In situ observation of pinewood nematode in wood
In situ observation of pinewood nematode in wood
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木材中松材线虫的原位观察
DOI：
10.1007/s10658-016-1013-8
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发表时间：
2017
影响因子：
1.8
通讯作者：
K.
中科院分区：
文献类型：
--
作者：
Umebayashi;T.;Yamada;T.;Fukuhara;K.;Endo;R.;Kusumoto;D.;Fukuda;K.
在松材线虫病中，木质部功能障碍与寄主组织中线虫的迁移和增殖有关，但松材线虫(PWN)在松茎组织中的检测准确性尚不清楚。本研究采用低温扫描电子显微镜(Cryo-SEM)和荧光素标记的小麦胚芽凝集素(F-WGA)染色检测PWN。松材线虫侵染后，松茎表面荧光频率自接种之日起至接种后3周内逐渐增加。但5周后，晚期荧光频率明显下降。因此，基于线虫体表外壳蛋白的荧光染色检测PWN在用于检查疾病症状的发展与线虫种群之间的相关性时可能具有误导性。相反，所有的切割体片段都是荧光的，无论接种后的时间如何，它们的荧光成分在松干横截面上都是常见的。此外，冷冻扫描下仅在空树脂管内观察到PWN，切片上的PWN F-WGA染色证实了这一分布。因此，基于F-WGA表面外壳蛋白荧光染色的PWN检测在松茎纵向切片上是不可靠的，因为线虫在疾病进展过程中染色能力发生了变化。为了检测受感染植物中的白粉病，我们得出结论，两种方法相结合是最有效的。
In pine wilt disease, xylem dysfunction occurs in relation to nematode migration and proliferation in host tissue, but the detection accuracy of pinewood nematode (PWN),Bursaphelenchus xylophilus, in pine stem tissue remains unclear. This study describes the use of cryo-scanning electron microscopy (cryo-SEM) and fluorescein-conjugated wheat germ agglutinin (F-WGA) staining to detect PWN. After PWN invasion, the frequency of surface fluorescence in PWN increased in pine stems from the day of inoculation to 3 weeks after inoculation. However, the fluorescence frequency decreased significantly during the advanced disease stage after 5 weeks. Thus, detecting PWN based on fluorescent staining of the nematode body surface coat protein can be misleading when used to examine the correlation between the development of disease symptoms and the nematode population. In contrast, all cut body segments were fluorescent, and their fluorescent components were common in pine-stem cross sections, regardless of the timing after inoculation. In addition, PWN were observed under cryo-SEM only in empty resin canals and this distribution was confirmed by F-WGA staining of PWN cut in a cross section. Thus, PWN detection based on fluorescent staining of surface coat proteins by F-WGA was not reliable in longitudinal sections of pine stems because of changes in nematode stainability during disease progression. To detect PWN in infected plants, we concluded that a combination of both methods is most effective.