, Volume 26, Issue 4, pp 333–343 | Cite as

The ectomycorrhizal status of a tropical black bolete, Phlebopus portentosus, assessed using mycorrhizal synthesis and isotopic analysis

  • Jaturong Kumla
  • Erik A. Hobbie
  • Nakarin Suwannarach
  • Saisamorn LumyongEmail author
Original Article


Phlebopus portentosus is one of the most popular wild edible mushrooms in Thailand and can produce sporocarps in the culture without a host plant. However, it is still unclear whether Phlebopus portentosus is a saprotrophic, parasitic, or ectomycorrhizal (ECM) fungus. In this study, Phlebopus portentosus sporocarps were collected from northern Thailand and identified based on morphological and molecular characteristics. We combined mycorrhizal synthesis and stable isotopic analysis to investigate the trophic status of this fungus. In a greenhouse experiment, ECM-like structures were observed in Pinus kesiya at 1 year after inoculation with fungal mycelium, and the association of Phlebopus portentosus and other plant species showed superficial growth over the root surface. Fungus-colonized root tips were described morphologically and colonization confirmed by molecular methods. In stable isotope measurements, the δ13C and δ15N of natural samples of Phlebopus portentosus differed from saprotrophic fungi. Based on the isotopic patterns of Phlebopus portentosus and its ability to form ECM-like structures in greenhouse experiments, we conclude that Phlebopus portentosus could be an ECM fungus.


Edible bolete Mycorrhizal synthesis Stable isotope Trophic status 



This work was supported by grants from the Thailand Research Fund for The Royal Golden Jubilee Ph.D. Program (PHD/0309/2550) and Research Team Association Grant RTA5580007, and Chiang Mai University, Thailand, and grant IOS-0843366 to Erik Hobbie from the US National Science Foundation. We thank Andrew Wilson and Jesse Sadowsky for very useful comments on the manuscript.

Supplementary material

572_2015_672_MOESM1_ESM.doc (1.3 mb)
ESM 1 (DOC 1352 kb)


