, Volume 21, Issue 1, pp 17–25

The AD-type ectomycorrhizas, one of the most common morphotypes present in truffle fields, result from fungi belonging to the Trichophaea woolhopeia species complex

  • Andrea Rubini
  • Beatrice Belfiori
  • Valentina Passeri
  • Leonardo Baciarelli Falini
  • Sergio Arcioni
  • Claudia Riccioni
  • Francesco Paolocci
Original Paper


Belowground ectomycorrhizal communities are often species rich. Characterization of the ectomycorrhizas (ECMs) underneath native truffle areas and/or cultivation sites is particularly relevant to identifying fungal species that might interfere with or promote truffle propagation and fruiting. Fungal identification at the genus/species level can now be achieved by combining detailed morphological and anatomical descriptions with molecular approaches. In a survey of the mycorrhizal biodiversity of Tuber melanosporum orchards and inoculated host plants in nurseries, we repeatedly sampled ECMs with morphological features resembling those of the ECMs widely known as the AD type. Despite the fact that the AD type is regarded as one of the most competitive fungal species towards Tuber spp., its taxonomical rank has yet to be resolved. By analyzing the 28S and internal transcribed spacer (ITS) rDNA regions, here, we show that AD-type ECMs result from host plant colonization by the pyronemataceous species Trichophaea woolhopeia. Further to this, the 28S and ITS phylogenetic trees built from the AD-type ECMs analyzed sustain the hypothesis that T. woolhopeia is a species complex.


Ectomycorrhiza Tuber Molecular identification LSU ITS Trichophaea woolhopeia 

Supplementary material

572_2010_308_MOESM1_ESM.doc (62 kb)
Fig. S1Multiple sequence alignment of the large subunit region. (DOC 61 kb)
572_2010_308_MOESM2_ESM.doc (62 kb)
Fig. S2Multiple sequence alignment of the internal transcribed spacer region. (DOC 61 kb)
572_2010_308_MOESM3_ESM.doc (240 kb)
Fig. S3Neighbor-joining tree based on large subunit sequences from AD-type samples and other Pyronemataceae. Numbers near the branches represent the bootstrap values (1,000 replicates). The sequences from AD-type samples obtained in this study are given in bold. (DOC 240 kb)
572_2010_308_MOESM4_ESM.doc (242 kb)
Fig. S4Maximum-likelihood tree based on large subunit sequences from AD-type samples and other Pyronemataceae. Numbers near the branches represent the bootstrap values (1,000 replicates). The sequences from AD-type samples obtained in this study are given in bold. (DOC 241 kb)


