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Mycorrhiza

, Volume 24, Issue 8, pp 635–644 | Cite as

Culturable fungal endophytes in roots of Enkianthus campanulatus (Ericaceae)

  • Keisuke Obase
  • Yosuke Matsuda
Original Paper

Abstract

Roots of plants in the genus Enkianthus, which belongs to the earliest diverging lineage in the Ericaceae, are commonly colonized by arbuscular mycorrhizal (AM) fungi. We documented the community of fungal root endophytes associated with Enkianthus species using a culture-based method for better understanding the members of root-colonizing fungi, except for AM fungi. Fungal isolates were successfully obtained from 610 out of 3,599 (16.9 %) root segments. Molecular analysis of fungal cultures based on ribosomal internal transcribed spacer (ITS) sequences yielded 63 operational taxonomical units (OTUs: 97 % sequence similarity cutoff) from 315 representative isolates. Further phylogenetic analysis showed that most (296 isolates) belonged to Ascomycota and were either members of Helotiales (Dermataceae, Hyaloscyphaceae, Phialocephala and Rhizoscyphus ericae aggregate), Oidiodendron, or other Pezizomycotina. Twenty-three out of 63 OTUs, which mainly consisted of Leotiomycetes, showed high similarities with reference sequences derived from roots of other ericaceous plants such as Rhododendron. The results indicated that Enkianthus houses variable root mycobionts including putative endophytic and mycorrhizal fungi in addition to AM fungi.

Keywords

Endophyte Ericaceous plant Evolution Molecular identification Mycorrhiza 

Notes

Acknowledgments

This study was carried out partly with the financial support of Grants-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan to YM (22688011, 23658124, and 25304026). We are grateful for sampling supports of Kyoto Botanical Gardens (Kyoto City, Kyoto Prefecture), Mie Prefecture Forestry Research Institute (Tsu City, Mie Prefecture), Mr. Keizo Kanazawa (Tsu City, Mie Prefecture), Dr. Hirosuke Oba (Nikko Botanical Gardens, Graduate School of Science, The University of Tokyo), and Ohmi-Fuji Karyoku Park (Yasu City, Shiga Prefecture), and for sequence analyses of Ms. Tomiko Chikada (Life Science Research Center, Center for Molecular Biology and Genetics, Mie University). We specially thank Dr. Shin-ichiro Ito (Graduate School of Bioresources, Mie University) and the members of the Laboratory of Forest Pathology and Mycology, Mie University, for their assistance and Dr. Matthew E Smith for critical comments and editing the manuscript.

Supplementary material

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Table S2 Numbers of isolates successfully obtained from each tree (DOCX 17 kb)

