Mycorrhiza

, Volume 24, Issue 7, pp 487–499

In situ seed baiting to isolate germination-enhancing fungi for an epiphytic orchid, Dendrobium aphyllum (Orchidaceae)

  • Xiao-Meng Zi
  • Chun-Ling Sheng
  • Uromi Manage Goodale
  • Shi-Cheng Shao
  • Jiang-Yun Gao
Original Paper

Abstract

Orchid conservation efforts, using seeds and species-specific fungi that support seed germination, require the isolation, identification, and germination enhancement testing of symbiotic fungi. However, few studies have focused on developing such techniques for the epiphytes that constitute the majority of orchids. In this study, conducted in Xishuangbanna Tropical Botanical Garden, Yunnan, China, we used seeds of Dendrobium aphyllum, a locally endangered and medicinally valuable epiphytic orchid, to attract germination promoting fungi. Of the two fungi isolated from seed baiting, Tulasnella spp. and Trichoderma spp., Tulasnella, enhanced seed germination by 13.6 %, protocorm formation by 85.7 %, and seedling development by 45.2 % (all P < 0.0001). Epulorhiza, another seed germination promoting fungi isolated from Cymbidium mannii, also enhanced seed germination (6.5 %; P < 0.05) and protocorm formation (20.3 %; P < 0.0001), but Trichoderma suppressed seed germination by 26.4 % (P < 0.0001). Tulasnella was the only treatment that produced seedlings. Light increased seed imbibition, protocorm formation, and two-leaved seed development of Tulasnella inoculated seeds (P < 0.0001). Because the germination stage success was not dependent on fungi, we recommend that Tulasnella be introduced for facilitating D. aphyllum seed germination at the protocorm formation stage and that light be provided for increasing germination as well as further seedling development. Our findings suggest that in situ seed baiting can be used to isolate seed germination-enhancing fungi for the development of seedling production for conservation and reintroduction efforts of epiphytic orchids such as D. aphyllum.

Keywords

Dendrobium aphyllum Orchid conservation Seed baiting Symbiotic germination Tulasnella 

Supplementary material

572_2014_565_MOESM1_ESM.docx (18 kb)
ESM 1(DOCX 18 kb)
572_2014_565_MOESM2_ESM.docx (19 kb)
ESM 2(DOCX 19 kb)

