采用共聚焦显微拉曼技术研究了炭疽病感染所致茶叶细胞壁结构和化学成分的变化。 对茶叶健康和染病组织细胞进行微米级空间分辨率的显微拉曼光谱扫描, 并结合透射电镜观察炭疽病侵染所致的细胞超微结构变化, 结果显示染病前后细胞壁的拉曼光谱位移和强度都有明显的差异, 表明炭疽病侵染导致细胞壁中化学成分发生了较大的变化。 其中由纤维素, 果胶, 酯类化合物产生的拉曼峰强度都有明显下降, 说明细胞壁中这些物质的含量在染病后减少了; 而木质素拉曼散射引起的拉曼峰强度有所上升, 说明木质素的含量在染病后有所增加。 随后基于纤维素的拉曼指纹波数和显微空间结构信息实现了茶叶健康组织和染病组织细胞壁中纤维素的化学成像分析, 结果显示炭疽病侵染不仅导致细胞壁中纤维素的含量大大减少, 而且纤维素的有序结构被破坏。 由此得出结论: 在无需对样本进行染色或复杂的化学处理的情况下, 共聚焦显微拉曼可以揭示由炭疽病侵染引起的茶叶细胞壁化学成分和结构的变化, 本研究是共聚焦显微拉曼技术首次用于植物病理学中寄主-病原物互作机制的研究, 将为深入研究寄主-病原物在细胞层面上的互作机制开辟蹊径。

Healthy tea and tea infected by anthracnose were first studied by confocal Raman microscopy to illustrate chemical changes of cell wall in the present paper. Firstly, Raman spectra of both healthy and infected sample tissues were collected with spatial resolution at micron-level, and ultrastructure of healthy and infected tea cells was got from scanning electron microscope. These results showed that there were significant changes in Raman shift and Raman intensity between healthy and infected cell walls, indicating that great differences occurred in chemical compositions of cell walls between healthy and infected samples. In details, intensities at many Raman bands which were closely associated with cellulose, pectin, esters were reduced after infection, revealing that the content of chemical compounds such as cellulose, pectin, esters was decreased after infection. Subsequently, chemical imaging of both healthy and infected tea cell walls were realized based on Raman fingerprint spectra of cellulose and microscopic spatial structure. It was found that not only the content of cellulose was reduced greatly after infection, but also the ordered structure of cellulose was destroyed by anthracnose infection. Thus, confocal Raman microscopy was shown to be a powerful tool to detect the chemical changes in cell wall of tea caused by anthracnose without any chemical treatment or staining. This research firstly applied confocal Raman microscopy in phytopathology for the study of interactive relationship between host and pathogen, and it will also open a new way for intensive study of host-pathogen at cellular level.