A multiplex one-step PCR method for the simultaneous identification of Bursaphelenchus xylophilus, B. mucronatus and B. doui– three species within the xylophilus group

Summary The pine wood nematode, Bursaphelenchus xylophilus, causes severe damage to pines in Eastern Asia. Bursaphelenchus mucronatus and B. doui resemble closely B. xylophilus morphologically, moreover they were found frequently in this area recently. It is necessary to identify the three species precisely and rapidly. In this study, we report the results of a multiplex one-step polymerase chain reaction (PCR) utilizing five primers to identify and discriminate the three Bursaphelenchus species simultaneously. The multiplex one-step PCR yielded one fragment of about 1000 bp for all Bursaphelenchus populations tested. Futhermore, B. xylophilus, B. mucronatus and B. doui produced another fragment of about 100, 350 and 600 bp respectively. This approach is simple and reliable to simultaneously identify the above three species within the xylophilus group usually encountered together in a nematode assay.