Skip to main content
SpringerLink
Log in

Comparison of molecular fingerprinting methods for analysis of soil microbial community structure

  • Technical Report
  • Published:
Ecological Research

Abstract

Denaturing gradient gel electrophoresis (DGGE), terminal-restriction fragment length polymorphism (T-RFLP) analysis, and automated ribosomal intergenic spacer analysis (ARISA) have been widely used as molecular fingerprinting methods for analysis of microbial communities. To find suitable methods, we compared the three fingerprinting methods by analyzing soil fungal communities in four differing land-use types: bare ground, crop fields, grasslands, and forests. We also examined optimal primer pairs for DGGE analysis by comparing single and mixed DNA samples of cultured fungal populations. Principal coordinate analysis (PCO), nonmetric multidimensional scaling method (NMDS), and analysis of similarities (ANOSIM), which are major multivariate statistical analyses for quantifying fingerprint patterns, were compared. All three fingerprinting methods yielded clear discrimination of soil fungal communities among the four land-use types, irrespective of statistical methods. The advantages and disadvantages of the three fingerprinting methods were discussed.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3

Similar content being viewed by others

References

  • Anderson IC, Campbell CD, Prosser JI (2003) Potential bias of fungal 18S rDNA and internal transcribed spacer polymerase chain reaction primers for estimating fungal biodiversity in soil. Environ Microbiol 5:36–47. doi:10.1046/j.1462-2920.2003.00383.x

    Article  CAS  PubMed  Google Scholar 

  • Bardgett RD (2005) The biology of soil. A community and ecosystem approach. Oxford Univ. Press, Oxford

    Google Scholar 

  • Borneman J, Hartin RJ (2000) PCR primers that amplify fungal rRNA genes from environmental samples. Appl Environ Microbiol 66:4356–4360. doi:10.1128/AEM.66.10.4356-4360.2000

    Article  CAS  PubMed  Google Scholar 

  • Buchan A, Newell SY, Butler M, Biers EJ, Hollibaugh JT, Moran MA (2003) Dynamics of bacterial and fungal communities on decaying salt marsh grass. Appl Environ Microbiol 69:6676–6687. doi:10.1128/AEM.69.11.6676-6687.2003

    Article  CAS  PubMed  Google Scholar 

  • Chandler DP, Fredrickson JK, Brockman FJ (1997) Effect of PCR template concentration on the composition and distribution of total community 16S rDNA clone libraries. Mol Ecol 6:475–482. doi:10.1046/j.1365-294X.1997.00205.x

    Article  CAS  PubMed  Google Scholar 

  • Collins RE, Rocap G (2007) REPK: an analytical web server to select restriction endonucleases for terminal restriction fragment length polymorphism analysis. Nucleic Acids Res 35:58–62. doi:10.1093/nar/gkm384

    Article  Google Scholar 

  • Edel-Hermann V, Dreumont C, Pérez-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 47:397–404. doi:10.1016/S0168-6496(04)00002-9

    Article  CAS  PubMed  Google Scholar 

  • Egger KN (1994) Molecular analysis of ectomycorrhizal fungal communities. Can J Bot 73:1415–1422. doi:10.1139/b95-405

    Article  Google Scholar 

  • Gardes M, Brums TD (1993) ITS primers with enhanced specificity for Basidiomycetes application to the identification of mycorrhizae and rusts. Mol Ecol 2:113–118. doi:10.1111/j.1365-294X.1993.tb00005.x

    Article  CAS  PubMed  Google Scholar 

  • Hansen MC, Tolker NT, Givskov M, Molin S (1998) Biased 16S rDNA PCR amplification caused by interference from DNA flanking the template region. FEMS Microbiol Ecol 26:141–149. doi:10.1111/j.1574-6941.1998.tb00500.x

    Article  CAS  Google Scholar 

  • Janssen PH, Yates PS, Grinton BE, Taylor PM, Sait M (2002) Improved culturability of soil bacteria and isolation in pure culture of novel members of the divisions acidobacteria, actinobacteria, proteobacteria, and verrucomicrobia. Appl Environ Microbiol 68:2391–2396. doi:10.1128/AEM.68.5.2391-2396.2002

    Article  CAS  PubMed  Google Scholar 

  • Jeewon R, Hyde KD (2007) Detection and diversity of fungi from environmental samples. In: Verma A, Oelmuller R (eds) Advanced techniques in soil microbiology. Springer, Berlin Heidelberg New York, pp 1–15

    Chapter  Google Scholar 

  • Jones CM, Thies JE (2007) Soil microbial community analysis using two-dimensional polyacrylamide gel electrophoresis of the bacterial ribosomal internal transcribed spacer regions. J Microbiol Methods 69:256–267. doi:10.1016/j.mimet.2006.12.024

    Article  CAS  PubMed  Google Scholar 

  • 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 69:6768–6776. doi:10.1128/AEM.69.11.6768-6776.2003

    Article  CAS  PubMed  Google Scholar 

  • 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 68:4370–4376. doi:10.1128/AEM.68.9.4370-4376.2002

