J. Asia-Pacific Entomol. 2(1): 61-67 (1999) Evaluation of Pakistanian Bacillus thuringiensis isolates against Scirpophaga incertulas and Cnaphalocrocis medinalis Shahid Karim'; Kausar Malik, Uzma Zafar and S. Riazuddin Abstract - A large number of Bacillus thuringiensis (Bt) isolates separated from different ecological regions of Pakistan were characterized for crystal protein gene composition and pesticidal activity against two lepidopteran rice insect pests, the yellow stem borer (Scripophaga incertulas) and the rice leaf folders (Cnaphalocrocis medinalis). A representative seventeen isolates were selected on the basis of initial screening and further characterization of pesticidal activity was performed according to following criteria; colony and parasporal inclusion morphology, SDS-PAGE, western blot analysis and comparative biotoxicity assays to determine LCso values. All isolates produced parasporal inclusion bodies and spores in their cells. Immunoblotting results showed that Pakistanian isolates synthesized entomocidal proteins belonging to CrylA and Cry2A toxin groups. The biological activity of local isolates demonstarted a wide range of LC so values against both target insects pests. The most potent isolates, INS 1.13, INS 2.25 and NW 4.1 against S. incertulas showed LC so values of 29.83, 30.37 and 24.77 ng/ml of toxin, respectively. The LCso values of 57.37 and 73.09 ng/ml of toxin were exhibited by local isolates, INS 2.25 and RL 4.8 against C. medinalis, respectively. Key Words - Bacillus thuringiensis, a-Endotoxin, Rice, Scirpophaga incertulas, Cnaphaiocrocis medinaiis, Biotoxicity assays, Immunoblot Introduction Bacillus thuringiensis (Bt), a gram positive, mo- tile, spore forming, rod-shaped bacterium, shows a broad range of pesticidal activity towards insect pests of agricultural, forestry, medical and veteri- nary importance. It is widely distributed in nature, specially in soil and insect-rich environments (Martin and Travers, 1989). Individual subspecies expresses insecticidal crystal proteins (also called 0- endotoxins or Cry proteins) that are quite specific for certain insect pests (Hofte and Whitely, 1989). Mode of action of Bt o-endotoxins consists of ingestion, solubilization, proteolytic activation in some cases, receptor binding, membrane insertion, ion channel formation and cell lysis (Karim and Riazuddin, 1997). Some Bt strains are widely used to control insect pests belonging to the orders Le- pidoptera, Diptera and Coleoptera (Schnepf et al., 1998). Moreover, within the same Bt pathotype, there are isolates with significantly different pesti- cidal activity (Dulmage, 198I). Today, the pesticidal spectrum of activity of Bt toxins expanded to at least six orders of insects (Lepidoptera, Diptera, Coleoptera, Hymenoptera, Homoptera and Mallo- phaga) as well as to another arthropod class (mites) and many invertebrate phyla (nematodes, flatworm and protozoa) (Schnepf et al., 1998). Thus, it seems advisable to search for new strains that can kill larvae that are relatively resistant to known varieties of Bt. There has been intense interest in recent years in collecting, separating and characterizing Bt isolates derived from different environmental sam- ples. Efforts to isolate Bt from different environ- ments worldwide are based on the assumption that * To whom correspondence should be addressed. National Centre of Excellence in Molecular Biology. Univ. Punjab, Canal Bank Road, Thokar Niaz Baig, Lahore-53700, Pakistan. e-mail, Shahid_97@ yahoo. com -61-