First Report of Bursaphelenchus doui on Tulip Tree (Liriodendron tulipifera) in Korea

Abstract

Tulip tree (Liriodendron tulipifera) is one the major tree species in Korean plantations, but it originated from North America. Bursaphelenchus doui was collected from the wood of dead tulip trees located at Wanju in Chonnam Province, Korea. Nematodes were maintained in fungal medium culture (Botrytis cinerae) on potato dextrose agar. B. doui is morphologically close to B. xylophilus, a causative organism of pine wilt disease, and identification to species is difficult. However, B. doui differs from the other species of the xyophilus group by the larger spicule (33.8 to 43.3 μm) (1). For accurate species identification, both morphology and molecular biological methods were applied. Morphological observations showed lip region 3.7 μm high and offset by a constriction, median bulb well developed, esophageal gland overlapping intestine dorsally, vulval flap present, tail terminus with distinct mucro. Males ventrally curved by fixation, spicules pared, and large with small, cucullus, oval-shaped bursa. Measurements of 20 females and 10 males (female; body length = 901.7 ± 60.5 μm, a = 34.3 ± 3.1, b = 10.4 ± 1.5, c = 23.0 ± 2.3, V = 77.1 ± 2.0, stylet length = 14.3 ± 0.4 μm, tail length = 39.5 ± 4.2 μm, length of postuterine sac = 104.6 ± 7.8: male; body length = 790.4 ± 40.6 μm, a = 34.4 ± 2.7, b = 10.8 ± 1.7, c = 22.7 ± 1.3, stylet length = 14.3 ± 0.3 μm, tail length = 34.8 ± 1.9 μm, spicules length = 39.5 ± 4.1 μm) were close to the original description of B. doui by Braasch et al. (1). For molecular diagnosis, DNA was extracted from more than 30 individual nematodes with a DNeasy Kit (Qiagen, Valencia, CA) and the internal transgenic spacers (ITS) 1, 2, and 5.8S regions in rDNA were amplified by PCR (US/PTC-0220; Bio Rad, Hercules, CA). ITS-restriction fragment length polymorphism (RFLP) can discriminate closely related species by using five different restriction enzymes (2). ITS-RFLP patterns obtained with these enzymes were the same as reported by Braasch et al. (1). B. doui previously has been detected in Korean wooden packages imported to China (1). However, most production of Korean wooden packages depends on imported coniferous trees from other countries. Although B. doui has been reported in Japanese red pine (Pinus densiflora) in Japan (3), the natural distribution of B. doui in Korea is unclear. On the basis of morphological and molecular biological information, we identify this nematode as B. doui. To our knowledge, this is the first report of B. doui on tulip tree and in a natural area in Korea.