Advertisement

Mycorrhiza

, Volume 26, Issue 3, pp 177–188 | Cite as

Mycorrhizal synthesis between Lactarius deliciosus and Arbutus unedo L.

  • Filomena GomesEmail author
  • Diego Suárez
  • Rita Santos
  • Márcia Silva
  • Daniel Gaspar
  • Helena Machado
Original Article

Abstract

Arbutoid mycorrhizae were synthesized in vitro between Arbutus unedo L. and two isolates of Lactarius deliciosus. The fungal isolates were obtained from sporocarps collected under Pinus sylvestris and in a mixed forest stand of Quercus suber and Pinus pinea. Synthesis tubes filled with a mixture of sterilized peat, vermiculite, and perlite imbibed with nutrient solution were used. Two inoculation methods using solid and liquid media were tested. Shoots from an adult selected clone of A. unedo were used after in vitro rooting by auxin dipping. After 3 months of shoots transfer to the substrate, the root systems were examined for arbutoid mycorrhizae formation and later on ex vitro conditions, 9 months after acclimatization. The inoculum treatment with liquid medium improved the mycorrhizal development for both isolates, in vitro. Sterilized substrate for plant acclimatization increased the mycorrhizal development. The arbutoid mycorrhizae were observed in vitro as well as 9 months after acclimatization. Standard arbutoid mycorrhiza features were observed: pale yellow mantle, typical cruciform appearance, Hartig net (HN), and intracellular hyphal complexes, both confined to the epidermis. L. deliciosus mycorrhizae synthetized in vitro persisted 9 months after plant acclimatization. Morphological observations were confirmed by molecular techniques.

Keywords

Arbutoid mycorrhizae Strawberry tree Pure culture synthesis Ex vitro persistence 

Notes

Acknowledgments

We thank Herbario Jaime Andrés, Universidad de León (Centro Nacional de Recursos Genéticos Forestales El Serranillo, Guadalajara, Spain) for kindly granting the L. deliciosus isolate collected in Léon (BO/2264). We thank Patricia Figueiredo for helping us in the laboratory. This work was supported by PRODER Projet, 4.1 Cooperation for Innovation ref. no. 43748 and PTDC/AGR-FOR/3746/2012 from the Portuguese Foundation for Science and Technology (FCT).

