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Soil Fungal Communities Underneath Willow Canopies on a Primary Successional Glacier Forefront: rDNA Sequence Results Can Be Affected by Primer Selection and Chimeric Data

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Abstract

Soil fungal communities underneath willow canopies that had established on the forefront of a receding glacier were analyzed by cloning the polymerase chain reaction (PCR)-amplified partial small subunit (18S) of the ribosomal (rRNA) genes. Congruence between two sets of fungus-specific primers targeting the same gene region was analyzed by comparisons of inferred neighbor-joining topologies. The importance of chimeric sequences was evaluated by Chimera Check (Ribosomal Database Project) and by data reanalyses after omission of potentially chimeric regions at the 5′- and 3′-ends of the cloned amplicons. Diverse communities of fungi representing Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota were detected. Ectomycorrhizal fungi comprised a major component in the early plant communities in primary successional ecosystems, as both primer sets frequently detected basidiomycetes (Russulaceae and Thelephoraceae) forming mycorrhizal symbioses. Various ascomycetes (Ophiostomatales, Pezizales, and Sordariales) of uncertain function dominated the clone libraries amplified from the willow canopy soil with one set of primers, whereas the clone libraries of the amplicons generated with the second primer set were dominated by basidiomycetes. Accordingly, primer bias is an important factor in fungal community analyses using DNA extracted from environmental samples. A large proportion (>30%) of the cloned sequences were concluded to be chimeric based on their changing positions in inferred phylogenies after omission of possibly chimeric data. Many chimeric sequences were positioned basal to existing classes of fungi, suggesting that PCR artifacts may cause frequent discovery of new, higher level taxa (order, class) in direct PCR analyses. Longer extension times during the PCR amplification and a smaller number of PCR cycles are necessary precautions to allow collection of reliable environmental sequence data.

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References

  1. SF Altschul, TL Madden, AA Schäffer, J Zhang, Z Zhang, DJ Miller and DJ Lipman, Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25 (1997) 3389-3402

    Article  PubMed  CAS  Google Scholar 

  2. IC Anderson, CD Campbell and JI Prosser, Potential bias of fungal 18S rDNA and internal transcribed polymerase chain reaction primers for estimating fungal biodiversity in soil. Environ Microbiol 5 (2003) 36-47

    Article  PubMed  CAS  Google Scholar 

  3. J Borneman and RJ Hartin, PCR primers that amplify fungal rRNA genes from environmental samples. Appl Environ Microbiol 66 (2000) 4356-4360

    Article  PubMed  CAS  Google Scholar 

  4. Cázares, E (1992) Mycorrhizal fungi and their relationship to plant succession in subalpine habitats. PhD Thesis, Oregon State University

  5. DP Chandler, JK Fredrickson and FJ Brockman, Effect of PCR template concentration on the composition and distribution of total community rDNA clone libraries. Mol Ecol 6 (1997) 475-482

    Article  PubMed  CAS  Google Scholar 

  6. JHC Cornelissen, R Aerts, MJA Cerabolini, MJA Werger and MGA derHeijden van, Carbon cycling traits of plant species are linked with mycorrhizal strategy. Oecologia 129 (2001) 611-619

    Google Scholar 

  7. JL Dahlstrom, JE Smith and NS Weber, Mycorrhiza-like interaction by Morchella with species of the Pinaceae in pure culture synthesis. Mycorrhiza 9 (2000) 279-285

    Article  Google Scholar 

  8. RM Danielson, Ectomycorrhiza formation by the operculate discomycete Sphaerosporella brunnea (Pezizales). Mycologia 76 (1984) 454-461

    Google Scholar 

  9. IA Dickie, B Xu and RT Koide, Vertical niche differentiation of ectomycorrhizal hyphae in soil as shown by T-RFLP analysis. New Phytol 156 (2002) 527-535

    Article  CAS  Google Scholar 

  10. KN Egger and JW Paden, Biotrophic associations between lodgepole pine seedlings and post-fire ascomycetes (Pezizales) in monoxenic culture. Can J Bot 64 (1986) 2719-2725

    Article  Google Scholar 

  11. S Egli, F Ayer and F Chatelain, Die Beschreibung der Diversität von Macromyceten. Erfahrungen aus pilzölologischen Langenzeitstudien im Pilzreservat La Chanéaz, FR. Mycol Helv 9 (1997) 19-32

    Google Scholar 

  12. V Farrelly, FA Rainey and E Stackebrandt, Effect of genome size and rrn gene copy number on PCR amplification of 16S rRNA genes from a mixture of bacterial species. Appl Environ Microbiol 91 (1995) 2798-2801

