A SCAR molecular marker to distinguish Bursaphelenchus mucronatus from the pinewood nematode, B. xylophilus

Bursaphelenchus mucronatus is closely related to the pinewood nematode Bursaphelenchus xylophilus, the causative agent of pine wilt disease. B. xylophilus became a devastating pest when it was introduced in the Far East; however, B. mucronatus is considered to have low virulence. Morphological similarities between B. xylophilus and B. mucronatus make the accurate morphological identification of both species difficult. Thus, it has become important to pay attention towards B. mucronatus impact and the need of discrimination of these two species. To distinguish among the two species, a B. mucronatus-specific sequence-characterized amplified region (SCAR) marker has been developed. The specific Random amplified polymorphic DNA (RAPD) fragment of B. mucronatus, OPY01-M-850 was excised from agarose gels and purified. The gel-purified fragment was cloned into the pGEM (R)-T Vector and subjected to sequencing. Based on the sequenced RAPD fragments, a number of SCAR primers were designed. It is demonstrated that OPY01-M-850 through primers Y01F/R can be transformed into a B. mucronatus-specific SCAR-Y-01-M-609 marker. Primers set Y01F/R had high specificity that could be used for the discriminative identification of B. mucronatus versus B. xylophilus.