, Volume 18, Issue 6–7, pp 339–354 | Cite as

Diversity and structure of ectomycorrhizal and co-associated fungal communities in a serpentine soil

  • Alexander UrbanEmail author
  • Markus Puschenreiter
  • Joseph Strauss
  • Markus Gorfer
Original Paper


The community of ectomycorrhizal (ECM) and co-associated fungi from a serpentine site forested with Pinus sylvestris and Quercus petraea was explored, to improve the understanding of ECM diversity in naturally metalliferous soils. ECM fungi were identified by a combination of morphotyping and direct sequencing of the nuclear ribosomal internal transcribed spacer region 2 and of a part of the large-subunit region. Co-associated fungi from selected ECM were identified by restriction fragment length polymorphism and sequencing of representative clones from libraries. Polymerase chain reaction with species-specific primers was applied to assess patterns of association of ECM and co-associated fungi. Twenty ECM species were differentiated. Aphyllophoralean fungi representing several basidiomycete orders and Russulaceae were dominant. Phialocephala fortinii was the most frequently encountered taxon from the diverse assemblage of ECM co-associated fungi. A ribotype representing a deeply branching ascomycete lineage known from ribosomal deoxyribonucleic acid sequences only was detected in some ECM samples. A broad taxonomic range of fungi have the potential to successfully colonise tree roots under the extreme edaphic conditions of serpentine soils. Distribution patterns of ECM-co-associated fungi hint at the importance of specific inter-fungal interactions, which are hypothesised to be a relevant factor for the maintenance of ECM diversity.


Pinus sylvestris Quercus petraea Ectomycorrhiza ECM-co-associated fungi Phialocephala fortinii Serpentine Rhizosphere Diversity Heavy metal toxicity 



This work was supported by grant P15357 from the Austrian Science Foundation (FWF) and grant LS149 from the Vienna Science and Technology Fund (WWTF) to JS. The authors thank Anton Russell and two anonymous reviewers for critical reading of the manuscript and helpful suggestions.

Supplementary material

572_2008_189_MOESM1_ESM.doc (143 kb)
Table S1 Comparison of ITS sequences from P. fortinii CSP. P. fortinii ITS sequences from Redlschlag and from Zürichberg (Grünig et al. 2004) were aligned with the ClustalW algorithm. Only positions with differences are shown with discriminating nucleotides highlighted in bold. Numbering of positions is according to sequence AY347406 (CSP3, strain 144-4). Classification into CSP is according to Grünig et al. (2004). Missing characters indicate missing sequence information, and ‘–’ indicates a gap. The poly-T stretch with variable length at the end of the ITS2-region (starting at position 500 in AY347406) is not included in the comparison. #: number of sequences with 100% identity from the Zürichberg sequence set (Grünig et al. 2004) (DOC 143 KB)
572_2008_189_MOESM2_ESM.pdf (9 kb)
Fig. S1 Phylogenetic tree for RSEM01_13 based on partial 18S rDNA sequences. A multiple alignment of a set of published partial 18S rDNA sequences from all recognised groups of fungi including a set of 18S rDNA sequences from AG1 was generated with the ClustalW algorithm in ARB. For construction of the phylogenetic tree, the Phylip package in ARB was used with a filter for positions covered by sequences from the unknown ascomycete lineage. Entomophaga conglomerata was used as the out-group for routing. Branches were collapsed for phyla, except for ascomycota, where branches were collapsed for sub-phyla (shaded triangles). The order Mucorales was separated from the remaining zygomycota. The phyla of chytridiomycota and zygomycota could not be separated based on partial 18S rDNA sequences. Separation of a group of hitherto uncultivated fungi—Ascomycota Group I (darkly shaded triangle)—including the enigmatic ascomycete RSEM01_13 was supported by different algorithms used for construction of the tree (neighbour joining, Bayesian analysis). Numbers inside or next to triangles indicate number of species included in the collapsed branch (PDF 9.23 KB)


