A taxonomic database for the pinewood nematode Bursaphelenchus xylophilus, and other Bursaphelenchus species

The main objective of this project is to develop an information database with the inclusion of text (research articles, keys, bibliography, etc.), images (jpeg files), video clips (mpeg files), in order to collect in one physical format (CD-ROM) all relevant information related to the taxonomy of pine wood nematode (PWN), Bursaphelenchus xylophilus, as well as other Bursaphelenchus species. Initially, all major research papers will be scanned in ‘pdf’ format. All available images of quality and importance to the subject will also be scanned from either journals, bulletins, reports, or obtained privately from colleagues’ image collections. In certain cases, image quality (brightness, contrast, colour) may be enhanced with Adobe Photoshop. Short video clips will be compressed to ‘mpeg’ format for inclusion in the CD-ROM. An updated and broad bibliography will be included, as well as an intuitive taxonomic key, based on classical dicotomous keys but with a better and more intuitive presentation. Pre-existing images will be used in most cases; however, nematode specimens may need to be observed and photographed (jpeg image) in the lab. All the incorporated elements will be interlinked for ease of search from text to image to video. The total collection of images may be viewed individually using ‘Extensis Portfolio’. The final product will be PC and Mac compatible. Future editions/updates will be developed. Ultimately, researchers, technicians and political decision-makers will have at their disposal a practical and readily available source of information on this extremely important pest and pathogen. Colleagues working with PWN are encouraged to provide materials for this task

First insights into the genetic diversity of the pinewood nematode in its native area and around the world.

The pinewood nematode (PWN), Bursaphelenchus xylophilus is the causal agent of the pine wilt disease and is currently considered as one of the most important pests and pathogens in forest ecosystems. Native to North America, it has been introduced and it has spread in pine forests in Asia and more recently in Europe where it has now considerable economic and environmental impacts (annual loss of millions of pine trees worldwide). Anticipating the possibility of expansion of the PWN in European forests is essential. It is therefore important to decipher the invasion routes and better understand the invasion process of this species. To do this, 16 microsatellite markers have been developed and the study of genetic variability of the PWN was undertaken

Pine wilt disease: a worldwide threat to forest ecosystems

Nematodes are the most abundant metazoans, comprising more than 80% of all animals alive today. Since 1743, when Needham (Needham, 1743) described the first nematode, approximately 20,000 - 30,000 species have been named, with estimates of species remaining to be described ranging from 100,000 to 1 million (Blaxter, 2004; De Ley, 2000). Unfortunately, the taxonomic community is woefully inadequate for this task. The number of taxonomists currently describing new species of nematodes around the world is less than 100, and significant increases are not expected. If each of these taxonomists were able to describe 10 new species every year, it would take between 100 to 1,000 years to name these yet to be described species

Bursaphelenchus hofmanni Braasch, 1998 associated with peat growth substrate in hops nurseries in the Czech Republic

