The Pinewood Nematode, Bursaphelenchus xylophilus

Proceedings of an International Workshop, University of Évora, Portugal, August 20-22, 2001

Edited by Manuel Mota and Paulo Vieira

According to the European Plant Protection Organization, the pinewood nematode (PWN), Bursaphelenchus xylophilus is a quarantine organism at the top of the list of the pathogenic species. PWN may be found in North America (Canada, USA and Mexico) and in East Asia (Japan, Korea, China and Taiwan) and has a highly destructive capability towards conifers, in a relatively short time, causing serious economic damage in Japan, China and Korea. This nematode surveying is extremely important and a correct identification of nematode species associated with conifers is essential. Several conifers species are very susceptible (Pinus sylvestris, Pinus nigra and Pinus pinaster), the last one (maritime pine) is a particulary important economic specie in Portugal, and in southern Europe.
In 1999, this nematode was found and identified for the first time in Portugal and in Europe. During 1999 and 2000, the Portuguese government, following an alert provided to European Community officials initiated an extensive national survey. During 2000 and 2001, research has been carried out regarding the morphobiometric as well as molecular (DNA: ITS-RFLP) characterization of the collected populations, as well as closely related species of Bursaphelenchus.
The book details the Proceedings of an international workshop held at the University of Évora in 2001, covering all major aspects of the bioecology of the pinewood nematode (PWN), Bursaphelenchus xylophilus, its insect vectors and effects on the tree.
Topics covered are: global issues and national surveys; morphology and molecular methods of identification of PWN; ecology and epidemiology; quarantine issues; tree physiology, resistance and histopathology; biology of PWN and relationships to its cerambycid vectors; control methods. General quarantine and political views are also presented.

Margarida Espada, John T. Jones and Manuel Mota

We have previously identified two secreted glutathione S-transferases (GST) expressed in the pharyngeal gland cell of Bursaphelenchus xylophilus, which are upregulated post infection of the host. This study examines the functional role of GSTs in B. xylophilus biology. We analysed the expression profiles of all predicted GSTs in the genome and the results showed that they belong to kappa and cytosolic subfamilies and the majority are upregulated post infection of the host. A small percentage is potentially secreted and none is downregulated post infection of the host. One secreted protein was confirmed as a functional GST and is within a cluster that showed the highest expression fold change in infection. This enzyme has a protective activity that may involve host defences, namely in the presence of terpenoid compounds and peroxide products. These results suggest that GSTs secreted into the host participate in the detoxification of host-derived defence compounds and enable successful parasitism.

Ana Rita Santos, Alexander Ryss, Manuel Mota and Paulo Vieira


The vulval pattern of six species of the genus Bursaphelenchus (B. abruptus, B. conicaudatus, B. fraudulentus, B. luxuriosae, B. mucronatus and B. xylophilus) was studied using scanning electron microscopy. A terminology for the vulval region structures observed is proposed herein and illustrated by micrographs and line drawings. It was shown that, of the studied species, only B. mucronatus and B. xylophilus share an identical morphology of the vulval region, all other species differing significantly from each other and from both B. mucronatus and B. xylophilus. This study indicates the diagnostic potential for variation in vulval morphology within Bursaphelenchus and it is recommended that such features are recorded in all future descriptions.

Oleg Kulinich, Manuel Mota, Paulo Vieira and Alexander Ryss


The 75 valid species of the genus Bursaphelenchus are listed together with their synonyms. Diagnostic characters and their states are discussed and illustrated. Tabular and traditional text keys are provided for the genus. Two new subspecies are proposed to distinguish populations of B. piniperdae and B. poligraphi, as described by Rühm (1956), from the original descriptions of these species published by Fuchs (1937). Known records of Bursaphelenchus species with their associated natural vectors, plants and plant families are given. Dendrograms of species relationships (UPGMA, standard distance: mean character difference) based on combined taxonomic characters and also on spicule characters only, are provided. Discussion as to whether the species groups are natural or artificial (and therefore purely diagnostic) is based on their relationships in the dendrogram and the vector and associated plant ranges of the species. Of the six species groups distinguished, two appear to represent natural assemblages, these being the xylophilus-group (with ten species) and the hunti-group (seven species), of which two, B. cocophilus and B. dongguanensis, form the cocophilus-cluster which is separated on the dendrogram from the main clusters. The remaining four species groups appear to be artificial and purely diagnostic in function, namely the aberrans-group (four species); the eidmanni-group (six species); the borealis-group (five species), and the piniperdae-group (43 species). Two new subspecies, both in the piniperdae-group, viz. B. piniperdae ruehmpiniperdae n. subsp. and B. poligraphi ruehmpoligraphi n. subsp., are proposed and diagnosed from B. piniperdae piniperdae and B. poligraphi poligraphi the respective type subspecies. Bursaphelenchus dongguanensis is regarded as being a valid member of the genus and its transfer to Parasitaphelenchus is rejected.

