Advertisement

Mycorrhiza

, Volume 25, Issue 8, pp 611–625 | Cite as

Preliminary findings on identification of mycorrhizal fungi from diverse orchids in the Central Highlands of Madagascar

  • Kazutomo Yokoya
  • Lawrence W. Zettler
  • Jonathan P. Kendon
  • Martin I. Bidartondo
  • Andrew L. Stice
  • Shannon Skarha
  • Laura L. Corey
  • Audrey C. Knight
  • Viswambharan Sarasan
Original Paper

Abstract

The Orchid flora of Madagascar is one of the most diverse with nearly 1000 orchid taxa, of which about 90 % are endemic to this biodiversity hotspot. The Itremo Massif in the Central Highlands of Madagascar with a Highland Subtropical climate range encompasses montane grassland, igneous and metamorphic rock outcrops, and gallery and tapia forests. Our study focused on identifying culturable mycorrhizae from epiphytic, lithophytic, and terrestrial orchid taxa to understand their diversity and density in a spatial matrix that is within the protected areas. We have collected both juvenile and mature roots from 41 orchid taxa for isolating their orchid mycorrhizal fungi (OMF), and to culture, identify, and store in liquid nitrogen for future studies. Twelve operational taxonomic units (OTUs), of three known orchid mycorrhizal genera, were recognized by analysis of internal transcribed spacer (ITS) sequences of 85 isolates, and, by comparing with GenBank database entries, each OTU was shown to have closely related fungi that were also found as orchid associates. Orchid and fungal diversity were greater in gallery forests and open grasslands, which is very significant for future studies and orchid conservation. As far as we know, this is the first ever report of detailed identification of mycorrhizal fungi from Madagascar. This study will help start to develop a programme for identifying fungal symbionts from this unique biodiversity hotspot, which is undergoing rapid ecosystem damage and species loss. The diversity of culturable fungal associates, their density, and distribution within the Itremo orchid hotspot areas will be discussed.

Keywords

Orchidaceae Fungal symbiont Rhizoctonia Tulasnella Ceratobasidium Sebacina In vitro 

Notes

Acknowledgments

We kindly thank the financial support received from Sainsbury Orchid Project, Bentham and Moxon Trust, and Margaret A Cargill Foundation. We acknowledge the invaluable assistance received from Gaëtan Ratovonirina and Landy Rajaovelona (KMCC), Solo Rapanarivo (PBZT) for field support during the collection; Korrie Edwards (Illinois College), and Helen Sandford, Margaret Ramsay, and Edward Jones (Kew) for technical assistance; Connie Gibas and Lynne Sigler (UAMH) for deposition of isolates; Stuart Cable (Kew) and Hanne Rasmussen for helpful suggestions; and Mike Fay and Robyn Cowan (Kew) for their advice on genetic fingerprinting of plant samples.

