, Volume 28, Issue 1, pp 17–28 | Cite as

Localization of helotialean fungi on ectomycorrhizae of Castanopsis cuspidata visualized by in situ hybridization

  • Noritaka Nakamura
  • Eiji Tanaka
  • Chihiro Tanaka
  • Yuko Takeuchi-Kaneko
Original Article


Non-ectomycorrhizal fungi that associate with typical ectomycorrhizae often remain hidden, and their localization inside ectomycorrhizal (ECM) roots has remained uncharacterized. In this study, the fungal community associated with the ectomycorrhizae of Castanopsis cuspidata was investigated using a culture-dependent isolation technique. Additionally, the species composition and localization were determined using molecular techniques. The results of the isolation and identification of fungal species revealed the predominance of a few species belonging to the order Helotiales. Furthermore, the fungal community structures were significantly different depending on the taxa of the ectomycorrhiza-forming fungi. A taxon-specific probe was developed to analyze the localization of one dominant Hyaloscyphaceae (Helotiales) species in ECM tissues by in situ hybridization. Hybridization signals were detected on the surface of the fungal mantle and around the ECM fungal cells within the mantle. Hyphal penetration into ECM hyphal cells of fungal mantles was also observed. Signals were not detected in the Hartig net or plant tissues inside the mantle in healthy ectomycorrhizae. These findings suggest that the analyzed species interact not only with host plant as root endophyte but also directly with the ECM fungi.


Fagaceae Fungal community Fungal root endophytes Helotiales Mycoparasite PCR-RFLP 



This work was financially supported by Japan Society for the Promotion of Science KAKENHI Grant Number 24380081.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.


