Discrimination of seven Pratylenchus species (Nematoda: Pratylenchidae) in Japan by PCR-RFLP analysis

Seven Pratylenchus species including P. brachyurus, P. coffeae, P. crenatus, P. neglectus, P. penetrans, P. zeae, and a Pratylenchus sp. which is morphologically indistinguishable from P. coffeae, were compared on the basis of the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis using a single nematode. The PCR primers used amplified the two internal transcribed spacer (ITS) regions, i.e. the 5.8S gene, and small portions of the 18S and 28S genes of the ribosomal DNA (rDNA). The isolates of P. penetrans, P. coffeae and Pratylenchus sp. yielded a single fragment after PCR amplification. Each fragment size was about 0.75 kb in P. penetrans, and about 1.1 kb in P. coffeae and Pratylenchus sp. The other nematodes, newly examined in this study, P. brachyurus, P. crenatus, P. neglectus and P. zeae, gave two PCR amplification products. The sizes of the two fragments were about 0.7 kb and 0.75 kb in P. brachyurus, P. neglectus and P. zeae, and about 0.8 kb and 0.9 kb in P. crenatus, respectively. These seven species were easily discriminated according to species-specific patterns by digesting the amplified products with endonucleases, AluI, HhaI, HinfI and TaqI.