Molecular Characterization of Isolates of the Bursaphelenchus sexdentati Group Using Ribosomal DNA Sequences and ITS-RFLP

Abstract

Species-specific ITS-RFLP patterns have been established for more than 30 Bursaphelenchus species including 5 species of the ‘sexdentati’ group (B. sexdentati, B. vallesianus, B. pinophilus, B. poligraphi and B. borealis). Morphological species differentiation in the ‘sexdentati’ group is based on the shape of female tail and the spicules. However, observations on different isolates of B. sexdentati have revealed considerable variability of these features suggesting the existence of intraspecific genetic types which could not be differentiated by ITS-RFLP analysis using five enzymes. To improve intraspecific differentiation, we have determined ITS1/2 sequences of 17 isolates belonging to five species of the ‘sexdentati’ group. In some isolates, sequence heterogeneity at a few sites of ITS2 was detected. In the sequence-based phylogenetic tree, branching of clusters confirmed the affiliation of isolates to their respective species. In addition, isolates of B. sexdentati were separated in two groups suggesting the existence of a Central European and a South European type of this species or two separate species. This was supported by the differences in shape of female tails and spicules between the two types. The information obtained from sequencing was used to select three additional enzymes for extending the scope of ITS-RFLP analysis. In this way, improved distinction of species and differentiation of the two types of B. sexdentati was achieved.