, Volume 23, Issue 5, pp 373–380 | Cite as

Using common mycorrhizal networks for controlled inoculation of Quercus spp. with Tuber melanosporum: the nurse plant method

  • Guillermo Pereira
  • Götz Palfner
  • Daniel Chávez
  • Laura M. Suz
  • Ángela Machuca
  • Mario Honrubia
Original Paper


The high cost and restricted availability of black truffle spore inoculum for controlled mycorrhiza formation of host trees produced for truffle orchards worldwide encourage the search for more efficient and sustainable inoculation methods that can be applied globally. In this study, we evaluated the potential of the nurse plant method for the controlled inoculation of Quercus cerris and Quercus robur with Tuber melanosporum by mycorrhizal networks in pot cultures. Pine bark compost, adjusted to pH 7.8 by liming, was used as substrate for all assays. Initially, Q. robur seedlings were inoculated with truffle spores and cultured for 12 months. After this period, the plants presenting 74 % mycorrhizal fine roots were transferred to larger containers. Nurse plants were used for two treatments of two different nursling species: five sterilized acorns or five 45-day-old, axenically grown Q. robur or Q. cerris seedlings, planted in containers around the nurse plant. After 6 months, colonized nursling plant root tips showed that mycorrhiza formation by T. melanosporum was higher than 45 % in the seedlings tested, with the most successful nursling combination being Q. cerris seedlings, reaching 81 % colonization. Bulk identification of T. melanosporum mycorrhizae was based on morphological and anatomical features and confirmed by sequencing of the internal transcribed spacer region of the ribosomal DNA of selected root tips. Our results show that the nurse plant method yields attractive rates of mycorrhiza formation by the Périgord black truffle and suggest that establishing and maintaining common mycorrhizal networks in pot cultures enables sustained use of the initial spore inoculum.


Périgord black truffle Pot culture Quercus robur Quercus cerris Bark compost Liming 


