Nematology, 2012, Vol. 14(8), 899-911 The pine wood nematode, Bursaphelenchus xylophilus, in Portugal: possible introductions and spread routes of a serious biological invasion revealed by molecular methods Vera VALADAS 1,MartaLARANJO 2,3,PedroBARBOSA 1,MargaridaESPADA 1, ∗ Manuel MOTA 1, and Solange OLIVEIRA 2 1 NemaLab-ICAAM & Departamento de Biologia, Universidade de Évora, Évora, Portugal 2 Lab. Microbiologia do Solo-ICAAM & Departamento de Biologia, Universidade de Évora, Évora, Portugal 3 IIFA (Instituto de Investigação e Formação Avançada), Universidade de Évora, Évora, Portugal Received: 15 December 2011; revised: 14 February 2012 Accepted for publication: 17 February 2012; available online: 2 May 2012 Summary – The pine wood nematode (PWN), Bursaphelenchus xylophilus, the causal agent of pine wilt disease (PWD), is a major world-wide pathogen and pest of pine, with impacts on forest health, natural ecosystem stability and international trade. In Portugal, PWN was first diagnosed in 1999, the first occurrence also for Europe. The disease was recently detected on the island of Madeira and in northern Spain. In an attempt to search for more reliable and robust molecular markers that enable the study of intraspecific variability of B. xylophilus from different geographic locations, the intergenic spacer (IGS) region of the 5S rRNA gene and inter-simple sequence repeats (ISSR) analysis were used to determine the genetic relationships among 43 B.xylophilus isolates from Portugal, China, Japan, South Korea and USA. IGS sequence analysis showed that this region can only be used to establish interspecific relationships, since no differences were detected among Portuguese isolates from different geographic locations. Fingerprints obtained with ISSR show high genetic variability among Portuguese isolates, except for the ones obtained prior to 2008. The ISSR dendrogram suggests the spread of the disease inside continental Portugal and to Madeira. Until 2008, B. xylophilus populations found in continental Portugal showed low genetic diversity, pointing to a single introduction, probably from Asia, whereas recent populations from continental Portugal (2009-2010) and Madeira show high genetic diversity, suggesting multiple introductions from different origins. Keywords – genetic diversity, IGS ISSR, intraspecific variability, ITS, molecular marker, pine wilt disease. The pine wood nematode (PWN), Bursaphelenchus xy- the most high-risk pathways of biological invasions, car- lophilus (Steiner & Buhrer, 1934; Fuchs, 1937), is indige- rying forest insects and pathogens into new environments nous to North America and is widespread in natural conif- (Evans et al., 1996; Tkacz, 2002). Many Bursaphelenchus erous forests in Canada and USA (Sutherland & Peterson, species, including the PWN, have been routinely inter- 1999). At present, B. xylophilus is considered one of the cepted in packaging and wood products in several coun- most important pests and pathogens in the world (Web- tries, e.g., Austria (Tomiczek et al., 2003), China (Gu et ster & Mota, 2008). The general fear of establishment al., 2006), Finland (Tomminen et al., 1991) and Germany of PWN, the causal agent of pine wilt disease (PWD), (Braasch et al., 2001). Furthermore, recent detection of into countries where conifer forests assume great impor- PWN in packaging wood imported from countries con- tance, stems from the devastating damage caused by this nematode to pine forests (Mamiya, 2004; Shin & Han, sidered free of this pest, due to the repeated use and cir- 2006). The introduction of PWN into non-native areas culation of this type of wood material, e.g., Brazil, Bel- (outside of North America) is primarily associated with gium, Italy and Spain (Gu et al., 2006), undoubtedly un- trade and the global flow of forest products (Bergdahl derlines the importance of trade globalisation for the po- & Halik, 1999; Webster, 2004). Non-manufactured wood, tential entry/establishment of this pathogen into endemic especially in raw log form, has been identified as one of forests worldwide. ∗ Corresponding author, e-mail: [email protected] © Koninklijke Brill NV, Leiden, 2012 DOI:10.1163/156854112X632673 899 V. Valadas et al. The introduction and spread of this nematode has re- are commonly used to study intraspecific variation and sulted in huge annual losses due to the rise in mortality evaluate genetic diversity. This method was described for and growth loss of pine forest, and to the increased costs eukaryotes in general (Zietkiewicz et al., 1994), but has of management procedures and disease control (Mamiya, been predominantly applied to plants (Reddy et al., 2002). 