Han H
Division of Forest Insect Pests and Diseases, Korea Forest Research Institute, Seoul, Korea.
Plant Dis. 2015 Jan;99(1):162. doi: 10.1094/PDIS-04-14-0385-PDN.
During a pinewood nematode survey in 2011, a dead pitch pine (Pinus rigida) was found in Daejeon city, Korea. Samples were taken from several regions of the trunk and nematodes were extracted from the wood samples by a modified Baermann funnel method (1). Recovered nematodes were then cultured on Botrytis cinerea grown in potato dextrose agar (PDA) medium. Males and females (n = 10) were examined under an inverted light microscope. Morphologically, these nematodes were identified as Bursaphelenchus yongensis sharing the characters of the genus Bursaphelenchus including a large median bulb, offset cephalic region, a well-developed stylet with small basal knobs, female with vulva at 70% of body length, and males with terminal bursa. Females of B. yongensis were similar to B. muconatus and B. fraudulentus by presence of tail mucron, but they were differentiated by a smaller vulval flap. Males had spicules with small cucullus, pointed rostrum, and with strongly dorsally bent condylus. The identity of the nematode was confirmed through molecular diagnosis. DNA was extracted from each of 10 nematodes using commercial DNeasy kits (Qiagen, Valencia, CA) and the ITS regions were amplified with a primer set by PCR (Bio Rad, US/PTC-0220) (3,5). Amplified ITS DNA was cloned and sequenced, and the sequence data was deposited to GenBank (Accession No. KJ857070). The ITS sequence of Korean isolate (KJ857070) showed 99% similarity to that from Chinese isolate in GenBank (AM180513). ITS-RFLP patterns using five different restriction enzymes, RsaI, HaeIII, MspI, HinfI, and AluI, matched 100% with those previously reported for B. yongensis (2,4). Host pathogenicity tests were conducted from June to August on saplings of five native and introduced conifer species found in Korea (Abies sp., Larix leptolepsis, P. koraiensis, P. strobus, and P. thunbergii). All saplings were 5 to 6 years old and 15 saplings were inoculated for each species. B. yongensis was cultures on B. cinerea for 10 days at 25°C and the inoculum concentration was calibrated at 5,000 nematodes/50 μl sterile water. Nematodes were artificially inoculated by making a 1-cm-long incision into the stem using a sterile razor blade. B. yongensis was pathogenic on L. leptolepsis, causing wilt and death in all inoculated saplings; however, the nematode had no effect on P. thunbergii, P. koraiensis, Abies sp., or P. strobus. To our knowledge, this is the first report of B. yongensis in Korea. This nematode is a potential threat to L. leptolepsis; however, the role of B. yongensis in the mortality of P. rigida is uncertain at this time. References: (1) S. M. Ayoub. Plant Nematology in Agricultural Training Aid. Department of Food and Agriculture, Division of Plant Industry, Sacramento, CA, 1977. (2) W. Burgermeister et al. Nematology 11:649, 2009. (3) V. R. Ferris et al. Fund. Appl. Nematol. 16:177, 1993. (4) J. Gu et al. Russian J. Nematol. 14:91, 2006. (5) T. C. Vrain. J. Nematol. 25:361, 1993.
2011年进行松材线虫调查期间，在韩国大田市发现一株死的刚松（Pinus rigida）。从树干的几个部位采集样本，并通过改良的贝尔曼漏斗法（1）从木材样本中提取线虫。然后将回收的线虫在生长于马铃薯葡萄糖琼脂（PDA）培养基上的灰葡萄孢上培养。在倒置光学显微镜下检查雌雄线虫（n = 10）。从形态学上看，这些线虫被鉴定为永氏伞滑刃线虫，具有伞滑刃属的特征，包括一个大的中食道球、偏移的头部区域、一个带有小基部瘤的发育良好的口针、雌虫阴门位于体长的70%处，以及雄虫具端交合伞。永氏伞滑刃线虫的雌虫因有尾尖突而与粘液伞滑刃线虫和欺诈伞滑刃线虫相似，但可通过较小的阴门盖加以区分。雄虫的交合刺具小的帽状突、尖的喙，且交合突强烈背弯。通过分子诊断确认了该线虫的身份。使用商用DNeasy试剂盒（Qiagen，加利福尼亚州瓦伦西亚）从10条线虫中分别提取DNA，并通过PCR使用引物对扩增ITS区域（Bio Rad，美国/PTC - 0220）（3,5）。扩增的ITS DNA被克隆并测序，序列数据存入GenBank（登录号KJ857070）。韩国分离株（KJ857070）的ITS序列与GenBank中中国分离株（AM180513）的序列相似性为99%。使用五种不同的限制性内切酶RsaI、HaeIII、MspI、HinfI和AluI的ITS - RFLP模式与先前报道的永氏伞滑刃线虫的模式100%匹配（2,4）。6月至8月对在韩国发现的五种本土和引进针叶树种（冷杉属、日本落叶松、红松、美国五针松和黑松）的树苗进行了寄主致病性测试。所有树苗均为5至6年生，每个树种接种15株树苗。将永氏伞滑刃线虫在灰葡萄孢上于25°C培养10天，接种物浓度校准为5000条线虫/50 μl无菌水。使用无菌剃须刀片在茎上划一个1厘米长的切口进行人工接种线虫。永氏伞滑刃线虫对日本落叶松致病，导致所有接种的树苗枯萎死亡；然而，该线虫对黑松、红松、冷杉属或美国五针松没有影响。据我们所知，这是永氏伞滑刃线虫在韩国的首次报道。这种线虫对日本落叶松是一种潜在威胁；然而，目前永氏伞滑刃线虫在刚松死亡中的作用尚不确定。参考文献：（1）S. M. Ayoub。《农业培训辅助中的植物线虫学》。加利福尼亚州萨克拉门托食品与农业部植物产业司，1977年。（2）W. Burgermeister等人。《线虫学》11:649，2009年。（3）V. R. Ferris等人。《基础与应用线虫学》16:177，1993年。（4）J. Gu等人。《俄罗斯线虫学杂志》14:91，2006年。（5）T. C. Vrain。《线虫学杂志》25:361，1993年。