  1. Agerer R (1991) Characterization of ectomycorrhiza. In: Norris JR, Read DJ, Varma AK (eds) Methods in microbiology: techniques for the study of mycorrhiza. Academic, San Diego, pp 25–73CrossRefGoogle Scholar
  2. Agerer R (2006) Fungal relationships and structural identity of their ectomycorrhizae. Mycol Prog 5:67–107. doi: 10.1007/s11557-006-0505-x CrossRefGoogle Scholar
  3. Bahram M, Põlme S, Kõljalg U, Tedersoo L (2011) A single European aspen (Populus tremula) tree individual may potentially harbour dozens of Cenococcum geophilum ITS genotypes and hundreds of species of ectomycorrhizal fungi. FEMS Microbiol Ecol 75:313–320. doi: 10.1111/j.1574-6941.2010.01000.x CrossRefPubMedGoogle Scholar
  4. Binder M, Hibbett DS (2006) Molecular systematics and biological diversification of Boletales. Mycologia 98:971–981. doi: 10.3852/mycologia.98.6.971 CrossRefPubMedGoogle Scholar
  5. Brundrett M (2004) Diversity and classification of mycorrhizal associations. Biol Rev 79:479–495. doi: 10.1017/S1464793103006316 CrossRefGoogle Scholar
  6. Brundrett MC, Kendrick B (1987) The relationship between the ash bolete (Boletinellus merulioides) and an aphid parasite on ash tree roots. Symbiosis 3:315–319Google Scholar
  7. Brundrett M, Bougher N, Dell B, Grove T, Malajczuk N (1996) Working with mycorrhizas in forestry and agriculture. ACIAR Monograph, CanberaGoogle Scholar
  8. Burgess T, Dell B, Malajczuk N (1994) Variation in mycorrhizal development and growth stimulation by 20 Pisolithus isolates inoculated onto Eucalyptus grandis W. Hill ex Maiden. New Phytol 127:731–739. doi: 10.1111/j.1469-8137.1994.tb02977.x CrossRefGoogle Scholar
  9. Chung HC, Kim DH, Lee SS (2002) Mycorrhizal formations and seedling growth of Pinus densiflora by in vitro synthesis with the inoculation of ectomycorrhizal fungi. Mycobiology 30:70–75CrossRefGoogle Scholar
  10. Dell B (2002) Role of mycorrhizal fungi in ecosystems. CMU J Nat Sci 1:47–60Google Scholar
  11. Den BHC, Gravendeel B, Kuyper TW (2004) An ITS phylogeny of Leccinum and an analysis of the evolution of minisatellite-like sequences within ITS1. Mycologia 96:102–118. doi: 10.2307/3761992 CrossRefGoogle Scholar
  12. Felsenstein J (1985) Confidence intervals on phylogenetics: an approach using bootstrap. Evolution 39:783–791CrossRefGoogle Scholar
  13. Feugey L, Strullu DG, Poupard P, Simoneau P (1999) Induced defence responses limit Hartig net formation in ectomycorrhizal birch roots. New Phytol 144:541–547. doi: 10.1046/j.1469-8137.1999.00538.x CrossRefGoogle Scholar
  14. Gardes M, Bruns TD (1993) ITS primers with enhanced specificity for basidiomycetes-application to the identification of mycorrhizae and rusts. Mol Ecol 2:113–118. doi: 10.1111/j.1365-294X.1993.tb00005.x CrossRefPubMedGoogle Scholar
  15. Gebauer G, Taylor AFS (1999) 15N natural abundance in fruit bodies of different functional groups of fungi in relation to substrate utilization. New Phytol 142:93–101CrossRefGoogle Scholar
  16. Gleixner G, Danier HJ, Werner RA, Schmidt HL (1993) Correlations between the 13C content of primary and secondary plant products in different cell compartments and that in decomposing basidiomycetes. Plant Physiol 102:1287–1290PubMedPubMedCentralGoogle Scholar
  17. Harley JL, Smith SE (1983) Mycorrhizal symbiosis. Academic, LondonGoogle Scholar
  18. Hart SC, Gehring CA, Selmants PC, Deckert RJ (2006) Carbon and nitrogen elemental and isotopic pattern in macrofungal sporocarps and trees in semiarid forests of the south-western USA. Funct Ecol 20:42–51. doi: 10.1111/j.1365-2435.2005.01058.x CrossRefGoogle Scholar
