Skip to main content
SpringerLink
Log in

Mycorrhizal synthesis between Pisolithus arhizus and adult clones of Arbutus unedo in vitro and in nursery

  • Original Paper
  • Published:
Journal of Forestry Research Aims and scope Submit manuscript

Abstract

Arbutoid mycorrhizae were synthesized between adult selected clones of Arbutus unedo L. and Pisolithus arhizus. Two micropropagated clones were tested: AL1, in vitro and C1 (acclimatized plants) in nursery and later in a field trial. In vitro, rooted shoots were transferred to test tubes containing the substrate previously inoculated with mycelium cultured on agar. In the nursery, two inoculation treatments were tested (vegetative inocula or dry sporocarps) and compared to control plants. In the field trial, plants from nursery inoculation treatments were compared and an additional control treatment using seedlings was implemented. Plant height was evaluated 4 months later in the nursery and 20 months later in the field trial. Roots were examined by morphological and histological studies: a) in vitro plantlets one month after inoculation and nine months after acclimatization; and b) 20 months after the field trial was established. Arbutoid mycorrhizae were observed in vitro one month after inoculation, indicating compatibility between A. unedo and P. arhizus. These showed the presence of a mantle, Hartig net, and intracellular hyphal complexes confined to the epidermal root cells. Arbutoid mycorrhizae were also observed nine months after acclimatization in inoculated and control plants. In order to confirm the identity of mycorrhizae, molecular techniques were used, in previously inoculated in vitro plants, 12 months after acclimatization. Thelephora and Hebeloma mycorrhizae, two types of highly competitive and widespread mycorrhizae on nurseries were identified. In the nursery, dry sporocarp treatment improved plant height after four months. In a field trial (20 months later), plants growth did not show significant differences. By this time, mycorrhized roots with Cenococcum geophilum and other types were identified. These results and their implications on A. unedo breeding program are discussed.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  • Agerer R, Rambold G. 2004–2010. DEEMY — An information system for characterization and determination of ectomycorrhizae. München, Germany. Available at: www.deemy.de Nov./2009.

    Google Scholar 

  • Águeda B, Parladé JF, Fernández-Toirán LM, Cisneros O, Miguel AM, Modrego MP, Martínez-Pena F, Pera J. 2008. Mycorrhizal synthesis between Boletus edulis species complex and rockroses (Cistus sp.). Mycorrhiza, 18: 443–449.

    Article  PubMed  Google Scholar 

  • 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 Research, 25: 3389–3402.

    Article  PubMed  CAS  Google Scholar 

  • Anderson WC. 1984. A revised tissue culture medium for shoot multiplication of Rhododendron. Journal of the American Society for Horticultural Science, 109: 343–347.

    Google Scholar 

  • Cantos M, Liñán J, Carretero CL, Troncoso A, Azcón R. 2009. Influence of mycorrhization during the hardening process of micropropagated plants. Acta Horticulturae, 812: 455–460.

    Google Scholar 

  • Chen YL, Kang LH, Dell B. 2006. Inoculation of Eucalyptus urophylla with spores of Scleroderma in a nursery in south China: Comparison of field soil and potting mix. Forest Ecology and Management, 222: 439–449.

    Article  Google Scholar 

  • De Fossard RA, Nitsch C, Cresswell RJ, Lee HCM. 1974. Tissue and organ culture of Eucalyptus. New Zealand Journal of Forest Science, 4: 267–278.

    Google Scholar 

  • Duncan DB. 1955. Multiple range and multiple F tests. Biometry, 11: 1–42.

    Article  Google Scholar 

  • Eccher T, Martinelli M. 2010 Inoculation of Rhododendron cultivars in vitro with different strains of ericoid endomycorrhizae. Acta Horticulturae, 865: 327–332.

    CAS  Google Scholar 

  • Eccher T, Noé N. 2002. Influence of Ericoid endomycorrhizae inoculated in vitro on rooting and early growth of micropropagated plants of Vaccinium corymbosum L. Acta Horticulturae, 574: 373–378.

    Google Scholar 

  • Fortin JA, Plenchette C, Piché Y. 2008. Les mycorhizes la nouvelle révolution verte. Ed. Multimondes, Ed. Quae, Québec.

