The Method Research on Tuber spp. DNA in Soil

Conference paper
Part of the Lecture Notes in Electrical Engineering book series (LNEE, volume 269)


Truffles are ascomata of ectomycorrhizal hypogeous fungi associated with endemic Pinus tabuliformis in Mount Helan National Nature Reserve in Inner Mongolia, China, was investigated f in soil cubes taken from pure P. tabuliformis stands. The objectives of this study were (i) to develop a molecular method to detect mycelia of Tuber spp. in soil and (ii) to test for mycelial distribution around two truffle-bearing P. tabuliformis trees in a truffle orchard. Isolation of total DNA from soil was performed, followed by PCR amplification with Tuber spp.-specific primers and restriction analysis. To address the detection sensitivity level, soil samples were inoculated with known amounts of gleba of Tuber spp. Mycelium was detected primarily within the area defined by the truffle burn and within the top 35 cm of the soil in all directions from the trees.


Tuber spp Soil mycelium PCR–RFLP Truffle orchard ITS 



This work was supported in part by the DMAH-GENCAT, by scholarship 2002FI-00711 to L.M. Suz from the DURSI-GENCAT, and by the research project FMI-REN2002-04068-CO2-01. We are indebted to M. Donate for the open access to his truffle orchard. The authors thank C.R. Fischer for helpful suggestions and English support.


  1. 1.
    Paolocci F, Rubini A, Granetti B et al (1997) Typing Tuber melanosporum and Chinese black truffle species by molecular markers. FEMS Microbiol Lett 153:255–260CrossRefGoogle Scholar
  2. 2.
    Paolocci F, Rubini A, Riccioni C et al (2000) Cloning and characterization of two repeated sequences in the symbiotic fungus Tuber melanosporum Vitt. FEMS Microbiol Ecol 34:139–146CrossRefGoogle Scholar
  3. 3.
    Gandeboeuf D, Dupr′e C, Roeckel-Devret P et al (1997a) Typing Tuber ectomycorrhizae by polymerase chain amplification of the internal transcribed spacer of rDNA and the sequence characterized amplified region markers. Can J Microbiol 43:723–728Google Scholar
  4. 4.
    Paolocci F, Rubini A, Riccioni C et al (2000) Cloning and characterization of two repeated sequences in the symbiotic fungus Tuber melanosporum vitt. FEMS Microbiol Ecol 34:139–146CrossRefGoogle Scholar
  5. 5.
    Riousset LG, Chevalier G, Bardet MC (2001) Truffes d’Europe et de Chine (Institut National de la Recherche Agronomique Eds). Centre Technique Interprofessionnel des Fruits et L′egumes, ParisGoogle Scholar
  6. 6.
    Karen O, Jonsson L, Jonsson M et al (1999) Cook-book for DNA extraction, restriction analysis (RFLP), agarose gel electrophoresis, PCR and sequencing. Preconference workshop, Uppsala June 29–July 4 1998. DNA based methods for identification of ectomycorrhiza. html DNA% 20 extraction
  7. 7.
    Martın MP, Winka K (2000) Alternative methods of extracting and amplifying DNA from lichens. Lichenologist 32:189–196CrossRefGoogle Scholar
  8. 8.
    White TJ, Bruns T, Lee S et al (1990) Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis MA, Gelfand DH, Sninsky JJ, White TJ (eds) PCR protocols, a guide to methods and applications, pp 315–322Google Scholar
  9. 9.
    Gardes M, Bruns TD (1993) ITS primers with enhanced specificity for basidiomycetes—application to the identification of mycorrhizae and rusts. Mol Ecol 2:113–118CrossRefGoogle Scholar
  10. 10.
    Mello A, Cantisani A, Vizzini A et al (2002) Genetic variability of Tuber uncinatum and its relatedness to other black truffles. Environ Microbiol 4:584–594CrossRefGoogle Scholar
  11. 11.
    Grebenc T, Piltaver A, Kraigher H (2000) Establishment of a PCR-RFLP library for basidiomycetes, ascomycetes and their ectomycorrhizae on Picea abies (L.) Karst. Phyton (Austria) 40:79–82Google Scholar
  12. 12.
    Pacioni G, Comandini O (1999) Tuber. Ectomycorrhizal fungi key genera in profile. In: Cairney WG, Chambers SM (eds). Springer Verlag, BerlinGoogle Scholar

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© Springer Science+Business Media Dordrecht 2014

Authors and Affiliations

  1. 1.The Biology Science and Technology Department of Baotou Teacher’s CollegeBaotouChina

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