First Report of the European Type of Bursaphelenchus mucronatus on Korean Pine (Pinus koraiensis) in Korea.

Abstract

More than 70 species of Bursaphelenchus have been reported and they are morphologically very similar. The pine wood nematode (Bursaphelenchus xylophilus) is a causal agent of pine wilt and accurate identification is essential for diagnosis of the disease. However, many other saprophytic nematodes are found in dead trees and some of them have high morphological similarity to B. xylophilus. B. mucronatus is commonly found in dead pine trees and is distinguished from B. xylophilus only by the presence of a distinct mucro in the female tail (3). However, natural populations of B. xylophilus show variation in tail morphology and some of them have a small lump on the end of the tail that looks like a short mucro, which could lead to misidentification. B. mucronatus has been reported to have two genotypes known as the East Asian type and the European type (2). The European type has a more rounded tail with a mucro, which is much more similar to B. xylophilus than the East Asian type. Molecular identification by internal transcribed spacer restriction fragment length polymorphism (ITS-RFLP) can distinguish these two genotypes clearly by using five different restriction enzymes (2). In Korea, we collected wood samples from dead Pinus koraiensis located at Pocheon, Gyeonggi Province in April, 2007 and nematodes were extracted by a modified Baermann funnel method. After 24 h, nematodes were collected and morphology was observed under an inverted light microscope (Leica DE/DMI 3000B, Wetzlar, Germany). Morphology was characterized by a typical Aphelenchoid-type esophagus, head constriction, female vulva flap, post uterine sac, female tail with mucro, and males with arched-shaped spicules. The tail was rounded with a long mucro >4 μm. For molecular diagnosis, DNA was extracted from single individual nematodes by using a DNeasy Kit (Qiagen, Valencia, CA) and the ITS 1 and 2 and 5.8S regions in rDNA were amplified by PCR (US/PTC-0220; Bio-Rad, Hercules, CA). ITS-RFLP patterns were analyzed and nucleotide sequences were compared with sequences of B. mucronatus already reported in GenBank (Accession Nos. AM179514, BMU93554, and DQ841162). The ITS-RFLP pattern of individuals matched the European type of B. mucronatus and was differentiated from the East Asian type by RsaI and HaeIII digestion (2). The ITS DNA sequence of nematodes from Korean pines was >98% identical to B. mucronatus in GenBank. The European type of B. mucronatus was consistently isolated from dead trees, but the pathogenicity of the European type of B. mucronatus on Korean pine is not yet clear. To our knowledge, this is the first report of the European type of B. mucronatus species on P. koraiensis in Korea, but it is already distributed in certain locations of Japan (4), China, and Europe (1).