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

  • Koji Matsunaga
  • Mitsuteru Akiba
  • Natsumi Kanzaki
  • Katsumi TogashiEmail author
Technical Note


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.


Cytochrome oxidase subunit I Discrimination Mitochondrial DNA PCR Pinewood nematode 



We are grateful to Drs. T. Aikawa and H. Kosaka of the Forestry and Forest Products Research Institute and Dr. S. Jikumaru of Hiroshima Prefectural Technology Research Institute for providing some isolates of B. xylophilus and B. mucronatus. We also thank Dr. A. Watanabe for his technical support. This work was supported in part by a Grant-in-aid for Young Scientists (B) from JSPS (no. 20780125) to KM and Grants-in-aid for Scientific Research from JSPS (nos. 18208013 and 22380081) to KT.


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Copyright information

© The Japanese Society of Applied Entomology and Zoology 2019

Authors and Affiliations

  1. 1.Kyushu Regional Breeding Office, Forest Tree Breeding CenterForestry and Forest Products Research InstituteKoshiJapan
  2. 2.Forestry and Forest Products Research InstituteTsukubaJapan
  3. 3.Kansai Research CenterForestry and Forest Products Research InstituteFushimiJapan
  4. 4.Graduate School of Agricultural and Life SciencesUniversity of TokyoTokyoJapan

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