Discrimination of the Main Pratylenchus Species (Nematoda: Pratylenchidae) in Japan by PCR-RFLP Analysis

Abstract

PCR-RFLP analysis of the internal transcribed spacer region of ribosomal DNA was performed to distinguish species on individual Pratylenchus penetrans, P. coffeae, P. vulnus, and Pratylenchus sp. (unidentified species near P. coffeae). A single nematode of each species was ruptured in a drop of lysis buffer, heat-treated, and added directly to a PCR reaction mixture in a microcentrifuge tube. This simple DNA extraction with lysis buffer was efficient for preparing template DNA in PCR amplification. This extraction method was applicable to all the developmental stages from egg to adult regardless of sex. The sizes of amplified products were about 0.8 kb in P. penetrans and P. vulnus, and about 1.1 kb in P. coffeae and Pratylenchus sp. These four species were easily discriminated from each other by digesting the amplified products with endonucleases, AluI, DdeI, HhaI, HinfI, and TaqI.