References

  1. Arditti J (1967) Factors affecting the germination of orchid seeds. Bot Rev 33:1–97CrossRefGoogle Scholar
  2. Arditti J (1992) Fundamentals of orchid biology. Wiley, New YorkGoogle Scholar
  3. Arditti J, Ernst R (1984) Physiology of germinating orchid seeds. In: Arditti J (ed) Orchid biology, reviews and perspectives, 3rd edn. Cornell University Press, New York, pp 170–222Google Scholar
  4. Arditti J, Ghani AKA (2000) Numerical and physical properties of orchid seeds and their biological implications. New Phytol 145:367–421CrossRefGoogle Scholar
  5. Atwood JT (1986) The size of the Orchidaceae and the systematic distribution of epiphytic orchids. Selbyana 9:171–186Google Scholar
  6. Bao XS, Shun QS, Chen LZ (2001) The medicinal plants of Dendrobium (Shi-Hu) in China, a coloured atlas. Fudan University Press, Shanghai (in Chinese)Google Scholar
  7. Batty AL, Dixon KW, Brundrett M, Sivasithamparam K (2001) Constraints to symbiotic germination of terrestrial orchid seed in a Mediterranean bushland. New Phytol 152:511–520CrossRefGoogle Scholar
  8. 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
  9. Bidartondo MI, Read DJ (2008) Fungal specificity bottlenecks during orchid germination and development. Mol Ecol 17:3707–3716PubMedGoogle Scholar
  10. Bretz F, Hothorn T, Westfall P (2010) Multiple comparisons using R. CRC, Boca RatonCrossRefGoogle Scholar
  11. Brundrett MC, Scade A, Batty AL, Dixon KW, Sivasithamparam K (2003) Development of in situ and ex situ seed baiting techniques to detect mycorrhizal fungi from terrestrial orchid habitats. Mycol Res 107:1210–1220PubMedCrossRefGoogle Scholar
  12. Catalano V, Vergara M, Hauzenberger JR, Seiboth B, Sarrocco S, Vannacci G, Kubicek CP, Seidl-Seiboth V (2011) Use of a non-homologous end-joining-deficient strain (delta-ku70) of the biocontrol fungus Trichoderma virens to investigate the function of the laccase gene lcc1 in sclerotia degradation. Curr Genet 57:13–23PubMedCentralPubMedCrossRefGoogle Scholar
  13. Chen XQ (1999) Orchidaceae. In: Flora of China 19. Science Press, Beijing, pp 106–108, in ChineseGoogle Scholar
  14. Chen XM, Guo SX (2005) Effects of four species of endophytic fungi on the growth and polysaccharide and alkaloid contents of Dendrobium nobile. Zhongguo Zhong Yao Za Zhi 30:253 (in Chinese)PubMedGoogle Scholar
  15. Chen J, Wang H, Guo SX (2012) Isolation and identification of endophytic and mycorrhizal fungi from seeds and roots of Dendrobium (Orchidaceae). Mycorrhiza 22:297–307PubMedCrossRefGoogle Scholar
  16. Chutima R, Dell B, Lumyong S (2011) Effects of mycorrhizal fungi on symbiotic seed germination of Pecteilis susannae (L.) Rafin (Orchidaceae), a terrestrial orchid in Thailand. Symbiosis 53:149–156CrossRefGoogle Scholar
  17. Cribb PJ, Kell SP, Dixon KW, Barrett RL (2003) Orchid conservation: a global perspective. In: Dixon KW, Kell SP, Barrett RL, Cribb PJ (eds) Orchid conservation. Natural History Publications, Kota Kinabalu, pp 1–24Google Scholar
  18. Dearnaley JDW (2007) Further advances in orchid mycorrhizal research. Mycorrhiza 17:475–486PubMedCrossRefGoogle Scholar
  19. Dearnaley JDW, Martos F, Selosse MA (2012) Orchid Mycorrhizas: molecular ecology, physiology, evolution and conservation aspects. In: Hock B (ed) Fungal associations. Springer, Berlin, pp 207–230CrossRefGoogle Scholar
  20. Doyle JJ, Doyle JL (1987) A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochem Bull 19:11–15 obtained from http://irc.igd.cornell.edu/Protocols/DoyleProtocol.pdf. Accessed 23 January 2011Google Scholar
  21. Dressler RL (1993) Phylogeny and classification of the orchid family. Cambridge University Press, New YorkGoogle Scholar
  22. Duan CF, Li ZL, Fang F, Yang GH, Chen SS, Hong QY (2010) Isolation and identification research of mycorrhizal isolated from several orchids in Yunnan Province. Xi Nan Nong Ye Xue Bao 23:756–759, in ChineseGoogle Scholar
  23. Eriksson O, Kainulainen K (2011) The evolutionary ecology of dust seeds. Perspect Plant Ecol Evol Syst 13:73–87CrossRefGoogle Scholar
  24. Fu LK (1992) China plant red data book: rare and endangered plants. I. Science Press, Beijing (in Chinese)Google Scholar
