A nested PCR assay targeting the DNA topoisomerase I gene to detect the pine wood nematode, Bursaphelenchus xylophilus

The pine wood nematode (PWN), Bursaphelenchus xylophilus, is a disastrous pathogen of the pine forests in East Asia and Europe. Plant quarantine is one of the most important ways to prevent its infection in current situation. A nested polymerase chain reaction (PCR) assay targeting the topoisomerse I gene has been developed to detect PWN in this study. To assess the specificity of the assay, 44 morphologically characterized nematode isolates including B. xylophilus, B. mucronatus, B. hofmanni, Seinura wuae, S. lii and Aphelenchoides macronucleatus were tested. Positive reactions characterized by amplification product of 509 bp were shown from all isolates of PWN. The nested PCR assay can detect 50 femtogram (fg) of template DNA or one individual nematode, as small as an egg. The validity was evaluated by analyzing the nematode samples extracted from the nematode-infested wood in the field. These results show that the assay is a specific, sensitive method for detection of PWN with the potential in relation to the pest risk assessment and quarantine regulations.