    Google Scholar 

  13. JE Ford, MG McHeyzer-Williams and MR Lieber, Chimeric molecules created by gene amplification interfere with the analyses of somatic hypermutation of murine immunoglobulin genes. Gene 142 (1994) 279-283

    Article  PubMed  CAS  Google Scholar 

  14. M Gardes and TD Bruns, ITS primers with enhanced specificity for higher fungi and basidiomycetes: application to identification of mycorrhizae and rusts. Mol Ecol 2 (1993) 113-118

    PubMed  CAS  Google Scholar 

  15. M Gardes and TD Bruns, ITS-RFLP matching for the identification of fungi. In: JP Clapp (ed.) Methods in Molecular Biology, Vol. 50: Species Diagnostics Protocols: PCR and Other Nucleic Acid Methods. Totowa, NJ: Humana Press Inc (1996) pp. 177-186

    Google Scholar 

  16. A Gargas, PT DePriest and JW Taylor, Positions of multiple insertion in SSU rDNA of lichen-forming fungi. Mol Biol Evol 12 (1995) 208-218

    PubMed  CAS  Google Scholar 

  17. A Holst-Jensen, M Vaage, T Schumacher and S Johansen, Structural characteristics and possible horizontal transfer of group I introns between closely related plant pathogenic fungi. Mol Biol Evol 16 (1999) 114-126

    PubMed  CAS  Google Scholar 

  18. P Hugenholtz, BM Goebel and NR Pace, Impact of culture-independent studies on the emerging phylogenetic view of bacterial diversity. J Bacteriol 180 (1998) 4765-4774

    PubMed  CAS  Google Scholar 

  19. A Jumpponen, Soil fungal community assembly in a primary successional glacier forefront ecosystem as inferred from rDNA sequence analyses. New Phytol 158 (2003) 569-578

    Article  Google Scholar 

  20. A Jumpponen, K Mattson, JM Trappe and R Ohtonen, Effects of established willows on primary succession on Lyman Glacier forefront: evidence for simultaneous canopy inhibition and soil facilitation. Arct Alp Res 30 (1998) 31-39

    Article  Google Scholar 

  21. A Jumpponen, JM Trappe and E Cázares, Ectomycorrhizal fungi in Lyman Lake Basin: a comparison between primary and secondary successional sites. Mycologia 91 (1999) 575-582

    Google Scholar 

  22. A Jumpponen, JM Trappe and E Cázares, Occurrence of ectomycorrhizal fungi on a receding glacier forefront. Mycorrhiza 12 (2002) 43-49

    Article  PubMed  Google Scholar 

  23. A Jumpponen, H Väre, KG Mattson, R Ohtonen and JM Trappe, Characterization of ‘safe sites’ for pioneers in primary succession on recently deglaciated terrain. J Ecol 87 (1999) 98-105

    Article  Google Scholar 

  24. ED Kopczynski, MM Bateson and DM Ward, Recognition of chimeric small-subunit ribosomal DNAs composed from genes from uncultivated microorganisms. Appl Environ Microbiol 63 (1994) 3614-3621

    Google Scholar 

  25. GA Kowalchuk, S Gerards and JW Woldendorp, Detection and characterization of fungal infections of Ammophila arenaria (marram grass) roots by denaturing gradient gel electrophoresis. Appl Environ Microbiol 63 (1997) 3858-3865

    PubMed  CAS  Google Scholar 

  26. BL Maidak, JR Cole, CT Parker Jr, GM Garrity, N Larsen, B Li, TG Lilburn, MJ McCaughey, GJ Olsen, R Overbeek, TM Pramanik, TM Schmidt, JM Tiedje and CR Woese, A new version of the RDP (Ribosomal Database Project). Nucleic Acids Res 27 (1999) 171-173

    Article  PubMed  CAS  Google Scholar 

  27. TE O'Dell, JE Smith, M Castellano and D Luoma, Diversity and conservation of forest fungi. In: D Pilz and R Molina (eds.) Managing Forest Ecosystems to Conserve Fungus Diversity and Sustain Wild Mushroom Harvests. U.S. Forest Service General Technical Reports, PNW-GTR-317. Portland, OR: US Department of Agriculture, Forest Service, Pacific Northwest Research Station (1996) pp. 5-18

    Google Scholar 

  28. T Pennanen, L Paavolainen and J Hantula, Rapid PCR-based method for the direct analysis of fungal communities in complex environmental samples. Soil Biol Biochem 33 (2001) 697-699

    Article  CAS  Google Scholar 

  29. S Perotto, P Nepote-Fus, L Saletta, C Bandi and JPW Young, A diverse population of introns in the nuclear ribosomal genes of ericoid mycorrhizal fungi includes elements with sequence similarity to endonuclease-coding genes. Mol Biol Evol 17 (2000) 44-59

    PubMed  CAS  Google Scholar 

  30. J Sambrook, . In: EF Fritsch and T Maniatis (eds.) Molecular Cloning—A Laboratory Manual. New York: Cold Spring Laboratory Press (1989) pp.