    Article  CAS  PubMed  Google Scholar 

  • 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 63:4516–4522

    CAS  PubMed  Google Scholar 

  • Marsh TL, Saxman P, Cole J, Tiedje J (2000) Terminal restriction fragment length polymorphism analysis program, a web-based research tool for microbial community analysis. Appl Environ Microbiol 66:3616–3620. doi:10.1128/AEM.66.8.3616-3620.2000

    Article  CAS  PubMed  Google Scholar 

  • May LA, Smiley B, Schmidt MG (2001) Comparative denaturing gradient gel electrophoresis of fungal communities associated with whole plant corn silage. Can J Microbiol 47:829–841. doi:10.1139/cjm-47-9-829

    Article  CAS  PubMed  Google Scholar 

  • Ramette A (2007) Multivariate analyses in microbial ecology. FEMS Microbiol Ecol 62:142–160. doi:10.1111/j.1574-6941.2007.00375.x

    Article  CAS  PubMed  Google Scholar 

  • Rolling WFM, Head LM (2005) Prokaryotic systematics: PCR and sequence analysis of amplified 16s rRNA genes. In: Osborn AM, Smith CJ (eds) Molecular microbial ecology. Taylor and Francis, New York, pp 25–64

    Google Scholar 

  • Schabereiter-Gurtner C, Piñar G, Lubitz W, Rölleke S (2001) Analysis of fungal communities on historical church window glass by denaturing gradient gel electrophoresis and phylogenetic 18s rDNA sequence analysis. J Microbiol Methods 47:345–354. doi:10.1016/S0167-7012(01)00344-X

    Article  CAS  PubMed  Google Scholar 

  • Schwarzenbach K, Enkerli J, Widmer F (2007) Objective criteria to assess representativity of soil fungal community profiles. J Microbiol Meth 68:358–366. doi:10.1016/j.mimet.2006.09.015

    Article  CAS  Google Scholar 

  • Singh BK, Munro S, Reid E, Ord B, Potts JM, Paterson E, Millard P, Ibekwe AM, Grieve CM (2006a) Investigating microbial community structure in soils by physiological, biochemical and molecular fingerprinting methods. Eur J Soil Sci 57:72–82. doi:10.1111/j.1365-2389.2005.00781.x

    Article  CAS  Google Scholar 

  • Singh BK, Nazaries L, Munro S, Anderson IC, Campbell CD (2006b) Use of multiplex terminal restriction fragment length polymorphism for rapid and simultaneous analysis of different components of the soil microbial community. Appl Environ Microbiol 72:7278–7285. doi:10.1128/AEM.00510-06

    Article  CAS  PubMed  Google Scholar 

  • Smit E, Leeflang P, Glandorf B, 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 65:2614–2621

    CAS  PubMed  Google Scholar 

  • Vainio EJ, Hantula J (2000) Direct analysis of wood-inhabiting fungi using denaturing gradient gel electrophoresis of amplified ribosomal DNA. Mycol Res 104:927–936. doi:10.1017/S0953756200002471

    Article  CAS  Google Scholar 

  • Wang GC-Y, Wang Y (1997) Frequency of formation of chimeric molecules as a consequence of PCR coamplification of 16S rRNA genes from mixed bacterial genomes. Appl Environ Microbiol 63:4645–4650

    CAS  PubMed  Google Scholar 

  • Wardle DA (2002) Communities and ecosystems. Linking the aboveground and belowground components. Princeton University Press, Princeton

    Google Scholar 

  • White TJ, Bruns T, Lee S, Taylor J (1990) Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innes MA, Gelfand DH, Sninsky JJ, White T (eds) PCR protocols: a guide to method and applications. Academic Press, San Diego, pp 315–322

    Google Scholar 

Download references

Acknowledgments

Pure cultures of microbial strains used for selection of primers were kindly provided by Kazuaki Tanaka (Plant Pathology, Hirosaki University), Sayaka Isono, and Teruo Sano (Plant Pathology, Hirosaki University). Part of this experiment was assisted by Hiroshi Ida and Liu Guangcheng.

Author information

Authors and Affiliations

  1. Faculty of Agriculture and Life Science, Hirosaki University, Hirosaki, 036-8561, Japan

    Atsushi Okubo & Shu-ichi Sugiyama

Authors
  1. Atsushi Okubo
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Shu-ichi Sugiyama
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Shu-ichi Sugiyama.

Appendix

Appendix

Tables 5, 6, 7

Table 5 Thermal cycles of PCR amplification on each primer pair
Full size table
Table 6 DGGE conditions for each primer pair
Full size table
Table 7 References investigating soil fungal community structure by T-RFLP
Full size table

About this article

Cite this article

Okubo, A., Sugiyama, Si. Comparison of molecular fingerprinting methods for analysis of soil microbial community structure. Ecol Res 24, 1399–1405 (2009). https://doi.org/10.1007/s11284-009-0602-9

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11284-009-0602-9

Keywords

Search

Navigation