  25. Gao JY, Liu Q, Yu DL (2014) Orchids in Xishuangbanna: diversity and conservation. China Forestry Publishing House, Beijing (in Chinese)Google Scholar
  26. Godo T, Komori M, Nakaoki E, Yukawa T, Miyoshi K (2010) Germination of mature seeds of Calanthe tricarinata Lindl., an endangered terrestrial orchid, by asymbiotic culture in vitro. In Vitro Cell Dev Biol Plant 46:323–328CrossRefGoogle Scholar
  27. Hágsater E, Dumont V (1996) Conservation threats. In: Prindgeon AM (ed) Orchids: status, survey, and conservation action plan. IUCN, Gland, pp 6–9Google Scholar
  28. Harley JL (1950) Recent progress in the study of endotrophic mycorrhiza. New Phytol 49:213–247CrossRefGoogle Scholar
  29. Harvais G (1973) Growth requirements and development of Cypripedium reginae in axenic culture. Can J Bot 51(2):327–332CrossRefGoogle Scholar
  30. Herre EA, Mejía LC, Kyllo DA, Rojas E, Maynard Z, Butler A, Van Bael SA (2007) Ecological implications of anti-pathogen effects of tropical fungal endophytes and mycorrhizae. Ecology 88:550–558PubMedCrossRefGoogle Scholar
  31. Hollick PS (2004) Mycorrhizal specificity in endemic Western Australian terrestrial orchids (tribe Diurideae): implications for conservation. Doctoral dissertation, Murdoch UniversityGoogle Scholar
  32. Hou XQ, Guo SX (2009) Interaction between a dark septate endophytic isolate from Dendrobium sp. and roots of D. nobile seedlings. J Integr Plant Biol 51:374–381PubMedCrossRefGoogle Scholar
  33. Huang L, He XR, Zheng LM, Cai J (2004) Preliminary studies on mycorrhizal fungi in promoting the growth of orchid seedlings from tissue culture. Re Dai Zuo Wu Xue Bao 25:36–38 (in Chinese)Google Scholar
  34. Johnson TR, Stewart SL, Dutra D, Kane ME, Richardson L (2007) Asymbiotic and symbiotic seed germination of Eulophia alta (Orchidaceae)—preliminary evidence for the symbiotic culture advantage. Plant Cell Tissue Organ Cult 90:313–323CrossRefGoogle Scholar
  35. Kartzinel TR, Trapnell DW, Shefferson RP (2013) Highly diverse and spatially heterogeneous mycorrhizal symbiosis in a rare epiphyte is unrelated to broad biogeographic or environmental features. Mol Ecol. doi:10.1111/mec.12536 Google Scholar
  36. Kauth PJ, Vendrame WA, Kane ME (2006) In vitro seed culture and seedling development of Calopogon tuberosus. Plant Cell Tissue Organ Cult 85:91–102CrossRefGoogle Scholar
  37. Keel BG, Zettler LW, Kaplin BA (2011) Seed germination of Habenaria repens (Orchidaceae) in situ beyond its range, and its potential for assisted migration imposed by climate change. Castanea 76:43–54CrossRefGoogle Scholar
  38. Knudson L (1922) Nonsymbiotic germination of orchid seeds. Bot Gaz 73:1–25CrossRefGoogle Scholar
  39. Koopowitz H, Hawkins BA (2012) Global climate change is confounding species conservation strategies. Integr Zool 7:158–164PubMedCrossRefGoogle Scholar
  40. Li H, Ma Y, Liu W, Liu W (2009) Clearance and fragmentation of tropical rain forest in Xishuangbanna, SW, China. Biodivers Conserv 18:3421–3440CrossRefGoogle Scholar
  41. Liu H, Luo Y, Liu H (2010) Studies of mycorrhizal fungi of Chinese orchids and their role in orchid conservation in China-a review. Bot Rev 76:241–262CrossRefGoogle Scholar
  42. Massey EE, Zettler LW (2007) An expanded role for in vitro symbiotic seed germination as a conservation tool: two case studies in North America (Platanthera leucophaea and Epidendrum nocturnum). Lankesteriana 7:303–308Google Scholar
  43. Masuhara G, Katsuya K (1994) In situ and in vitro specificity between Rhizoctonia spp. and Spiranthes sinensis (Persoon) Ames, var. amoena (M. Bieberstein) Hara (Orchidaceae). New Phytol 127:711–718CrossRefGoogle Scholar
  44. McCormick MK, Whigham DF, Sloan D, O'Malley K, Hodkinson B (2006) Orchid-fungus fidelity: a marriage meant to last? Ecology 87:903–911PubMedCrossRefGoogle Scholar
  45. McCormick MK, Lee Taylor D, Juhaszova K, Brunett RK, Whigham DF, O'Neill JP (2012) Limitations on orchid recruitment: not a simple picture. Mol Ecol 21:1511–1523PubMedCrossRefGoogle Scholar
  46. Moore RT (1987) The genera of Rhizoctonia-like fungi: Ascorhizoctonia, Ceratorhiza gen. nov., Epulorhiza gen. nov., Moniliopsis, and Rhizoctonia. Mycotaxon 29:91–99Google Scholar