References

  1. Agerer R, Rambold G (2004–2010) [first posted on 2004-06-01; most recent update: 2004-06-11]. DEEMY—an information system for characterization and determination of ectomycorrhizae. www.deemy.de. München, Germany
  2. 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 18:443–449CrossRefPubMedGoogle Scholar
  3. Arnan X, Quevedo L, Rodrigo A (2013) Forest fire occurrence increases the distribution of a scarce forest type in the Mediterranean Basin. Acta Oecol 46:39–47CrossRefGoogle Scholar
  4. Ayaz FA, Kucukislamoglu M, Reunanen M (2000) Sugar, non-volatile and phenolic acids composition of strawberry tree (Arbutus unedo L. var.ellipsoidea ) fruits. J Food Compos Anal 13(2):171–177CrossRefGoogle Scholar
  5. Cavuşoğlu A, Sulusoglu M, Erkal S (2015) Biotechnological approaches in strawberry tree (Arbutus unedo L.) breedi̇ng. Ekin J Crop Breed Gen 1(1):36–41Google Scholar
  6. Celikel G, Demirsoy L, Demirsoy H (2008) The strawberry tree (Arbutus unedo L.) selection in Turkey. Sci Hortic 118:115–119CrossRefGoogle Scholar
  7. Dalla Serra A, Franco MA, Mattivi F, Ramponi M, Vacca V, Versini G (1999) Aroma characterization of Sardinian strawberry tree (Arbutus unedo L.) honey. Ital J Food Sci 11(1):47–56Google Scholar
  8. Dickie IA, Fitzjohn RG (2007) Using terminal restriction fragment length polymorphism (T-RFLP) to identify mycorrhizal fungi: a methods review. Mycorrhiza 17:259–270CrossRefPubMedGoogle Scholar
  9. Duarte A, Gomes F, Figueiredo P, Santos R, Clemente M, (2014) Hydric stress tolerance of Arbutus unedo L. selected trees. In: IUFRO Forest Tree Breeding Conference. Program and Abstract Book. pp 45, ISBN: 978-980-213-2471-2478Google Scholar
  10. Duncan DB (1955) Multiple range and multiple F tests. Biometry 11:1–42CrossRefGoogle Scholar
  11. El-Mahrouk ME, Dewir YH, Omar AMK (2010) In vitro propagation of adult strawberry tree (Arbutus unedo L.) through adventitious shoots and somatic embryogenesis. Propagag Ornam Plants 10(2):93–98Google Scholar
  12. Figueiredo P, Gomes F, Santos R, Pop RL, (2013) Rapid propagation of Arbutus unedo L. adult selected plants using ex vitro rooting. In: 8th International Symposium on In Vitro Culture and Horticultural Breeding. Program and Abstract Book. p 157Google Scholar
  13. Flores R, Díaz G, Honrubia M (2005) Mycorrhizal synthesis of Lactarius indigo (Schw.) Fr. with five Neotropical pine species. Mycorrhiza 15:563–570. doi: 10.1007/s00572-005-0004-y CrossRefPubMedGoogle Scholar
  14. Floris I, Satta A, Ruiu R (2007) Honeys of Sardinia (Italy). J Apic Res 46:198–209CrossRefGoogle Scholar
  15. Fortin JA, Plenchette C, Piché Y (2008) Les mycorhizes la nouvelle révolution verte. Ed. Multimondes, Ed. Quae, QuébecGoogle Scholar
  16. Gautheret RJ (1959) La culture des tissues végétaux, techniques et réalisations. Masson & Cie, ParisGoogle Scholar
  17. Giovannetti M, Lioi L, Picci G (1989) Host and non-host relationships between Arbutus unedo L. and mycorrhizal fungi. Agric Ecosyst Environ 29:169–172CrossRefGoogle Scholar
  18. 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 edn. Springer, Berlin, pp 209–240. doi: 10.1007/978-3-540-78826-3 Google Scholar
  19. Goltapeh EM, Danesh YR, Prasad R, Varma A (2008) Mycorrhizal fungi: what we know and what should we know? In: Varma A (ed) Mycorrhiza genetics and molecular biology, eco-function, biotechnology, eco-physiology, structure and systematics, 3rd edn. Springer, Berlin, pp 3–27. doi: 10.1007/978-3-540-78826-3 Google Scholar