Koichi Hasegawa, Manuel M. Mota, Kazuyoshi Futai and Johji Miwa


Chromosome structure and behaviour in both meiosis of the germ cells and mitosis of the embryo from fertilisation to the two-cell stage in Bursaphelenchus xylophilus were examined by DAPI staining and three-dimensional reconstruction of serial-section images from confocal laser-scanning microscopy. By this method, each chromosome's shape and behaviour were clearly visible in early embryogenesis from fertilisation through the formation and fusion of the male and female pronuclei to the first mitotic division. The male pronucleus was bigger than that of the female, although the oocyte is larger and richer in nutrients than the sperm. From the shape of the separating chromosomes at anaphase, the mitotic chromosomes appeared to be polycentric or holocentric rather than monocentric. Each chromosome was clearly distinguishable in the male and female germ cells, pronuclei of the one-cell stage embryo, and the early embryonic nuclei. The haploid number of chromosomes (N) was six (2n = 12), and all chromosomes appeared similar. The chromosome pair containing the ribosomal RNA-coding site was visualised by fluorescence in situ hybridisation. Unlike the sex determination system in Caenorhabditis elegans (XX in hermaphrodite and XO in male), the system for B. xylophilus may consist of an XX female and an XY male.

Manuel Mota, Vera Valadas, Ana Catarina Penas and Kai Metge


Bursaphelenchus antoniae sp. n. is described and illustrated. Dauer juveniles were isolated from the body of the large pine weevil, Hylobius sp., collected from maritime pine (Pinus pinaster) stumps, in Portugal. Bursaphelenchus antoniae sp. n. was reared and maintained in P. pinaster wood segments and on Petri dish cultures of the fungi Botrytis cinerea and Monilinia fructicola. The new species is characterised by a relatively small body length of ca 583 μ m (females) and 578 μ m (males), a lateral field with two incisures, presence of a small vulval flap and a conoid female tail with a rounded or pointed terminus. Males have stout spicules with a disc-like cucullus and seven caudal papillae arranged as a single midventral precloacal papilla, one precloacal pair and two postcloacal pairs. In the character of the lateral field, B. antoniae sp. n. comes close to B. abietinus, B. rainulfi and B. hylobianum, whilst spicule characters place it within the piniperdae-group sensu Ryss et al. Morphologically, B. antoniae sp. n. is closest to B. hylobianum; the spicules of these two species having flattened, wing-like, alae on the distal third of the lamina. Bursaphelenchus antoniae sp. n. is distinguished from B. hylobianum on the arrangement of the caudal papillae (two vs three pairs). ITS-RFLP profiles and the failure to hybridise support the separation of the two species. Phylogenetic analysis of the new species, based on the 18S rDNA sequence, supports the inclusion of this new species in the B. hylobianum-group sensu Braasch. Sequence analysis of the 28S rDNA D2/D3 domain did not place the new species in a definite group.

Carlos Esplugues Mota, Rosa Lapiedra Alcamí, Carmen Azcárraga Monzonis, Manuel de Lorenzo Segrelles, José Juan Castelló Pastor and Carlos Esplugues Mota

Carlos Esplugues Mota, Rosario Espinosa Calabuig, Carmen Azcárraga Monzonis, Manuel de Lorenzo Segrelles, José Juan Castelló Pastor and Carlos Esplugues Mota

Wolfgang Burgermeister, Helen Braasch, Edmundo Sousa, Ana Catarina Penas, Manuel Mota, Kai Metge and Maria Antonia Bravo


A survey of aphelenchid nematodes (Nematoda: Aphelenchida) associated with maritime pine, Pinus pinaster, was conducted in Portugal in 1996 and 1999. A Bursaphelenchus species has been identified for the first time in the Iberian Peninsula. B. xylophilus is reported for the first time in Europe. It was found in very high numbers - up to 38 000 per 10 g of pine wood - inside a few declining trees infested with curculionid, cerambycid and scolytid beetles. Morphological observations, including shape of spicules, bursa, vulva, female tail end and stylet as well as morphometrics, were in accordance with the species description. Species-specific DNA fragment patterns were obtained using ITS-RFLP analysis, with five different restriction enzymes. The importance and implications of this finding are discussed. Premiere signalisation de Bursaphelenchus xylophilus au Portugal, at en Europe - Une enquete sur les nematodes Aphelenchides (Nematoda: Aphelenchida) associes au pin maritime (Pinus pinaster) a ete realisee au Portugal de 1996 a 1999. Une espece de Bursaphelenchus a ete identifiee pour la premiere fois dans la Peninsule Iberique. B. xylophilus est signale pour la premiere fois en Europe. Il a ete trouve en tres grand nombre - jusqu'a 38 000 individus pour 10 g de bois de pin - dans des arbres deperissants infestes par des Coleopteres Curculionides, Cerambycides et Scolytides. Les observations concernant la morphologie - en particulier la forme des spicules, la bourse, la vulve, l'extremite de la queue de la femelle et le stylet - de meme que les donnees morphometriques correspondent a la description de l'espece. Des sequences de fragments d'ADN specifique de l'espece ont ete obtenus par analyse ITS-RFLP a l'aide de cinq enzymes de restriction. L'importance et les implications de cette decouverte sont discutees.

Pedro Naves, Manuel Mota, Joana Pires, Ana Catarina Penas, Edmundo Sousa, Luís Bonifácio and Maria Antónia Bravo