A rapid β-Myrcene-attractant assisted wood sampling method for PCR-based detection of Bursaphelenchus xylophilus in Pinus massoniana wood tissue

Pine wood nematode (PWN) Bursaphelenchus xylophilus is listed as a quarantine pest in the legislation of more than 40 countries. PWN density is usually too low in wood tissue for successful detection and species identification by existing methods. The objective of this study is to increase PWN density in the wood sample through using β-Myrcene for the subsequent PWN detection by PCR assay without separate nematode extraction steps. A hole (of 1cm depth and 1cm diameter) was drilled in a PWN infesting wood sample of P. massoniana, into which 1000 μl of 10-2 mol-L-1 β-Myrcene solution was sprayed, kept for 5 min, then a sterilized cotton ball holding 600 μl 10-2 mol-L-1 β-Myrcene solution was also placed into the hole, before it was covered by a parafilm and left for 1.5 h at 20 in an air conditioned chamber. The wood sample was then boiled for 15 min to kill nematodes in it. Nematode density in the wood tissue around treated hole was significantly increased, compared with that in the control hole, which was not treated by β-Myrcene. Five mg wood tissue from bottom part of the treatment hole was successfully used for detecting PWN by the PCR assay. The entire procedure only takes 7 hrs. This study provides a rapid β-Myrcene assisted wood sampling method for PCR-based detection of PWN in P. massoniana wood tissue, particularly when the PWN density is low.