Nematology, 2011, Vol. 13(6), 755-757
Short communication
Incidence of the pinewood nematode Bursaphelenchus xylophlius Steiner &
Buhrer, 1934 (Nickle, 1970) in Spain
Lee ROBERTSON 1,∗ , Susana C OBACHO A RCOS 1 , Miguel E SCUER 2 ,
Remedios S ANTIAGO M ERINO 3 , Guadaloupe E SPARRAGO 3 ,
Adela A BELLEIRA 4 and Alfonso NAVAS 1
The pine wood nematode (PWN) Bursaphelenchus
xylophilus Steiner & Buhrer, 1934 (Nickle, 1970), is
a pathogenic species in the genus Bursaphelenchus Fuchs,
1937. The introduction and establishment of PWN in
the Setubal Peninsula, Portugal (Mota et al., 1999),
and the evidence that Monochamus galloprovincialis is
its insect vector in this country (Sousa et al., 2001),
has stimulated studies on its further distribution and its
potential vector insects in Europe, including Spain. There
is a clear risk of spread of pine wood nematode into
Spanish territory from Portugal, both as a result of natural
spread and due to the important timber trade between the
two countries. The occurrence of Bursaphelenchus spp. in
Spain has been previously studied (Abelleira et al., 2002,
2003; Escuer et al., 2003, 2004; Robertson et al., 2008).
These studies have shown, in addition, the distribution
of insects belonging to the Cerambycidae, Curculionidae
and Scolytidae families (Cobos, 1986; Gil Sánchez &
Pajares Alonso, 1986; Vives, 2000; Verdugo Paez, 2004),
which are known to be vectors of Bursaphelenchus
spp. other than B. xylophilus and are often found to
be associated with stressed, dead or decaying conifers.
Although it is generally accepted that B. xylophilus is
the only truly pathogenic species (reviewed by Jones
et al., 2008), other species may be involved in pine
forest decline in some European regions or, alternatively,
may simply be opportunistic infectors of stressed trees
(Mamiya, 1999; Caroppo et al., 2000; Skarmoutsos et
al., 2000). Pinus pinaster is the most representative
conifer of the indigenous Spanish vegetation, followed
by P. halepensis, P. nigra, P. pinea, P. radiata and P.
sylvestris (MAPA, 2001), with P. nigra, P. pinaster and P.
sylvestris being the most susceptible species to PWN, and
P. halepensis, P. pinea and P. radiata with an intermediate
susceptibility (Evans et al., 1996). This, coupled with the
Spanish climate, suggests that there is the potential for
serious disease development in the event of B. xylophilus
becoming established.
In October 2008, a wood sample collected by the regional plant health laboratory (Consejeria de Agricultura
and Desarrollo Rural, Junta de Extremadura, Spain) from
‘Villanueva de la Sierra’ 20 km from the Portuguese border was analysed and the extracted nematodes identified
as B. xylophilus. The nematode population was subsequently multiplied on Botrytis cinerea and the nematodes
were extracted from the agar plates using a modified Baermann funnel technique. Fifty nematodes were fixed in
FA (formalin/acetic acid, 95/5), mounted in anhydrous
glycerin and studied under a Dialux microscope (Leica,
BMLB, Wetzlar, Germany) equipped with Nomarski interference in order to allow identification by morphology
and morphometrics. The same multiplied population was
also analysed using molecular diagnostics by polymerase
chain reaction restriction fragment length polymorphism
(PCR-RFLP) analysis. Twenty nematodes were placed in
lysis buffer (1× PCR buffer, 60 μg ml−1 protease K),
heated to 60◦ C for 1 h and then heated to 94◦ C for 15 min.
The nematodes were centrifuged and 5 μl of the su-
1
Museo Nacional de Ciencias Naturales, Dpto Biodiversidad y Biología Evolutiva, CSIC, C/José Gutiérrez Abascal 2,
Madrid 28006, Spain
2
Instituto de Ciencias Agrarias, CISC, C/Serrano 115dpdo, Madrid 28006, Spain
3
Servicio de Sanidad Vegetal Laboratorio de Diagnóstico Ctra, San Vicente 3, Badajoz 06071, Spain
4
Estación Fitopatológica do Areeiro, Subida a la Robleda s/n, Pontevedra 36153, Spain
∗
Corresponding author, e-mail: lee.r@mncn.csic.es
Received: 2 March 2011; revised: 12 May 2011
Accepted for publication: 12 May 2011; available online: 8 July 2011
Keywords: conifer, pine wilt, Pinus pinaster.
© Koninklijke Brill NV, Leiden, 2011
Also available online - www.brill.nl/nemy
DOI:10.1163/138855411X578888
755
Short communication
Fig. 1. Morphological characteristics of Bursaphelenchus xylophilus 197 from Extremadura, Spain. A: Female tail; B: Vulval flap;
C: Male spicule.
pernatant was used for PCR amplification. The forward
primer 5 -CGTAACAAGGTAGCTGTAG-3 (Ferris et al.,
1993) and reverse primer 5 -TTTCACTCGCCGTTACTA
AGG-3 (Vrain, 1993) were used in reactions containing, 10 mm Tris-HCl (pH 8.3), 1.5 mm MgCl2 , 50 mm
KCl, 200 mm each of dATP, dCTP, dGTP and dTTP and
1 unit of Taq DNA polymerase (Biotools B and M labs,
Madrid, Spain). Initial denaturalisation was carried out
for a period of 2 min at 94◦ C followed by 35 cycles of
94◦ C for 1 min, 55◦ C for 1 min, 72◦ C for 1 min and followed by a final 5-min extension period at 72◦ C. Amplified DNA fragments were digested with the restriction enzymes RsaI, HaeIII, MspI, HinfI and AluI (New England
Biolabs, Massachusetts, MA, USA) following the manufacturer’s instructions and separated by a 2% agarose gel.
This analysis was repeated three times. Digestion products were compared with previously published data for B.
xylophilus and B. mucronatus (Hoyer et al., 1998). Based
on morphological characters (Fig. 1) and molecular analysis (Fig. 2), the nematode population was confirmed to
be B. xylophilus.
Since 2008, preventative measures have been carried
out based on an official contingency programme headed
by the Spanish Ministry of Environmental Affairs in
756
Fig. 2. ITS-RFLP analysis of Bursaphelenchus xylophilus.
M) 100-bp molecular markers. 1) ITSAmplicon, 2) RsaI, 3)
HaeIII, 4) MspI, 5) HinfI, 6) AluI.
agreement with the European Union. As a consequence
of systematic sampling along the Portuguese border and
national territory, B. xylophilus was found in Sierra de
las Nieves (Lugo province in Galicia) in 2010, 10 km
from the Portuguese border. This finding was officially
reported to the European authorities. This last population
was morphologically and molecularly identified as B.
xylophilus following the EPPO protocols. In the future,
Nematology
Short communication
molecular studies are planned to compare the Spanish outbreaks with intercepted material from Portugal. Vector
studies are also planned given that M. galloprovincialis
is also widely spread throughout Spain.
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