  1. Adriaensen K, Vralstad T, Noben JP, Vangronsveld J, Colpaert JV (2005) Copper-adapted Suillus luteus, a symbiotic solution for pines colonizing Cu mine spoils. Appl Environ Microbiol 71:7279–7284PubMedCrossRefGoogle Scholar
  2. Adriaensen K, Vangronsveld J, Colpaert JV (2006) Zinc-tolerant Suillus bovinus improves growth of Zn-exposed Pinus sylvestris seedlings. Mycorrhiza 16:553–558PubMedCrossRefGoogle Scholar
  3. Agerer R (1991) Characterization of ectomycorrhiza. In: Norris JR, Read DJ, Varma AK (eds) Methods Microbiol Techniques for the study of mycorrhiza, 23:25–73Google Scholar
  4. Aggangan NS, Dell B, Malajczuk N (1998) Effects of chromium and nickel on growth of the ectomycorrhizal fungus Pisolithus and formation of ectomycorrhizas on Eucalyptus urophylla S.T. Blake. Geoderma 84:15–27CrossRefGoogle Scholar
  5. Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25:3389–3402PubMedCrossRefGoogle Scholar
  6. Amir H, Pineau R (1998) Effects of metals on the germination and growth of fungal isolates from New Caledonian ultramafic soils. Soil Biol Biochem 30:2043–2054CrossRefGoogle Scholar
  7. 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 5:36–47PubMedCrossRefGoogle Scholar
  8. Barceloux DG (1999) Nickel. J Toxicol Clin Toxicol 37:239–258PubMedCrossRefGoogle Scholar
  9. Bellion M, Courbot M, Jacob C, Blaudez D, Chalot M (2006) Extracellular and cellular mechanisms sustaining metal tolerance in ectomycorrhizal fungi. FEMS Microbiol Lett 254:173–181PubMedGoogle Scholar
  10. Berbee ML (2001) The phylogeny of plant and animal pathogens in the Ascomycota. Phys Mol Plant Pathol 59:165–187CrossRefGoogle Scholar
  11. Blaudez D, Jacob C, Turnau K, Colpaert JV, Ahonen-Jonnarth U, Finlay R, Botton B, Chalot M (2001) Differential responses of ectomycorrhizal fungi to heavy metals in vitro. Mycol Res 104:1366–1371CrossRefGoogle Scholar
  12. Brady KU, Kruckeberg AR, Bradshaw Jr HD (2005) Evolutionary ecology of plant adaptation to serpentine soils. Ann Rev Ecol Evol Syst 36:243–266CrossRefGoogle Scholar
  13. Bruns T (1995) Thoughts on the processes that maintain local species diversity of ectomycorrhizal fungi. Plant Soil 170:63–73CrossRefGoogle Scholar
  14. Castelli JP, Casper BB (2003) Intraspecific AM fungal variation contributes to plant-fungal feedback in a serpentine grassland. Ecology 84:323–336CrossRefGoogle Scholar
  15. Chen DM, Cairney JW (2002) Investigation of the influence of prescribed burning on ITS profiles of ectomycorrhizal and other soil fungi at three Australian sclerophyll forest sites. Mycol Res 106:532–540CrossRefGoogle Scholar
  16. Colpaert JV, Muller LAH, Lambaerts M, Adriaensen K, Vangronsveld J (2004) Evolutionary adaptation to Zn toxicity in populations of Suilloid fungi. New Phytol 162:549–559CrossRefGoogle Scholar
  17. Courty P-E, Pritsch K, Schloter M, Hartmann A, Garbaye J (2005) Activity profiling of ectomycorrhiza communities in two forest soils using multiple enzymatic tests. New Phytol 167:309–319PubMedCrossRefGoogle Scholar
  18. Crous PW, Schubert K, Braun U, de Hoog GS, Hocking AD, Shin H-D, Groenewald JZ (2007) Opportunistic, human-pathogenic species in the Herpotrichiellaceae are phenotypically similar to saprobic or phytopathogenic species in the Venturiaceae. Stud Mycol 58:185–217PubMedCrossRefGoogle Scholar
  19. Danielson RM, Pruden M (1989) The ectomycorrhizal status of urban spruce. Mycology 8:335–341CrossRefGoogle Scholar
