Dong Xiuli, Zhao Bin
State Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070, China.
Sci China C Life Sci. 2006 Aug;49(4):354-61. doi: 10.1007/s11427-006-2008-z.
Plant can be infected by different arbuscular mycorrhizal fungi, but little is known about the interaction between them within root tissues mainly because different species cannot be distinguished on the basis of fungal structure. Accurate species identification of Arbuscular mycorrhizal fungi (AMF) colonized in plant roots is the cornerstone of mycorrhizal study, yet this fundamental step is impossible through its morphological character alone. For accurate, rapid and inexpensive detection of partial mycorrhizal fungal community in plant roots, a nested multiplex polymerase chain reaction (PCR) was developed in this study. Five discriminating primers designed based on the variable region of the 5' end of the large ribosomal subunit were used in the experiment for testing their specificity and the sensitivity in nested PCR by using spores from Glomus mosseae (BEG12), Glomus intraradices (BEG141), Scutellospora castaneae (BEG1) and two unidentified Glomus sp. HAUO3 and HAUO4. The feasibility assay of nested multiplex PCR was conducted by use of spore mixture, Astragalus sinicum roots co-inoculated with 4 species of arbuscular mycorrhizal fungi from pot cultures and 15 different field-growing plant roots respectively after analyses of the compatibility of primers. The result indicated that the sensitivity was in the same range as that of the corresponding single PCR reaction. Overall accuracy was 95%. The efficiency and sensitivity of this multiplex PCR procedure provided a rapid and easy way to simultaneously detect several of arbuscular mycorrhiza fungal species in a same plant root system.
植物可被不同的丛枝菌根真菌感染，但对于它们在根组织内的相互作用却知之甚少，主要原因是基于真菌结构无法区分不同的物种。准确鉴定定殖于植物根系中的丛枝菌根真菌（AMF）物种是菌根研究的基石，但仅通过其形态特征无法完成这一基本步骤。为了准确、快速且低成本地检测植物根系中的部分菌根真菌群落，本研究开发了一种巢式多重聚合酶链反应（PCR）。基于大核糖体亚基5'端可变区设计的5条鉴别引物用于实验，通过使用摩西球囊霉（BEG12）、根内球囊霉（BEG141）、栗色盾巨孢囊霉（BEG1）的孢子以及两种未鉴定的球囊霉属真菌HAUO3和HAUO4来测试其在巢式PCR中的特异性和灵敏度。在分析引物兼容性后，分别使用孢子混合物、盆栽接种4种丛枝菌根真菌的紫云英根以及15种不同田间生长植物的根进行巢式多重PCR的可行性分析。结果表明，灵敏度与相应的单重PCR反应处于同一范围。总体准确率为95%。这种多重PCR方法的效率和灵敏度为同时检测同一植物根系中的多种丛枝菌根真菌物种提供了一种快速简便的方法。