  19. Hasselquist NJ, Douhan GW, Allen MF (2011) First report of ectomycorrhizal status of boletes on Northern Yucatan Peninsula, Mexico determined using isotopic method. Mycorrhiza 21:456–471. doi: 10.1007/s00572-010-0355-x CrossRefGoogle Scholar
  20. Heinemann P, Rammeloo J (1982) Observations sur le genre Phlebopus (Boletineae). Mycotaxon 15:384–404Google Scholar
  21. Henn MR, Chapela IH (2001) Ecophysiology of 13C and 15N isotope fractionation in forest fungi and the roots of the saprotrophic mycorrhizal divide. Oecologia 128:480–487CrossRefGoogle Scholar
  22. Hobbie EA (2005) Using isotopic tracers to follow carbon and nitrogen cycling of fungi. In: Dighton J, Oudemans P, White J (eds) The fungal community: its organization and role in the ecosystem. Marcel Dekker, pp 361–381Google Scholar
  23. Hobbie EA, Agerer R (2010) Nitrogen isotopes in ectomycorrhizal sporocarps correspond to belowground exploration types. Plant Soil 327:71–83. doi: 10.1007/s11104-009-0032-z CrossRefGoogle Scholar
  24. Hobbie EA, Diepen LT, Lilleskov EA, Ouimette AP, Finzi AC, Hofmockel KS (2014) Fungal functioning in a pine forest: evidence from a 15N-labeled global change experiment. New Phytol 201:1431–1439CrossRefPubMedGoogle Scholar
  25. Hobbie EA, Jumpponen A, Trappe J (2005) Foliar and fungal 15N:14N ratios reflect development of mycorrhizae and nitrogen supply during primary succession: testing analytical models. Oecologia 146:258–268. doi: 10.1007/s00442-005-0208-z CrossRefPubMedGoogle Scholar
  26. Hobbie EA, Macko SA, Shugart HH (1999) Insights into nitrogen and carbon dynamics of ectomycorrhizal and saprotrophic fungi from isotopic evidence. Oecologia 118:353–360CrossRefGoogle Scholar
  27. Hobbie EA, Wallander H (2006) Integrating ectomycorrhizal fungi into quantitative frameworks of forest carbon and nitrogen cycling. In: Gadd GM (ed) Fungi in biogeochemical cycles. Cambridge University Press, New York, pp 98–128CrossRefGoogle Scholar
  28. Hobbie EA, Weber NS, Trappe JM (2001) Mycorrhizal vs. saprotrophic status of fungi: the isotopic evidence. New Phytol 150:601–610. doi: 10.1046/j.1469-8137.2001.00134.x CrossRefGoogle Scholar
  29. Hou W, Lian B, Dong H, Jiang H, Wu X (2012) Distinguishing ectomycorrhizal and saprophytic fungi using carbon and nitrogen isotopic compositions. Geosci Front 3:351–356. doi: 10.1016/j.gsf.2011.12.005 CrossRefGoogle Scholar
  30. Ji KP, Cao Y, Zhang CX, He MX, Liu J, Wang WB, Wang Y (2011) Cultivation of Phlebopus portentosus in southern China. Mycol Prog 10:293–300. doi: 10.1007/s11557-010-0700-7 CrossRefGoogle Scholar
  31. Ji KP, He MX, Zhang CX, Liu J, Wang WB, Hou JY (2009) Semi-artificial simulate cultivation of Phlebopus portentosus and the durability of hyphae on host roots. Microbiology 36:377–382Google Scholar
  32. Kikuchi K, Matsushita N, Suzuki K (2009) Fruit body formation of Tylopilus castaneiceps in pure culture. Mycoscience 50:313–316. doi: 10.1007/s10267-009-0481-5 CrossRefGoogle Scholar
  33. Kirk PM, Cannon PF, Minter DW, Stalpers JA (2008) Dictionary of the fungi, 10th edn. CABI Europe, WallingfordGoogle Scholar
  34. Kohzu A, Yoshioka T, Ando T, Takahashi M, Koba K, Wada E (1999) Natural 13C and 15N abundance of field-collected fungi and their ecological implications. New Phytol 144:323–330. doi: 10.1046/j.1469-8137.1999.00508.x CrossRefGoogle Scholar
  35. Kumla J, Bussaban B, Suwannarach N, Lumyong S, Danell E (2012) Basidiome formation of an edible wild, putatively ectomycorrhizal fungus, Phlebopus portentosus without host plant. Mycologia 104:597–603. doi: 10.3852/11-074 CrossRefPubMedGoogle Scholar
