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

Abstract

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.

In vitro co-cultures of Pinus pinaster with Bursaphelenchus xylophilus: a biotechnological approach to study pine wilt disease

“…In 1999, the nematode was detected in Portugal (Mota et al 1999) endangering European pine forests and has progressed throughout large areas of the country (Mota and Vieira 2008). In 2010, it was also found in Madeira island (Fonseca et al 2012), and in 2011 for the first time in Spain (Abelleira et al 2011). It was classified as an A2 type quarantine pest by the European and Mediterranean Plant Protection Organization (EPPO 2012).…”

Evidence for the involvement of ACC deaminase from Pseudomonas putida UW4 in the biocontrol of pine wilt disease caused by Bursaphelenchus xylophilus

“…In Europe, PWD was first reported in 1999 in Portugal (Mota et al 1999). Since then, the disease has been observed throughout the Portuguese mainland and, more recently, PWD foci were found in the island of Madeira (Fonseca et al 2012) and also in Spain (Abelleira et al 2011). Considering the potential climate change scenarios and possible introduction points in Eastern and Northern Europe (Robinet et al 2011), PWD may become one of the most common coniferous diseases in European forests in the near future.…”

Bioactivity against Bursaphelenchus xylophilus: Nematotoxics from essential oils, essential oils fractions and decoction waters

“…In 1999, Portugal became its entry point to the European pine forests (Mota et al, 1999). Since then, this phytoparasite has been progressing through the country, having been found in Madeira Island, in 2010 (Fonseca et al, 2012), and in Spain, in 2011 (Abelleira et al, 2011). It has been classified as an A2 type quarantine pest by the European Plant Protection Organization (EPPO, 2012).…”