  20. Gomes F, Botelho G, Franco J, Gama J, João C, Santos R, Figueiredo P, (2014) Assessment of Arbutus unedo L. clonal plants in a field clonal trial. In: IUFRO Forest Tree Breeding Conference. Program and Abstract Book. pp 29, ISBN: 978-980-213-2471-2478Google Scholar
  21. Gomes F, Canhoto JM (2009) Micropropagation of strawberry tree (Arbutus unedo L.) from adult plants. In Vitro Cell Dev Biol Plant 45:72–82CrossRefGoogle Scholar
  22. Gomes F, Costa R, Ribeiro M, Figueiredo E, Canhoto J (2013a) Analysis of genetic relationship among Arbutus unedo L. genotypes using RAPD and SSR markers. J For Res 24(2):227–236. doi: 10.1007/s11676-012-0302-0 CrossRefGoogle Scholar
  23. Gomes F, Lopes ML, Agrela J, Canhoto JM, (2007) Micropropagation of selected adult trees of Arbutus unedo. In: IUFRO Tree Biotechnology-07. Program and Abstract Book. p 17Google Scholar
  24. Gomes F, Machado H, San Martin E, Portugal A, Canhoto J (2013b) Mycorrhizal synthesis between Pisolithus arhizus and adult clones of Arbutus unedo in vitro and in nursery. J Forestry Res 24(2):1–12. doi: 10.1007/s11676-013-0364-7 CrossRefGoogle Scholar
  25. 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 Biotechnol 27(6):882–892. doi: 10.1016/j.nbt.2010.02.009 CrossRefGoogle Scholar
  26. Guerin-Laguette A, Conventi S, Ruiz G, Plassard C, Mousain D (2003) The ectomycorrhizal symbiosis between Lactarius deliciosus and Pinus sylvestris in forest soil samples: symbiotic efficiency and development on roots of a rDNA internal transcribed spacer-selected isolate of L. deliciosus. Mycorrhiza 13:17–25. doi: 10.1007/s00572-002-0191-8 CrossRefPubMedGoogle Scholar
  27. Guerin-Laguette A, Cummings N, Butler RC, Willows A, Hesom-Williams N, Li S, Wang Y (2014) Lactarius deliciosus and Pinus radiata in New Zealand: towards the development of innovative gourmet mushroom orchards. Mycorrhiza 24:511–523. doi: 10.1007/s00572-014-0570-y CrossRefPubMedGoogle Scholar
  28. Guerin-Laguette A, Plassard C, Mousain D (2000) Effects of experimental conditions on mycorrhizal relationships between Pinus sylvestris and Lactarius deliciosus and unprecedented fruit-body formation of the Saffron milk cap under controlled soilless conditions. Can J Microbiol 46:790–799. doi: 10.1139/w00-059 CrossRefPubMedGoogle Scholar
  29. Guerreiro AC, Gago CML, Faleiro ML, Miguel MGC, Antunes MDC (2015) The effect of alginate based edible coatings enriched with essential oils constituents on Arbutus unedo L. fresh fruit storage. Postharvest Biol Technol 100:226–233. doi: 10.1016/j.postharvbio.2014.09.002 CrossRefGoogle Scholar
  30. Guerreiro AC, Gago CML, Miguel MGC, Antunes MDC (2013) The effect of temperature and film covers on the storage ability of Arbutus unedo L. fresh fruit. Sci Hortic 159:96–102CrossRefGoogle Scholar
  31. Hall TA (1999) BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp Ser 41:95–98Google Scholar
  32. Honrubia M (2009) Las micorrizas: una relación planta-hongo que dura más de 400 millones de años. An Jard Bot Madr 66(1):133–144. doi: 10.3989/ajbm.2226 CrossRefGoogle Scholar
  33. Hortal S, Pera J, Galipienso L, Parladé J (2006) Molecular identification of the edible ectomycorrhizal fungus Lactarius deliciosus in the symbiotic and extraradical mycelium stages. J Biotechnol 126:123–134. doi: 10.1016/j.jbiotec.2006.04.011 CrossRefPubMedGoogle Scholar
