Molecular Biological Characterization of Bursaphelenchus xylophilus and Bursaphelenchus mucronatus in Korea

Pine wood nematode (PWN), Bursaphelenchus xylophilus, is a causal organism to induce pine wilt disease (PWD) in many varieties of pine trees. PWD was first introduced to Korea in 1988 but, the damage has been dramatically increased since 2000. Recently PWD is newly reported in Korean pine tree (Pinus koraiensis) and is considered one of the most important forest diseases in Korea. Fifteen isolates of B. xylophilus, 2 isolates of B. mucronatus, and 6 unidentified Bursaphelenchus spp. were collected from different geographical locations and hosts in Korea. All the isolates were cultured in fungal medium and nematodes were harvested for further experiment. First, all the isolates were characterized by both ITS and D2D3 rDNA sequence analysis. Template DNA was prepared by DNA extraction from single female nematode. ITS and D2D3 region were amplified by PCR and followed by cloning and sequence. As a result, all the sequences of ITS and D2D3 from B. xyophilus isolates were identical and there is no intraspecific variation. However, 2 genotypes of B. mucronatus were found, which one from P. thunbergii was East Asian type and the other one from P. koraiensis was European type. ITS-RFLP phenotype was also proposed to discriminate different species and genotypes and 5 enzymes, Hinf I, Alu I, Msp I, Hae III, and Rsa I. were selectively treated.