  20. Denny HJ, Wilkins DA (1987) Zinc tolerance in Betula spp. IV. The mechanism of ectomycorrhizal amelioration of zinc toxicity. New Phytol 106:545–553Google Scholar
  21. di Pietro M, Churin JL, Garbaye J (2007) Differential ability of ectomycorrhizas to survive drying. Mycorrhiza 17:547–550PubMedCrossRefGoogle Scholar
  22. Dixon RK (1988) Response of ectomycorrhizal Quercus rubra to soil cadmium, nickel and lead. Soil Biol Biochem 20:555–559CrossRefGoogle Scholar
  23. Dixon RK, Buschena CA (1988) Response of ectomycorrhizal Pinus banksiana and Picea glauca to heavy metals in soil. Plant Soil 105:265–271CrossRefGoogle Scholar
  24. Ernst WHO (2000) Evolution of metal hyperaccumulation and phytoremediation hype. New Phytol 146:357–358CrossRefGoogle Scholar
  25. Furnier GR, Adams WT (1986) Geographic patterns of allozyme variation in Jeffrey pine. Am J Bot 73:1009–1015CrossRefGoogle Scholar
  26. Gadd GM (1993) Interactions of fungi with toxic metals. New Phytol 124:25–60CrossRefGoogle Scholar
  27. 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
  28. Gehring CA, Theimer TC, Whitham TG, Keim P (1998) Ectomycorrhizal fungal community structure of pinyon pines growing in two environmental extremes. Ecol 79:1562–1572CrossRefGoogle Scholar
  29. Genney DR, Anderson IC, Alexander IJ (2006) Fine-scale distribution of pine ectomycorrhizas and their extramatrical mycelium. New Phytol 170:381–390PubMedCrossRefGoogle Scholar
  30. Gonçalves S, Portugal A, Gonçalves M, Vieira R, Martins-Loução M, Freitas H (2007) Genetic diversity and differential in vitro responses to Ni in Cenococcum geophilum isolates from serpentine soils in Portugal. Mycorrhiza 17:677–686PubMedCrossRefGoogle Scholar
  31. Gorfer M, Klaubauf S, Bandian D, Strauss J (2007) Cadophora finlandia and Phialocephala fortinii: Agrobacterium-mediated transformation and functional GFP-expression. Mycol Res 111:850–855PubMedCrossRefGoogle Scholar
  32. Grünig CR, McDonald BA, Sieber TN, Rogers SO, Holdenrieder O (2004) Evidence for subdivision of the root-endophyte Phialocephala fortinii into cryptic species and recombination within species. Fungal Genet Biol 41:676–687PubMedCrossRefGoogle Scholar
  33. Grünig CR, Duo A, Sieber TN (2006) Population genetic analysis of Phialocephala fortinii s.l. and Acephala applanata in two undisturbed forests in Switzerland and evidence for new cryptic species. Fungal Genet Biol 43:410–421PubMedCrossRefGoogle Scholar
  34. Hartley J, Cairney JWG, Meharg AA (1997) Do ectomycorrhizal fungi exhibit adaptive tolerance to potentially toxic metals in the environment? Plant Soil 189:303–319CrossRefGoogle Scholar
  35. Huber T, Faulkner G, Hugenholtz P (2004) Bellerophon: a program to detect chimeric sequences in multiple sequence alignments. Bioinformatics 20:2317–2319PubMedCrossRefGoogle Scholar
  36. Ishida TA, Nara K, Hogetsu T (2007) Host effects on ectomycorrhizal fungal communities: insight from eight host species in mixed conifer–broadleaf forests. New Phytol 174:430–440PubMedCrossRefGoogle Scholar
  37. Izzo A, Agbowo J, Bruns TD (2005) Detection of plot-level changes in ectomycorrhizal communities across years in an old-growth mixed-conifer forest. New Phytol 166:619–630PubMedCrossRefGoogle Scholar
  38. Jones MD, Hutchinson TC (1986) The effect of mycorrhizal infection on the response of Betula papyrifera to nickel and copper. New Phytol 102:429–442CrossRefGoogle Scholar
