Abstract
Tricholoma matsutake is an ectomycorrhizal basidiomycete that produces prized, yet uncultivable, “matsutake” mushrooms along densely developed mycelia, called “shiro,” in the rhizosphere of coniferous forests. Pinus densiflora is a major host of this fungus in Japan. Measuring T. matsutake biomass in soil allows us to determine the kinetics of fungal growth before and after fruiting, which is useful for analyzing the conditions of the shiro and its surrounding mycorrhizosphere, predicting fruiting timing, and managing forests to obtain better crop yields. Here, we document a novel method to quantify T. matsutake mycelia in soil by quantifying a single-copy DNA element that is uniquely conserved within T. matsutake but is absent from other fungal species, including close relatives and a wide range of ectomycorrhizal associates of P. densiflora. The targeted DNA region was amplified quantitatively in cultured mycelia that were mixed with other fungal species and soil, as well as in an in vitro co-culture system with P. densiflora seedlings. Using this method, we quantified T. matsutake mycelia not only from shiro in natural environments but also from the surrounding soil in which T. matsutake mycelia could not be observed by visual examination or distinguished by other means. It was demonstrated that the core of the shiro and its underlying area in the B horizon are predominantly composed of fungal mycelia. The fungal mass in the A or A0 horizon was much lower, although many white mycelia were observed at the A horizon. Additionally, the rhizospheric fungal biomass peaked during the fruiting season.
Similar content being viewed by others
References
Águeda B, Zambonelli A, Molina R (2014) Tuber 2013: scientific advances in sustainable truffle culture. Mycorrhiza 24:S1–S4
Amend A, Keeley S, Garbelotto M (2009) Forest age correlates with fine-scale spatial structure of Matsutake mycorrhizas. Mycol Res 113:541–551
Amend A, Fang Z, Yi C, McClatchey WC (2010) Local perceptions of Matsutake mushroom management, in NW Yunnan China. Biol Conserv 143:165–172
Babasaki K, Masuno K, Murata H (2003) Interactions of heterologous mycelia colonized in the substrate govern fruit body production in the cultivated homobasidiomycete Pholiota nameko. Biosci Biotechnol Biochem 67:100–106
Babasaki K, Neda H, Murata H (2007) megB1, a novel macroevolutionary genomic marker of the fungal phylum Basidiomycota. Biosci Biotechnol Biochem 71:1927–1939
Bustin SA, Benes V, Garson JA, Hellemans J, Huggett J, Kubista M, Mueller R, Nolan T, Pfaffl MW, Shipley GL, Vandesompele J, Wittwer CT (2009) The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin Chem 55:611–622
De la Varga H, Águeda B, Martínez-Peña F, Parladé J, Pera J (2012) Quantification of extraradical soil mycelium and ectomycorrhizas of Boletus edulis in a Scots pine forest with variable sporocarp productivity. Mycorrhiza 22:59–68
De la Varga H, Águeda B, Águeda T, Martínez-Peña F, Parladé J, Pera J (2013) Seasonal dynamics of Boletus edulis and Lactarius deliciosus extraradical mycelium in pine forests of central Spain. Mycorrhiza 23:391–402
De Miguel AM, Águeda B, Sánchez S, Parladé J (2014) Ectomycorrhizal fungus diversity and community structure with natural and cultivated truffle hosts: applying lessons learned to future truffle culture. Mycorrhiza 24:S5–S18
Hamada M (1970) Diaries on Armillaria matsutake (5). Trans Mycol Soc Jpn 11:81–86
Hall IR, Wang Y, Amicucci A (2003) Cultivation of edible ectomycorrhizal mushrooms. Trends Biotechnol 21:433–438
Hall IR, Brown GT, Zambonelli A (2012) Taming the truffle: the history, lore, and science of the ultimate mushroom. Timber Press, London
Hortal S, Pera J, Parladé J (2008) Tracking mycorrhizas and extraradical mycelium of the edible fungus Lactarius deliciosus under field competition with Rhizopogon spp. Mycorrhiza 18:69–77
Kawai M, Ogawa M (1976) Studies on the artificial reproduction of Tricholoma matsutake (S. Ito et Imai) Sing. IV: studies on a seed culture and a trial for the cultivation on solid media. Trans Mycol Soc Jpn 17:499–505
