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Comparative molecular analysis of Bursaphelenchus vallesianus,a wood‐inhabiting nematode isolated from declining pine trees in the Czech Republic
Authors:M Marek  M Zouhar  O Douda  V Gaar  P Ry?ánek
Institution:1. Department of Plant Protection, Faculty of Agrobiology, Food and Natural Resources, Czech University of Life Sciences (CULS) Prague, , 165 21 Prague 6– Suchdol, Czech Republic;2. Division of Plant Health, Department of Entomology, Crop Research Institute, , 161 06 Prague 6– Ruzyne, Czech Republic;3. Division of Diagnostics, State Phytosanitary Administration, , 779 00 Olomouc, Czech Republic
Abstract:The Bursaphelenchus genus (Nematoda: Parasitaphelenchidae) comprises mostly wood‐inhabiting nematodes that feed on various tree‐colonizing fungi. One species of the genus, Bxylophilus, has been proven as an agent causing pine wilt disease (PWD). However, involvement of other Bursaphelenchus species in the PWD remains enigmatic. In the current paper, comparative molecular analysis is performed based on nuclear ribosomal DNA (rDNA) of Bvallesianus, a species that was recently isolated from pine trees (Pinus sylvestris) exhibiting wilting and declining symptoms in the Czech Republic. Sequencing of the nuclear‐encoded ITS1–5·8S–ITS2 rDNA region confirmed previous taxonomic conclusions based on morphology. Evolutionary reconstructions resulted in a phylogenetic tree, where the Czech isolate of Bvallesianus occupied a common clade together with other species belonging to the so‐called Bsexdentati group. Unexpectedly, comprehensive analysis of the sequence data revealed a genetic variation distinguishing the Czech isolate of Bvallesianus from all other species of the Bsexdentati group. This dissimilarity consists of the presence of a four nucleotide exchange found in the 5·8S rRNA‐coding gene. The newly identified genetic variation appears to affect the 5·8S rRNA folding, as deduced from secondary structure models. Additionally, it is shown that for the first time, to the authors’ knowledge, both bursaphelenchid internal transcribed spacers (ITS1 and ITS2) fold into the multibranched closed loops. While the ITS2 closed loop is formed with help of canonical 5·8S‐28S rRNA pairing, the ITS1 forms the thermodynamically stable closed loop with no support of flanking rRNA sequences. The current information on bursaphelenchid ITS rDNA sequence diversity and structure is further discussed.
Keywords:  8S rRNA  ITS rDNA  nematode  phylogeny  secondary structure
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