  34. Hortal S, Pera J, Parladé J (2008) Tracking mycorrhizas and extraradical mycelium of the edible fungus Lactarius deliciosus under field competition with Rhizopogon spp. Mycorrhiza 18:69–77. doi: 10.1007/s00572-007-0160-3 CrossRefPubMedGoogle Scholar
  35. Ingleby K, Mason PA, Last FT, Fleming V (1990) Identification of ectomycorrhizas. London, HMSO (ITE Research Publication no 5), 112pp. ISBN: 0 11 701461 3Google Scholar
  36. 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. Eur J Biochem 268:3223–3232CrossRefPubMedGoogle Scholar
  37. Kennedy P, Smith D, Horton T, Molina R (2012) Arbutus menziesii (Ericaceae) facilitates regeneration dynamics in mixed evergreen forests by promoting mycorrhizal fungal diversity and host connectivity. Am J Bot 99:1691–1701CrossRefPubMedGoogle Scholar
  38. Krpata D, Mühlmann O, Kuhnert R, Ladurner H, Göbl F, Peintner U (2007) High diversity of ectomycorrhizal fungi associated with Arctostaphylos uva-ursi in subalpine and alpine zones: potential inoculum for afforestation. For Ecol Manag 250:167–175. doi: 10.1016/j.foreco.2007.05.012 CrossRefGoogle Scholar
  39. Lancellotti E, Iotti M, Zambonelli A, Franceschini A (2014) Characterization of Tuber borchii and Arbutus unedo mycorrhizas. Mycorrhiza 24:481–486. doi: 10.1007/s00572-014-0564-9 CrossRefPubMedGoogle Scholar
  40. Lopes L, Sá O, Pereira J, Baptista P (2012) Genetic diversity of Portuguese Arbutus unedo L. populations using leaf traits and molecular markers: An approach for conservation purposes. Sci Hort 142:57–67CrossRefGoogle Scholar
  41. Martins A (2004) Micorrização controlada de Castanea sativa Mill.: Aspectos fisiológicos da micorrização in vitro e ex vitro. University of Lisbon.Google Scholar
  42. 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 3:1–11CrossRefGoogle Scholar
  43. Meireles C, Gonçalves P, Rego F, Silveira S (2005) Estudo da regeneração natural das espécies arbóreas autóctones na Reserva Natural da Serra da Malcata. Silva Lusit 13(2):217–231Google Scholar
  44. Miguel MG, Faleiro ML, Guerreiro AC, Antunes MD (2014) Arbutus unedo L.: chemical and biological properties. Molecules 19:15799–15823. doi: 10.3390/molecules191015799 CrossRefPubMedGoogle Scholar
  45. Molina M, Pardo-de-Santayana M, Aceituno L, Morales R, Tardio J (2011) Fruit production of strawberry tree (Arbutus unedo L.) in two. Span For Forestry 84(4):419–429CrossRefGoogle Scholar
  46. Molina R (1979) Pure culture synthesis and host specificity of red alder mycorrhizae. Can J Bot 57:1223–1228CrossRefGoogle Scholar
  47. Molina R, Trappe JM (1982) Lack of mycorrhizal specificity by the Ericaceous hosts Arbutus menziesii and Arctostaphylos uva-ursi. New Phytol 90:495–509CrossRefGoogle Scholar
  48. Moser M (1960) Die Bedeutung der Mykorrhiza bei Aufforstungen unter besonderer Berücksichtigung von Hochlagen. In: Mykorrhiza. Internat. Symposium. pp 407–424Google Scholar
  49. Mühlmann O, Göbl F (2006) Mycorrhiza of the host-specific Lactarius deterrimus on the roots of Picea abies and Arctostaphylos uva-ursi. Mycorrhiza 16:245–250. doi: 10.1007/s00572-006-0038-9 CrossRefPubMedGoogle Scholar
  50. Mulas M, Cani MR, Brigaglia N, Deidda P (1998) Selezione varietale da popolazioni spontanee per la coltivazione di mirto e coberzzolo in Sardegna. Rev Bras Frutic 3:45–50Google Scholar
  51. Münzenberger E, Kottke I, Oberwinkler F (1992) Ultrastructural investigations of Arbutus unedo -Laccaria amethystea mycorrhiza synthesized in vitro. Trees 7:40–47CrossRefGoogle Scholar
  52. 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 21:53–64CrossRefGoogle Scholar
