, Volume 17, Issue 4, pp 259–270 | Cite as

Using terminal restriction fragment length polymorphism (T-RFLP) to identify mycorrhizal fungi: a methods review

  • I. A. DickieEmail author
  • R. G. FitzJohn


Terminal restriction fragment length polymorphism (T-RFLP) is an increasingly widely used technique in mycorrhizal ecology. In this paper, we review the technique as it is used to identify species of mycorrhizal fungi and distinguish two different versions of the technique: peak-profile T-RFLP (the original version) and database T-RFLP. We define database T-RFLP as the use of T-RFLP to identify individual species within samples by comparison of unknown data with a database of known T-RFLP patterns. This application of T-RFLP avoids some of the pitfalls of peak-profile T-RFLP and allows T-RFLP to be applied to polyphyletic functional groups such as ectomycorrhizal fungi. The identification of species using database T-RFLP is subject to several sources of potential error, including (1) random erroneous matches of peaks to species, (2) shared T-RFLP profiles across species, and (3) multiple T-RFLP profiles within a species. A mathematical approximation of the risk of the first type of error as a function of experimental parameters is discussed. Although potentially less accurate than some other methods such as clone libraries, the high throughput of database T-RFLP permits much greater replication and may, therefore, be preferable for many ecological questions, particularly when combined with other techniques such as cloning.


Arbuscular mycorrhiza Ectomycorrhiza Molecular identification Terminal restriction fragment length polymorphism (T-RFLP) 



Valuable discussions and sharing of data and concepts with P. Avis, S. Branco, I. Edwards, G. Forrester, D. Park, R. Smissen, and many others have contributed to this review. R. Molina and two reviewers provided helpful comments. The authors were supported by funds from the Foundation for Research, Science and Technology (FRST, NSOF) of New Zealand.


