Ye Weimin, Giblin-Davis Robin M
Nematode Assay Section, Agronomic Division, North Carolina Department of Agriculture & Consumer Services, Raleigh, North Carolina, United States of America.
PLoS One. 2013 Nov 11;8(11):e78804. doi: 10.1371/journal.pone.0078804. eCollection 2013.
Bursaphelenchus xylophilus, the pine-wood nematode (PWN), is the causal agent of pine wilt disease, one of the most damaging emerging pest problems to forests around the world. It is native to North America where it causes relatively minor damage to native conifers but is labeled an EPPO-A-2 pest and a quarantine nematode for many countries outside of the United States because of its potential for destruction to their native conifers. Exports of wood logs and commodities involving softwood packaging materials now require a lab test for the presence/absence of this regulated nematode species. We characterized the DNA sequences on the ribosomal DNA small subunit, large subunit D2/D3, internal transcribed spacer (ITS) and mitochondrial DNA cytochrome oxidase subunit one on the aphelenchid species and described the development of a real-time-PCR method for rapid and accurate identification of PWN targeting the ITS-1. A total of 97 nematode populations were used to evaluate the specificity and sensitivity of this assay, including 45 populations of B. xylophilus and 36 populations of 21 other species of Bursaphelenchus which belong to the abietinus, cocophilus, eggersi, fungivorus, hofmanni, kevini, leoni, sexdentati, and xylophilus groups and one unassigned group from a total of 13 groups in the genus Bursaphelenchus; 15 populations of Aphelenchoides besseyi, A. fragariae, Aphelenchoides species and Aphelenchus avenae; and one population of mixed nematode species from a soil sample. This assay proved to be specific to B. xylophilus only and was sensitive to a single nematode specimen regardless of the life stages present. This approach provides rapid species identification necessary to comply with the zero-tolerance export regulations.
松材线虫（Bursaphelenchus xylophilus）是松材线虫病的病原体，松材线虫病是全球森林面临的最具破坏性的新出现的害虫问题之一。它原产于北美，在那里对当地针叶树造成相对较小的损害，但由于其对许多美国以外国家的本土针叶树具有潜在的破坏能力，被欧洲和地中海植物保护组织（EPPO）列为A2类有害生物以及许多国家的检疫性线虫。现在，原木和涉及软木包装材料的商品出口需要进行实验室检测，以确定是否存在这种受管制的线虫物种。我们对滑刃线虫物种核糖体DNA小亚基、大亚基D2/D3、内转录间隔区（ITS）和线粒体DNA细胞色素氧化酶亚基I的DNA序列进行了特征分析，并描述了一种针对ITS-1的实时荧光定量PCR方法，用于快速准确地鉴定松材线虫。总共使用了97个线虫种群来评估该检测方法的特异性和灵敏度，其中包括45个松材线虫种群、21种其他滑刃线虫的36个种群，这些滑刃线虫属于冷杉滑刃线虫、椰树滑刃线虫、埃氏滑刃线虫、食真菌滑刃线虫、霍夫曼滑刃线虫、凯氏滑刃线虫、莱奥尼滑刃线虫、六齿滑刃线虫和松材线虫组，以及滑刃线虫属13个组中的一个未分类组；15个草莓滑刃线虫、草莓滑刃线虫、滑刃线虫属物种和燕麦真滑刃线虫种群；以及一个来自土壤样本的混合线虫物种种群。该检测方法仅对松材线虫具有特异性，并且无论线虫处于何种生活阶段，对单个线虫标本都很敏感。这种方法为符合零容忍出口规定提供了必要的快速物种鉴定。