  39. Jones MD, Hutchinson TC (1988) Nickel toxicity in mycorrhizal birch seedlings infected with Lactarius rufus or Scleroderma flavidum. I. Effects on growth, photosynthesis, respiration and transpiration. New Phytol 108:451–459CrossRefGoogle Scholar
  40. Jonsson L, Dahlberg A, Nilsson M-C, Zackrisson O, Karen OLA (1999) Ectomycorrhizal fungal communities in late-successional Swedish boreal forests, and their composition following wildfire. Mol Ecol 8:205–215CrossRefGoogle Scholar
  41. Jumpponen A, Johnson LC (2005) Can rDNA analyses of diverse fungal communities in soil and roots detect effects of environmental manipulations—a case study from tallgrass prairie. Mycology 97:1177–1194CrossRefGoogle Scholar
  42. Jumpponen A, Trappe JM (1998a) Dark septate endophytes: a review of facultative biotrophic root-colonizing fungi. New Phytol 140:295–310CrossRefGoogle Scholar
  43. Jumpponen A, Trappe JM (1998b) Performance of Pinus contorta inoculated with two strains of root endophytic fungus, Phialocephala fortinii: effects of synthesis system and glucose concentration. Can J Bot 76:1205–1213CrossRefGoogle Scholar
  44. Kaldorf M, Renker C, Fladung M, Buscot F (2004) Characterization and spatial distribution of ectomycorrhizas colonizing aspen clones released in an experimental field. Mycorrhiza 14:295–306PubMedCrossRefGoogle Scholar
  45. Kalendar R (2006) FastPCR: a PCR primer design and repeat sequence searching software with additional tools for the manipulation and analysis of DNA and protein.
  46. Katoh K, Kuma K-i, Toh H, Miyata T (2005) MAFFT version 5: improvement in accuracy of multiple sequence alignment. Nucleic Acids Res 33:511–518PubMedCrossRefGoogle Scholar
  47. Kayama M, Choi D, Tobita H, Utsugi H, Kitao M, Maruyama Y, Nomura M, Koike T (2006) Comparison of growth characteristics and tolerance to serpentine soil of three ectomycorrhizal spruce seedlings in northern Japan. Trees Struct Funct 20:430–440Google Scholar
  48. Koide RT, Xu B, Sharda J (2005a) Contrasting below-ground views of an ectomycorrhizal fungal community. New Phytol 166:251–262PubMedCrossRefGoogle Scholar
  49. Koide RT, Xu B, Sharda J, Lekberg Y, Ostiguy N (2005b) Evidence of species interactions within an ectomycorrhizal fungal community. New Phytol 165:305–316PubMedCrossRefGoogle Scholar
  50. Korkama T, Pakkanen A, Pennanen T (2006) Ectomycorrhizal community structure varies among Norway spruce (Picea abies) clones. New Phytol 171:815–824PubMedCrossRefGoogle Scholar
  51. Kruckeberg A (1967) Ecotypic response to ultramafic soils by some plant species of northwestern United States. Brittonia 19:133–151CrossRefGoogle Scholar
  52. Krupa P, Kozdrój J (2007) Ectomycorrhizal fungi and associated bacteria provide protection against heavy metals in inoculated pine (Pinus sylvestris L.) seedlings. Water Air Soil Pollut 182:83–90CrossRefGoogle Scholar
  53. Landeweert R, Leeflang P, Smit E, Kuyper T (2005) Diversity of an ectomycorrhizal fungal community studied by a root tip and total soil DNA approach. Mycorrhiza 15:1–6PubMedCrossRefGoogle Scholar
  54. Leyval C, Turnau K, Haselwandter K (1997) Effect of heavy metal pollution on mycorrhizal colonization and function: Physiological, ecological and applied aspects. Mycorrhiza 7:139–153CrossRefGoogle Scholar
  55. Ludwig W, Strunk O, Westram R, Richter L, Meier H, Yadhukumar, Buchner A, Lai T, Steppi S, Jobb G, Forster W, Brettske I, Gerber S, Ginhart AW, Gross O, Grumann S, Hermann S, Jost R, Konig A, Liss T, Lussmann R, May M, Nonhoff B, Reichel B, Strehlow R, Stamatakis A, Stuckmann N, Vilbig A, Lenke M, Ludwig T, Bode A, Schleifer K-H (2004) ARB: a software environment for sequence data. Nucleic Acids Res 32:1363–1371PubMedCrossRefGoogle Scholar
