| A Simple PCR-RFLP for Idenficiation of Bursaphelenchus spp. Collected from Korea |
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Hye Rim Han, Bo Young Han, Yeong Jin Chung, Sang Chul Shin |
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| Abstract |
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Accurate identification of pine wood nematode, Bursaphelenchus xylophilus is a prerequisite to diagnose the pine wilt disease. However, a fungivorous nematode, B. mucronatus is highly similar to B. xylophilus and it is difficult to differentiate these two species by morphological features. A molecular diagnosis method, ITSRFLP was applied for the identification of B. xylophilus and B. mucronatus from Korea. Genomic DNA was extracted from a single individual nematode and ITS DNA was amplified by PCR. The size of PCR product was approximately 900 bp and the sequence data were obtained after cloning. Amplified ITS was digested by 5 different restriction enzymes (Rsa I, Hae III, Msp I, Hinf I, and Alu I) and provided a discriminatory profile for B. xylophilus and B. mucronatus. Besides, B. mucro- natus was determined to have 2 different genotypes, East Asian type and European type also clearly separated by Rsa I and Hae III digestion. European type of B. mucronatus is recently collected from Pinus koraiensis and has not been reported before. ITS sequnce data were analyzed by Restriction Mapper program and the result supported ITS-RFLP pattern. These data indicated that PCRRFLP method is an accurate and simple way for identification of Bursaphelenchus species. |
| Key Words:
Bursaphelenchus xylophilus, B. mucronatus, identification, ITS, PCR-RFLP, pine wood nematode |
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