References

  1. Abe JP (2005) An arbuscular mycorrhizal genus in the Ericaceae. Inoculum 56:6Google Scholar
  2. Allen TR, Millar T, Berch SM, Berbee ML (2003) Culturing and direct DNA extraction find different fungi from the same ericoid mycorrhizal roots. New Phytol 160:255–272CrossRefGoogle Scholar
  3. 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 Res 25:3389–3402PubMedCrossRefPubMedCentralGoogle Scholar
  4. Berch SM, Allen TR, Berbee ML (2002) Molecular detection, community structure and phylogeny of ericoid mycorrhizal fungi. Plant Soil 244:55–66CrossRefGoogle Scholar
  5. Bergero R, Perotto S, Girlanda M, Vidano G, Luppi AM (2000) Ericoid mycorrhizal fungi are common root associates of a Mediterranean ectomycorrhizal plant (Quercus ilex). Mol Ecol 9:1639–1649PubMedCrossRefGoogle Scholar
  6. Bougoure DS, Cairney JWG (2005a) Assemblages of ericoid mycorrhizal and other root associated fungi from Epacris pulchella (Ericaceae) as determined by culturing and direct DNA extraction from roots. Environ Microbiol 7:819–827PubMedCrossRefGoogle Scholar
  7. Bougoure DS, Cairney JWG (2005b) Fungi associated with hair roots of Rhododendron lochiae (Ericaceae) in an Australian tropical cloud forest revealed by culturing and culture-independent molecular methods. Environ Microbiol 7:1743–1754PubMedCrossRefGoogle Scholar
  8. Bougoure DS, Parkin PI, Cairney JWG, Alexander IJ, Anderson IC (2007) Diversity of fungi in hair roots of Ericaceae varies along a vegetation gradient. Mol Ecol 16:4624–4636PubMedCrossRefGoogle Scholar
  9. Chambers SM, Curlevski NJA, Cairney JWG (2008) Ericoid mycorrhizal fungi are common root inhabitants of non-Ericaceae plants in a south-eastern Australian sclerophyll forest. FEMS Microbiol Ecol 65:263–270PubMedCrossRefGoogle Scholar
  10. Couture M, Fortin JA, Dalpe Y (1983) Oidiodendron griseum Robak: an endophyte of ericoid mycorrhiza in Vaccinium spp. New Phytol 95:375–380CrossRefGoogle Scholar
  11. Curlevski NJA, Chambers SM, Anderson IC, Cairney JWG (2009) Identical genotypes of an ericoid mycorrhiza-forming fungus occur in roots of Epacris pulchella (Ericaceae) and Leptospermum polygalifolium (Myrtaceae) in an Australian sclerophyll forest. FEMS Microbiol Ecol 67:411–420PubMedCrossRefGoogle Scholar
  12. Felsenstein J (1985) Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39:783–791CrossRefGoogle Scholar
  13. Fukuchi S, Obase K, Tamai Y, Yajima T, Miyamoto T (2011) Vegetation and colonization status of mycorrhizal and endophytic fungi in plant species on acidic barren at crater basin of volcano Esan in Hokkaido, Japan. Eurasian J For Res 14:1–11Google Scholar
  14. Gallaud I (1905) Études sur les mycorrhizes endotrophes. Rev Gen Bot 17:5–48, 66–83, 123–135, 223–239, 313–325, 425–433, 479–500Google Scholar
  15. 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
  16. Gorman NR, Starrett MC (2003) Host range of a select isolate of the ericoid mycorrhizal fungus Hymenoschyphus ericae. HortSci 38:1163–1166Google Scholar
  17. Gorzelak MA, Hambleton S, Massicotte HB (2012) Community structure of ericoid mycorrhizas and root-associated fungi of Vaccinium membranaceum across an elevation gradient in the Canadian Rocky Mountains. Fungal Ecol 5:36–45CrossRefGoogle Scholar
  18. Hammer Ø, Harper DAT, Ryan PD (2001) PAST: Paleontological statistics software package for education and data analysis. Palaeontol Electron 4:1–9Google Scholar
  19. Hynson NA, Bruns TD (2009) Evidence of a myco-heterotroph in the plant family Ericaceae that lacks mycorrhizal specificity. Proc R Soc B 276:4053–4059PubMedCrossRefPubMedCentralGoogle Scholar
  20. Ishida TA, Nordin A (2010) No evidence that nitrogen enrichment affect fungal communities of Vaccinium roots in two contrasting boreal forest types. Soil Biol Biochem 42:234–243Google Scholar
  21. Jumpponen A, Trappe JM (1998) Dark septate endophytes: a review of facultative biotrophic root-colonizing fungi. New Phytol 140:295–310CrossRefGoogle Scholar
  22. Jumpponen A, Mattson KG, Trappe JM (1998) Mycorrhizal functioning of Phialocephala fortinii with Pinus contorta on glacier forefront soil: interactions with soil nitrogen and organic matter. Mycorrhiza 7:261–265PubMedCrossRefGoogle Scholar
  23. Katenin AE (1964) Mycorrhiza of arctic plants. Proble Sev 8:148–154 [paper in Russian]Google Scholar
  24. Kennedy PG, Smith DP, Horton TR, Molina RJ (2012) Arbutus menziesii (Ericaceae) facilitates regeneration dynamics in mixed evergreen forests by promoting mycorrhizal fungal diversity and host connectivity. Am J Bot 99:1691–1701PubMedCrossRefGoogle Scholar
