MOLECULAR IDENTIFICATION OF ENTOMOPATHOGENIC NEMATODE SPECIES (STEINERNEMA AND HETERORHABDITIS)BY RFLP ANALYSIS OF PCR AMPLIFIED rDNA INTERNAL TRANSCRIBED SPACER (ITS)REGION

A molecular technique for identification of 7 species of entomopathogenic nematodes (EPN)currently used in insect biocontrol was provided. The rDNA internal transcribed spacer (ITS)regions of S.feltiae, S.glaseri,H.bacteriophora, H.zealandica, H.indicus, H.megidi and two strains of S.carpocapsae were amplified with universal primers from the single worm or the purified genomic DNA. The RFLP analysis was generated with six restriction endonucleases(Alu Ⅰ,Hinf Ⅰ,Mbo Ⅰ,Hae Ⅲand PvuⅡ)following by 2.0% agarose gel electrophoresis.Four enzymes (AluⅠ,HinfⅠ,RsaⅠ and MboⅠ)yielded patterns that could be used for differentiating all 7 species of EPN examined, while Hae Ⅲand PvuⅡ provided little taxonomic information. Two strains of S.carpocapsae (All and A24) gave the same patterns for the six enzymes examined, so do the axenic and monoxenic H.bacteriophora. This study showed for the first time that the presence of symbiotic bacteria had no effect on the PCR-RFLP pattern of ITS of nematodes. It also demonstrated that ITS was an ideal marker for species identification and PCR-RFLP technique provided an accurate and reliable molecular means for the taxonomy of EPN. With this technique, unambiguous and rapid identification of EPN could be achieved even using single infective juvenile. Therefore, the technique provided a simpler and more rapid means for monitoring field release of EPN and assessing biologic virulence of nematodes than the bioassay test used previously.