Han H, Chung Y-J, Shin S-C
Division of Forest Insect Pests and Diseases, Korea Forest Research Institute, Seoul, Korea.
Plant Dis. 2009 Dec;93(12):1354. doi: 10.1094/PDIS-93-12-1354C.
The genus Bursaphelenchus Fuchs, 1937 contains approximately 90 species (3) that are morphologically similar. Pine wood nematode, Bursaphelenchus xylophilus (4) Nickle, 1970, is the causal organism of pine wilt disease and accurate identification is essential for diagnosis of the disease. In Korea, pine wilt disease was first reported in 1988 and devastated 6,800 ha of pine forest through 2008. For a survey of trees with pine wilt disease, wood samples were taken randomly from dead Pinus koraiensis in Namyangju, Gyeonggi Province in Korea. The extracted nematodes from dead trees were maintained in culture on Botrytis cinerea and morphological characteristics were observed with an inverted light microscope (Leica DE/DMI 3000B). Identification of Bursaphelenchus spp. based on morphological characteristics is difficult, especially for identification of juveniles that carry few morphological features for species identification. The internal transcribed spacer (ITS) region in ribosomal DNA provides useful molecular diagnostic markers for this genus (1). The nematodes were provisionally identified as Bursaphelenchus pinophilus based on the characteristic long and arcuate body shape, male spicule with distinctive rostrum and small cucullus, female vulval flap, and mucronate conical tail. Other Bursaphelenchus spp. with vulval flaps and spicules with cucullus are B. xylophius, B. mucronatus, B. abruptus, and B. pinophilus. For molecular diagnosis, DNA was extracted from more than 30 individual nematodes with a DNeasy Kit (Qiagen, Valencia, CA) and ITS regions 1, 2, and 5.8S in rDNA were amplified by PCR (US/PTC-0220; Bio Rad, Hercules, CA). The ITS-restriction fragment length polymorphism pattern was consistent with that of B. pinophilus (2). The ITS rDNA sequence of B. pinophilus from Korean pines had a 98% sequence homology to that of B. pinophilus in GenBank (Accession No. AM160664). The pathogenicity of B. pinophilus has not been determined. To our knowledge, this is the first report of B. pinophilus on P. koraiensis, but it was previously reported from Poland, Germany, and Portugal on P. sylvestris and P. pinaster (1). References: (1) H. Braasch. EPPO Bull. 31:127, 2001. (2) W. Burgermeister et al. Russ. J. Nematol. 13:29, 2005. (3) R. Sriwati et al. Nematology 10:1, 2008. (4) G. Steiner and E. M. Buhrer. J. Agric. Res. 48:946, 1934.
滑刃属（Bursaphelenchus）由富克斯于1937年建立，包含约90个形态相似的物种。松材线虫（Bursaphelenchus xylophilus），由尼克于1970年命名，是松材线虫病的致病生物，准确鉴定对于该病的诊断至关重要。在韩国，松材线虫病于1988年首次报道，截至2008年已毁坏了6800公顷的松林。为了调查感染松材线虫病的树木，从韩国京畿道南杨州市死亡的红松中随机采集木材样本。从死树中提取的线虫在灰葡萄孢上进行培养，并使用倒置光学显微镜（徕卡DE/DMI 3000B）观察其形态特征。基于形态特征鉴定滑刃属线虫很困难，尤其是鉴定幼虫时，幼虫几乎没有可用于物种鉴定的形态特征。核糖体DNA中的内转录间隔区（ITS）为该属提供了有用的分子诊断标记。根据其特征性的长而弯曲的体型、具有独特喙和小尾端膜的雄虫交合刺、雌虫阴门盖以及具短尖的锥形尾部，这些线虫初步被鉴定为嗜松滑刃线虫。其他具有阴门盖和具尾端膜交合刺的滑刃属线虫有松材线虫、拟松材线虫、短体滑刃线虫和嗜松滑刃线虫。为进行分子诊断，使用DNeasy试剂盒（Qiagen公司，加利福尼亚州瓦伦西亚）从30多条线虫个体中提取DNA，并通过PCR（美国/PTC - 0220；伯乐公司，加利福尼亚州赫尔克里士）扩增rDNA中的ITS区域1、2和5.8S。ITS - 限制性片段长度多态性模式与嗜松滑刃线虫一致。来自韩国松树的嗜松滑刃线虫的ITS rDNA序列与GenBank中嗜松滑刃线虫（登录号AM160664）的序列同源性为98%。嗜松滑刃线虫的致病性尚未确定。据我们所知，这是嗜松滑刃线虫在红松上的首次报道，但此前在波兰、德国和葡萄牙曾报道其寄生于欧洲赤松和海岸松。参考文献：（1）H. Braasch. EPPO Bull. 31:127, 2001.（2）W. Burgermeister等人。Russ. J. Nematol. 13:29, 2005.（3）R. Sriwati等人。Nematology 10:1, 2008.（4）G. Steiner和E. M. Buhrer。J. Agric. Res. 48:946, 1934.