  25. 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 250:167–175CrossRefGoogle Scholar
  26. Kron KA, Judd WS, Stevens PF, Crayn DM, Anderberg AA, Gadek PA, Quinn CJ, Luteyn JL (2002) Phylogenetic classification of Ericaceae: molecular and morphological evidence. Bot Rev 68:335–423CrossRefGoogle Scholar
  27. Mandyam K, Jumpponen A (2005) Seeking the elusive function of the root-colonising dark septate endophytic fungi. Stud Mycol 53:173–189CrossRefGoogle Scholar
  28. Matsuda Y, Okochi S, Katayama T, Yamada A, Ito S (2011) Mycorrhizal fungi associated with Monotropastrum humile (Ericaceae) in central Japan. Mycorrhiza 21:569–576PubMedCrossRefGoogle Scholar
  29. Matsuda Y, Shimizu S, Mori M, Ito S, Selosse MA (2012) Seasonal and environmental changes of mycorrhizal associations and heterotrophy levels in mixotrophic Pyrola japonica growing under different light environments. Am J Bot 99:1177–1188PubMedCrossRefGoogle Scholar
  30. Moyersoen B (2006) Pakaraimaea dipterocarpacea is ectomycorrhizal, indicating an ancient Gondwanaland origin for the ectomycorrhizal habit in Dipterocarpaceae. New Phytol 172:753–762PubMedCrossRefGoogle Scholar
  31. Obase K, Lee JK, Lee SK, Lee SY, Chun KW (2010) Variation in sodium chloride resistance of Cenococcum geophilum and Suillus granulatus isolates in liquid culture. Mycobiology 38:225–228PubMedCrossRefPubMedCentralGoogle Scholar
  32. Obase K, Matsuda Y, Ito S (2013) Enkianthus campanulatus (Ericaceae) is commonly associated with arbuscular mycorrhizal fungi. Mycorrhiza 23:199–208PubMedCrossRefGoogle Scholar
  33. Oberwinkler F, Riess K, Bauer R, Selosse MA, Weiß M, Garnica S, Zuccaro A (2013) Enigmatic Sebacinales. Mycol Prog 12:1–27CrossRefGoogle Scholar
  34. Raup DM, Crick RE (1979) Measurement of faunal similarity in paleontology. J Paleontol 53:1213–1227Google Scholar
  35. Redman RS, Sheehan KB, Stout RG, Rodriguez RJ, Henson JM (2002) Thermotolerance generated by plant/fungal symbiosis. Science 298:1581PubMedCrossRefGoogle Scholar
  36. 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 166:1011–1023PubMedCrossRefGoogle Scholar
  37. Rillig MC, Wendt S, Antonovics J, Hempel S, Kohler J, Wehner J, Caruso T (2014) Interactive effects of root endophytes and arbuscular mycorrhizal fungi on an experimental plant community. Oecologia 174:263–270PubMedCrossRefGoogle Scholar
  38. Schulz B, Boyle C, Draeger S, Römmert AK, Krohn K (2002) Endophytic fungi: a source of novel biologically active secondary metabolites. Mycol Res 106:996–1004CrossRefGoogle Scholar
  39. Schulz BJE, Boyle CJC, Sieber TN (2006) Microbial root endophytes. Vol. 9. SpringerGoogle Scholar
  40. Schoch CL et al (2012) Nuclear ribosomal internal transcribed spacer (ITS) region as a universal DNA barcode marker for fungi. Proc Natl Acad Sci 109:6241–6246PubMedCrossRefPubMedCentralGoogle Scholar
  41. Selosse MA, Setaro S, Glatard F, Richard F, Urcelay C, Weiß M (2007) Sebacinales are common mycorrhizal associates of Ericaceae. New Phytol 174:864–878PubMedCrossRefGoogle Scholar
  42. Schüßler A, Gehrig H, Schwarzott D, Walker C (2001) Analysis of partial Glomales SSU rRNA gene sequences: implications for primer design and phylogeny. Mycol Res 105:5–15CrossRefGoogle Scholar
  43. Sharples JM, Chambers SM, Meharg AA, Cairney JWG (2000) Genetic diversity of root-associated fungal endophytes from Calluna vulgaris at contrasting field sites. New Phytol 148:153–162CrossRefGoogle Scholar
  44. Shen M, Zhang CQ, Ma YP, Welti S, Moreau PA, Selosse MA (2012) Mycorrhizal features and fungal partners of four mycoheterotrophic Monotropoideae (Ericaceae) species from Yunnan, China. Symbiosis 57:1–13CrossRefGoogle Scholar
  45. Smith FA, Smith SE (1997) Tansley Review No. 96 Structural diversity in (vesicular)–arbuscular mycorrhizal symbioses. New Phytol 137:373–388CrossRefGoogle Scholar
  46. Smith ME, Douhan GW, Rizzo DM (2007) Intra-specific and intra-sporocarp ITS variation of ectomycorrhizal fungi as assessed by rDNA sequencing of sporocarps and pooled ectomycorrhizal roots from a Quercus woodland. Mycorrhiza 18:15–22PubMedCrossRefGoogle Scholar
  47. Stevens PF (2014) Angiosperm Phylogeny Website. Version 12, July 2012 [and more or less continuously updated since]." will do. http://www.mobot.org/MOBOT/research/APweb/. Accessed Jan 2014