  1. Aldrich-Wolfe L (2007) Distinct mycorrhizal fungal communities on new and established host species in a transitional tropical plant community. Ecology 88:559–566CrossRefPubMedGoogle Scholar
  2. Allmer J, Vasiliauskas R, Ihrmark K, Stenlid J, Dahlberg A (2006) Wood-inhabiting fungal communities in woody debris of Norway spruce (Picea abies (L.) Karst.), as reflected by sporocarps, mycelial isolations, and T-RFLP analysis. FEMS Microbiol Ecol 55:57–67CrossRefPubMedGoogle Scholar
  3. Anderson IC, Cairney JWG (2004) Diversity and ecology of soil fungal communities: increased understanding through the application of molecular techniques. Environ Microbiol 6:769–779CrossRefPubMedGoogle Scholar
  4. Artursson V, Finlay RD, Jansson JK (2005) Combined bromodeoxyuridine immunocapture and terminal-restriction fragment length polymorphism analysis highlights differences in the active soil bacterial metagenome due to Glomus mosseae inoculation or plant species. Environ Microbiol 7:1952–1966CrossRefPubMedGoogle Scholar
  5. Avis PG, Feldheim KA (2005) A method to size DNA fragments from 50 to 800 bp on a DNA analyzer. Mol Ecol Notes 5:969–970CrossRefGoogle Scholar
  6. Avis PG, Dickie IA, Mueller G (2006) A “dirty” business: testing the limitations of TRFLP analysis of soil fungi. Mol Ecol 15:873–882CrossRefPubMedGoogle Scholar
  7. Blackwood CB, Marsh T, Kim S-H, Paul EA (2003) Terminal restriction fragment length polymorphism data analysis for quantitative comparison of microbial communities. Appl Environ Microbiol 69:926–932CrossRefPubMedPubMedCentralGoogle Scholar
  8. Bougoure DS, Cairney JWG (2005) Fungi associated with hair roots of Rhododendron lochiae (Ericaceae) in an Australian tropical cloud forest revealed by culturing and culture-independent molecular methods. Environ Microbiol 7:1743–1754CrossRefPubMedGoogle Scholar
  9. Brodie E, Edwards S, Clipson N (2003) Soil fungal community structure in a temperate upland grassland soil. FEMS Microbiol Ecol 45:105–114CrossRefPubMedGoogle Scholar
  10. Bruns TD (1995) Thoughts on the processes that maintain local species diversity of ectomycorrhizal fungi. Plant Soil 170:63–73CrossRefGoogle Scholar
  11. Burke DJ, Martin KJ, Rygiewicz PT, Topa MA (2005) Ectomycorrhizal fungi identification in single and pooled root samples: terminal restriction fragment length polymorphism (TRFLP) and morphotyping compared. Soil Biol Biochem 37:1683–1694CrossRefGoogle Scholar
  12. Burke DJ, Kretzer AM, Rygiewicz PT, Topa MA (2006a) Soil bacterial diversity in a loblolly pine plantation: influence of ectomycorrhizas and fertilization. FEMS Microbiol Ecol 57:409–419CrossRefPubMedGoogle Scholar
  13. Burke DJ, Martin KJ, Rygiewicz PT, Topa MA (2006b) Relative abundance of ectomycorrhizas in a managed loblolly pine (Pinus taeda) genetics plantation as determined through terminal restriction fragment length polymorphism profiles. Can J Bot 84:924–932CrossRefGoogle Scholar
  14. Dickie IA, Reich PB (2005) Ectomycorrhizal fungal communities at forest edges. J Ecol 93:244–255CrossRefGoogle Scholar
  15. Dickie IA, Xu W, Koide RT (2002) Vertical niche differentiation of ectomycorrhizal hyphae in soils as shown by T-RFLP analysis. New Phytol 156:527–535CrossRefGoogle Scholar
  16. Dollhopf SL, Hashsham SA, Tiedje JM (2001) Interpreting 16S rDNA T-RFLP data: application of self-organizing maps and principal component analysis to describe community dynamics and convergence. Microb Ecol 42:495–505CrossRefPubMedGoogle Scholar
  17. Douhan GW, Petersen C, Bledsoe CS, Rizzo DM (2005) Contrasting root associated fungi of three common oak-woodland plant species based on molecular identification: host specificity or non-specific amplification. Mycorrhiza 15:365–372CrossRefPubMedGoogle Scholar