  1. 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 9:1639–1649. CrossRefPubMedGoogle Scholar
  2. Bergero R, Girlanda M, Bello F, Luppi A, Perotto S (2003) Soil persistence and biodiversity of ericoid mycorrhizal fungi in the absence of the host plant in a Mediterranean ecosystem. Mycorrhiza 13:69–75. CrossRefPubMedGoogle Scholar
  3. Bödeker IT, Nygren CM, Taylor AF, Olson Å, Lindahl BD (2009) Class II peroxidase-encoding genes are present in a phylogenetically wide range of ectomycorrhizal fungi. ISME J 3:1387–1395. CrossRefPubMedGoogle Scholar
  4. Bödeker I, Clemmensen KE, Boer W, Martin F, Olson Å, Lindahl BD (2014) Ectomycorrhizal Cortinarius species participate in enzymatic oxidation of humus in northern forest ecosystems. New Phytol 203:245–256. CrossRefPubMedGoogle Scholar
  5. Gardes M, Bruns TD (1993) ITS primers with enhanced specificity for basidiomycetes-application to the identification of mycorrhizae and rusts. Mol Ecol 2:113–118. CrossRefPubMedGoogle Scholar
  6. Grelet GA, Johnson D, Paterson E, Anderson IC, Alexander IJ (2009) Reciprocal carbon and nitrogen transfer between an ericaceous dwarf shrub and fungi isolated from Piceirhiza bicolorata ectomycorrhizas. New Phytol 182:359–366. CrossRefPubMedGoogle Scholar
  7. Grünig CR, Duò A, Sieber TN, Holdenrieder O (2008) Assignment of species rank to six reproductively isolated cryptic species of the Phialocephala fortinii s.l.-Acephala applanata species complex. Mycologia 100:47–67. CrossRefPubMedGoogle Scholar
  8. Hashimoto Y, Hyakumachi M (2001) Effects of isolates of ectomycorrhizal fungi and endophytic Mycelium radicis atrovirens that were dominant in soil from disturbed sites on growth of Betula platyphylla var. japonica seedlings. Ecol Res 16:117–125. CrossRefGoogle Scholar
  9. Heinonsalo J, Buée M, Vaario LM (2016) Root-endophytic fungi cause morphological and functional differences in Scots pine roots in contrast to ectomycorrhizal fungi. Botany 95:203–210. CrossRefGoogle Scholar
  10. Hobbie EA, Ouimette AP, Schuur EA, Kierstead D, Trappe JM, Bendiksen K, Ohenoja E (2013) Radiocarbon evidence for the mining of organic nitrogen from soil by mycorrhizal fungi. Biogeochemistry 114:381–389. Google Scholar
  11. Hosoya T (2002) Hyaloscyphaceae in Japan (6)**: the genus Hyphodiscus in Japan and its anamorph Catenulifera gen. nov. Mycoscience 43:47–57. CrossRefGoogle Scholar
  12. Hosoya T (2013) Enumeration of remarkable Japanese discomycetes (3): first records of three inoperculate helotialean discomycetes in Japan. Bull Natl Mus Nat Sci 35:113–121Google Scholar
  13. Huang J, Nara K, Zong K, Wang J, Xue S, Peng K, Shen Z, Lian C (2014) Ectomycorrhizal fungal communities associated with Masson pine (Pinus massoniana) and white oak (Quercus fabri) in a manganese mining region in Hunan Province, China. Fungal Ecol 9:1–10. CrossRefGoogle Scholar
  14. Huhtinen S, Hawksworth DL, Ihlen PG (2008) Observations on two glassy-haired lichenicolous discomycetes. Lichenologist 40:549–557. CrossRefGoogle Scholar
  15. Izumitsu K, Hatoh K, Sumita T, Kitade Y, Morita A, Gafur A, Ohta A, Kawai M, Yamanaka T, Neda H, Ota Y, Tanaka C (2012) Rapid and simple preparation of mushroom DNA directly from colonies and fruiting bodies for PCR. Mycoscience 53:396–401. CrossRefGoogle Scholar
  16. Kernaghan G, Patriquin G (2011) Host associations between fungal root endophytes and boreal trees. Microb Ecol 62:460–473. CrossRefPubMedGoogle Scholar
  17. Kernaghan G, Patriquin G (2015) Diversity and host preference of fungi co-inhabiting Cenococcum mycorrhizae. Fungal Ecol 17:84–95. CrossRefGoogle Scholar
  18. Kernaghan G, Sigler L, Khasa D (2003) Mycorrhizal and root endophytic fungi of containerized Picea glauca seedlings assessed by rDNA sequence analysis. Microbial Ecol 45:128–136. CrossRefGoogle Scholar
