Characterization ofBacillus thuringiensismutants and natural isolates by molecular methods

Abstract

Two Bacillus thuringiensis var. kurstaki HD-1 mutants, two Bacillus thuringiensis 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 – polyacrylamide gel electrophoresis. Specific detection of cryI- and cryIV-type genes was performed in a polymerase chain reaction using cryI and cryIV-specific oligonucleotide primers. The cry-type genes under study consisted of cryIA(a), cryI(A)b, cryI(A)c, cryIB, and cryIV. Presence or absence of the cryI- and cryIV-type genes was further confirmed by Southern blotting followed by hybridization with specific cryI and cryIV gene fragments. A genetically modified strain of B. thuringiensis var. kurstaki HD-1, called OZK-13 and obtained following mutagenesis with ozone, was shown to contain cryIA(a), cryIA(b), and cryIA(c) genes. Another kurstaki HD-1 mutant, called NGK-13 and obtained following treatment with N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), was shown to have lost the cryIA(b) gene while retaining the cryIA(a) and cryIA(c) genes. NGI-23-1, an oligosporogenous–multicrystalliferous mutant of B. thuringiensis var. israelensis (Bti) HD-500, obtained following treatment with MNNG contained cryIV-type genes. NGI-22, an oligosporogenous–acrystalliferous mutant of Bti HD-500, contained no cryI- nor cryIV-type 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, whereas isolate BT-209 contained cryIA(a), cryIA(b), and cryIB genes. Isolate BT-205 contained no cryI- nor cryIV-type genes. Bacillus thuringiensis mutants and natural isolates shown to contain cryI-type genes were tested for their insecticidal activities in a series of bioassays against Hyphantria cunea Drury (Lepidoptera: Arctiidae). All cryI-carrying strains were toxic against the insect larvae. BT-205 was also tested and exhibited no toxicity against the insect larvae.Key words: Bacillus thuringiensis, δ-endotoxin crystal, cry-type genes, polymerase chain reaction.