A fast and sensitive multiplex real-time PCR assay for simultaneous identification of Bursaphelenchus xylophilus, B. mucronatus and B. fraudulentus – three closely related species from the xylophilus group

Abstract

The quarantine nematode Bursaphelenchus xylophilus is the causal agent of pine wilt disease in Asia and Europe. Differentiation between B. xylophilus and other related, non-pathogenic species can be ambiguous when based exclusively on morphological characters. The morphology of B. mucronatus and B. fraudulentus most closely resembles that of B. xylophilus. Moreover, all of these nematodes are found in both Asia and Europe and can colonise various species of pine. Therefore, for phytosanitary purposes it is necessary to identify the three species precisely and rapidly. In this paper we described the results of the optimized multiplex real-time PCR protocol that utilizes two universal primers and three specific TaqMan probes to identify and differentiate the three Bursaphelenchus species, simultaneously. Species-specific real-time PCR reactions gave expected products for B. xylophilus, B. mucronatus and B. fraudulentus templates. The optimized primer combination together with designed species-specific probes has produced reliable results in multiplex PCR assays with eleven geographically distant isolates of B. xylophilus, eleven of B. mucronatus, seven of B. fraudulentus, and no cross-reactions were observed between tested samples and with other species (i.e., B. piniperdae, B. pinophilus, B. populi and Parasitaphelenchus papillatus). All species-specific real-time PCR reactions performed on DNA extracted from a single nematode individual consistently gave specific amplicons, confirming the reproducibility of the method. Moreover, the conducted study revealed that 30 fg was the lowest DNA input still detected in multiplex real-time PCR analysis. The described approach is simple, time-saving, and reliable in comparison to conventional PCR, and it can be used for simultaneous identification of the above three species within the xylophilus group.