References

  1. Alvarado ST, Buisson E, Rabarison H, Rajeriarison C, Birkinshaw C, Lowry PP (2014) Comparison of plant communities on the Ibity and Itremo massifs, Madagascar, with contrasting conservation histories and current status. Plant Ecol Divers 7:497–508Google Scholar
  2. Arditti J (1992) Fundamentals of orchid biology. Wiley, New YorkGoogle Scholar
  3. Batty AL, Brundrett MC, Dixon KW, Sivasithamparam K (2006) In situ symbiotic seed germination and propagation of terrestrial orchid seedlings for establishment at field sites. Aust J Bot 54:375–381CrossRefGoogle Scholar
  4. Bidartondo MI, Burghardt B, Gebauer G, Bruns TD, Read DJ (2004) Changing partners in the dark: isotopic and molecular evidence of ectomycorrhizal liaisons between forest orchids and trees. Proc R Soc Lond B 271:1799–1806CrossRefGoogle Scholar
  5. Bonnardeaux Y, Brundrett M, Batty A, Dixon K, Koch J, Sivasithamparam K (2007) Diversity of mycorrhizal fungi of terrestrial orchids: compatibility webs, brief encounters, lasting relationships and alien invasions. Mycol Res 111:51–61CrossRefPubMedGoogle Scholar
  6. Clements MA, Ellyard RK (1979) The symbiotic germination of Australian terrestrial orchids. Am Orchid Soc Bull 48:810–816Google Scholar
  7. Cribb P, Hermans J (2009) Field guide to the orchids of Madagascar. Kew Publishing. Royal Botanic Gardens, KewGoogle Scholar
  8. Dearnaley JDW (2007) Further advances in orchid mycorrhizal research. Mycorrhiza 17:475–486CrossRefPubMedGoogle Scholar
  9. Dixon KW (1987) Raising terrestrial orchids from seed. In: Harris WK (ed) Modern orchid growing for pleasure and profit. Orchid Club of S Australia, Inc, Adelaide, pp 47–100Google Scholar
  10. Doyle JJ, Doyle JL (1987) A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochem Bull 19:11–15Google Scholar
  11. du Puy D, Moat J (1996) A refined classification of the primary vegetation of Madagascar based on the underlying geology: using GIS to map its distribution and to assess its conservation status. In: Lourenço WR (ed) Proceedings of the International Symposium on the ‘Biogeography de Madagascar’, Paris, September 1995, pp 205–218Google Scholar
  12. Fenu G, Mattana E, Congiu A, Bacchetta G (2010) The endemic vascular flora of Supramontes (Sardinia), a priority plant conservation area. Candollea 65:347–358CrossRefGoogle Scholar
  13. Gale SW, Yamazaki J, Hutchings MJ, Yukawa T, Miyoshi K (2010) Constraints on establishment in an endangered terrestrial orchid: a comparative study of in vitro and in situ seed germinability and seedling development in Nervilia nipponica. Bot J Lin Soc 163:166–180CrossRefGoogle Scholar
  14. Gardes M, Bruns TD (1993) ITS primers with enhanced specificity for basidiomycetes: application to the identification of mycorrhizae and rusts. Mol Ecol 2:113–118CrossRefPubMedGoogle Scholar
  15. Girlanda M, Segreto R, Cafasso D, Liebel HT, Rodda M, Ercole E, Cozzolino S, Gebauer G, Perotto S (2011) Photosynthetic Mediterranean meadow orchids feature partial mycoheterotrophy and specific mycorrhizal associations. Am J Bot 98:1148–1163CrossRefPubMedGoogle Scholar
  16. Jacquemyn H, Deja A, De hert K, Bailarote BC, Lievens B (2012) Variation in mycorrhizal associations with tulasnelloid fungi among populations of five Dactylorhiza species. PLoS One 7(8):e42212PubMedCentralCrossRefPubMedGoogle Scholar
  17. Jonsson L, Nylund JE (1979) Favolaschia dybowskyana (Singer) Singer (Aphyllophorales), a new orchid mycorrhizal fungus from tropical Africa. New Phytol 83:121–128CrossRefGoogle Scholar
