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

Abstract

Pinewood nematode (PWN), Bursaphelenchus xylophilus, the causal agent of pine wilt disease is a major worldwide pathogen and pest of pine, with impacts on forest health, natural ecosystem stability and international trade. In Portugal, the first diagnosis of PWN was in 1999, a first occurrence also for Europe of this major biological invader. The disease was recently detected in Madeira Island, in the center of Continental Portugal and in the 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, intergenic spacer (IGS) region of the 5.8S rDNA gene was used, combined with inter-simple sequence repeats (ISSR), to determine the genetic relationships among 45 B. xylophilus isolates from Portugal, China, Japan, South Korea and USA. IGS sequences revealed that this region can only be used to establish interspecific correlations. IGS is not a good marker to establish genetic diversity of B. xylophilus, since no differences were detected among Portuguese isolates from different geographic locations. Fingerprints obtained with ISSR detected a low genetic variability among Portuguese isolates, except for the ones isolated after 2008. ISSR dendrograms suggest the possibility of multiple introductions in Madeira Island, from Asia and/or North America. Recently, collected isolates, from the center of the country, showed a greater similarity with Asian or USA isolates, suggesting multiple introductions from different origins, contrary to previous information which suggests a single introduction from Asia in the initial affected area.