  18. Dunbar J, Ticknor LO, Kuske CR (2000) Assessment of microbial diversity in four southwestern United States soils by 16S rRNA gene terminal restriction fragment analysis. Appl Environ Microbiol 66:2943–2950CrossRefPubMedPubMedCentralGoogle Scholar
  19. Edel-Hermann W, Dreumont C, Perez-Piqueres A, Steinberg C (2004) Terminal restriction fragment length polymorphism analysis of ribosomal RNA genes to assess changes in fungal community structure in soils. FEMS Microbiol Ecol 47:397–404CrossRefGoogle Scholar
  20. Edwards IP, Turco RF (2005) Inter- and intraspecific resolution of nrDNA TRFLP assessed by computer-simulated restriction analysis of a diverse collection of ectomycorrhizal fungi. Mycol Res 109:212–226CrossRefPubMedGoogle Scholar
  21. Edwards IP, Cripliver JL, Gillespie AR, Johnsen KH, Scholler M, Turco RF (2004) Nitrogen availability alters macrofungal basidiomycete community structure in optimally fertilized loblolly pine forests. New Phytol 162:755–770CrossRefGoogle Scholar
  22. FitzJohn RG, Dickie IA (2007) TRAMPR: an R package for analysis and matching of terminal-restriction fragment length polymorphism (TRFLP) profiles. Mol Ecol Notes (in press). DOI 10.1111/j.1471-8286.2007.01744.x
  23. Gardes M, Bruns TD (1996) Community structure of ectomycorrhizal fungi in a Pinus muricata forest: above- and below-ground views. Can J Bot 74:1572–1583CrossRefGoogle Scholar
  24. Genney DR, Anderson IC, Alexander IJ (2006) Fine-scale distribution of pine ectomycorrhizas and their extramatrical mycelium. New Phytol 170:381–390CrossRefPubMedGoogle Scholar
  25. Haskins KE, Gehring CA (2004) Interactions with juniper alter the abundance and composition of pinyon pine ectomycorrhizal fungal communities. Ecology 85:2687–2692CrossRefGoogle Scholar
  26. Hibbett DS, Gilbert L-B, Donoghue MJ (2000) Evolutionary instability of ectomycorrhizal symbioses in basidiomycetes. Nature 407:506–508CrossRefPubMedGoogle Scholar
  27. Horton TR, Bruns TD (2001) The molecular revolution in ectomycorrhizal ecology: peeking into the black-box. Mol Ecol 10:1855–1871CrossRefPubMedGoogle Scholar
  28. Johnson D, Vandenkoornhuyse PJ, Leake JR, Gilbert L, Booth RE, Grime JP, Young PW, Read DJ (2003) Plant communities affect arbuscular mycorrhizal fungal diversity and community composition in grassland microcosms. New Phytol 161:503–515CrossRefGoogle Scholar
  29. Kanchanaprayudh J, Zhou Z, Yomyart S, Sihanonth P, Hogetsu T (2003) Molecular phylogeny of ectomycorrhizal Pisolithus fungi associated with pine, dipterocarp, and eucalyptus trees in Thailand. Mycoscience 44:287–294CrossRefGoogle Scholar
  30. Kaplan CW, Kitts CL (2003) Variation between observed and true terminal restriction fragment length is dependent on true TRF length and purine content. J Microbiol Methods 54:121–125CrossRefPubMedGoogle Scholar
  31. Kent AD, Smith DJ, Benson BJ, Triplett EW (2003) Web-based phylogenetic assignment tool for analysis of terminal restriction fragment length polymorphism profiles of microbial communities. Appl Environ Microbiol 69:6768–6776CrossRefPubMedPubMedCentralGoogle Scholar
  32. Kitts CL (2001) Terminal restriction fragment patterns: a tool for comparing microbial communities and assessing community dynamics. Curr Issues Intest Microbiol 2:17–25PubMedGoogle Scholar
  33. Klamer M, Hedlund K (2004) Fungal diversity in set-aide [sic] agricultural soil investigated using terminal-restriction fragment length polymorphism. Soil Biol Biochem 36:983–988CrossRefGoogle Scholar
  34. Klamer M, Roberts MS, Levine LH, Drake BG, Garland JL (2002) Influence of elevated CO2 on the fungal community in a coastal scrub oak forest soil investigated with terminal-restriction fragment length polymorphism analysis. Appl Environ Microbiol 68:4370–4376CrossRefPubMedPubMedCentralGoogle Scholar