2004; Shimazu, 2006). In addition, the introduction of Studies of animal ISSR polymorphisms have been use- PWN has resulted in vast and irreversible changes to na- ful in analysing the pathway of introduced species (Ab- tive forest ecosystems, including tree species conversions, bot, 2001; Metge et al., 2006). ISSR provides informa- wildlife habitat destruction, soil and water conservation tive markers because they are highly polymorphic. ISSR and loss of biodiversity (Kiyohara & Bolla, 1990; Suzuki, markers are highly reproducible due to stringent annealing 2002). The PWN has already been established for more temperatures, long primers, and low primer-template mis- then 100 years in Japan (Yano, 1913) and, in the past two match (semi-arbitrary primers that anchor on SSR loci) decades, new reports of PWD came mainly from East Asia (Jones et al., 1997). (Cheng et al., 1983; Yi et al., 1989). In this study, our main goal was to understand the In 1999, PWN was reported for the first time in widespread patterns of B. xylophilus isolates from Portu- Portugal and in Europe (Mota et al., 1999), collected from gal and Madeira, comparing them with foreign isolates by dead maritime pine (Pinus pinaster) trees located in the using two distinct approaches: sequence analysis of IGS Setúbal Peninsula, 30 km south east of Lisbon. During region and ISSR analysis. approximately 10 years, PWN was confined to the Setúbal Peninsula, occupying 510 000 ha of the continental area. Materials and methods Despite efforts implemented by the Portuguese authorities to control this quarantine nematode, by 2008 new areas of the disease were established in other regions of the NEMATODE SAMPLING AND CULTURE country (Rodrigues, 2008), and, more recently, PWN was In the present study, 34 B. xylophilus isolates from detected on Madeira, 1000 km south west of continental Portugal (continental and the island of Madeira; Fig. 1) Portugal (Fonseca et al., 2010). Recently, it has also been were used (Table 1). Madeira isolates were provided by detected in Spain (Abelleira et al., 2011; Robertson et al., the Nematology Laboratory, IMAR-CMA (Department of 2011). Therefore, it is of major importance to determine Life Sciences, University of Coimbra), and continental the origin of the new isolates and their spread routes Portugal isolates were provided by AFN (National Forest to prevent further dissemination of the disease across Authority) from the national surveys made each year. Europe. Each isolate was obtained from a wood sample collected Sequence analysis of specific regions of genomic DNA from one tree. A Portuguese isolate of the closely related has proved to be an effective approach for species iden- species B. mucronatus was used as outgroup. Nine foreign tification. Ribosomal internal transcribed spacer regions isolates from China, Japan, South Korea and USA were ITS1 and ITS2 are positioned between the small rRNA used as reference (Table 1). Isolates were maintained subunit (SSU), the 5.8S rRNA gene and the large rRNA in the laboratory in barley seed cultures grown with subunit (LSU). ITS is an excellent marker for quickly dis- Botrytis cinerea at 25°C (Y. Mamiya, pers. commun.). tinguishing among known species, due to its low level The nematodes were extracted from cultures using the of intraspecific polymorphisms and sequence variation Baermann funnel technique (Southey, 1986). After this, (Blouin, 2002). On the other hand, the intergenic spacer nematodes were collected and concentrated in 1.5 ml (IGS) region of the 5S rRNA gene is known to be highly microtubes in a minimum amount of distilled water. variable, even between closely related species such as B. xylophilus B. mucronatus et al. and (Kang , 2004). It has DNA EXTRACTION also been useful to study species at the intraspecific level, such as, for example, the fungus Fusarium oxysporum Genomic DNA was obtained from a 50 μl suspension (Kim et al., 2001). Inter-simple sequence repeats (ISSR) containing more than 10 000 nematodes from each isolate Fig. 1. Map of the geographical distribution of Bursaphelenchus xylophilus isolates in continental Portugal and Madeira Island, showing the sampling sites of isolates collected until 2008 and in 2009-2010. Isolates code and symbol, representing sampling year, are shown in Table 1. 900 Nematology Molecular studies on invasion routes of Bursaphelenchus xylophilus in Portugal Vol. 14(8), 2012 901 V. Valadas et al. Table 1. List of Bursaphelenchus xylophilus isolates used in the present study; location, year of culture, map symbol (sampling year), code, accession numbers of ITS and IGS sequences. Country Geographic location District Year of Map symbol/Code Accession numbers culture ITS sequences IGS sequences Portugal Alcácer do Sal Setúbal 2005 BxPt4AS JN684822 JN684853 Alcácer do Sal Setúbal 2005 BxPt11AS JN684827 JN684854 Grândola Setúbal 2006 BxPt12G JN684828 JN684856