Comparative molecular analysis of B ursaphelenchus vallesianus , a wood‐inhabiting nematode isolated from declining pine trees in the C zech R epublic

The B ursaphelenchus genus ( N ematoda: P arasitaphelenchidae) comprises mostly wood‐inhabiting nematodes that feed on various tree‐colonizing fungi. One species of the genus, B .  xylophilus , has been proven as an agent causing pine wilt disease ( PWD ). However, involvement of other B ursaphelenchus species in the PWD remains enigmatic. In the current paper, comparative molecular analysis is performed based on nuclear ribosomal DNA (r DNA ) of B .   vallesianus , a species that was recently isolated from pine trees ( P inus sylvestris ) exhibiting wilting and declining symptoms in the C zech R epublic. Sequencing of the nuclear‐encoded ITS 1–5·8S– ITS 2 r DNA region confirmed previous taxonomic conclusions based on morphology. Evolutionary reconstructions resulted in a phylogenetic tree, where the C zech isolate of B .  vallesianus occupied a common clade together with other species belonging to the so‐called B .  sexdentati group. Unexpectedly, comprehensive analysis of the sequence data revealed a genetic variation distinguishing the C zech isolate of B .  vallesianus from all other species of the B .  sexdentati group. This dissimilarity consists of the presence of a four nucleotide exchange found in the 5·8S r RNA ‐coding gene. The newly identified genetic variation appears to affect the 5·8S r RNA 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 ( ITS 1 and ITS 2) fold into the multibranched closed loops. While the ITS 2 closed loop is formed with help of canonical 5·8 S ‐28 S r RNA pairing, the ITS 1 forms the thermodynamically stable closed loop with no support of flanking r RNA sequences. The current information on bursaphelenchid ITS r DNA sequence diversity and structure is further discussed.