  36. Kumla J, Danell E, Lumyong S (2015) Improvement of yield for a tropical black bolete, Phlebopus portentosus, cultivation in northern Thailand. Mycoscience 56:114–117. doi: 10.1016/j.myc.2014.04.005 CrossRefGoogle Scholar
  37. Langer I, Krpata D, Peintner U, Wenzel WW, Schweiger P (2008) Media formulation influences in vitro ectomycrrhizal synthesis on the European aspen Populus tremula L. Mycorrhiza 18:297–307. doi: 10.1007/s00572-008-0182-5 CrossRefPubMedGoogle Scholar
  38. Lei QY, Zhou JJ, Wang QB (2009) Notes on three bolete species from China. Mycosystema 28:56–59Google Scholar
  39. Lilleskov E, Hobbie E, Horton T (2011) Conservation of ectomycorrhizal fungi: exploring the linkages between functional and taxonomic responses to anthropogenic N deposition. Fungal Ecol 4:174–183. doi: 10.1016/j.funeco.2010.09.008 CrossRefGoogle Scholar
  40. Lindahl BD, Ihrmark K, Boberg J, Trumbore SE, Högberg P, Stenlid J, Finlay RD (2007) Spatial separation of litter decomposition and mycorrhizal nitrogen uptake in a boreal forest. New Phytol 173:611–620. doi: 10.1111/j.1469-8137.2006.01936.x CrossRefPubMedGoogle Scholar
  41. Lumyong S, Sanmee R, Lumyong P (2007) Is large scale cultivation of boletes possible? Opera Mycol 1:34–37Google Scholar
  42. Mayor JR, Schuur EAG, Henkel TW (2009) Elucidating the nutritional dynamics of fungi using stable isotopes. Ecol Lett 12:171–183. doi: 10.1111/j.1461-0248.2008.01265.x CrossRefPubMedGoogle Scholar
  43. McKenzie EHC, Buchanan PK, Johnston PR (2000) Checklist of fungi on Nothofagus species in New Zealand. NZ J Bot 38:635–720CrossRefGoogle Scholar
  44. Miller OK Jr, Lodge DJ, Baroni TJ (2000) New and interesting ectomycorrhizal fungi from Puerto Rico, Mona, and Guana Islands. Mycologia 92:558–570. doi: 10.2307/3761516 CrossRefGoogle Scholar
  45. Molina R, Massicotte H, Trappe JM (1992) Specificity phenomena in mycorrhizal symbioses: community ecological consequences and practical implications. In: Allen MF (ed) Mycorrhizal functioning: an integrated plant-fungal process. Chapman & Hall, New York, pp 357–423Google Scholar
  46. Mortimer PE, Karunarathna SC, Li Q, Gui H, Yang X, Yang X, He J, Ye L, Guo J, Li H, Sysouphanthong P, Zhou D, Xu J, Hyde KD (2012) Prized edible Asian mushrooms: ecology, conservation and sustainability. Fungal Divers 56:31–47. doi: 10.1007/s13225-012-0196-3 CrossRefGoogle Scholar
  47. Nouhra E, Urcelay C, Becerra A, Dominguez L (2008) Mycorrhizal status of Phlebopus bruchii (Boletaceae): does it form ectomycorrhizas with Fagara coco (Rutaceae)? Symbiosis 46:113–120Google Scholar
  48. Ohta A (1994a) Production of fruit-bodies of a mycorrhizal fungus, Lyophyllum shimeji, in pure culture. Mycoscience 35:147–151. doi: 10.1007/BF02318492 CrossRefGoogle Scholar
  49. Ohta A (1994b) Some cultural characteristics of mycelia of a mycorrhizal fungus, Lyophyllum shimeji. Mycoscience 35:83–87. doi: 10.1007/BF02268533 CrossRefGoogle Scholar
  50. Ohta A (1998) Fruit-body production of two ectomycorrhizal fungi in the genus Hebeloma in pure culture. Mycoscience 39:15–19. doi: 10.1007/BF02461573 CrossRefGoogle Scholar
  51. Ohta A, Fujiwara N (2003) Fruit-body production of an ectomycorrhizal fungus in genus Boletus in pure culture. Mycoscience 44:295–300. doi: 10.1007/s10267-003-0120-5 CrossRefGoogle Scholar
  52. Paolocci F, Rubini A, Granetti B, Arcioni S (1999) Rapid molecular approach for a reliable identification of Tuber spp. ectomycorrhizae. FEMS Microbiol Ecol 28:23–30. doi: 10.1016/S0168-6496(98)00088-9 CrossRefGoogle Scholar
