Extraction of Bursaphelenchus xylophilus from wood and bark using zonal centrifugation.

The pine wood nematode, Bursaphelenchus xylophilus, is a quarantine organism in the EU. Legislation requires that samples from surveys in standing trees and imported wood products are analyzed for the presence of this nematode. The extraction method recommended by EPPO is incubation on Baermann funnel. We use the automated zonal centrifuge, an apparatus designed at ILVO, for routine extraction of nematodes from soil and plants. Earlier research (EU project FAIR1-CT95-0034) showed that the extraction efficiency of B. xylophilus from wood with the zonal centrifuge is similar to that with the Baermann funnel. Lately, we have been using the zonal centrifuge to detect B. xylophilus in imported (ornamental) bark, a commodity inspected more frequently in recent years. Extraction of B. xylophilus from bark with the zonal centrifuge was optimized using “spiked” bark (soaked in water infested with cultured B. xylophilus). Blending bark prior to nematode extraction, as done for other plant materials, resulted in cut pieces of nematodes, making subsequent morphological identification impossible. Therefore, similar to wood, 50 g bark is chopped in small pieces and incubated for 24 h in water. This material is then passed over an 850-µm sieve and nematodes are extracted from the resulting aqueous suspension, using zonal centrifugation. The extraction efficiency of B. xylophilus from spiked bark was > 85% for both the zonal centrifuge and the Baermann funnel in mistifier. A proficiency test with naturally infested bark and wood could tell us more about the detection efficiency of the zonal centrifuge compared with other techniques.