  53. 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. Environ Exp Bot 66:362–371CrossRefGoogle Scholar
  54. Neppi M (2001) Alberi ed arbusti della flora mellifera della regione mediterrânea. In: Propagazione per seme di alberi e arbusti della flora mediterranea. Dipart. Prev. Risanam. Ambient., Manuale ANPA (Agenz. Naz. per la Protezi. dell’ Ambiente), pp 44–49Google Scholar
  55. Nuytinck J, Verbeken A (2007) Species delimitation and phylogenetic relationships in Lactarius section Deliciosi in Europe. Mycol Res 111:1285–1297. doi: 10.1016/j.mycres.2007.09.001 CrossRefPubMedGoogle Scholar
  56. Oddoux L (1957) Recherches sur les mycéliums secondaires des Homobasidiés en culture pure: morphologie, cytologie, exigences alimentaires. Impremerie de Trevoux, LyonGoogle Scholar
  57. Oliveira I, Baptista P, Bento A, Pereira JA (2011) Arbutus unedo L. and its benefits on human health. J Food Nutr Res 50(2):73–85Google Scholar
  58. Pallauf K, Rivas-Gonzalo JC, Del Castillo MD, Cano MP, Pascual-Teresa S (2008) Characterization of the antioxidant composition of strawberry tree (Arbutus unedo L.) fruits. J Food Compos Anal 21(4):273–281CrossRefGoogle Scholar
  59. Parladé J, Hortal S, Pera J, Galipienso L (2007) Quantitative detection of Lactarius deliciosus extraradical soil mycelium by real-time PCR and its application in the study of fungal persistence and interspecific competition. Journal Biotechnology 128:14–23. doi: 10.1016/j.jbiotec.2006.09.010 CrossRefGoogle Scholar
  60. 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 14:171–176CrossRefPubMedGoogle Scholar
  61. Parladé X, Pera J, De La Varga H, Hortal S, (2009) Tracking inoculated ectomycorrhizal fungi by real-time PCR. In: 14th European Congress on Biotechnology. Program and Abstract Book. p 375Google Scholar
  62. Pawlowska AM, Marinella DL, Braca A (2006) Phenolics of Arbutus unedo L. (Ericaceae) fruits: identification of anthocyanins and gallic acid derivatives. J Agric Food Chem 54(26):10234–10238CrossRefPubMedGoogle Scholar
  63. Pedro JG (1994) Portugal Atlas do Ambiente. Notícia Explicativa II.6 Carta da distribuição de figueira e medronheiro. D.G.A., Ministério do Ambiente e Recursos Naturais, Lisboa, ISBN: 972-9392-37-4 (col.) 972-9392-39-0Google Scholar
  64. Peterson RL, Massicotte HB (2004) Exploring structural definitions of mycorrhizas, with emphasis on nutrient-exchange interfaces. Can J Bot 82:1074–1088CrossRefGoogle Scholar
  65. Piotto B, Piccini C, Arcadu P (2001) La ripresa della vegetazione dopo gli incendi nella regione mediterrânea. In: Piotto B, Noi A (eds) Propagazione per seme di alberi e arbusti della flora mediterranea, vol Dipart. Prev. Risanam. Ambient., Manuale ANPA (Agenz. Naz. per la Protezi. dell’ Ambiente). pp 32–38Google Scholar
  66. Poitou N, Mamoun M, Ducamp M, Guinberteau J, Olivier J (1989) Controlled mycorrhization and experimental cultivation in the field of Boletus (=Suillus) granulatus and Lactarius deliciosus. Mushroom Sci 12 (Part II):551–564Google Scholar
  67. Quinteiro SL (2005) Respuesta de siete orígenes ibéricos de Pinus pinaster Aiton frente a la inoculación en vivero con Pisolithus tinctorius y Paxillus involutus. University of Santiago de Compostela.Google Scholar
  68. Rai MK (2001) Current advances in mycorrhization in micropropagation. In Vitro Cell Dev Biol-Plant 37:158–167CrossRefGoogle Scholar