  35. Koide RT, Xu B, Sharda J (2005a) Contrasting below-ground views of an ectomycorrhizal fungal community. New Phytol 166:251–262CrossRefPubMedGoogle Scholar
  36. Koide RT, Xu B, Sharda J, Lekberg Y, Ostiguy N (2005b) Evidence of species interactions within an ectomycorrhizal fungal community. New Phytol 165:305–316CrossRefPubMedGoogle Scholar
  37. Kowalchuk GA, De Souza FA, Van Veen JA (2002) Community analysis of arbuscular mycorrhizal fungi associated with Ammophila arenaria in Dutch coastal sand dunes. Mol Ecol 11:571–581CrossRefPubMedGoogle Scholar
  38. Landeweert R, Leeflang P, Kuyper TW, Hoffland E, Rosling A, Wernars K, Smit E (2003) Molecular identification of ectomycorrhizal mycelium in soil horizons. Appl Environ Microbiol 69:327–333CrossRefPubMedPubMedCentralGoogle Scholar
  39. Landeweert R, Leeflang P, Smit E, Kuyper TW (2005) Diversity of an ectomycorrhizal fungal community studied by a root tip and total soil DNA approach. Mycorrhiza 15:1–6CrossRefPubMedGoogle Scholar
  40. Leckie SE (2005) Methods of microbial community profiling and their application to forest soils. For Ecol Manag 220:88–106CrossRefGoogle Scholar
  41. Lekberg Y, Koide RT, Rohr JR, Aldrich-Wolfe L, Morton JB (2007) Role of niche restrictions and dispersal in the composition of arbuscular mycorrhizal fungal communities. J Ecol 95:95–105CrossRefGoogle Scholar
  42. Lindahl BD, Ihrmark K, Boberg J, Trumbore SE, Högberg P, Stenlid J, Finlay RD (2006) Spatial separation of litter decomposition and mycorrhizal nitrogen uptake in a boreal forest. New Phytol 173:611–620CrossRefGoogle Scholar
  43. Liu WT, Marsh TL, Cheng H, Forney LJ (1997) Characterization of microbial diversity by determining terminal restriction fragment length polymorphisms of genes encoding 16S rRNA. Appl Environ Microbiol 63:4516–4522PubMedPubMedCentralGoogle Scholar
  44. Lord NS, Kaplan CW, Shank P, Kitts CL, Elrod SL (2002) Assessment of fungal diversity using terminal restriction fragment (TRF) pattern analysis: comparison of 18S and ITS ribosomal regions. FEMS Microbiol Ecol 42:327–337CrossRefPubMedGoogle Scholar
  45. Lukow T, Dunfield PF, Liesack W (2000) Use of the T-RFLP technique to assess spatial and temporal changes in the bacterial community structure within an agricultural soil planted with transgenic and non-transgenic potato plants. FEMS Microbiol Ecol 32:241–247CrossRefGoogle Scholar
  46. Ma WK, Siciliano SD, Germida JJ (2005) A PCR–DGGE method for detecting arbuscular mycorrhizal fungi in cultivated soils. Soil Biol Biochem 37:1589–1597CrossRefGoogle Scholar
  47. Maleszka R, Clark-Walker GD (1993) Yeasts have a four-fold variation in ribosomal DNA copy number. Yeast 9:53–58CrossRefPubMedGoogle Scholar
  48. Marsh TL (1999) Terminal restriction fragment length polymorphism (T-RFLP): an emerging method for characterizing diversity among homologous populations of amplification products. Curr Opin Microbiol 2:323–327CrossRefPubMedGoogle Scholar
  49. Martin KJ (2007) Introduction to molecular analysis of ectomycorrhizal communities. Soil Sci Soc Am J 71:601–610CrossRefGoogle Scholar
  50. Mummey DL, Rillig MC (2006) The invasive plant species Centaurea maculosa alters arbuscular mycorrhizal fungal communities in the field. Plant Soil 288:81–90CrossRefGoogle Scholar
  51. Mummey DL, Rillig MC, Holben WE (2005) Neighboring plant influences on arbuscular mycorrhizal fungal community composition as assessed by T-RFLP analysis. Plant Soil 271:83–90CrossRefGoogle Scholar
  52. Nara K (2005) Ectomycorrhizal networks and seedling establishment during early primary succession. New Phytol 169:169–178CrossRefGoogle Scholar
  53. Nara K, Nakaya H, Wu B, Zhou Z, Hogetsu T (2003) Underground primary succession of ectomycorrhizal fungi in a volcanic desert on Mount Fuji. New Phytol 159:743–756CrossRefGoogle Scholar