  18. Martos F, Munoz F, Pailler T, Kottke I, Gonneau C, Selosse MA (2012) The role of epiphytism in architecture and evolutionary constraint within mycorrhizal networks of tropical orchids. Mol Ecol 21:5098–5109CrossRefPubMedGoogle Scholar
  19. McKendrick SL, Leake JR, Taylor DL, Read DJ (2000) Symbiotic germination and development of myco-heterotrophic plants in nature: ontogeny of Corallorhiza trifida and characterization of its mycorrhizal fungi. New Phytol 145:523–537CrossRefGoogle Scholar
  20. Mitchell RB (1989) Growing hardy orchids from seeds at Kew. Plantsman 2:152–169Google Scholar
  21. Mittermeier RA, Robles PG, Hoffman M, Pilgrim J, Brooks T, Mittermeier CG, Lamoreux J, da Fonseca GAB (2005) Hotspots revisited: earth’s biologically richest and most endangered terrestrial ecoregions. University of Chicago Press, ChicagoGoogle Scholar
  22. Moat J, Smith P (2007) Atlas of the vegetation of Madagascar. Royal Botanic Gardens, KewGoogle Scholar
  23. Mugambi GK (2001) Ensuring survival of Kenyan orchids: the ex situ conservation interventions. International Orchid Conservation Congress I (Conference Proceedings), p 102Google Scholar
  24. Murray-Smith C, Brummitt NA, Oliviera-Filho AT, Bachman S, Moat J, Lughadha EM, Lucas EJ (2009) Plant diversity hotspots in the Atlantic coastal forests of Brazil. Conserv Biol 23:151–163CrossRefPubMedGoogle Scholar
  25. Nilsson LA, Rabakonandrianina E (1988) Hawk-moth scale analysis and pollination specialization in the epilithic malagasy endemic Aerangis ellisii (Reichenb fil) Schltr (Orchidaceae). Bot J Linn Soc 97:49–61CrossRefGoogle Scholar
  26. Nilsson LA, Rabakonandrianina E, Pettersson B (1992a) Exact tracking of pollen transfer and mating in plants. Nature 360:666–668CrossRefGoogle Scholar
  27. Nilsson LA, Rabakonandrianina E, Rotaharivelo R, Randriamanindry JJ (1992b) Long pollinia on eyes: hawk-moth pollination of Cynorkis uniflora Lindley (Orchidaceae) in Madagascar. Bot J Linn Soc 109:145–160CrossRefGoogle Scholar
  28. Nontachaiyapoom S, Sasirat S, Manoch L (2010) Isolation and identification of Rhizoctonia-like fungi from roots of three orchid genera, Paphiopedilum, Dendrobium, and Cymbidium, collected in Chiang Rai and Chiang Mai provinces of Thailand. Mycorrhiza 20:459–471CrossRefPubMedGoogle Scholar
  29. Otero JT, Ackerman JD, Bayman P (2002) Diversity and host specificity of endophytic Rhizoctonia-like fungi from tropical orchids. Am J Bot 89:1852–1858CrossRefGoogle Scholar
  30. Phillips RD, Barrett MD, Dixon KW, Hopper SD (2011) Do mycorrhizal symbioses cause rarity in orchids? J Ecol 99:858–869CrossRefGoogle Scholar
  31. Rasmussen HN (2002) Recent developments in the study of orchid mycorrhiza. Plant Soil 244:149–163CrossRefGoogle Scholar
  32. Rasmussen HN, Whigham DF (1993) Seed ecology of dust seeds in situ: a new study technique and its application to terrestrial orchids. Am J Bot 80:1374–1378CrossRefGoogle Scholar
  33. Richardson KA, Currah RS, Hambleton S (1993) Basidiomycetous endophytes from the roots of neotropical epiphytic Orchidaceae. Lindleyana 8:127–137Google Scholar
  34. Roos MC, Kessler PJA, Gradstein SR, Baas P (2004) Species diversity and endemism of five major Malesian islands: diversity–area relationships. J Biogeogr 31:1893–1908CrossRefGoogle Scholar
  35. Samarakoon T, Wang SY, Alford MH (2013) Enhancing PCR amplification of DNA from recalcitrant plant specimens using a trehalose-based additive. Appl Plant Sci 1:1200236Google Scholar
