Analysis of Rhizosphere Fungal Communities Using rRNA and rDNA

Part of the Soil Biology book series (SOILBIOL, volume 18)


Microbial communities in soil and rhizosphere are diverse. Use of sequence data derived from environmental samples has become somewhat of a standard to explore the microbial diversity and community composition in soils. While the DNA encoding the ribosomal RNA genes can be easily PCR-amplified from many environmental samples, this DNA may carry a historical fingerprint, as naked DNA or as dormant, but inactive organisms. The use of ribosomal RNA may provide a tool to focus on the active organisms. This chapter describes kit-based methods to simultaneously extract DNA and RNA from environmental samples, and outlines a protocol for reverse-transcribing ribosomal RNA for PCR amplification of the produced cDNA. The fungal communities observed via sequencing the ribosomal RNA or the ribosomal RNA encoding DNA from Andropogon gerardii rhizosphere are compared, and the results briefly discussed.


Clone Library Panicum Virgatum Fungal Community Tallgrass Prairie Andropogon Gerardii 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.



This material is based upon work supported by the National Science Foundation under Grants 0344838 and 0516456. Stacie Kageyama assisted in sampling Andropogon gerardii roots. Konza Prairie Biological Station and its personnel maintain the research site, and are funded partly through the National Science Foundation Long Term Ecological Research program.


  1. Altschul SF, Madden TL, Schäffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucl Acids Res 25:3389–3402CrossRefPubMedGoogle Scholar
  2. Anderson IC, Parkin PI (2007) Detection of active soil fungi by RT-PCR amplification of precursor rRNA molecules. J Microbiol Meth 68:248–253CrossRefGoogle Scholar
  3. Bentivenga SP, Hetrick BAD (1992) Seasonal and temperature effects on mycorrhizal activity and dependence of cool- and warm-season tallgrass prairie grases. Can J Bot 70:1596–1602Google Scholar
  4. Borneman J, Hartin RJ (2000) PCR primers that amplify fungal rRNA genes from environmental samples. Appl Environ Microbiol 66:4356–4360CrossRefPubMedGoogle Scholar
  5. Domsch KH, Gams W, Anderson T-H (1980) Compendium of soil fungi. Academic, New YorkGoogle Scholar
  6. Duineveld BM, Kowalchuk GA, Keijzer A, van Elsas JD, van Veen JA (2001) Analysis of bacterial communities in the rhizosphere of chrysanthemum via denaturing gradient gel electrophoresis of PCR-amplified 16S rRNA as well as DNA fragments coding for 16S rRNA. Appl Environ Microbiol 67:172–178CrossRefPubMedGoogle Scholar
  7. Girvan MS, Bullimore J, Ball AS, Pretty JN, Osborn AM (2004) Responses of active bacterial and fungal communities in soils under winter wheat to different fertilizer and pesticide regimens. Appl Environ Microbiol 70:2692–2701CrossRefPubMedGoogle Scholar
  8. Helgason T, Daniell TJ, Husband R, Fitter AH, Young JPW (1998) Ploughing up the wood-wide web? Nature 394:431CrossRefPubMedGoogle Scholar
  9. Horton TR, Bruns TD (2001) The molecular revolution in ectomycorrhizal ecology: peeking into the black-box. Mol Ecol 10:1855–1871CrossRefPubMedGoogle Scholar
  10. Jumpponen A (2003) Soil fungal community assembly in a primary successional glacier forefront ecosystem as inferred from rDNA sequence analyses. New Phytol 158:569–578CrossRefGoogle Scholar
  11. Jumpponen A (2007) Soil fungal communities underneath willow canopies on a primary successional glacier forefront: rDNA sequence results can be affected by primer selection and chimeric data. Microbial Ecol 53:233–246CrossRefGoogle Scholar
  12. Kowalchuk GA, Drigo B, Yergeau E, van Veen JA (2006) Assessing bacterial and fungal community structure in soil using ribosomal RNA and other structural gene markers. In: Nannipieri P, Smalla K (eds) Nucleic acids and proteins in soil, vol 7. Springer, Berlin, pp 159–188Google Scholar
  13. Poulsen LK, Ballard G, Stahl DA (1993) Use of ribosomal-RNA fluorescence in situ hybridization for measuring the activity of single cells in young and established biofilms. Appl Environ Microbiol 59:1354–1360PubMedGoogle Scholar
  14. Prosser JI (2002) Molecular and functional diversity in soil microorganisms. Plant Soil 244:9–17CrossRefGoogle Scholar
  15. Rambelli A, Persiani AM, Maggi O, Lunghini D, Onofri S, Riess S, Dowgiallo G, Puppi G (1983) Comparative studies on microfungi in tropical ecosystems. UNESCO, RomeGoogle Scholar
  16. Smit E, Leeflang P, Glandorf B, van Elsas JD, Wernars K (1999) Analysis of fungal diversity in the wheat rhizosphere by sequencing of cloned PCR-amplified genes encoding 18S rRNA and temperature gradient gel electrophoresis. Appl Environ Microbiol 65:2614–2621PubMedGoogle Scholar
  17. von Wintzingerode F, Göbel UB, Stackebrandt E (1997) Determination of microbial diversity in environmental samples: pitfalls of PCR-based rRNA analysis. FEMS Microbiol Rev 21:213–229CrossRefGoogle Scholar
  18. White TJ, Bruns TD, Lee SB, Taylor JW (1990) Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis MA, Gelfand DH, Sninsky JJ, White TJ (eds) PCR protocols: a guide to methods and applications. Academic, New York, pp 315–322Google Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2009

Authors and Affiliations

  1. 1.Division of BiologyKansas State UniversityManhattanUSA

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