  54. Opik M, Moora M, Liira J, Koljalg U, Zobel M, Sen R (2003) Divergent arbuscular mycorrhizal fungal communities colonize roots of Pulsatilla spp. in boreal Scots pine forest and grassland soils. New Phytol 160:581–593CrossRefGoogle Scholar
  55. Pennanen T, Heiskanen J, Korkama U (2005) Dynamics of ectomycorrhizal fungi and growth of Norway spruce seedlings after planting on a mounded forest clearcut. For Ecol Manag 213:243–252CrossRefGoogle Scholar
  56. R Development Core Team (2006) R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. www.R-project.orgGoogle Scholar
  57. Råberg U, Högberg NOS, Land CJ (2005) Detection and species discrimination using rDNA T-RFLP for identification of wood decay fungi. Holzforschung 59:696–702CrossRefGoogle Scholar
  58. Råberg U, Brischke C, Rapp AO, Högberg NOS, Land CJ (2007) External and internal fungal flora of pine sapwood (Pinus sylvestris L.) specimens in above-ground field tests at six different sites in south-west Germany. Holzforschung 61:104–111CrossRefGoogle Scholar
  59. Renker C, Heinrichs J, Kaldorf M, Buscot F (2003) Combining nested PCR and restriction digest of the internal transcribed spacer region to characterize arbuscular mycorrhizal fungi on roots from the field. Mycorrhiza 13:191–198CrossRefPubMedGoogle Scholar
  60. Renker C, Weißhuhn K, Kellner H, Buscot F (2006) Rationalizing molecular analysis of field-collected roots for assessing diversity of arbuscular mycorrhizal fungi: to pool, or not to pool, that is the question. Mycorrhiza 16:525–531CrossRefPubMedGoogle Scholar
  61. Saari TA, Saari SK, Campbell CD, Alexander IJ, Anderson IC (2007) FragMatch—a program for the analysis of DNA fragment data. Mycorrhiza 17:133–136CrossRefPubMedGoogle Scholar
  62. Schüßler A, Schwarzott D, Walker C (2001) A new fungal phylum, the Glomeromycota: phylogeny an evolution. Mycol Res 105:1413–1421CrossRefGoogle Scholar
  63. Singh BK, Munro S, Reid E, Ord B, Potts JM, Paterson E, Millard P (2006) Investigating microbial community structure in soils by physiological, biochemical and molecular fingerprinting methods. Eur J Soil Sci 57:72–82CrossRefGoogle Scholar
  64. Sogin ML, Morrison HG, Huber JA, Welch DA, Huse SM, Neal PR, Arrieta JM, Herndl GJ (2006) Microbial diversity in the deep sea and the underexplored “rare biosphere.” Proc Natl Acad Sci U S A 103:12115–12120CrossRefPubMedPubMedCentralGoogle Scholar
  65. Thies JE (2007) Soil microbial community analysis using terminal restriction fragment length polymorphisms. Soil Sci Soc Am J 71:579–591CrossRefGoogle Scholar
  66. Tonin C, Vandenkoornhuyse P, Joner EJ, Straczek J, Leyval C (2001) Assessment of arbuscular mycorrhizal fungi diversity in the rhizosphere of Viola calaminaria and effect of these fungi on heavy metal uptake by clover. Mycorrhiza 10:161–168CrossRefGoogle Scholar
  67. Trowbridge J, Jumpponen A (2004) Fungal colonization of shrub willow roots at the forefront of a receding glacier. Mycorrhiza 14:283–293CrossRefPubMedGoogle Scholar
  68. Vandenkoornhuyse P, Ridgway KP, Watson IJ, Fitter AH, Young JPW (2003) Co-existing grass species have distinctive arbuscular mycorrhizal communities. Mol Ecol 12:3085–3095CrossRefPubMedGoogle Scholar
  69. Widmer F, Hartmann M, Frey B, Kölliker R (2006) A novel strategy to extract specific phylogenetic sequence information from community T-RFLP. J Microbiol Methods 66:512–529CrossRefPubMedGoogle Scholar
  70. Zhou ZH, Hogetsu T (2002) Subterranean community structure of ectomycorrhizal fungi under Suillus grevillei sporocarps in a Larix kaempferi forest. New Phytol 154:529–539CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2007

Authors and Affiliations

  1. 1.Landcare ResearchLincolnNew Zealand

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