    Google Scholar 

  • Fusconi A, Bonfante-Fasolo P. 1984. Ultrastructural aspects of host endophyte relationships in Arbutus unedo L. mycorrhizas. New Phytologist, 96: 397–410.

    Article  Google Scholar 

  • Garbaye J. 1990. Pourquoi et comment observer l’état mycorhizien des plants forestiers. Rev. Revue Forestière Française, Biologie et Forêt, XLII 1: 35–47.

    Article  Google Scholar 

  • Gardes M, Bruns TD. 1993. ITS primers with enhanced specificity for basidiomycetes: application to the identification of mycorrhizae and rusts. Molecular Ecology, 2: 113–118.

    Article  PubMed  CAS  Google Scholar 

  • Gautheret RJ. 1959. La culture des tissues végétaux, techniques et réalisations. Masson & Cie, Paris.

    Google Scholar 

  • Gautry JY, Jouvenceau S, Thivolle-Cazat A. 1991 Mycorrhization artificielle du Douglas et de l’épicéa et interaction clone x champignon. Annais AFOCEL, 125–158.

    Google Scholar 

  • Giovannetti M, Lioi L, Picci G. 1989 Host and non-host relationships between Arbutus unedo L. and mycorrhizal fungi. Agriculture, Ecosystems & Environment, 29: 169–172.

    Article  Google Scholar 

  • Gobert A, Plassard C. 2008. The beneficial effect of mycorrhizae on N utilization by host-plant: myth or reality? In: Varma A. (ed.), Mycorrhiza Genetics and Molecular Biology, Eco-function, Biotechnology, Eco-physiology, Structure and Systematic. 3rd ed. Berlin: Springer, pp. 209–240

    Google Scholar 

  • Gomes F, Canhoto JM. 2009. Micropropagation of strawberry tree (Arbutus unedo L.) from adult plants. In Vitro Cellular & Developmental Biology — Plant, 45: 72–82.

    Article  CAS  Google Scholar 

  • Gomes F, Lopes ML, Santos T, Canhoto JM. 2009 Micropropagation of selected trees of Arbutus unedo L. trough axillary shoot proliferation and somatic embryogenesis. Acta Horticulturae, 839: 111–116.

    CAS  Google Scholar 

  • Gomes F, Simões M, Lopes ML, Canhoto JM. 2010 Effect of plant growth regulators and genotype on the micropropagation of adult trees of Arbutus unedo L. (strawberry tree). New Biotechnology, 27: 882–892.

    Article  PubMed  CAS  Google Scholar 

  • Harrison MJ. 1999. Molecular and cellular aspects of the arbuscular mycorrhizal symbiosis. Annual Review of Plant Physiology and Plant Molecular Biology, 50: 361–389.

    Article  PubMed  CAS  Google Scholar 

  • Ingleby K, Mason PA, Last FT, Fleming V. 1990. Identification of ectomycorrhizas, ITE Research Publication n° 5. NE Research Council, HMSO Publ., London.

    Google Scholar 

  • Jacob C, Courbot M, Brun A, Steinman HM, Jacquot JP, Botton B, Chalot M. 2001. Molecular cloning, characterization and regulation by cadmium of a superoxide dismutase from the ectomycorrhizal fungus Paxillus involutus. European Journal of Biochemistry, 268: 3223–3232.

    Article  PubMed  CAS  Google Scholar 

  • Kosola KR, Workmaster BAA, Spada PA. 2007. Inoculation of cranberry (Vaccinium macrocarpon) with the ericoid mycorrhizal fungus Rhizoscyphus ericae increases nitrate influx. New Phytologist, 176: 184–196.

    Article  PubMed  Google Scholar 

  • Lopes ML, Gomes F, Canhoto JM. 2011. Somatic embryogenesis induction and plant conversion from young leaves of adult strawberry tree-derived shoots: role of the genotype. XXXVI Jornadas Portuguesas de Genética, Abst, 68.

    Google Scholar 

  • Martins A. 2004. Micorrização controlada de Castanea sativa Mill.: Aspectos fisiológicos da micorrização in vitro e ex vitro. PhD Thesis. University of Lisbon.

