Distribution ofcryV-Type Insecticidal Protein Genes inBacillus thuringiensisand Cloning ofcryV-Type Genes fromBacillus thuringiensissubsp.kurstakiandBacillus thuringiensis subsp.entomocidus

DNA dot blot hybridizations with acryV-specific probe and acryI-specific probe were performed to screen 24 Bacillus thuringiensis strains for their cryV-type (lepidopteran- and coleopteran-specific) and cryI-type (lepidopteran-specific)insecticidalcrystalproteingenecontents,respectively.ThecryV-specificprobehybridizedto 12 of theB. thuringiensisstrains examined. Most of thecryV-positive strains also hybridized to thecryI-specific probe, indicating that thecryVgenes are closely related tocryIgenes. TwocryV-type genes,cryV1andcryV465, were cloned fromB. thuringiensissubsp.kurstakiHD-1 andB. thuringiensissubsp.entomocidusBP465, respectively, and their nucleotide sequences were determined. The CryV1 protein was toxic to Plutella xylostella and Bombyx mori, whereas the CryV465protein was toxic only toPlutella xylostella. Bacillus thuringiensis is a gram-positive bacterium that produces proteinaceous crystals during sporulation. Many differentB. thuringiensiscrystal protein genes have been identified, and the toxicity of the encoded proteins has been investigated. At least four major classes of insecticidal protein genes have been identified: cryI, cryII, cryIII, and cryIV. These genes encode lepidopteran-specific (CryI), lepidopteran- and dipteranspecific (CryII), coleopteran-specific (CryIII), and dipteranspecific (CryIV) proteins, respectively (11). After the discovery of various B. thuringiensis strains that have toxicity against a wide spectrum of insect pests, these bacteria have been developed as the most successful commercialmicrobialinsecticideforthecontrolofvariousinsectpests. However, several insect pest species of globally important crops have little or no susceptibility to the existing insecticidal protein of variousB. thuringiensisstrains. Development of resistanceininsectpestsisanothersignificantproblemintheuse ofB. thuringiensisas a successful microbial insecticide.B. thuringiensis had been used commercially for more than two decades without reports of substantial resistance development in field populations. However, the diamondback moth, Plutella xylostella,hasevolvedresistanceintheopenfieldinHawaiiand