鉴定分离株。根据在针对棉铃虫三龄幼虫的昆虫生物测定中获得的半数致死浓度(LC50)和时间(LT50),选择最具毒力的绿僵菌菌株。使用大米作为底物通过固态发酵(SSF)对所选菌株进行孢子的大规模生产,持续14天。使用0.1%吐温-80从产孢生物量中提取孢子,并制备孢子的不同制剂。通过随机区组设计对用于防治木豆中棉铃虫侵染的制剂进行田间试验。油剂和水剂制剂获得的侵染控制水平(分别为78.0%和70.9%)优于化学农药获得的63.