    Google Scholar 

  • Martins A. 2010. Mycorrhizal inoculation of Chestnut seedlings: effect on survival and growth after transplantation. Acta Horticulturae, 866: 325–334.

    Google Scholar 

  • Martins A, Barroso J, Pais MS. 1996 Effect of ectomycorrhizal fungi on survival and growth of micropropagated plants and seedlings of Castanea sativa Mill. Mycorrhiza, 6: 265–270.

    Article  Google Scholar 

  • Massicotte HB, Melville LH, Molina R, Peterson RL. 1993. Structure and histochemistry of mycorrhizae synthesized between Arbutus menziesii (Ericaceae) and two basidiomycetes Pisolithus tinctorius (Pisolithaceae) and Piloderma bicolor (Corticiaceae). Mycorrhiza, 3: 1–11.

    Article  Google Scholar 

  • Mirabelli C, Tullio M, Pierandrei F, Rea E. 2009. Effect of arbuscular mycorrhizal fungi on micropropagated hazelnut (Corylus avellana L.) plants. Acta Horticulturae, 812: 467–472.

    Google Scholar 

  • Molina R. 1979. Pure culture synthesis and host specificity of red alder mycorrhizae. Canadian Journal of Botany, 57: 1223–1228.

    Article  Google Scholar 

  • Molina R, Trappe JM. 1982 Lack of mycorrhizal specificity by the Ericaceous hosts Arbutus menziesii and Arctostaphylos uva-ursi. New Phytologist, 90: 495–509.

    Article  Google Scholar 

  • Münzenberger E, Kottke I, Oberwinkler F. 1992. Ultrastructural investigations of Arbutus unedo -Laccaria amethystea mycorrhiza synthesized in vitro. Trees, 7: 40–47.

    Article  Google Scholar 

  • Navarro A, Banón S, Morte A, Sánchez-Blanco MJ. 2011. Effects of nursery preconditioning through mycorrhizal inoculation and drought in Arbutus unedo L. plants. Mycorrhiza, 21: 53–64.

    Article  Google Scholar 

  • Navarro A, Sánchez-Blanco MJ, Morte A, Banón S. 2009. The influence of mycorrhizal inoculation and paclobutrazol on water and nutritional status of Arbutus unedo L. Environmental Experimental Botany, 66: 362–371.

    Article  CAS  Google Scholar 

  • Niemi K, Scagel C, Häggman H. 2004. Application of ectomycorrhizal fungi in vegetative propagation of conifers. Plant Cell, Tissue and Organ Culture, 78: 83–91.

    Article  Google Scholar 

  • Noé N, Eccher T, Borra M. 2002. The use of selected mycorrhizae for quality improvement of Highbush Blueberry (Vaccinium corymbosum L.) plant production. Acta Horticulturae, 574: 387–392.

    Google Scholar 

  • Oliveira P, Barriga J, Cavaleiro C, Peixe A, Potes AZ. 2003. Sustained in vitro root development obtained in Pinus pinea L. inoculated with ectomycorrhizal fungi. Forestry, 76: 579–587.

    Article  Google Scholar 

  • Parladé J, Pera J, Luque J. 2004. Evaluation of mycelial inocula of edible Lactarius species for the production of Pinus pinaster and P. sylvestris mycorrhizal seedlings under greenhouse conditions. Mycorrhiza, 14: 171–176.

    Article  PubMed  Google Scholar 

  • Parladé X, Pera J, De La Varga H, Hortal S. 2009. Tracking inoculated ectomycorrhizal fungi by Real-Time PCR. 25, Supplement 1, 14 th European Congress on Biotechnology, Abst S375.

    Google Scholar 

  • Peterson RL, Massicotte HB. 2004. Exploring structural definitions of mycorrhizas, with emphasis on nutrient-exchange interfaces. Canadian Journal of Botany, 82: 1074–1088.

    Article  Google Scholar 

  • Piotto B, Piccini C, Arcadu P. 2001. La ripresa della vegetazione dopo gli incendi nella regione mediterranea. In: Piotto B, Noi A. (eds), Propagazione per seme di alberi e arbusti della flora mediterranea, Roma, pp. 32–38.