  69. Ribeiro MM, Quinta-Nova L, Roque N, Ricardo A, Gaspar D, Costa R, Vendramin GG, (2014) Application of biophysical factors and molecular markers to explain spatial genetic structure in strawberry tree using GIS tools. In: IUFRO Forest Tree Breeding Conference. Program and Abstract Book. p 41Google Scholar
  70. 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 Phytol 166:1011–1023CrossRefPubMedGoogle Scholar
  71. Richard F, Selosse M-A, Gardes M (2009) Facilitated establishment of Quercus ilex in shrub-dominated communities within a Mediterranean ecosystem: do mycorrhizal partners matter? FEMS Microbiol Ecol 68:14–24CrossRefPubMedGoogle Scholar
  72. 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. Ann For Sci 62:817–822CrossRefGoogle Scholar
  73. Ruiz-Rodríguez BM, Morales P, Fernández-Ruiz V, Sánchez-Mata MC, Cámara M, Díez-Marqués C, Pardo-de-Santayana M, Molina M, Tardío J (2011) Valorization of wild strawberry-tree fruits (Arbutus unedo L.) through nutritional assessment and natural production data. Food Res Int 44:1244–1253CrossRefGoogle Scholar
  74. Scanu R, Spano N, Panzanelli A, Pilo MI, Piu PC, Sanna G, Tapparo A (2005) Direct chromatographic methods for the rapid determination of homogentisic acid in strawberry tree (Arbutus unedo L.) honey. J Chromatogr A 1090(1–2):76–80CrossRefPubMedGoogle Scholar
  75. Smith SE, Read DJ (2008) Mycorrhizal Symbiosis. 3th ed. Academic Press, Elsevier LtdGoogle Scholar
  76. Takrouni MM, Boussaid M (2010) Genetic diversity and population's structure in Tunisian strawberry tree (Arbutus unedo L.). Sci Hort 126:330–337. doi: 10.1016/j.scienta.2010.07.031 CrossRefGoogle Scholar
  77. Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG (1997) The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 25:4876–4882CrossRefPubMedPubMedCentralGoogle Scholar
  78. Wang Y, Cummings N, Guerin-Laguette A (2012) Cultivation of Basidiomycete edible ectomycorrhizal mushrooms: Tricholoma, Lactarius and Rhizopogon. In: Zambonelli A, Bonito G (eds) Edible ectomycorrhizal mushrooms, vol 34, Soil Biology. Springer-Verlag, Berlin, pp 281–304. doi: 10.1007/978-3-642-33823-6_16 CrossRefGoogle Scholar
  79. Wang Y, Hall I (2004) Edible mycorrhizal mushrooms: challenges and achievements. Can J Bot 82(8):1063–1073. doi: 10.1139/B04-051 CrossRefGoogle Scholar
  80. Wang Y, Hall I, Dixon C, Hance-Halloy M, Strong G, Brass P, (2002) The cultivation of Lactarius deliciosus (saffron milk cap) and Rhizopogon rubescens (shoro) in New Zealand. In: Hall I, Wang Y, Danell E, Zambonelli A (eds) Second International Conference on Edible Mycorrhizal Mushrooms. Program and Abstract Book. pp 0–6. ISBN: 0-478-10829-XGoogle Scholar
  81. 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, Sninski JJ, White TJ (eds) PCR protocols: a guide to methods and applications. Academic Press, New York, pp 315–322Google Scholar
  82. Zak B (1974) Ectendomycorrhiza of Pacific madrone (Arbutus menziesii). Trans Br Mycol Soc 62:202–204CrossRefGoogle Scholar
  83. Zak B (1976) Pure culture synthesis of Pacific Madrone Ectendomycorrhizae. Mycologia 68(2):362–369CrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2015

Authors and Affiliations

  • Filomena Gomes
    • 1
    Email author
  • Diego Suárez
    • 1
  • Rita Santos
    • 1
    • 2
  • Márcia Silva
    • 3
  • Daniel Gaspar
    • 3
  • Helena Machado
    • 3
  1. 1.Instituto Politécnico de CoimbraEscola Superior AgráriaCoimbraPortugal
  2. 2.GREENCLON LDACoimbraPortugal
  3. 3.Instituto Nacional de Investigação Agrária e VeterináriaOeirasPortugal

Personalised recommendations