  19. Kluber LA, Tinnesand KM, Caldwell BA, Dunham SM, Yarwood RR, Bottomley PJ, Myrold DD (2010) Ectomycorrhizal mats alter forest soil biogeochemistry. Soil Biol Biochem 42:1607–1613. CrossRefGoogle Scholar
  20. Kluber LA, Smith JE, Myrold DD (2011) Distinctive fungal and bacterial communities are associated with mats formed by ectomycorrhizal fungi. Soil Biol Biochem 43:1042–1050. CrossRefGoogle Scholar
  21. Komon-Zelazowska M, Bissett J, Zafari D, Hatvani L, Manczinger L, Woo S, Lorito M, Kredics L, Kubicek CP, Druzhinina IS (2007) Genetically closely related but phenotypically divergent Trichoderma species cause green mold disease in oyster mushroom farms worldwide. Appl Environ Microb 73:7415–7426. CrossRefGoogle Scholar
  22. Leski T, Pietras M, Rudawska M (2010) Ectomycorrhizal fungal communities of pedunculate and sessile oak seedlings from bare-root forest nurseries. Mycorrhiza 20:179–190. CrossRefPubMedGoogle Scholar
  23. Mandyam K, Jumpponen A (2008) Seasonal and temporal dynamics of arbuscular mycorrhizal and dark septate endophytic fungi in a tallgrass prairie ecosystem are minimally affected by nitrogen enrichment. Mycorrhiza 18:145–155. CrossRefPubMedGoogle Scholar
  24. Martin KJ, Rygiewicz PT (2005) Fungal-specific PCR primers developed for analysis of the ITS region of environmental DNA extracts. BMC Microbiol 5:28. CrossRefPubMedPubMedCentralGoogle Scholar
  25. Menkis A, Vasiliauskas R, Taylor AF, Stenlid J, Finlay R (2005) Fungal communities in mycorrhizal roots of conifer seedlings in forest nurseries under different cultivation systems, assessed by morphotyping, direct sequencing and mycelial isolation. Mycorrhiza 16:33–41. CrossRefPubMedGoogle Scholar
  26. Olsson PA, Münzenberger B, Mahmood S, Erland S (2000) Molecular and anatomical evidence for a three-way association between Pinus sylvestris and the ectomycorrhizal fungi Suillus bovinus and Gomphidius roseus. Mycol Res 104:1372–1378. CrossRefGoogle Scholar
  27. Pöder R, Scheuer C (1994) Moserella radicicola gen. et sp. nov., a new hypogeous species of Leotiales on ectomycorrhizas of Picea abies. Mycol Res 98:1334–1338. CrossRefGoogle Scholar
  28. Rice AV, Currah RS (2002) New perspectives on the niche and holomorph of the myxotrichoid hyphomycete, Oidiodendron maius. Mycol Res 106:1463–1467. CrossRefGoogle Scholar
  29. Rosling A, Landeweert R, Lindahl BD, Larsson KH, Kuyper TW, Taylor AFS, Finlay RD (2003) Vertical distribution of ectomycorrhizal fungal taxa in a podzol soil profile. New Phytol 159:775–783. CrossRefGoogle Scholar
  30. Rubner A (1996) Revision of predacious hyphomycetes in the Dactylella-Monacrosporium complex. Stud Mycol 39:1–134Google Scholar
  31. Summerbell RC (1987) The inhibitory effect of Trichoderma species and other soil microfungi on formation of mycorrhiza by Laccaria bicolor in vitro. New Phytol 105:437–448. CrossRefGoogle Scholar
  32. Tagawa H (1995) Distribution of lucidophyll oak-laurel forest formation in Asia and other areas. Tropics 5(1/2):1–40. CrossRefGoogle Scholar
  33. Tanaka E (2009) Specific in situ visualization of the pathogenic endophytic fungus Aciculosporium take, the cause of witches’ broom in bamboo. Appl Environ Microb 75:4829–4834. CrossRefGoogle Scholar
  34. Tanaka E, Kumagawa T, Ito N, Nakanishi A, Ohta Y, Suzuki E, Adachi N, Hamada A, Ashizawa T, Ohara T, Tsuda M (2016) Colonization of the vegetative stage of rice plants by the false smut fungus Villosiclava virens, as revealed by a combination of species-specific detection methods. Plant Pathol.
  35. Tedersoo L, Jairus T, Horton BM, Abarenkov K, Suvi T, Saar I, Kõljalg U (2008) Strong host preference of ectomycorrhizal fungi in a Tasmanian wet sclerophyll forest as revealed by DNA barcoding and taxon-specific primers. New Phytol 180:479–490. CrossRefPubMedGoogle Scholar