  56. Markkola AM, Ahonen JU, Roitto M, Strommer R, Hyvarinen M (2002) Shift in ectomycorrhizal community composition in Scots pine (Pinus sylvestris L.) seedling roots as a response to nickel deposition and removal of lichen cover. Environ Pollut 120:797–803PubMedGoogle Scholar
  57. Marschner H (1995) Mineral nutrition of higher plants. Academic, LondonGoogle Scholar
  58. Marschner H, Dell B (1994) Nutrient uptake in mycorrhizal symbiosis. Plant Soil 159:89–102Google Scholar
  59. Meharg A, Cairney J (2000) Co-evolution of mycorrhizal symbionts and their hosts to metal-contaminated environments. Adv Ecol Res 30:69–112CrossRefGoogle Scholar
  60. Menkis A, Vasiliauskas R, Taylor A, Stenlid J, Finlay R (2005) Fungal communities in mycorrhizal roots of conifer seedlings in forest nurseries under different cultivation systems, assessed by morphotyping, direct sequencing and mycelial isolation. Mycorrhiza 16:33–41PubMedCrossRefGoogle Scholar
  61. Miller SP, Cumming JR (2000) Effects of serpentine soil factors on Virginia pine (Pinus virginiana) seedlings. Tree Physiol 20:1129–1135PubMedGoogle Scholar
  62. Moser AM, Petersen CA, D’Allura JA, Southworth D (2005) Comparison of ectomycorrhizas of Quercus garryana (Fagaceae) on serpentine and non-serpentine soils in southwestern Oregon. Am J Bot 92:224–230CrossRefGoogle Scholar
  63. Murat C, Vizzini A, Bonfante P, Mello A (2005) Morphological and molecular typing of the below-ground fungal community in a natural Tuber magnatum truffle-ground. FEMS Microbiol Lett 245:307–313PubMedCrossRefGoogle Scholar
  64. O’Brien HE, Parrent JL, Jackson JA, Moncalvo J-M, Vilgalys R (2005) Fungal community analysis by large-scale sequencing of environmental samples. Appl Environ Microbiol 71:5544–5550PubMedCrossRefGoogle Scholar
  65. Oline DK, Mitton JB, Grant MC (2000) Population and subspecific genetic differentiation in the foxtail pine (Pinus balfouriana). Evol 54:1813–1819Google Scholar
  66. Panaccione D, Sheets N, Miller S, Cumming J (2001) Diversity of Cenococcum geophilum isolates from serpentine and non-serpentine soils. Mycol 93:645–652CrossRefGoogle Scholar
  67. Porter TM, Schadt CW, Rizvi L, Martin AP, Schmidt SK, Scott-Denton L, Vilgalys R, Moncalvo JM (2008) Widespread occurrence and phylogenetic placement of a soil clone group adds a prominent new branch to the fungal tree of life. Mol Phylogenet Evol 46:635–644PubMedCrossRefGoogle Scholar
  68. Pringle A, Moncalvo JM, Vilgalys R (2000) High levels of variation in ribosomal DNA sequences within and among spores of a natural population of the arbuscular mycorrhizal fungus Acaulospora colossica. Mycology 92:259–268CrossRefGoogle Scholar
  69. Rosling A, Landeweert R, Lindahl BD, Larsson KH, Kuyper TW, Taylor AFS, Finlay RD (2003) Vertical distribution of ectomycorrhizal fungal taxa in a podzol soil profile. New Phytol 159:775–783CrossRefGoogle Scholar
  70. Schadt CW, Martin AP, Lipson DA, Schmidt SK (2003) Seasonal dynamics of previously unknown fungal lineages in tundra soils. Science 301:1359–1361PubMedCrossRefGoogle Scholar
  71. Sen R (2001) Multitrophic interactions between a Rhizoctonia sp. and mycorrhizal fungi affect Scots pine seedling performance in nursery soil. New Phytol 152:543–553CrossRefGoogle Scholar