  47. 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 20:459–471PubMedCrossRefGoogle Scholar
  48. 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
  49. Park EJ, Lee WY (2013) In vitro symbiotic germination of myco-heterotrophic Gastrodia elata by Mycena species. Plant Biotechnol Rep 7:185–191CrossRefGoogle Scholar
  50. Perkins AJ, McGee PA (1995) Distribution of the orchid mycorrhizal fungus, Rhizoctonia solani, in relation to its host, Pterostylis acuminata, in the field. Aust J Bot 43:565–575CrossRefGoogle Scholar
  51. Phillips RD, Barrett MD, Dixon KW, Hopper SD (2011) Do mycorrhizal symbioses cause rarity in orchids? J Ecol 99:585–869CrossRefGoogle Scholar
  52. R Development Core Team (2011) R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. http://www.R-project.org/. Accessed 30 June 2013
  53. Rasmussen HN (2002) Recent developments in the study of orchid mycorrhiza. Plant Soil 244:149–163CrossRefGoogle Scholar
  54. Rasmussen HN, Rasmussen FPN (1991) Climatic and seasonal regulation of seed plant establishment in Dactylorhiza majalis inferred from symbiotic experiments in vitro. Lindleyana 6:221–227Google Scholar
  55. Rasmussen HN, Rasmussen FN (2009) Orchid mycorrhiza: implications of a mycophagous life style. Oikos 118:334–345CrossRefGoogle Scholar
  56. Rasmussen HN, Whigham DF (1993) Seed ecology of dust seeds in situ: a new study technique and its application in terrestrial orchids. Am J Bot 80:1374–1378CrossRefGoogle Scholar
  57. Rasmussen HN, Whigham DF (1998a) Importance of woody debris in seed germination of Tipularia discolor (Orchidaceae). Am J Bot 85:829–834PubMedCrossRefGoogle Scholar
  58. Rasmussen HN, Whigham DF (1998b) The underground phase: a special challenge in studies of terrestrial orchid populations. Bot J Linn Soc 126:49–64CrossRefGoogle Scholar
  59. Romand-Monnier F (2013) Dendrobium aphyllum. IUCN 2013. IUCN red list of threatened species. Version 2013.1. http://www.iucnredlist.org/details/15053780/0. Accessed 19 November 2013.
  60. Sanchez MS, Bills GF, Zabalgogeazcoa I (2008) Diversity and structure of the fungal endophytic assemblages from two sympatric coastal grasses. Fungal Divers 33:87–100Google Scholar
  61. Seaton P, Kendon JP, Pritchard HW, Puspitaningtyas DM, Marks TR (2013) Orchid conservation: the next ten years. Lankesteriana 13:93–101Google Scholar
  62. Selosse MA, Boullard B, Richardson D (2011) Noël Bernard (1874–1911): orchids to symbiosis in a dozen years, one century ago. Symbiosis 54:61–68CrossRefGoogle Scholar
  63. Sheng CL, Lee YI, Gao JY (2012) Ex situ symbiotic seed germination, isolation and identification of effective symbiotic fungus in Cymbidium mannii (Orchidaceae). Zhi Wu Sheng Tai Xue Bao 36:859–869 (in Chinese)Google Scholar
  64. Shimura H, Sadamoto M, Matsuura M, Kawahara T, Naito S, Koda Y (2009) Characterization of mycorrhizal fungi isolated from the threatened Cypripedium macranthos in a northern island of Japan: two phylogenetically distinct fungi associated with the orchid. Mycorrhiza 19:525–534PubMedCrossRefGoogle Scholar
  65. Sileshi GW (2012) A critique of current trends in the statistical analysis of seed germination and viability data. Seed Sci Res 22:145–159CrossRefGoogle Scholar
  66. Stewart SL, Kane ME (2006) Symbiotic seed germination of Habenaria macroceratitis (Orchidaceae), a rare Florida terrestrial orchid. Plant Cell Tissue Organ Cult 86:159–167CrossRefGoogle Scholar
  67. Stewart SL, Zettler LW (2002) Symbiotic germination of three semi-aquatic rein orchids Habenaria repens, H. quinquiseta, H. macroceratitis from Florida. Aquat Bot 72:25–35CrossRefGoogle Scholar
  68. Subedi A, Kunwar B, Gravendeel B et al (2013) Collection and trade of wild-harvested orchids in Nepal. J Ethnobiol Ethnomed 9:64PubMedCentralPubMedCrossRefGoogle Scholar
  69. Swarts ND, Dixon KW (2009a) Terrestrial orchid conservation in the age of extinction. Ann Bot 104:543–556PubMedCentralPubMedCrossRefGoogle Scholar
  70. Swarts ND, Dixon KW (2009b) Perspectives on orchid conservation in botanic gardens. Trends Plant Sci 14:590–598PubMedCrossRefGoogle Scholar
  71. Swarts ND, Sinclair EA, Francis A, Dixon KW (2010) Ecological specialization in mycorrhizal symbiosis leads to rarity in an endangered orchid. Mol Ecol 19:3226–3242PubMedCrossRefGoogle Scholar