  53. Pereira MF, Betancourth BML, Teixeira JA, Zubieta MP, Queiroz MV, Kauya MCM, Costa MD, Araújo EF (2014) In vitro Scleroderma laeve and Eucalyptus grandis mycorrhization and analysis of atp6, 17S rDNA, and ras gene expression during ectomycorrhizal formation. J Basic Microbiol 54:1358–1366. doi: 10.1002/jobm.201400253 CrossRefGoogle Scholar
  54. Peterson RL, Massicotte HB, Melville LH (2004) Mycorrhizas: anatomy and cell biology. National Research Council Research Press, OttawaGoogle Scholar
  55. Pham NDH, Yamada A, Shimizu K, Noda K, Dang LAT, Suzuki A (2012) A sheathing mycorrhiza between the tropical bolete Phlebopus spongiosus and Citrus maxima. Mycoscience 53:347–353. doi: 10.1007/s10267-011-0177-5 CrossRefGoogle Scholar
  56. Pruett GE, Bruhn JN, Mihail JD (2009) Greenhouse production of burgundy truffle mycorrhizae on oak roots. New For 37:43–52. doi: 10.1007/s11056-008-9108-5 CrossRefGoogle Scholar
  57. Rinaldi AC, Comandini O, Kuyper TW (2008) Ectomycorrhizal fungal diversity: separating the wheat from the chaff. Fungal Divers 33:1–45Google Scholar
  58. Repác I (2007) Ectomycorrhiza formation and growth of Picea abies seedlings inoculated with alginate-bead fungal inoculum in peat and bark compost substrates. Forestry 5:517–530. doi: 10.1093/forestry/cpm036 CrossRefGoogle Scholar
  59. Sanmee R, Dell B, Lumyong P, Izumori K, Lumyong S (2003) Nutritive value of popular wild edible mushrooms from northern Thailand. Food Chem 82:527–532. doi: 10.1016/S0308-8146(02)00595-2 CrossRefGoogle Scholar
  60. Sanmee R, Lumyong R, Dell B, Lumyong S (2010) In vitro cultivation and fruit body formation of the black bolete, Phlebopus portentosus, a popular edibl ectomycorrhizal fungus in Thailand. Mycoscience 51:15–22. doi: 10.1007/s10267-009-0010-6 CrossRefGoogle Scholar
  61. Singer R (1986) The Agaricales in modern taxonomy, 4th edn. Koeltz Scientific Books, KönigsteinGoogle Scholar
  62. Singer R, Araujo I, Ivory MH (1983) The ectotrophically mycorrhizal fungi of the neotropical lowlands, especially central Amazonia. Beih. Nova Hedwigia 77:1–352Google Scholar
  63. Stone GN, Schönrogge K (2003) The adaptive significance of insect gall morphology. Trends Ecol Evol 18:512–522. doi: 10.1016/S0169-5347(03)00247-7 CrossRefGoogle Scholar
  64. Seitzman BH, Ouimette A, Mixon RL, Hobbie EA, Hibbett DS (2011) Conservation of biotrophy in Hygrophoraceae inferred from combined satble isotope and phylogenetic analyses. Mycologia 103:280–290. doi: 10.3852/10-195 CrossRefPubMedGoogle Scholar
  65. Swofford DL (2002) PAUP*: Phylogenetic analysis using parsimony (*and other methods), beta version 4.0b10. Sinauer Associates, SunderlandGoogle Scholar
  66. Taylor AFS, Alexander I (2005) The ectomycorrhizal symbiosis: life in the real world. Mycologist 19:102–112. doi: 10.1017/S0269915X05003034 CrossRefGoogle Scholar
  67. Taylor AFS, Fransson PM, Högberg P, Högberg MN, Plamboeck AH (2003) Species level patterns in 13C and 15N abundance of ectomycorrhizal and saprotrophic fungal sporocarps. New Phytol 159:757–774. doi: 10.1046/j.1469-8137.2003.00838.x CrossRefGoogle Scholar
  68. Tedersoo L, May TW, Smith ME (2010) Ectomycorrhizal lifestyle in fungi: global diversity, distribution, and evolution of phylogenetic lineages. Mycorrhiza 20:217–263. doi: 10.1007/s00572-009-0274-x CrossRefPubMedGoogle Scholar
  69. Theodorou C, Reddell P (1991) In vitro synthesis of ectomycorrhizas on Casuarinaceae with a range of mycorrhizal fungi. New Phytol 118:279–288. doi: 10.1111/j.1469-8137.1991.tb00978.x CrossRefGoogle Scholar
