CHARACTERIZATION OF BACILLUS-THURINGIENSIS MUTANTS AND NATURAL ISOLATES BY MOLECULAR METHODS

Two Bacillus thuringiensis var. kurstaki HD-1 mutants, two Bacillus th uringiensis var. israelensis HD-500 mutants, and four rice grain dust isolates were characterized using microscopic examination and protein profiles of purified crystals on sodium dodecyl sulfate - polyacrylami de gel electrophoresis. Specific detection of cryI- and cryIV-type gen es was performed in a polymerase chain reaction using cryI and cryIV-s pecific oligonucleotide primers. The cry-type genes under study consis ted of cryIA(a), cryI(A)b, cryI(A)c, cryIB, and cryIV. Presence or abs ence of the cryI- and cryIV-type genes was further confirmed by Southe rn blotting followed by hybridization with specific cryI and cryIV gen e fragments. A genetically modified strain of B. thuringiensis var. ku rstaki HD-I, called OZK-13 and obtained following mutagenesis with ozo ne, was shown to contain cryIA(a), cryIA(b), and cryIA(c) genes. Anoth er kurstaki HD-1 mutant, called NGK-13 and obtained following treatmen t with N-methyl-N-1 -nitro-N-nitrosoguanidine (MNNG), was shown to hav e lost the cryIA(b) gene while retaining the cryIA(a) and cryIA(c) gen es. NGI-23-1, an oligosporogenous-multicrystalliferous mutant of B. th uringiensis var. israelensis (Bti) HD-500, obtained following treatmen t with MNNG contained cryIV-type genes. NGI-22, an oligosporogenous -a crystalliferous mutant of Bti HD-500, contained no cryI- nor cryIV-typ e genes. The rice grain dust isolate BT-285 contained the cryIA(a) and cryIA((c) genes. Isolate BT-14 contained only the cryIA(c) gene, wher eas isolate BT-209 contained cryIA(a), cryIA(b), and cryIB genes. Isol ate BT-205 contained no cryI- nor cryIV-type genes. Bacillus thuringie nsis mutants and natural isolates shown to contain cryI-type genes wer e tested for their insecticidal activities in a series of bioassays ag ainst Hyphantria cunea Drury (Lepidoptera: Arctiidae). All cryI-carryi ng strains were toxic against the insect larvae. BT-205 was also teste d and exhibited no toxicity against the insect larvae.