Han H, Chung Y-J, Shin S-C
Division of Forest Insect Pests and Diseases, Korea Forest Research Institute, Seoul, Korea.
Plant Dis. 2009 Nov;93(11):1221. doi: 10.1094/PDIS-93-11-1221C.
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. References: (1) H. Braasch et al. Russ. J. Nematol. 12:19, 2004. (2) W. Burgermeister et al. Russ. J. Nematol. 13:29, 2005. (3) N. Kanzaki et al. Nematology. 10:69, 2008.
鹅掌楸（北美鹅掌楸）是韩国人工林中的主要树种之一，但它原产于北美。豆氏伞滑刃线虫是从韩国全罗南道完州的死鹅掌楸木材中采集到的。线虫在马铃薯葡萄糖琼脂培养基上的真菌培养基（灰葡萄孢）中培养。豆氏伞滑刃线虫在形态上与松材线虫相近，松材线虫是松材线虫病的致病生物，因此很难鉴定到种。然而，豆氏伞滑刃线虫与嗜木伞滑刃线虫组的其他物种不同，其交合刺较大（33.8至43.3μm）（1）。为了进行准确的物种鉴定，同时应用了形态学和分子生物学方法。形态学观察显示，唇区高3.7μm，有缢缩，中食道球发达，食道腺在肠的背侧重叠，有阴门盖，尾端有明显的尖突。雄虫因固定而腹弯，交合刺成对，大且有小的、帽状的、椭圆形的交合伞。对20只雌虫和10只雄虫的测量结果（雌虫；体长=901.7±60.5μm，a=34.3±3.1，b=10.4±1.5，c=23.0±2.3，V=77.1±2.0，口针长=14.3±0.4μm，尾长=39.5±4.2μm，子宫后囊长=104.6±7.8；雄虫；体长=790.4±40.6μm，a=34.4±2.7，b=10.8±1.7，c=22.7±1.3，口针长=14.3±0.3μm，尾长=34.8±1.9μm，交合刺长=39.5±4.1μm）与布拉施等人（1）对豆氏伞滑刃线虫的原始描述相近。为了进行分子诊断，用DNeasy试剂盒（Qiagen，加利福尼亚州瓦伦西亚）从30多个线虫个体中提取DNA，并通过PCR扩增rDNA中的内部转录间隔区（ITS）1、2和5.8S区域（美国/PTC - 0220；伯乐公司，加利福尼亚州赫尔克里士）。ITS - 限制性片段长度多态性（RFLP）可以通过使用五种不同的限制酶来区分亲缘关系相近的物种（2）。用这些酶获得的ITS - RFLP图谱与布拉施等人（1）报道的相同。此前在进口到中国的韩国木质包装中检测到了豆氏伞滑刃线虫（1）。然而，韩国大多数木质包装的生产依赖于从其他国家进口的针叶树。尽管在日本的日本赤松（黑松）中报道过豆氏伞滑刃线虫（3），但其在韩国的自然分布尚不清楚。基于形态学和分子生物学信息，我们将这种线虫鉴定为豆氏伞滑刃线虫。据我们所知，这是关于豆氏伞滑刃线虫在鹅掌楸上以及在韩国自然区域的首次报道。参考文献：（1）H. 布拉施等人，《俄罗斯线虫学杂志》12:19，2004年。（2）W. 伯格迈斯特等人，《俄罗斯线虫学杂志》13:29，2005年。（3）N. 神崎等人，《线虫学》10:69，2008年。
