Taxonomy And Molecular Identification Of Some Bursaphelenchus And Ektaphelenchoides Species

The pinewood nematode (PWN), Bursaphelenchus xylophilus is the major causal agent of pine wilt disease and caused world concern. Many countries have taken it as a quarantine pest. B. mucronatus, a species morphologically very similar to B. xylophilus, often be detected in the dead pine trees, but has no pathogenicity to pine trees. Otherwise, many species in the genus are associated with coniferous trees. Exact identification of these species is very important. In our studies, some species of Bursaphelenchus were identified with both morphological and molecular methods. The diagnostic systems for B. xylophilus were constructed with different methods, such as PCR-RFLP based on rDNA, duplex PCR, and monoclonal antibody, etc. While surveying for the nematodes in the genus Bursaphelenchus, 2 species of Ektaphelenchoides were isolated and identified.Among seventeen Bursaphelenchus populations, six of which were selected for identification with the aid of PCR-RFLP of ITS region of rDNA based on the morphological comparison. It was found that two populations, one from Zhenhai, Zhejiang, China and the other from imported coniferous wood packages in Japan were identified as B. xylophilus. The other four populations including populations from Fuyang, Zhejiang province, Korea, Hangkong and Japan, were identified as B. mucronatus. All the four restriction enzymes, Hinf I, Hae III, Msp I, and Cfo I, could be used to differentiate the two morphologically very similar species. There were two Bursaphelenchus populations, one from dead pine trees in Shangyu, Zhejiang and the other from coniferous wood packages in HongKong, were identified as B. rainulfi and B. thailandae, respectively, both of which were recorded for the first time in China. Additionally, sequences of 12 Bursaphelenchus species were compared by Clustal X 1.8 and the phylogenetic tree were constructed based on the ITS region of rDNA available in our study and the GenBank, with MEGA 2.1 software. 12 species of Bursaphelenchus were grouped into 3 clades, which showed the genetic relationship among the Bursaphelenchus species.Duplex PCR with species-specific primers designed was developed for identification of B. xylophilus based on the differences of ITS between B. xylophilus and other species of Bursaphelenchus from Genbank. Using duplex PCR, All B. xylophilus populations produced two fragments, including one species-specific fragment for B. xylophilus, with the size of 580 bp, and the other fragment with the length of about 770 bp, which was amplified the D2D3 region of 28S RNA gene by the universal primers. The other species andpopulations, no matter intra- or inter- genus of Bursaphelenchus, only amplified D2D3 region of 28S RNA gene. Comparing to the standard molecular diagnostic methods, such as PCR-RFLP of ITS region of rDNA and species-specific amplification with only one pair of primers used for identification, using duplex PCR to identify B. xylophilus is more rapid, exact, and the procedure is simpler.The present studies also explored the possibility that 28S RNA gene on the systematics and identification of Bursaphelenchus and Ektaphelenchoides species. It was found that no distinct differences on the length of D2D3 region of 28S RNA gene of Bursaphelenchus, all of which yielded a fragment with the length of 750 bp. The PCR-RFLP patterns showed distinct differences among 3 Bursaphelenchus species, B. doui, B. rainulfi, and B. thailandae, but B. xylophilus and B. mucronatus have similar digestion pattern, one restriction enzyme, Bshl236 I, could differentiate B. xylophilus and B. mucronatus. Further studies confirmed that all the species and populations of Bursaphelenchus and Ektaphelenchoides used in this study were differentiated by digestion of D2D3 region of 28S RNA gene with Bshl236 I. The phylogenetic tree were constructed according to the 12 sequences of D2D3 region of 28S RNA gene of Bursaphelenchus species obtained from our study and Genbank, the result corresponded well with the phylogenetic analysis based on ITS region.Using monoclonal antibody to identify B. xylophilus was tested. One hybrid cell lines, named 1D3, resistant to homogenates of B. xylophilus, were developed by fusing splenocytes of BALB/c mice to mouse myeloma cell line SP2/0. The ELISA titers was 5.12 X105. This specific Mab was identified as IgGl. Western - blot analysis showed that the Mab could react with homogenates of B. xylophilus. Mab raised successfully against B. xylophilus provided another rapid and sensitive method for diagnosis.This is the first report about the monoclonal antibody to B. xylophilus.Two species in Genus Ektaphelenchoides, E. pini and E. compasi, were identified on the basis of morphology, and the rDNA characters of these two species were first reported. PCR-RFLP of ITS region and D2D3 expension region of 28S RNA gene indicated that there are clear differences on the fragment length and the digestion patterns, and which can be used for differentiation the speices in Ektaphelenchoides.