  72. Takahashi K, Ogiwara I, Hakoda N (2000) Seed germination of Habenaria (pecteilis) radiata (Orchidaceae: Orchideae) in vitro. Lindleyana 15:59–63Google Scholar
  73. Wang H, Zhu H, Li BG (1997) Vegetation on limestone in Xishuangbanna Southwest China. Guangxi Zhi Wu 17:101–117 (in Chinese)Google Scholar
  74. Wang D, Jia SH, Zhang ZX, Cai YP, Lin Y (2007) Isolation and culture of an endophytic fungus associated with Dendrobium huoshanense and its effects on the growth of plantlets. J Fungal Res 5:84–88 (in Chinese)Google Scholar
  75. Wang H, Fang H, Wang Y, Duan L, Guo S (2011) In situ seed baiting techniques in Dendrobium officinale Kimuraet Migo and Dendrobium nobile Lindl.: the endangered Chinese endemic Dendrobium (Orchidaceae). World J Microbiol Biotechnol 27:2051–2059CrossRefGoogle Scholar
  76. Warcup JH, Talbot PHB (1967) Perfect states of Rhizoctonias associated with orchids. New Phytol 66:631–641CrossRefGoogle Scholar
  77. Warcup JH, Talbot PHB (1971) Perfect states of rhizoctonias associated with orchids. II. New Phytol 70:35–40CrossRefGoogle Scholar
  78. Warcup JH, Talbot PHB (1980) Perfect states of rhizoctonias associated with orchids. III. New Phytol 86:267–272CrossRefGoogle Scholar
  79. White TJ, Bruns T, 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, New York, pp 315–322CrossRefGoogle Scholar
  80. Xing X, Ma X, Deng Z, Chen J, Wu F, Guo S (2012) Specificity and preference of mycorrhizal associations in two species of the genus Dendrobium (Orchidaceae). Mycorrhiza 23:317–324. doi:10.1007/s00572-012-0473-8 PubMedCrossRefGoogle Scholar
  81. Yoder JA, Zettler LW, Stewart SL (2000) Water requirements of terrestrial and epiphytic orchid seeds and seedlings, and evidence for water uptake by means of mycotrophy. Plant Sci 156:145–150PubMedCrossRefGoogle Scholar
  82. Yonzone R, Lama D, Bhujel RB, Rai S, Kendra DKV, Viswavidyalaya UBK, Kalimpong PO (2011) Epiphytic orchid species diversity of Darjeeling Himalaya of West Bengal, India. Asian J Pharm Life Sci ISSN 2231:4423Google Scholar
  83. Yuan ZL, Chen YC, Yang Y (2009) Diverse non-mycorrhizal fungal endophytes inhabiting an epiphytic, medicinal orchid (Dendrobium nobile): estimation and characterization. World J Microbiol Biotechnol 25:295–303CrossRefGoogle Scholar
  84. Zettler LW, Hofer CJ (1998) Propagation of the little club-spur orchid Platanthera clavellata by symbiotic seed germination and its ecological implications. Environ Exp Bot 39:189–195CrossRefGoogle Scholar
  85. Zettler LW, McInnis JTM (1994) Light enhancement of symbiotic seed germination and development of an endangered terrestrial orchid, Platanthera integrilabia. Plant Sci 102:133–138CrossRefGoogle Scholar
  86. Zettler LW, Piskin KA (2011) Mycorrhizal fungi from protocorms, seedlings, and mature plants of the eastern prairie fringed orchid, Platanthera leucophaea: a comprehensive list to augment conservation. Am Midl Nat 166:29–39CrossRefGoogle Scholar
  87. Zhang J, Cao M (1995) Tropical forest vegetation of Xishuangbanna, SW China and its secondary changes, with special reference to some problems in local nature conservation. Biol Conserv 73:229–238CrossRefGoogle Scholar
  88. Zhu JM, Zhao YM, Bai J, Li YT, Zhang KM, Chen JW, Wu JB, Wang HZ (2011) Preliminary studies on the antagonism of Dendrobium mycorrhizal fungi (Trichoderma persoon). J Hangzhou Normal Univ (Nat Sci Ed) 10:340–344 (in Chinese)Google Scholar
  89. Ziegler AD, Fox JM, Xu J (2009) The rubber juggernaut. Science 324(5930):1024–1025PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2014

Authors and Affiliations

  • Xiao-Meng Zi
    • 1
    • 2
  • Chun-Ling Sheng
    • 1
    • 2
    • 4
  • Uromi Manage Goodale
    • 1
  • Shi-Cheng Shao
    • 3
  • Jiang-Yun Gao
    • 1
    • 3
  1. 1.Key Laboratory of Tropical Forest Ecology, Xishuangbanna Tropical Botanical GardenChinese Academy of SciencesMenglaChina
  2. 2.University of Chinese Academy of SciencesBeijingChina
  3. 3.Center for Integrative Conservation, Xishuangbanna Tropical Botanical GardenChinese Academy of SciencesMenglaChina
  4. 4.China Forestry Publishing HouseBeijingChina

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