Diagnosis and quantification of the pine wood nematode, Bursaphelenchus xylophilus (Steiner & Buhner), in wood of Pinus thunbergii with real-time PCR

Abstract

A new diagnostic and quantitative real-time PCR assay was developed for the pine wood nematode, Bursaphelenchus xylophilus, which causes a serious epidemic wilt disease of susceptible landscape and forest pine trees. One, 10, 50, 100, 200, and 400 individuals of B. xylophilus in 100 mg of pine tissues were detected and quantified with two sets of primers coding the rDNA-ITS region and β-tubulin (tbb) gene of B. xylophilus, avoiding the need to extract nematodes with Baermann funnels. The assay was efficient, specific for pine wood nematode and sensitive enough to detect a single individual in pine tissues. To test the applicability of the assay for infested hosts, seedlings of the susceptible host species, Pinus thunbergii, were inoculated with B. xylophilus, and the dispersal and multiplication of B. xylophilus inside the seedlings were monitored by both Baermann funnel extraction and realtime PCR. Quantification by real-time PCR gave similar trends in estimates of the nematode population changes inside the host to those obtained by Baermann funnel extraction, although the size of the population estimated was larger for the new method than for the Baermann funnel extraction. The method developed in this study, which was expected to detect and quantify all life stages of the pine wood nematode should be useful for quarantine and field diagnosis.