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ECTOMYCORRHIZAL FUNGI IDENTIFICATION IN SINGLE AND POOLED ROOT SAMPLES: TERMINAL RESTRICTION FRAGMENT LENGTH POLYMORPHISM (TRFLP) AND MORPHOTYPING COMPARED
Citation:
Burke, D. J., K. J. Martin, P T. Rygiewicz, AND M. A. Topa. ECTOMYCORRHIZAL FUNGI IDENTIFICATION IN SINGLE AND POOLED ROOT SAMPLES: TERMINAL RESTRICTION FRAGMENT LENGTH POLYMORPHISM (TRFLP) AND MORPHOTYPING COMPARED. SOIL BIOLOGY AND BIOCHEMISTRY. Elsevier Science Ltd, New York, NY, 37(9):1683-1694, (2005).
Description:
PCR-TRFLP methodology targeting rRNA genes has effectively been used to discriminate between microbial communities but to date has not been used specifically for the analysis of ectomycorrhizal communities colonizing plant roots. We describe here results of a study conducted to assess the efficacy of a PCR-TRFLP approach for determination of ectomycorrhizal communities colonizing the roots of loblolly pine (Pinus taeda L.). Root tips separated from soil cores were morphotyped; DNA was extracted separately from each morphotype and amplified with novel primers capable of amplifying both ascomycetes as well as basidiomycetes. Labeled amplicon was used to generate terminal restriction fragments (TRF) for molecular fingerprinting of root colonizing fungi. A fixed proportion of the DNA extracted from each morphotype from the same core was pooled, amplified, and used to assess presence of fungal species types within the mixed community and results compared to traditional morphotyping techniques. Approximately 5.0 ? 0.3 phylotypes were detected per core with TRFLP corrected morphotyping, whereas 4.0 ? 0.4 phylotypes were detected using TRFLP on pooled community samples. Pooled community TRFLP detected 80% of phylotypes in whole soil cores, constituting more than 93% of colonized root tips, as compared to TRFLP corrected morphotyping. Although some morphotypes share TRFs for the ITS1 region, they can be distinguished based upon analysis of ITS2 TRFs and dual analysis of both regions permits accurate fingerprinting of fungal species types. The method appears to be viable for rapid analysis of mycorrhizal communities using pooled root tips collected from soil cores.