Nested multiplex PCR—A feasible technique to study partial community of arbuscular mycorrhizal fungi in field-growing plant root |
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Authors: | DONG Xiuli ZHAO Bin |
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Institution: | State Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070, China |
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Abstract: | 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 comerstone 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 mycorrhizal fungal species in a
same plant root system. |
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Keywords: | arbuscular mycorrhiza nested PCR multiplex PCR mycorrhizal community specific primers |
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