  36. Selosse MA, Faccio A, Scappaticci G, Bonfante P (2004) Chlorophyllous and achlorophyllous specimens of Epipactis microphylla (Neottieae, Orchidaceae) are associated with ectomycorrhizal septomycetes, including truffles. Microb Ecol 47:416–426PubMedGoogle Scholar
  37. Swarts ND, Dixon KW (2009) Terrestrial orchid conservation in the age of extinction. Ann Bot 104:543–556PubMedCentralCrossRefPubMedGoogle Scholar
  38. Taberlet P, Gielly L, Pautou G, Bouvet J (1991) Universal primers for amplification of three non-coding regions of chloroplast DNA. Plant Mol Biol 17:1105–1109CrossRefPubMedGoogle Scholar
  39. Taylor DL, McCormick MK (2008) Internal transcribed spacer primers and sequences for improved characterization of basidiomycetous orchid mycorrhizas. New Phytol 177:1020–1033CrossRefPubMedGoogle Scholar
  40. Tyson P (2000) The eighth continent: life, death and discovery in the lost world of Madagascar. William Morrow (Harper Collins) Publishers, New YorkGoogle Scholar
  41. Vorontsova MS, Ratovonirina G, Randriamboavonjy T (2013) Revision of Andropogon and Diectomis (Poaceae: Sacchareae) in Madagascar and the new Andropogon itremoensis from the Itremo Massif. Kew Bull 68:1–15CrossRefGoogle Scholar
  42. Warcup JH, Talbot PHB (1980) Perfect states of Rhizoctonias associated with orchids III. New Phytol 86:267–272CrossRefGoogle Scholar
  43. Waterman RJ, Bidartondo MI, Stofberg J, Combs JK, Gebauer G, Savolainen V, Barraclough TG, Pauw A (2011) The effects of above- and belowground mutualisms on orchid speciation and coexistence. Am Nat 177:54–68CrossRefGoogle Scholar
  44. Weiss M, Selosse MA, Rexer KH, Urban A, Oberwinkler F (2004) Sebacinales: a hitherto overlooked cosm of heterobasidiomycetes with a broad mycorrhizal potential. Mycol Res 108:1003–1010CrossRefPubMedGoogle Scholar
  45. White TJ, Bruns TD, Lee S, Taylor JW (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, San Diego, pp 315–322Google Scholar
  46. Whitman M, Medler M, Randriamanindry JJ, Rabakonandrianina E (2011) Conservation of Madagascar’s granite outcrop orchids: the influence of fire and moisture. Lankesteriana 11:55–67CrossRefGoogle Scholar
  47. Zettler LW, Piskin KA (2011) Mycorrhizal fungi from protocorms, seedlings and mature plants of the Eastern Prairie Fringed Orchid, Platanthera leucophaea (Nutt.) Lindl.: a comprehensive list to augment conservation. Am Midl Nat 166:29–39CrossRefGoogle Scholar
  48. Zettler LW, Sharma J, Rasmussen F (2003) Mycorrhizal diversity. In: Dixon KW, Kell SP, Barrett RL, Cribb PJ (eds) Orchid conservation. Natural History Publications (Borneo), Kota, pp 185–203Google Scholar
  49. Zettler LW, Corey LL, Jacks AL, Gruender LT, Lopez AM (2013) Tulasnella irregularis (Basidiomycota: Tulasnellaceae) from roots of Encyclia tampensis in south Florida, and confirmation of its mycorrhizal significance through symbiotic seed germination. Lankesteriana 13:119–128Google Scholar
  50. Zi XM, Sheng CL, Goodale UM, Shao SC, Gao JY (2014) In situ seed baiting to isolate germination-enhancing fungi for an epiphytic orchid, Dendrobium aphyllum (Orchidaceae). Mycorrhiza 24:487–499CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2015

Authors and Affiliations

  • Kazutomo Yokoya
    • 1
  • Lawrence W. Zettler
    • 2
  • Jonathan P. Kendon
    • 1
  • Martin I. Bidartondo
    • 1
    • 3
  • Andrew L. Stice
    • 2
  • Shannon Skarha
    • 2
  • Laura L. Corey
    • 2
  • Audrey C. Knight
    • 2
  • Viswambharan Sarasan
    • 1
  1. 1.Royal Botanic GardensRichmondUK
  2. 2.Department of BiologyIllinois CollegeJacksonvilleUSA
  3. 3.Department of Life SciencesImperial College LondonLondonUK

Personalised recommendations