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Both native European species, Bursaphelenchus mucronatus and B. fraudulentus, are relatively common and harmless to trees. They belong to the xylophilus group, which includes also the quarantine pest B. xylophilus – the causal agent of the pine wilt disease. They can be difficult to distinguish morphologically from each other and from B. xylophilus. Therefore, reliable methods of taxonomic identification of these species are therefore of particular interest to plant quarantine services. PCR amplification with specific primers enables rapid and precise species identification, even from a single nematode. In 2004, Matsunaga and Togashi designed specific primers for B. xylophilus and B. mucronatus. The aim of this study was to design specific primers for B. fraudulentus. The specificity of the newly designed primers was tested on eight isolates of B. fraudulentus which originated from different parts in Europe (Austria, Germany, Poland, Russia and Ungarn). For all isolates, PCR amplification resulted in products of identical length of 617 bp, but no PCR products were generated for B. xylophilus and B. mucronatus. The PCR amplification with primer sets specific for B. xylophilus (XF and XR), B. mucronatus (MF and MR) and those for B. fraudulentus (FF and FR) designed in this study resulted in amplicons of different lengths (557, 210 and 617 bp, respectively), which can be easily distinguished in agarose gels.
Economic significance of the quarantine nematode Bursaphelenchus xylophilus (Steiner & Buhrer, 1934) Nickle, 1970 in eastern Asia and a growing threat of its dispersion in forest stands across Europe have recently created the urgent need for greater precision of taxonomic identification of nematodes in the genus Bursaphelenchus and increased scientific interest in this group. The genus Bursaphelenchus includes over 80 species with the majority living under the bark and wood of coniferous or deciduous trees in association with wood-boring insects. In natural conditions these nematodes are not pathogenic to the host-tree or vector-insect, with the only exception of B. xylophilus, which can kill even mature trees within a few week period. Up to the present 18 species of to the genus Bursaphelenchus were isolated in Poland. Extensive sampling of trees in forest stands and in shipments of timber did not reveal the presence of the quarantine pest B. xylophilus, however other Bursaphelenchus species were frequently recorded. Four of these species turned to be new to science and were described subsequently as B. glochis, B. pinophilus, B. populi and B. trypophloei. Comparative study focused on geographically distant populations of members of the xylophilus group revealed a great intraspecific morphological variation among examined populations. The range of variation observed in morphology of female tail in B. fraudulentus and B. populi widely overlapped the range present in B. xylophilus, thus making these species morphologically undistinguishable from the quarantine pest. The obtained results showed that nematodes from the genus Bursaphelenchus are common in forest stands in Poland. The wide morphological variation and partial overlapping of essential characters among species of the xylophilus group make additional molecular examination of DNA a necessary component of the nematodes’ identification.
Four species of Bursaphelenchus were found in survey of pine wood are described: B. mucronatus, B. naujaci, B. pinophiliis sp. n. and B. glochis sp. n. The analysis of various morphological characters brought to the conclusion the followinga are constant and useful as the main diagnostic features: shape of spicules, shape and size of anterior vulval lip, arrangement of cells in gonads, thickenings of stylet base and shape of female tail.
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