  1. Agerer R (1986) Colour Atlas of ectomycorrhizae. Einhorn, Schwäbisch-GmündGoogle Scholar
  2. Agerer R (1991) Characterization of ectomycorrhiza. In: Norris JR, Read DJ, Varma AK (eds) Techniques for the study of mycorrhiza. Academic, London, pp 25–73CrossRefGoogle Scholar
  3. Agerer R (2001) Exploration types of ectomycorrhizae. Mycorrhiza 11:107–114CrossRefGoogle Scholar
  4. Agueda B, Agerer R, De Miguel AM, Parlade J (2008a) Querciriza quadratum + Quercus ilex L. Subsp ballota (Scop.) Desf. Samp. In: Agerer R, Danielson RM, Egli S, Ingleby K, Luoma D, Treu R (eds) Descriptions of ectomycorrhizae, vol 12. Einhorn-Verlag, Scwäbisch Gmünd, pp 113–123Google Scholar
  5. Agueda B, Agerer R, De Miguel AM, Parlade J (2008b) Querciriza quadratum: a revision of the characters and identity of AD-type ectomycorrhiza. In Abstracts of Third International Spoleto Congress on Truffles. Spoleto 25–28 Nov 2008, p 39Google Scholar
  6. Baciarelli Falini L, Rubini A, Riccioni C, Paolocci F (2006) Morphological and molecular analyses of ectomycorrhizal diversity in a man-made T. melanosporum plantation: description of novel truffle-like morphotypes. Mycorrhiza 16:475–484CrossRefPubMedGoogle Scholar
  7. Buée M, Reich M, Murat C, Morin E, Nilsson RH, Uroz S, Martin F (2009) 454 Pyrosequencing analyses of forest soils reveal an unexpectedly high fungal diversity. New Phytol 184:449–456CrossRefPubMedGoogle Scholar
  8. De Miguel AM, Sáez R (2005) Algunas micorrizas competidoras de plantaciones truferas. Publ Biol Univ Navarra, Ser Bot 16:1–18Google Scholar
  9. Donnini D, Bencivenga M (1995) Micorrize inquinanti frequenti nelle piante tartufigene. Nota 2- inquinanti in campo. Micol Ital 2:185–207Google Scholar
  10. Edgar RC (2004) MUSCLE: a multiple sequence alignment method with reduced time and space complexity. BMC Bioinformatics 5:113CrossRefPubMedGoogle Scholar
  11. Giraud M (1988) Prélévement et analyse de mycorhizes. Bull. FNTP, 10, 49–63. In: La Truffe. CTIFL, ParisGoogle Scholar
  12. Granetti B, Angelini P (1992) Competizione tra alcuni funghi micorrizici e T. melanosporum Vitt. in una tartufaia coltivata. Micol Veget Mediterr 7:173–188Google Scholar
  13. Granetti B, Baciarelli Falini L (1997) Competizione tra le micorrize di T. melanosporum Vitt. e quelle di altri funghi in una tartufaia coltivata a Quercus ilex L. Micol Ital 1:45–49Google Scholar
  14. Guindon S, Gascuel O (2003) A simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood. Syst Biol 52:696–704CrossRefPubMedGoogle Scholar
  15. Horton TR, Bruns TD (2001) The molecular revolution in ectomycorrhizal ecology: peeking into the black-box. Mol Ecol 10:1855–1871CrossRefPubMedGoogle Scholar
  16. Izzo AD, Agbowo J, Bruns TD (2005) Detection of plot-level changes in ectomycorrhizal communities across years in an old-growth mixed-conifer forest. New Phytol 166:619–630CrossRefPubMedGoogle Scholar
  17. Kimura M (1980) A simple method for estimating evolutionary rates of nucleotide substitution through comparative studies of nucleotide sequences. J Mol Evol 16:111–120CrossRefPubMedGoogle Scholar
  18. Kumar S, Tamura K, Jakobsen IB, Nei M (2001) MEGA 2: molecular evolutionary genetics analysis software. Arizona State University, TempeGoogle Scholar
  19. Luppi AM (1972) La microflora della rizosfera nelle tartufaie. III Analisi micologiche di terreni tartufiferi dell’Italia centrale. Allionia 22:105–113Google Scholar
  20. Luppi AM, Fontana A (1977) Studi sull’ecologia del Tuber melanosporum. IV. Analisi micologiche di terreni tartufiferi francesi. Allionia 18:33–40Google Scholar
  21. Mamoun M, Oliver JM (1997) Mycorrhizal inoculation of cloned hazels by Tuber melanosporum: effect of soil disinfestation and co-culture with Festuca ovina. Plant Soil 188:221–226CrossRefGoogle Scholar
  22. Moncalvo JM, Lutzoni FM, Rehner SA, Johnson J, Vilgalys R (2000) Phylogenetic relationships of agaric fungi based on nuclear large subunit ribosomal DNA sequences. Syst Biol 49:278–305CrossRefPubMedGoogle Scholar
  23. Nei M, Kumar S (2000) Molecular evolution and phylogenetics. Oxford University Press, New YorkGoogle Scholar
  24. Paolocci F, Rubini A, Granetti B, Arcioni S (1999) Rapid molecular approach for a reliable identification of Tuber spp. ectomycorrhizae. FEMS Microbiol Ecol 28:23–30CrossRefGoogle Scholar
  25. Peay KG, Kennedy PG, Bruns TD (2008) Fungal community ecology: a hybrid beast with a molecular master. Bioscience 58:799–810CrossRefGoogle Scholar
  26. Perry BA, Hanseny K, Pfister DH (2007) A phylogenetic overview of the family Pyronemataceae (Ascomycota, Pezizales). Mycol Res 111:549–571CrossRefPubMedGoogle Scholar
  27. Posada D, Crandal KA (1998) Modeltest: testing the model of DNA substitution. Bioinformatics 14:817–818CrossRefPubMedGoogle Scholar
  28. Saitou N, Nei M (1987) The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Bio Evol 4:406–425Google Scholar
  29. Smith ME, Douhan GW, Rizzo DM (2007) Intra-specific and intra-sporocarp ITS variation of ectomycrrhizal fungi assessed by rDNA sequencing of sporocarps and pooled ectomycorrhizal roots from a Quercus woodland. Mycorrhiza 18:15–22CrossRefPubMedGoogle Scholar
  30. Sourzat P, Sanchez A, Ourzick A, Roux JJ (1999) Analyse du statut mycorhizien de brules steriles dans trois regions de France : Midi-Pyrenees (Lot), Paca (Var), Charente Poitou (Vienne). In Proceedings of the 5th International Congress on the Science and Cultivation of Truffle and Other Edible Hypogeous Mushrooms, Aix-en-Provence, France, 4–6 Mar 1999, pp 474–479Google Scholar
  31. Tedersoo L, Köljalg U, Hallenberg N, Larsson K (2003) Fine scale distribution of ectomycorrhizal fungi and roots across substrate layers including course woody debris in a mixed forest. New Phytol 159:153–165CrossRefGoogle Scholar
  32. Tedersoo L, Hansen K, Perry BA, Kjøller R (2006) Molecular and morphological diversity of pezizalean ectomycorrhiza. New Phytol 170:581–596CrossRefPubMedGoogle Scholar
  33. White TJ, Bruns TD, Lee S, Taylor J (1990) Analysis of phylogenetic relationship by amplification and direct sequencing of ribosomal RNA genes. 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 2010

Authors and Affiliations

  • Andrea Rubini
    • 1
  • Beatrice Belfiori
    • 1
  • Valentina Passeri
    • 1
  • Leonardo Baciarelli Falini
    • 2
  • Sergio Arcioni
    • 1
  • Claudia Riccioni
    • 1
  • Francesco Paolocci
    • 1
  1. 1.National Research Council, Plant Genetics Institute, Perugia DivisionPerugiaItaly
  2. 2.Department of Applied BiologyUniversity of PerugiaPerugiaItaly

Personalised recommendations