  36. Tedersoo L, Pärtel K, Jairus T, Gates G, Põldmaa K, Tamm H (2009) Ascomycetes associated with ectomycorrhizas: molecular diversity and ecology with particular reference to the Helotiales. Environ Microbiol 11:3166–3178. CrossRefPubMedGoogle Scholar
  37. Toju H, Yamamoto S, Sato H, Tanabe AS (2013a) Sharing of diverse mycorrhizal and root-endophytic fungi among plant species in an oak-dominated cool-temperate forest. PLoS One 8:e78248. CrossRefPubMedPubMedCentralGoogle Scholar
  38. Toju H, Yamamoto S, Sato H, Tanabe AS, Gilbert GS, Kadowaki K (2013b) Community composition of root-associated fungi in a Quercus-dominated temperate forest: “codominance” of mycorrhizal and root-endophytic fungi. Ecol Evol 3:1281–1293. CrossRefPubMedPubMedCentralGoogle Scholar
  39. Toju H, Sato H, Tanabe AS (2014) Diversity and spatial structure of belowground plant-fungal symbiosis in a mixed subtropical forest of ectomycorrhizal and arbuscular mycorrhizal plants. PLoS One 9:e86566. CrossRefPubMedPubMedCentralGoogle Scholar
  40. Urban A, Puschenreiter M, Strauss J, Gorfer M (2008) Diversity and structure of ectomycorrhizal and co-associated fungal communities in a serpentine soil. Mycorrhiza 18:339–354. CrossRefPubMedGoogle Scholar
  41. Vilela R, Mendoza L, Rosa PS, Belone AFF, Madeira S, Opromolla DVA, de Resende MA (2005) Molecular model for studying the uncultivated fungal pathogen Lacazia loboi. J Clin Microbiol 43(8):3657–3661. CrossRefPubMedPubMedCentralGoogle Scholar
  42. Villarreal-Ruiz L, Anderson IC, Alexander IJ (2004) Interaction between an isolate from the Hymenoscyphus ericae aggregate and roots of Pinus and Vaccinium. New Phytol 164:183–192. CrossRefGoogle Scholar
  43. 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 6:281–292. CrossRefGoogle Scholar
  44. Vrålstad T, Fossheim T, Schumacher T (2000) Piceirhiza bicolorata—the ectomycorrhizal expression of the Hymenoscyphus ericae aggregate? New Phytol 145:549–563. CrossRefGoogle Scholar
  45. Vrålstad T, Schumacher T, Taylor AF (2002a) Mycorrhizal synthesis between fungal strains of the Hymenoscyphus ericae aggregate and potential ectomycorrhizal and ericoid hosts. New Phytol 153:143–152. CrossRefGoogle Scholar
  46. Vrålstad T, Myhre E, Schumacher T (2002b) Molecular diversity and phylogenetic affinities of symbiotic root-associated ascomycetes of the Helotiales in burnt and metal polluted habitats. New Phytol 155:131–148. CrossRefGoogle Scholar
  47. Wang Z, Binder M, Schoch CL, Johnston PR, Spatafora JW, Hibbett DS (2006) Evolution of helotialean fungi (Leotiomycetes, Pezizomycotina): a nuclear rDNA phylogeny. Mol Phylogenet Evol 41:295–312. CrossRefPubMedGoogle Scholar
  48. Wang Q, Gao C, Guo LD (2011) Ectomycorrhizae associated with Castanopsis fargesii (Fagaceae) in a subtropical forest, China. Mycol Prog 10:323–332. CrossRefGoogle Scholar
  49. White TJ, Bruns T, Lee SJWT, Taylor JL (1990) Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. PCR protocol 18(1):315–322Google Scholar
  50. Yamada A, Katsuya K (1995) Mycorrhizal association of isolates from sporocarps and ectomycorrhizas with Pinus densiflora seedlings. Mycoscience 36:315–323. CrossRefGoogle Scholar
  51. Yamamoto S, Sato H, Tanabe AS, Hidaka A, Kadowaki K, Toju H (2014) Spatial segregation and aggregation of ectomycorrhizal and root-endophytic fungi in the seedlings of two Quercus species. PLoS One 9:e96363. CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany 2017

Authors and Affiliations

  1. 1.Laboratory of Terrestrial Microbial Ecology, Division of Environmental Science and Technology, Graduate School of AgricultureKyoto UniversityKyotoJapan
  2. 2.Department of Environmental Science and EngineeringIshikawa Prefectural UniversityNonoichiJapan

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