The pine wood nematode, Bursaphelenchus xylophilus, in Portugal: possible introductions and spread routes of a serious biological invasion revealed by molecular methods

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.