The pine wood nematode Bursaphelenchus xylophilus is the causal agent of pine wilt disease. It is a quarantine pest for most countries in the world. Surveys for the occurrence of the nematode may be necessary in the framework of internationally agreed phytosanitary standards, in order to delimit infested areas or to demonstrate the absence of B. xylophilus and thus to identify pest free areas. Import inspections of wood and wood products also need sound monitoring systems. It is obvious that the quality of survey/monitoring, as well as the results of diagnosis, are strongly dependent on the sampling procedure. Although, in the literature, numerous results of surveys and monitoring are documented, sampling procedures vary and there is no scientific or statistically based sampling system published for B. xylophilus. The current paper relates background information concerning the biology of the nematode, its vector beetles and the development of pine wilt disease and the influence of these factors on sampling procedures, and attempts to define a more systematic methodology.

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The Bursaphelenchus genus (Nematoda: Parasitaphelenchidae) 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 Bursaphelenchus species in the PWD remains enigmatic. In the current paper, comparative molecular analysis is performed based on nuclear ribosomal DNA (rDNA) of B. vallesianus, 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 ITS1-5Á8S-ITS2 rDNA region confirmed previous taxonomic conclusions based on morphology. Evolutionary reconstructions resulted in a phylogenetic tree, where the Czech 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 Czech 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 5Á8S rRNA-coding gene. The newly identified genetic variation appears to affect the 5Á8S 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 5Á8S-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.

Composition and vertical distribution of soil nematode communities within soil profile were investigated in eight hop gardens in Czech Republic. In total, the presence of 78 nematode genera was confirmed. Genus Drilocephalobus (Coomans & Coomans, 1990) is new for fauna of the Czech Republic. The highest abundance of soil nematodes was found at a depth of 0–10 cm and declined with increasing depth of soil profile. The most dominant genus was Bitylenchus, followed by genera Acrobeloides, Ditylenchus, Chiloplacus and Cervidelus. Ten genera of plant parasitic nematodes were recorded: Bitylenchus (with prevalence of B. dubius), Helicotylenchus, Heterodera (with absolute prevalence of H. humuli), Geocenamus, Longidorella, Longidorus (only L. elongatus), Merlinius (with prevalence of M. brevidens), Paratylenchus and Pratylenchus. Low population densities of predators and omnivores, low values of the community indices (MI, ΣMI, SI, and CI), and high values of NCR, EI, and PPI/MI ratio indicated disturbed nematode communities in hop gardens and bacteria-dominated decomposition pathways in the soil food web.

SummaryAn overview of the genus Bursaphelenchus in the Czech Republic is presented, based on a recent survey for monitoring the presence of the pinewood nematode, Bursaphelenchus xylophilus, as well as on previous reports of this genus in the country. In addition, we provide a morphological and molecular characterization of four Bursaphelenchus species (B. eremus, B. pinophilus, B. vallesianus and B. borealis) found during the monitoring programme for forest pests, conducted during 2006-2010, within the Moravian and Bohemian regions. Nematodes were extracted from over 1917 insects and 1493 wood samples collected from deciduous and coniferous trees exhibiting wilting and declining symptoms. Bursaphelenchus species were found only in 0.73% of insects and 0.47% of the total number of wood samples. Bursaphelenchus borealis and B. pinophilus dauer juveniles were found associated with the insect vectors Dryocetes autographus and Pityogenes bidentatus, respectively. While a total of seven Bursaphelenchus species are now reported from the Czech Republic, the status of B. xylophilus remains as absent.