  70. Thoen D, Ducouso M (1989) Mycorrhizal habit and sclerogenesis of Phlebopus sudanicus (Gyrodontaceae) in Senegal. Agric Ecosyst Environ 28:519–523. doi: 10.1016/0167-8809(90)90091-Q CrossRefGoogle Scholar
  71. Thomson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG (1997) The CLUSTAL X windows interface: flexible strategies formultiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 25:4876–4882CrossRefGoogle Scholar
  72. Thongklang N, Hyde KD, Bussaban B, Lumyong S (2010) Culture condition, inoculum production and host response of a wild mushroom, Phlebopus portentosus strain CMUHH121-005. Maejo Int J Sci Technol 5:413–425Google Scholar
  73. Vaario LM, Gill WM, Lapeyrie F, Matsushita N, Suzuki K (2000) Aseptic ectomycorrhizal synthesis between Abies firma and Cenococcum geophilum in artificial cuture. Mycoscience 41:395–399. doi: 10.1007/BF02463953 CrossRefGoogle Scholar
  74. Watling R (2001) The relationships and possible distributional patterns of boletes in south-east Asia. Mycol Res 105:1440–1448. doi: 10.1017/S0953756201004877 CrossRefGoogle Scholar
  75. Watling R (2006) The sclerodermatoid fungi. Mycoscience 47:18–24. doi: 10.1007/s10267-005-0267-3 CrossRefGoogle Scholar
  76. White TJ, Bruns TD, Lee S, Taylaor 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
  77. Williams DJ (2004) Mealybugs of southern asia. The Natural History Museum, London, and Southdene SDN, BHD, Kuala LumpurGoogle Scholar
  78. Wilson AW, Binder M, Hibbett DS (2011) Effects of gasteroid fruiting body morphology on diversification rates in three independent clades of fungi estimated using binary state speciation and extinction analysis. Evolution 65:1305–1322. doi: 10.1111/j.1558-5646.2010.01214.x CrossRefPubMedGoogle Scholar
  79. Wilson AW, Hobbie EA, Hibbett DS (2007) The ectomycorrhizal status of Calostoma cinnabarinum determined using isotopic, molecular, and morphological method. Can J Bot 85:385–393. doi: 10.1139/B07-026 CrossRefGoogle Scholar
  80. Yamanaka K, Namba K, Tajiri A (2000) Fruit body formation of Boletus reticulatus in pure culture. Mycoscience 41:189–191. doi: 10.1007/BF02464330 CrossRefGoogle Scholar
  81. Yamanaka T, Ota Y, Konno M, Kawai M, Ohta A, Neda H, Terashima Y, Yamada A (2014) The host range of conifer-associatied Tricholoma matsutake, Fagaceae-assoviated T. bakamatsutake and T. fulvocastaneum are wider in vitro than in nature. Mycologia 106:397–406. doi: 10.3852/13-197 CrossRefPubMedGoogle Scholar
  82. Zeller B, Brechet C, Maurice J-P, Le Tacon F (2007) 13C and 15N isotopic fractionation in trees, soils and fungi in a natural forest stand and a Norway spruce plantation. Ann For Sci 64:419–429. doi: 10.1051/forest:2007019 CrossRefGoogle Scholar
  83. Zhang CX, He MX, Cao Y, Liu J, Gao F, Wang WB, Ji KP, Shao SC, Wang Y (2015) Fungus-insect gall of Phlebopus portentosus. Mycologia 107:12–20. doi: 10.3852/13-267 CrossRefPubMedGoogle Scholar
  84. Zhang CX, Ji KP, He MX, Cao Y, Liu J, Wang WB (2010) Analysis on nutrient components of Phlebopus portentosus fruit bodies. J Yunnan Univ 32:702–704Google Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2015

Authors and Affiliations

  • Jaturong Kumla
    • 1
  • Erik A. Hobbie
    • 2
  • Nakarin Suwannarach
    • 1
  • Saisamorn Lumyong
    • 1
    Email author
  1. 1.Department of Biology, Faculty of ScienceChiang Mai UniversityChiang MaiThailand
  2. 2.Earth Systems Research Center, Morse HallUniversity of New HampshireDurhamUSA

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