Molecular Characterization of Isolates of the Bursaphelenchus sexdentati Group Using Ribosomal DNA Sequences and ITS-RFLP
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.
KeywordsITS2 Sequence Pine Wilt Disease Sequence Heterogeneity Female Tail ITS2 Fragment
Unable to display preview. Download preview PDF.
- Ambrogioni, L. and Caroppo, S. (1998). Morphology and morphometrics of Italian populations of Bursaphelenchus species. Nematologia Mediterranea 26: 97–116.Google Scholar
- Blok, V.C., Malloch, G., Harrower, B., Phillips, M.S. and Vrain, T.C. (1998). Intraspecific variation in ribosomal DNA in populations of the potato cyst nematode Globodera pallida. Journal of Nematology 30: 262–274.Google Scholar
- Braasch, H. (2001). Bursaphelenchus species in conifers in Europe: distribution and morphological relationships. Bulletin OEPP/EPPO Bulletin 31: 127–142Google Scholar
- Braasch, H., Brandstetter, M. and Burgermeister, W. (2006). Supplementary characters of Bursaphelenchus lini Braasch, 2004 (Nematoda: Parasitaphelenchidae) and remarks on this nematode. Zootaxa 1141: 55–61.Google Scholar
- Burgermeister, W., Metge, K., Braasch, H. and Buchbach, E. (2005). ITS-RFLP patterns for differentiation of 26 Bursaphelenchus species (Nematoda: Parasitaphelenchidae) and observations on their distribution. Russian Journal of Nematology 13: 29–42.Google Scholar
- Gu, J., Zhang, J., Braasch, H. and Burgermeister, W. (2005). Bursaphelenchus singaporensis sp. n. (Nematoda: Parasitaphelenchidae) in packaging wood from Singapore – a new species of the B. xylophilus group. Zootaxa 988: 1–12.Google Scholar
- Hall, T.A. (1999). BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symposium Series 41: 95–98.Google Scholar
- Iwahori, H., Tsuda, K., Kanzaki, N., Izui, K. and Futai, K. (1998). PCR-RFLP analysis of ribosomal DNA of Bursaphelenchus nematodes related to pine wilt disease. Fundamental and Applied Nematology 21: 655–666.Google Scholar
- Lange, C., Burgermeister, W., Metge, K. and Braasch, H. (2006). Phylogenetic analysis of isolates of the Bursaphelenchus sexdentati group using ribosomal intergenic transcribed spacer DNA sequences. Journal of Nematode Morphology and Systematics 9: 95–109.Google Scholar
- Rühm, W. (1960). Ein Beitrag zur Nomenklatur und Systematik einiger mit Scolytiden vergesellschafteter Nematodenarten. Zoologischer Anzeiger 164: 201–213.Google Scholar
- Zheng, J., Subbotin, S.A., He, S., Gu, J. and Moens, M. (2003). Molecular characterization of some Asian isolates of Bursaphelenchus xylophilus and B. mucronatus using PCR-RFLPs and sequences of ribosomal DNA. Russian Journal of Nematology 11: 17–22.Google Scholar