    Google Scholar 

  31. CW Schadt, AW Martin, DA Lipson and SK Schmidt, Seasonal dynamics of previously unknown fungal lineages in tundra soils. Science 301 (2003) 1359-1361

    Article  PubMed  CAS  Google Scholar 

  32. E Smit, P Leeflang, B Glandorf, JD vanElsas and K Wernars, 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 (1999) 2614-2621

    PubMed  CAS  Google Scholar 

  33. E Smit, C Veenman and J Baar, Molecular analysis of ectomycorrhizal basidiomycete communities in Pinus sylvestris L. stand reveals long-term increased diversity after removal of litter and humus layers. FEMS Microbiol Ecol 45 (2003) 49-57

    Article  CAS  PubMed  Google Scholar 

  34. SE Smith and DJ Read, Mycorrhizal Symbiosis. London: Academic Press (1997).

    Google Scholar 

  35. G Straatsma and I Krisai-Greilhuber, Assemblage structure, species richness, abundance, and distribution of fungal fruitbodies in a seven year plot-based survey near Vienna. Mycol Res 107 (2003) 632-640

    Article  PubMed  Google Scholar 

  36. MT Suzuki and SJ Giovannoni, Bias caused by template annealing in the amplification mixtures of 16S rRNA genes by PCR. Appl Environ Microbiol 62 (1996) 625-630

    PubMed  CAS  Google Scholar 

  37. DL Swofford, PAUP, Phylogenetic Analysis Using Parsimony (and Other Methods), Version 4. Sunderland, MA: Sinauer Associates (2001).

    Google Scholar 

  38. V Torsvik, J Goksøyr and FL Daae, High diversity on DNA of soil bacteria. Appl Environ Microbiol 56 (1990) 782-787

    PubMed  CAS  Google Scholar 

  39. J Trowbridge and A Jumpponen, Fungal colonization of shrub willow roots at the forefront of a receding glacier. Mycorrhiza 14 (2004) 283-293

    Article  PubMed  Google Scholar 

  40. EJ Vainio and J Hantula, Direct analysis of wood-inhabiting fungi using denaturing gradient gel electrophoresis of amplified ribosomal DNA. Mycol Res 104 (2000) 927-936

    Article  CAS  Google Scholar 

  41. P Vandenkoornhuyse, SL Baldauf, C Leyval, J Straczek and JPW Young, Extensive fungal diversity in plant roots. Science 295 (2002) 2051

    Article  PubMed  Google Scholar 

  42. F vonWintzingerode, UB Göbel and E Stackebrandt, Determination of microbial diversity in environmental samples: pitfalls of PCR-based rRNA analysis. FEMS Microbiol Rev 21 (1997) 213-229

    Article  CAS  Google Scholar 

  43. GCY Wang and Y Wang, The frequency of chimeric molecules as a consequence of PCR co-amplification of 16S rRNA genes from different bacterial species. Microbiology 142 (1996) 1107-1114

    Article  PubMed  CAS  Google Scholar 

  44. D Zheng, EW Alm, DA Stahl and L Raskin, Characterization of universal small-subunit rRNA hybridization probes for quantitative molecular microbial ecology studies. Appl Environ Microbiol 62 (1996) 4504-4513

    PubMed  CAS  Google Scholar 

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Acknowledgments

This work was supported by Kansas State University BRIEF program, National Science Foundation EPSCoR Grant No. 9874732 with matching support from the State of Kansas, and National Science Foundation Grant No. OPP-0221489. I am grateful to Dr. Francesco T. Gentili, Nicolo Gentili, Anna Jumpponen, and Dr. James M. Trappe for their assistance during sample collection, transport, and preparation in August 2001 and to Emily L. King and Justin Trowbridge for their assistance in clone library screening and plasmid preparation. Dr. Charles L. Kramer, Nicholas B. Simpson, and Dr. James M. Trappe provided helpful comments on early drafts of this manuscript. Nicholas B. Simpson edited the manuscript.

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  1. Division of Biology, Kansas State University, 125 Ackert Hall, Manhattan, KS, 66506, USA

    Ari Jumpponen

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Jumpponen, A. Soil Fungal Communities Underneath Willow Canopies on a Primary Successional Glacier Forefront: rDNA Sequence Results Can Be Affected by Primer Selection and Chimeric Data. Microb Ecol 53, 233–246 (2007). https://doi.org/10.1007/s00248-004-0006-x

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