    Google Scholar 

  • Puthur JT, Prasad KVSK, Sharmila P, Saradhi PP. 1998. Vesicular arbuscular mycorrhizal fungi improves establishment of micropropagated Leucaena leucocephala plantlets. Plant Cell, Tissue and Organ Culture, 53: 41–47.

    Article  Google Scholar 

  • Quinteiro SL. 2005. Respuesta de siete orígenes ibéricos de Pinus pinaster Aiton frente a la inoculación en vivero con Pisolithus tinctorius y Paxillus involutus. PhD Thesis. University of Santiago de Compostela.

    Google Scholar 

  • Rai MK. 2001 Current advances in mycorrhization in micropropagation. In Vitro Cellular & Developmental Biology — Plant, 37: 158–167.

    Article  Google Scholar 

  • Ratnaparkhe MB. 2007. Blueberry. In: Kole C. (ed) Genome mapping and molecular breeding in plants, Fruits and Nuts, Berlin: Springer, pp. 217–227.

    Google Scholar 

  • Richard F, Millot S, Gardes M, Selosse MA. 2005. Diversity and specificity of ectomycorrhizal fungi retrieved from an old-growth Mediterranean forest dominated by Quercus ilex. New Phytologist, 166: 1011–1023.

    Article  PubMed  CAS  Google Scholar 

  • Rincón A, Alvarez IF, Pera J. 2001. Inoculation of containerized Pinus pinea L. seedlings with seven ectomycorrhizal fungi. Mycorrhiza, 11: 265–271.

    Article  Google Scholar 

  • Rincón A, Parladé J, Pera J. 2005. Effects of ectomycorrhizal inoculation and the type of substrate on mycorrhization, growth and nutrition of containerized Pinus pinea L. seedlings produced in a commercial nursery. Annals of Forest Science, 62: 817–822.

    Article  Google Scholar 

  • Robertson DC, Robertson JA. 1985. Ultrastructural aspects of Pyrola mycorrhizae. Canadian Journal of Botany, 63: 1089–1098.

    Article  Google Scholar 

  • Smith SE, Read DJ. 1997. Arbutoid and monotropoid mycorrhizas. Mycorrhizal Symbiosis, 2nd ed. San Diego: Academic Press, Harcourt Brace and Company Publishers.

    Google Scholar 

  • Subhan S, Sharmila P, Saradhi PP. 1998 Glomus fasciculatum alleviates transplantation shock of micropropagated Sesbania sesban. Plant Cell Reports, 17: 268–272.

    Article  CAS  Google Scholar 

  • White TJ, Bruns TD, Lee S, Taylor JW. 1990. Amplification and direct sequencing of fungal ribossomal RNA genes for phylogenetics. In: Innis MA, Gelfand DH, Sninsky JJ, White TJ. (eds), PCR Protocols: a guide to methods and amplifications. London: Academic Press, pp. 315–322.

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. CERNAS, Dep. Recursos Florestais, Escola Superior Agrária Coimbra, Bencanta, 3040-316, Coimbra, Portugal

    Filomena Gomes & Esteban San Martin

  2. INIAV, Instituto Nacional de Investigação Agrária e Veterinária, IP., Av. República, Quinta do Marquês, 2780-159, Oeiras, Portugal

    Helena Machado

  3. Centre of Functional Ecology, Department of Life Sciences, University of Coimbra, Ap. 3046, 3001-401, Coimbra, Portugal

    A. Portugal & Jorge M. Canhoto

Authors
  1. Filomena Gomes
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Helena Machado
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Esteban San Martin
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. A. Portugal
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Jorge M. Canhoto
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Filomena Gomes.

Additional information

Fund project: This work was supported by a PhD fellowship (SFRH/BD/37170/2007) from the Portuguese Foundation for Science and Technology (FCT)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Gomes, F., Machado, H., San Martin, E. et al. Mycorrhizal synthesis between Pisolithus arhizus and adult clones of Arbutus unedo in vitro and in nursery. Journal of Forestry Research 24, 659–670 (2013). https://doi.org/10.1007/s11676-013-0364-7

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11676-013-0364-7

Keywords

Search

Navigation