Han H, Chung Y-J, Shin S-C
Division of Forest Insect Pests and Diseases, Korea Forest Research Institute. Seoul, Korea.
Plant Dis. 2008 Aug;92(8):1251. doi: 10.1094/PDIS-92-8-1251A.
Pine wilt disease is one of the most important forest tree diseases, especially in the East Asian countries of Japan, China, and Korea. The causal agent, Bursaphelenchus xylophilus (3), is transmitted by the insect vectors, pine sawyer beetles (Monochamus spp.). The vectors mainly attack coniferous trees and the infected trees die within several weeks. In Korea, pine wilt disease was first reported in Busan City in 1988, and now, the damaged area covers 7,820 ha and more than 60 cities in Korea. The main host trees are Pinus densiflora and P. thunbergii, which are the most common pines distributed in Korea. In 2006, however, we found pine wilt disease in a forest of P. koraiensis located in Gwangju City in Gyeonggi Province. Symptoms were systemic and leaves turned brown or yellowish. Wood samples were collected from the affected trees, and nematodes were extracted by a modified Baermann's funnel method. After 24 h, nematodes were collected from wood chips, and morphology was observed with an inverted light microscope (Leica DE/DMI 3000B, Wetzlar, Germany). Morphology was characterized by a typical Aphelenchoid-type esophagus, head constriction, female vulva flap, female tail, and a male spicule shape similar to B. xylophilus. For more accurate identification, DNA was extracted from individual nematodes with a DNeasy Kit (Qiagen, Valencia, CA). The internal transcribed spacers (ITS) 1 and 2 and 5.8S regions were amplified by PCR (1). Sequenced nucleotide information was compared with the sequences of B. xylophilus already reported in GenBank (Accession Nos. AB294736, AB277208, AM 157747, AY 347913, and BXU92464). ITS DNA sequences of the nematode from Korean pine was >99% identical to B. xylophilus in GenBank. The B. xylophilus from Korean pine tree was also successfully cultured in Botrytis cinerea medium and pathogenicity was tested from June to October 2007. More than 95% mortality was observed with the inoculation of 20 replicate 15-year-old trees of P. koraiensis and P. densiflora with 15,000 nematodes per tree. Therefore, we confirmed that pine wilt disease occurred in P. koraiensis in Korea. P. koraiensis is an endemic species in Korea and distribution is limited to the northern Korean Peninsula and some locations in Russia. It has been shown that P. koraiensis is susceptible to the pine wood nematode by an inoculation test in Japan (2); however, to our knowledge, this is the first report of pine wilt disease on P. koraiensis under field conditions in Korea. References: (1) W. K. Burgermeister et al. Russ. J. Nematol. 13:29, 2005. (2) K. Futai and T. Furuno. Bull. Kyoto Univ. For. 51:3, 1979. (3) G. Steiner and E. M. Buhrer. J. Agr. Res. 48:946, 1934.
松材线虫病是最重要的森林病害之一，在东亚国家日本、中国和韩国尤为严重。其病原为松材线虫（Bursaphelenchus xylophilus），由昆虫媒介松墨天牛（Monochamus spp.）传播。媒介主要侵害针叶树，受感染的树木会在几周内死亡。在韩国，1988年釜山首次报告松材线虫病，如今，受灾面积达7820公顷，涉及韩国60多个城市。主要寄主树种为赤松（Pinus densiflora）和黑松（P. thunbergii），它们是韩国分布最广的松树品种。然而，2006年我们在京畿道广州市一片红松（P. koraiensis）林中发现了松材线虫病。症状为全株性，叶片变为褐色或淡黄色。从患病树木采集木材样本，采用改良的贝尔曼漏斗法提取线虫。24小时后，从木片中收集线虫，用倒置光学显微镜（德国韦茨拉尔徕卡DE/DMI 3000B）观察其形态。形态特征为典型的滑刃型食道、头部缢缩、雌虫阴门盖、雌虫尾部以及与松材线虫相似的雄虫交合刺形状。为进行更准确的鉴定，用DNeasy试剂盒（Qiagen，加利福尼亚州瓦伦西亚）从单个线虫中提取DNA。通过PCR扩增内部转录间隔区（ITS）1、2和5.8S区域。将测序得到的核苷酸信息与GenBank中已报道的松材线虫序列（登录号AB294736、AB277208、AM 157747、AY 347913和BXU92464）进行比较。来自韩国红松的线虫ITS DNA序列与GenBank中的松材线虫序列相似度超过99%。来自韩国红松的松材线虫也成功在灰葡萄孢培养基中培养，并于2007年6月至10月进行了致病性测试。给20株重复的15年生红松和赤松每株接种15000条线虫，观察到死亡率超过95%。因此，我们证实韩国红松林中发生了松材线虫病。红松是韩国的特有物种，分布仅限于朝鲜半岛北部和俄罗斯的一些地区。在日本通过接种试验已表明红松对松材线虫敏感；然而，据我们所知，这是韩国野外条件下红松发生松材线虫病的首次报告。参考文献：(1) W. K. Burgermeister等人，《俄罗斯线虫学杂志》13:29，2005年。(2) K. Futai和T. Furuno，《京都大学森林学公报》51:3，1979年。(3) G. Steiner和E. M. Buhrer，《农业研究杂志》48:946，1934年。
