EXTENDED SCREENING BY PCR FOR 7 CRY-GROUP GENES FROM FIELD-COLLECTED STRAINS OF BACILLUS-THURINGIENSIS

An extended multiplex PCR method was established to rapidly identify a nd classify Bacillus thuringiensis strains containing cry (crystal pro tein) genes toxic to species of Lepidoptera, Coleoptera, and Diptera. The technique enriches current strategies and simplifies the initial s tages of large-scale screening of cry genes by pinpointing isolates th at contain specific genes or unique combinations of interest with pote ntial insecticidal activities, thus facilitating subsequent toxicity a ssays. Five pairs of universal primers were designed to probe the high ly conserved sequences and classify most (34 of about 60) genes known in the following groups: 20 cry1, 3 cry2, 4 cry3, 2 cry4, 2 cry7, and 3 cry8 gens. The DNA of each positive strain was probed with a set of specific primers designed for 20 of these genes and for cry11A. Twenty -two distinct cry-type profiles were identified from 126 field-collect ed B. thuringiensis strains. Several of them were found to be differen t from all published profiles. Some of the field-collected strains, bu t none of the 16 standard strains, were positive for cry2Ac. Three sta ndard and 38 field-collected strains were positive by universal primer s but negative by specific primers for all five known genes of cry7 an d cry8. These field-collected strains seem to contain a new gene or ge nes that seem promising for biological control of insects and manageme nt of resistance.