  72. Smit E, Veenman C, Baar J (2003) Molecular analysis of ectomycorrhizal basidiomycete communities in a Pinus sylvestris L. stand reveals long-term increased diversity after removal of litter and humus layers. FEMS Microbiol Ecol 45:49–57CrossRefPubMedGoogle Scholar
  73. Stamatakis A (2006) RAxML-VI-HPC: maximum likelihood-based phylogenetic analyses with thousands of taxa and mixed models. Bioinformatics 22:2688–2690PubMedCrossRefGoogle Scholar
  74. Summerbell RC (2005) Root endophyte and mycorrhizosphere fungi of black spruce, Picea mariana, in a boreal forest habitat: influence of site factors on fungal distributions. Stud Mycol 53:121–145CrossRefGoogle Scholar
  75. Suresh B, Ravishankar G (2004) Phytoremediation—a novel and promising approach for environmental clean-up. Crit Rev Biotech 24:97–124CrossRefGoogle Scholar
  76. Taylor DL, Bruns TD (1999) Community structure of ectomycorrhizal fungi in a Pinus muricata forest: minimal overlap between the mature forest and resistant propagule communities. Mol Ecol 8:1837–1850PubMedCrossRefGoogle Scholar
  77. Tedersoo L, Hansen K, Perry BA, Kjoller R (2006) Molecular and morphological diversity of pezizalean ectomycorrhiza. New Phytol 170:581–596PubMedCrossRefGoogle Scholar
  78. Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG (1997) The ClustalX windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 24:4876–4882CrossRefGoogle Scholar
  79. Turnau K, Przybylowicz WJ, Mesjasz-Przybylowicz J (2001) Heavy metal distribution in Suillus luteus mycorrhizas—as revealed by micro-PIXE analysis. Nucl Instrum Methods Phys Res B 181:649–658CrossRefGoogle Scholar
  80. Urban A, Weiss M, Bauer R (2003) Ectomycorrhizae involving sebacinoid mycobionts. Mycol Res 107:3–1PubMedCrossRefGoogle Scholar
  81. Vralstad T, Myhre E, Schumacher T (2002) Molecular diversity and phylogenetic affinities of symbiotic root-associated ascomycetes of the Helotiales in burnt and metal polluted habitats. New Phytol 155:131–148CrossRefGoogle Scholar
  82. Wenzel WW, Jockwer F (1999) Accumulation of heavy metals in plants grown on mineralised soils of the Austrian Alps. Environ Pollut 104:145–155CrossRefGoogle Scholar
  83. Wenzel WW, Bunkowski M, Puschenreiter M, Horak O (2003) Rhizosphere characteristics of indigenously growing nickel hyperaccumulator and excluder plants on serpentine soil. Environ Pollut 123:131–138PubMedCrossRefGoogle Scholar
  84. Wilcox HE, Wang CJK (1987) Mycorrhizal and pathological associations of dematiaceous fungi in roots of 7-month-old tree seedlings. Can J Forest Res 17:884–899CrossRefGoogle Scholar
  85. Wilkinson D, Dickinson N (1995) Metal resistance in trees: The role of mycorrhizae. Oikos 72:298–300CrossRefGoogle Scholar
  86. Wright J (2007) Local adaptation to serpentine soils in Pinus ponderosa. Plant Soil 293:209–217CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2008

Authors and Affiliations

  • Alexander Urban
    • 1
    • 2
    • 3
    Email author
  • Markus Puschenreiter
    • 4
  • Joseph Strauss
    • 1
    • 2
  • Markus Gorfer
    • 1
    • 2
  1. 1.Fungal Genomics UnitAustrian Research CentersViennaAustria
  2. 2.Institute of Applied Genetics and Cell BiologyUniversity of Natural Resources and Applied Life SciencesViennaAustria
  3. 3.Department for Systematic and Evolutionary Botany, Mycology Research GroupUniversity of ViennaViennaAustria
  4. 4.Department of Forest and Soil SciencesUniversity of Natural Resources and Applied Life SciencesViennaAustria

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