Detection of Bursaphelenchus Xylophilus, Causal Agent of Pine Wilt Disease on Pinus pinaster in Northwestern Spain.

The pine wood nematode, Bursaphelenchus xylophilus (Steiner & Buhrer) Nickle, a quarantine organism, causes serious damage to pines worldwide. In Europe, it was first detected in Portugal in 1999 (3) and the pathogen was thought to be restricted to this area. However, in 2008, B. xylophilus was isolated from a single tree in the Cáceres Region (Extremadura) of Spain, bordering Portugal (2). The region of Galicia has approximately 383,632 ha of Pinus pinaster Aiton that constitutes more than 40% of the surface of Spain. Since 1999, we have analyzed 5,155 samples to monitor the presence of the pathogen. In 2008, a Spanish national contingency plan established three delimiting sampling areas, including a high risk area 20 km from the border with Portugal (2 × 2 km grid), a medium risk area 80 km wide (4 × 4), and another area covering the whole region (8 × 8). The plan required collecting samples from symptomatic trees. In 2010, in the high risk area, 307 sites were surveyed in coniferous forests. At each site, wood chip samples were collected from five pine trees. The collected wood chips were then incubated for 15 days in the lab and nematodes were extracted by Baermann's funnel method. B. xylophilus was detected from a decayed mass of P. pinaster from the As Neves Municipality (Pontevedra, Galicia). Affected specimens showed typical symptoms associated with pine wilt, including needle discoloration and death of branches. B. xylophilus was identified by morphological and molecular methods. Morphological characteristics included high lips, constricted heads, and short stylets with reduced basal knobs. Females had rounded tails, some with a short mucro, and flat vulva, while males had spicules curved with a cucullus. Measurements of these nematodes (10 females: body length = 720.99 ± 123.87 μm, a = 41.07 ± 5.83, b = 9.22 ± 3.44, c = 26.57 ± 4.13, V = 73.2, stylet length = 14.91 ± 1.65 μm; 10 males: body length = 576.4 ± 88.16 μm, a = 38.12 ± 5.36, b = 7.83 ± 0.39, c = 23.07 ± 2.59, stylet length = 14.63 ± 1.95 μm, spicules length = 22.5 ± 2.21 μm) were similar to the isolates found in Portugal described by Penas et al.(4) and smaller than described by Mota et al. (3). Molecular diagnosis was done following the protocols recommended by the EPPO (1): (i) Amplification of satellite DNA of B. xylophilus by PCR obtaining fragments of 160, 320, and 480 bp; (ii) PCR amplification of a region of 77 bp satellite DNA of B. xylophilus by Taqman Real Time; and (iii) PCR-restriction fragment length polymorphism of the internal transcribed spacer (ITS) region of Bursaphelenchus spp. nrDNA obtaining the restriction pattern for B. xylophilus. The ITS product amplified by PCR was also sequenced, showing a 99% homology with the sequences of B. xylophilus deposited in GenBank. A sequence of this nematode was submitted to GenBank database and assigned the number HQ646254. On the basis of these diagnostic characteristics, we have confirmed that B. xylophilus is now present in Galicia (northwestern Spain), which is one of the most productive and important region of Spain for forestry. References: (1) EPPO Bull. 39:344, 2009. (2) EPPO Rep. Serv. 3:2010/051, 2010. (3) M. M. Mota et al. Nematology 1:727, 1999. (4) A. C. Penas et al. J. Nematol. Morphol. Syst. 10:137, 2008.