  1. Agerer R (1986) Studies on ectomycorrhizae. II. Introductory remarks on their characterizations and identification. Mycotaxon 26:473–492Google Scholar
  2. Agerer R (1991) Characterization of ectomycorrhiza. In Norris JR, Read DJ, Varma AK (eds) Techniques for the study of ectomycorrhiza. Meth in Microbiol. 23: 25–73Google Scholar
  3. Águeda B, Fernández-Toirán L, De Miguel A, Martínez-Peña F (2010) Ectomycorrhizal status of a nature productive black truffle plantation. For Syst 19(1):89–97Google Scholar
  4. Altschul SF, Madden TL, Schäffer A, Zhang J, Zhang Z, Miller W, Lipman DJ (1997) Gapped LAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25:3389–3402PubMedCrossRefGoogle Scholar
  5. Amnón B, Bentura I, Kagan-Zur V, Roth-Vejerano N (2006) Optimized conditions for mycorrhiza formation between the pink rockrose (Cistus incanus) and the black Périgord truffle (Tuber melanosporum). Isr J Plant Sci 54(2):87–96CrossRefGoogle Scholar
  6. Bertini L, Amicucci A, Agostini D, Polidori E, Potenza L, Guidi Ch, Stocchi V (1999) Enhanced amplification of ITS region for the identification of Tuber species. Actes du Ve Congrès Internacional. Science et Culture de la Truffe. Federation Fancaise Des Trufficulteurs. 4 au 6 mars. 1999. Aix-en Provence. France. pp 2.51–2.54Google Scholar
  7. Bonito G, Trappe JM, Donovan S, Vilgalys R (2011) The Asian black truffle Tuber indicum can form ectomycorrhizas with North American host plants and complete its life cycle in non-native soils. Fungal Ecol 4(1):83–93CrossRefGoogle Scholar
  8. Bradshaw B (2005) Physiological aspects of Corylus avellana associated with the French black truffle fungus Tuber melanosporum and the consequence for commercial production of black truffles in Western Australia. PhD Thesis. School of Biological Sciences and Biotechnology Murdoch University Perth, Western AustraliaGoogle Scholar
  9. Brundrett M, Bougher N, Grove T, Malajczuk N (1996) Working with mycorrhizas in forestry and agriculture. ACIAR, CanberraGoogle Scholar
  10. Callot G (1999) La truffe, la terre, la vie. Éditions INRA. ParísGoogle Scholar
  11. Cartié G, Palazón C, Delgado I, Barriuso J (1999) Influencia del método de inoculación, del tipo de substrato y de la procedencia de la trufa, en la micorrización de Q. ilex L. por Tuber melanosporum Vitt, y en la supervivencia de las plantas. Actes du Ve Congrès Internacional. Science et Culture de la Truffe. Federation Fancaise Des Trufficulteurs. 4 au 6 mars. 1999. Aix-en Provence. France. pp 6.296–6.299Google Scholar
  12. Chevalier G, Frochot H (1997) La truffe de Bourgogne (Tuber uncinatum Chatin). Éditions Pétrarque, Levallois-PerretGoogle Scholar
  13. De Miguel AM, Sáez R (1997) Análisis de micorrizas en truferas cultivadas de Navarra (España). Publicaciones de Biología. Universidad de Navarra. España. Serie Botánica No. 10. pp 11–18Google Scholar
  14. Domínguez J, Rodríguez J, Saiz J (2005) Ectomicorrizas en dos plantaciones truferas de encina (Quercus ilex L. subsp. ballota (Desf.) Samp.) en Castellón. Bol San Veg Plagas 31:147–157Google Scholar
  15. García S, Reyna S, Pérez R, Rodríguez J (2007) Ecología de la trufa y las áreas truferas. In: Domenech R (ed) Truficultura. Fundamentos y técnicas. Ediciones Mundi-Prensa, España, pp 153–208Google Scholar
  16. García-Montero L, Pascual C, García-Abril A, García-Cañete J (2007) Problems of using rockroses in Tuber melanosporum culture: soil and truffle harvest associated with Cistus laurifolius. Agrofor Syst 70:252–258Google Scholar
  17. Gardes M, Bruns TD (1993) ITS primers with enhanced specificity for basidiomycetes—application to the identification of mycorrhizae and rusts. Mol Ecol 2:113–118PubMedCrossRefGoogle Scholar
  18. Gent L, Wang X, Yu F, Deng X, Tian X, Shi X, Xie X, Liu P, Shen Y (2009) Mycorrhizal synthesis of Tuber indicum with two indigenous hosts, Castanea mollissima and Pinus armandii. Mycorrhiza 19:461–467CrossRefGoogle Scholar
  19. Giomaro G, Sisti D, Zambonelli A, Amicucci A, Cecchini M, Comandini O, Stocchi V (2002) Comparative study and molecular characterization of ectomycorrhizas in Tilia americana and Quercus pubescens with Tuber brumale. Microbiol Lett 216:9–14CrossRefGoogle Scholar
  20. Hall I, Brown G, Zambonelli A (2009) Trufas. Historia, Ciencia y Recolección. Ediciones Tutor. MadridGoogle Scholar
  21. Henrion B, Chevalier G, Martin F (1994) Typing truffle species by PCR amplification of the ribosomal DNA spacers. Mycol Res 98:37–43CrossRefGoogle Scholar
  22. Honrubia M, Torres P, Díaz G, Cano A (1992) Manual para micorrizar plantas en viveros forestales. Ministerio de Agricultura, Pesca y Alimentación. ICONA, MadridGoogle Scholar