To date, seven Bursaphelenchus species have been re- ported in surveys of the Czech Republic (Cˇ ermák et al., 2013). However, the occurrence of some species, such as Bursaphelenchus hofmanni Braasch, 1998, is limited to single detections in imported coniferous wood (un- publ. data in Braasch, 2001). During a survey to deter- mine pathogenic agents on hops (Humulus lupulus L.) conducted by State Phytosanitary Administration (CZ) in hops fields and nurseries in 2012, B. hofmanni was found in a mixture of peat and soil in a hops seedling nurs- ery in Žatec (Bohemia, Czech Republic). The occurrence of Bursaphelenchus species in non-woody plants or sub- strates has been previously reported for species of the fungivorus group, such as B. gonzalezi Loof, 1964, B. hunti Giblin & Kaya, 1983 and B. fungivorus Franklin & Hooper, 1962. Species belonging to the hofmanni group are often associated with wood products such as packag- ing material (Gu et al., 2006) and, to our knowledge, this is the first time that this species has been found associated with peat substrate and soil. Nematodes were isolated from 60 g of peat substrate (3 parts peat to 1 part soil) associated with hops seedlings, and extracted using the Baermann funnel technique. Specimens belonging to Bursaphelenchus were killed and fixed in hot 4% formalin and transferred to pure glycerin according to De Grisse (1969). Two mature females and two males were used to establish a culture maintained in Botryotinia fuckeliana, growing on 5% malt extract agar (MEA). Nematode identification was confirmed by both mor- phological and molecular analyses (sequencing of the ITS, 18S and 28S rDNA loci). DNA was extracted as fol- lows: single specimens were collected into 20 μl of ex- traction buffer (10 mM Tris-HCl, pH 8.8; 1 mM EDTA; 1% Triton X-100 (v/v); 100 μg ml−1 Proteinase K) in a 1.5 ml Eppendorf tube. Each sample was ground using a micropestle and incubated at 55°C for 1 h and subse- quently at 95°C for 10 min. The mixture was used as a DNA template for PCR. The SSU, LSU and ITS regions of rDNA were amplified using a Phusion High Fidelity DNA polymerase (having 3′ → 5′ exonuclease activity) (NEB). GoTaq DNA polymerase (Promega) was added to a terminal 10 min/72°C elongation step to create an A- overhang for subsequent TA cloning

Worldwide invasion routes of the pinewood nematode:What can we infer from population genetics analyses?

Identifying the invasion routes and deter- mining the origin of new outbreaks of invasive species are of crucial importance if we are to understand the invasion process, improve or establish regulatory measures and, potentially, limit the damage. We focused here on the invasion of Europe by the pinewood nematode (PWN), Bursaphelenchus xylo- philus (Steiner & Buhrer, 1934; Nickle 1970; Nem- atoda: Aphelenchoididae), a major pest of forest ecosystems, native to North America and already invasive in Asia since the beginning of the twentieth century. We evaluated the genetic diversity and structure of worldwide field PWN samples by classical and Bayesian population genetics methods to Electronic supplementary material The online version of this article (doi:10.1007/s10530-014-0788-9) contains supple- mentary material, which is available to authorized users. S. Mallez (&) C. Castagnone P. Castagnone-Sereno T. Guillemaud UMR 1355 Institut Sophia Agrobiotech, INRA, 06903 Sophia Antipolis, France e-mail: [email protected] S. Mallez C. Castagnone P. Castagnone-Sereno T. Guillemaud UMR Institut Sophia Agrobiotech, Universite ́ de Nice Sophia Antipolis, 06903 Sophia Antipolis Cedex, France S. Mallez C. Castagnone P. Castagnone-Sereno T. Guillemaud UMR 7254 Institut Sophia Agrobiotech, CNRS, 06903 Sophia Antipolis Cedex, France determine the source of the European invasive pop- ulations and the number of introduction events in Europe. We found (1) a very strong spatial genetic structure in native PWN populations, (2) a very low level of polymorphism in each of the invaded areas and (3) contrasted results concerning the origin of European invasive populations. Our findings provide evidence for: (1) a large effect of genetic drift on the biological cycle of the PWN, due to intense demo- graphic bottlenecks during tree infections, not com- pensated for by effective dispersal of its vector; (2) a single introduction event for each of the invaded areas in Japan and Europe and a small effective size for the introduced populations and (3) a mainland Portuguese origin for PWN populations from Madeira. However, more sophisticated methods of invasion route infer- ence and broader sampling are required to conclu- sively determine the origin of the European outbreak. M. Espada P. Vieira M. Mota NemaLab/ICAAM – Instituto de Cieˆncias Agra ́rias e Ambientais Mediterraˆnicas, Universidade de E ́ vora, Nu ́ cleo da Mitra, Ap. 94, 7002-554 E ́ vora, Portugal J. D. Eisenback Department of Plant Pathology, Physiology, and Weed Science, Virginia Tech, Blacksburg, VA 24061, USA M. Harrell Nebraska Forest Service, University of Nebraska, Lincoln, NE 68583-0815, US