Ribotyping of entomopathogenic Verticillium lecanii in Japan

To estimate the genetic diversity in 30 isolates of Verticillium lecanii from aphids, whiteflies, mite and black pine in Japan, including two commercialized strains (Mycotal and Vertalec), DNA polymorphisms in ribosomal DNA of those isolates were analyzed using polymerase chain reaction (PCR). The internal transcribed spacer (ITS) and intergenic spacer (IGS) regions of the nuclear ribosomal RNA gene of each isolate were analyzed by PCR-RFLP (restriction fragment length polymorphism). The size of the PCR product from the ITS region was ∼ 580 bp in 27 of the isolates. A 600 bp ITS product was detected in Mycotal and Vertalec. One Japanese isolate produced both the 580 bp and 600 bp products. Enzymatic digestion of the ITS region with Sau3A I, Msp I, Hae III and Rsa I revealed RFLPs that consisted of eight haplotypes. Mycotal and Vertalec were specific haplotypes that differed from other isolates. The Japanese isolates had a complex relationship with the original host, but we identified several specific haplotypes common to an aphid origin. Ten distinct IGS haplotypes were detected in the IGS region, some of which were associated with aphid and whitefly origins. These results suggest that the haplotype of rDNA RFLP analysis can be used for studying genetic diversity in V. lecanii.