  48. Sun L, Pei K, Wang F, Ding Q, Bing Y, Gao B, Zheng Y, Liang Y, Ma K (2012) Different distribution patterns between putative ercoid mycorrhizal and other fungal assemblages in roots of Rhododendron decorum in the Southwest of China. PLoS One 7:e49867Google Scholar
  49. Tamura K, Nei M, Kumar S (2004) Prospects for inferring very large phylogenies by using the neighbor-joining method. Proc Natl Acad Sci 101:11030–11035PubMedCrossRefPubMedCentralGoogle Scholar
  50. Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S (2011) MEGA5: Molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28:2731–2739PubMedCrossRefPubMedCentralGoogle Scholar
  51. Tian W, Zhang CQ, Qiao P, Milne R (2011) Diversity of culturable ericoid mycorrhizal fungi of Rhododendron decorum in Yunnan, China. Mycologia 103:703–709Google Scholar
  52. Toftegaard T, Iason GR, Alexander IJ, Rosendahl S, Taylor AFS (2010) The threatened plant intermediate wintergreen (Pyrola media) associates with a wide range of biotrophic fungi in native Scottish pine woods. Biodivers Conserv 19:3963–3971Google Scholar
  53. Upson R, Read DJ, Newsham KK (2007) Widespread association between the ericoid mycorrhizal fungus Rhizoscyphus ericae and a leafy liverwort in the maritime and sub-Antarctic. New Phytol 176:460–471Google Scholar
  54. Usuki F, Abe JP, Kakishima M (2003) Diversity of ericoid mycorrhizal fungi isolated from hair roots of Rhododendron obtusum var. kaempferi in a Japanese red pine forest. Mycoscience 44:97–102Google Scholar
  55. Vandenkoornhuyse P, Baldauf SL, Leyval C, Straczek J, Young JPW (2002) Extensive fungal diversity in plant roots. Science 15:2051CrossRefGoogle Scholar
  56. Villarreal-Ruiz L, Neri-Luna C, Anderson IC, Alexander IJ (2012) In vitro interactions between ectomycorrhizal fungi and ericaceous plants. Symbiosis 56:67–75CrossRefGoogle Scholar
  57. Vohník M, Sadowsky JJ, Kohout P, Lhotáková Z, Nestby R, Kolařík M (2012) Novel root-fungus symbiosis in Ericaceae: sheathed ericoid mycorrhiza formed by a hitherto undescribed Basidiomycete with affinities to Trechisporales. PLoS One 7:e39524PubMedCrossRefPubMedCentralGoogle Scholar
  58. Vohník M, Mrnka L, Lukešová T, Bruzone MC, Kohout P, Fehrer J (2013) The cultivable endophytic community of Norway spruce ectomycorrhizas from microhabitats lacking ericaceous hosts is dominated by ericoid mycorrhizal Meliniomyces variabilis. Fungal Ecol 6:281–292Google Scholar
  59. Vrålstad T, Myhre E, Schumacher T (2002) Molecular diversity and phylogenetic affinities of symbiotic root-associated ascomycetes of the Helotiales in burnt and metal polluted habitats. New Phytol 155:131–148CrossRefGoogle Scholar
  60. Walker JF, Aldrich-Wolfe L, Riffel A, Barbare H, Simpson NB, Trowbridge J, Jumpponen A (2011) Diverse Helotiales associated with the roots of three species of Arctic Ericaceae provide no evidence for host specificity. New Phytol 191:515–527PubMedCrossRefGoogle Scholar
  61. Wang B, Qiu YL (2006) Phylogenetic distribution and evolution of mycorrhizas in land plants. Mycorrhiza 16:299–363PubMedCrossRefGoogle Scholar
  62. White TJ, Bruns TD, Lee S, Taylor J (1990) Analysis of phylogenetic relationships by amplification and direct sequencing of ribosomal RNA genes. 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
  63. Yang S, Pfister DH (2006) Monotropa uniflora plants of eastern Massachusetts form mycorrhizae with a diversity of russulacean fungi. Mycologia 98:535–540Google Scholar
  64. Young BW, Massicotte HB, Tackaberry LE, Baldwin QF, Egger KN (2002) Monotropa uniflora: morphological and molecular assessment of mycorrhizae retrieved from sites in the Sub-Boreal Spruce biogeoclimatic zone in central British Columbia. Mycorrhiza 12:75–82Google Scholar
  65. Yukawa T, Ogura-Tsujita Y, Shefferson R, Yokoyama J (2009) Mycorrhizal diversity in Apostasia (Orchidaceae) indicates the origin and evolution of orchid mycorrhiza. Am J of Bot 96:1997–2009Google Scholar
  66. Zhang C, Yin L, Dai S (2009) Diversity of root-associated fungal endophytes in Rhododendron fortunei in subtropical forests of China. Mycorrhiza 19:417–423Google Scholar
  67. Zhuang X, Chan W (1997) Investigation of plant mycorrhizae in secondary forests of Hong Kong. Chin Biodivers 5:287–292Google Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2014

Authors and Affiliations

  1. 1.Department of Plant PathologyUniversity of FloridaGainesvilleUSA
  2. 2.Laboratory of Forest Pathology and Mycology, Graduate School of BioresourcesMie UniversityTsuJapan

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