  23. Honrubia M, Fernández A, Moya D, González A, De las Heras J (2006) Potencialidad de la trufa negra (Tuber nigrum Bull.) en la provincia de Albacete. Montes, Vol. 83: 35–40Google Scholar
  24. Iotti M, Amicucci A, Stocchi V, Zambonelli A (2002) Morphological and molecular characterization of mycelia of some Tuber species in pure culture. New Phytol 155(3):499–505CrossRefGoogle Scholar
  25. Martín MP, Winka K (2000) Alternative methods of extracting and amplifying DNA from lichens. Lichenologist 32:189–196CrossRefGoogle Scholar
  26. Meotto F (1994) Guida illustrata coltivazione e cerca del tartufo. Supplemento a Vita in Campagna 6/94. 26 pGoogle Scholar
  27. Oliach D, Bonet J, Fischer C, Olivera A, Martínez de Aragón J, Colinas C (2008) El cultiu de la tofona negra. Dossier Técnic No. 26. 18 pGoogle Scholar
  28. Palazón C, Barriuso J (2007) Vivero y producción de plantas micorrizadas. In: Domenech R (ed) Fundamentos y técnicas. Ediciones Mundi-Prensa, España, pp 209–236Google Scholar
  29. Pereira G, Chávez D, Machuca A, Martínez-Suz L, Honrubia M (2010) Trufas: Alternativa de cultivo para agroforestadores de la Provincia del Bío Bío. Universidad de Concepción. ChileGoogle Scholar
  30. Pinkas Y, Maimon M, Shabi E, Elisha S, Shmulewich Y, Fremman S (2000) Inoculation, isolation and identification of Tuber melanosporum from old and new oak hosts in Israel. Mycol Res 104:472–477CrossRefGoogle Scholar
  31. Rauscher T, Chevalier G (1995) Tuber melanosporum. In: Agerer R (ed) Colour atlas of ectomycorrhizae, plate 87. Einhorn-Verlag, Schwäbisch GmündGoogle Scholar
  32. Rauscher T, Agerer R, Chevalier G (1995) Ektomykorrhizen von Tuber melanosporum, Tuber mesentericum und Tuber rufum (Tuberales) an Corylus avellana. Nova Hedwig 61:281–322Google Scholar
  33. Reyna R (2000) Trufas, truficultura y selvicultura trufera. Ediciones Mundi-Prensa, BarcelonaGoogle Scholar
  34. Reyna S (2007) Sostenibilidad de La truficultura: aspectos ecológicos, económicos y sociales. Truficultura. Fundamentos y técnicas. Reyna Domenech (Coordinador). Ediciones Mundi-Prensa. España. pp: 50–73Google Scholar
  35. Reyna S, Colina C (2007) Truficultura. Fundamentos y técnicas. Reyna Domenech (Coordinador). Ediciones Mundi-Prensa. España. pp 237–276Google Scholar
  36. Reyna S, De Miguel A (2007) Las micorrizas. Ciclo biológico de la trufa. Truficultura. Fundamentos y técnicas. Reyna Domenech (Coordinador). Ediciones Mundi-Prensa. España. pp 107–152Google Scholar
  37. Riousset L, Chevalier G, Bardet MC (2001) Truffes d’Europe et de Chine. Institut National de la Recherche Agronomique (INRA). Centre Technique interprofessionnel des Fruits et Légumes, París, FranciaGoogle Scholar
  38. Sáez R (1991) Trufas y Truficultura. Navarra Agraria. Revista de Agricultura, Ganadería y Montes. No. 67. pp 55–11Google Scholar
  39. Sáez R, De Miguel A (2008) La trufa. Guía de truficultura. Instituto Técnico y de Gestión Agrícola, Navarra-EspañaGoogle Scholar
  40. Santelices R, Palfner G (2010) Controlled rhizogenesis and mycorrhization of hazelnut (Corylus avellana L.) cuttings with black truffle (Tuber melanosporum Vitt.). Chil J Agric Res 70(2):204–212Google Scholar
  41. Selosse MA, Richard F, He X, Simard SW (2006) Mycorrhizal networks: des liaisons dangereux? Trends Ecol Evol 11:621–628CrossRefGoogle Scholar
  42. Steel R, Torrie J (1989) Bioestadística: Principios y procedimientos, 2nd edn. McGraw Hill, MéxicoGoogle Scholar
  43. Turgeman T, Sitrit Y, Danai O, Luzzati Y, Bustan A, Roth-Bejerano N, Kagan-Zur V, Masaphy S (2012) Introduced Tuber aestivum replacing introduced Tuber melanosporum: a case study. Agrofor Syst 84(3):337–343CrossRefGoogle Scholar
  44. White TJ, Bruns T, Lee S, Taylor J (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. Academic, New York, pp 315–322Google Scholar
  45. Zambonelli A, Salomoni S, Pisi A (1993) Caratterizzazione anatomo-morfológica delle micorrize di Tuber spp. su Quercus pubescens Willd. Mic Ital 3:73–90Google Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2013

Authors and Affiliations

  • Guillermo Pereira
    • 1
  • Götz Palfner
    • 2
  • Daniel Chávez
    • 1
  • Laura M. Suz
    • 3
  • Ángela Machuca
    • 1
  • Mario Honrubia
    • 3
  1. 1.Departamento de Ciencias y Tecnología Vegetal, Campus Los ÁngelesUniversidad de ConcepciónLos ÁngelesChile
  2. 2.Departamento de Botánica, Facultad de Ciencias Naturales y OceanográficasUniversidad de ConcepciónConcepciónChile
  3. 3.Departamento de Biología Vegetal, Facultad de BiologíaUniversidad de MurciaMurciaSpain

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