A simple method for distinguishing Bursaphelenchus xylophilus, B. mucronatus mucronatus, and B. m. kolymensis (Nematoda: Aphelenchoididae) by polymerase chain reaction with specific primers designed based on cytochrome oxidase subunit I genes

Based on DNA sequences of mitochondrial cytochrome oxidase subunit I (mtCOI) genes, three PCR forward primers specific for Bursaphelenchus mucronatus mucronatus , B. m. kolymensis , and B. xylophilus (Nematoda: Aphelenchoididae), and one common PCR reverse primer were developed to determine the interspecific hybrids in a population. PCR amplification with the new primer pairs was expected to produce DNA fragments of 286 bp, 117 bp and 479 bp for B. m. mucronatus , B. m. kolymensis , and B . xylophilus , respectively. Electrophoresis of PCR products with the new primer pairs distinguished the two B. mucronatus subspecies and B. xylophilus collected in Japan. It also exhibited a band-free image for Bursaphelenchus doui , B. niphades , B. sinensis , B. firmae , Bursaphelenchus sp. (NK230), and Aphelenchoides stammeri , whereas it exhibited one (ca. 100-bp DNA fragment) and two (ca. 100 and 500-bp DNA fragments) bands for B. poligraphi and B. yongensis , respectively. As PCR products with other primer pairs for nuclear ribosomal RNA (rRNA) gene distinguished B. mucronatus (210 bp DNA fragment), B. xylophilus (557 bp fragment), and the eight other nematode species (no amplification), primer pairs for mtCOI and rRNA genes can determine whether B. poligraphi resides in specified trees or in study pine stands. Thus, those primer pairs would be helpful to find out hybrids between B. mucronatus and B. xylophilus and to analyze the dynamics of hybrids and introgressants in hybrid-derived populations in field and laboratory.