Kennedy PG, Bergemann SE, Hortal S, Bruns TD (2007) Determining the outcome of field-based competition between two Rhizopogon species using real-time PCR. Mol Ecol 16:881–890
Kikuchi K, Matsushita N, Alexis GL, Ohta A, Suzuki K (2000) Detection of Tricholoma matsutake by specific ITS primers. Mycol Res 104:1427–1430
Kobayashi H, Watahiki T, Kuramochi M, Onose S, Yamada A (2007) Production of pine seedlings with the shiro-like structure of the matsutake mushroom (Tricholoma matsutake (S. Ito et Imai) Sing.) in a large culture bottle. Mushroom Sci Biotech 15:151–155
Kurth F, Zeitler K, Feldhahn L, Neu TR, Weber T, Kristufek V, Wubet T, Herrmann S, Buscot F, Tarkka MT (2013) Detection and quantification of a mycorrhization helper bacterium and a mycorrhizal fungus in plant-soil microcosms at different levels of complexity. BMC Microbiol 13:205
Landeweert R, Veenman C, Kuyper TW, Fritze H, Wernars K, Smit E (2003) Quantification of ectomycorrhizal mycelium in soil by real-time PCR compared to conventional quantification techniques. FEMS Microbiol Ecol 45:283–292
Lian C, Hogetsu T, Matsushita N, Guerin-Laguette A, Suzuki K, Yamada A (2003) Development of microsatellite markers from an ectomycorrhizal fungus, Tricholoma matsutake, by an ISSR-suppression-PCR method. Mycorrhiza 13:27–31
Lian C, Narimatsu M, Nara K, Hogetsu T (2006) Tricholoma matsutake in a natural Pinus densiflora forest: correspondence between above- and below-ground genets, association with multiple host trees and alteration of existing ectomycorrhizal communities. New Phytol 171:825–836
Lindahl BD, de Boer W, Finlay RD (2010) Disruption of root carbon transport into forest humus stimulates fungal opportunists at the expense of mycorrhizal fungi. ISME J 4:872–881
Marx DH (1969) The influence of ectotrophic mycorrhizal fungi on the resistance of pine roots to pathogenic infections. Phytopathology 59:153–163
Murata H, Yamada A, Babasaki K (1999) Identification of repetitive sequences containing motifs of retrotransposons in the ectomycorrhizal basidiomycete Tricholoma matsutake. Mycologia 91:766–775
Murata H, Yamada A (2000) marY1, a member of the gypsy group of long terminal repeat retroelements from the ectomycorrhizal basidiomycete Tricholoma matsutake. Appl Environ Microbiol 66:3642–3645
Murata H, Babasaki K, Yamada A (2005a) Highly polymorphic DNA markers to specify strains of the ectomycorrhizal basidiomycete Tricholoma matsutake based on σ marY1 , the long terminal repeat of gypsy-type retroelement marY1. Mycorrhiza 15:179–186
Murata H, Ohta A, Yamada A, Narimatsu M, Futamura N (2005b) Genetic mosaics in the massive persisting rhizosphere colony “shiro” of the ectomycorrhizal basidiomycete Tricholoma matsutake. Mycorrhiza 15:505–512
Murata H, Babasaki K (2005c) Intra- and inter-specific variations in the copy number of two types of retrotransposons from the ectomycorrhizal basidiomycete Tricholoma matsutake. Mycorrhiza 15:381–386
Murata H, Babasaki K, Saegusa T, Takemoto K, Yamada A, Ohta A (2008) Traceability of Asian matsutake, specialty mushrooms produced by the ectomycorrhizal basidiomycete Tricholoma matsutake, on the basis of retroelement-based DNA markers. Appl Environ Microbiol 74:2023–2031
Murata H, Ota Y, Yamaguchi M, Yamada A, Katahata S, Ohtsuka Y, Babasaki K, Neda H (2013) Mobile DNA distributions refine the phylogeny of “matsutake” mushrooms, Tricholoma sect Caligata. Mycorrhiza 23:447–461
Narimatsu M, Koiwa T, Masaki T, Sakamoto Y, Ohmori H, Tawaraya K (2015) Relationship between climate, expansion rate, and fruiting in fairy rings (‘shiro’) of an ectomycorrhizal fungus, Tricholoma matsutake, in a Pinus densiflora forest. Fungal Ecol 15:18–28
Ogawa M (1975) Microbial ecology of mycorrhizal fungus, Tricholoma matsutake Ito et Imai (Sing.) in pine forest I: fungal colony (‘shiro’) of Tricholoma matsutake. Bull Gov For Exp Sta 272:79–121
Ohta A (1997) Ability of ectomycorrhizal fungi to utilize starch and related substrates. Mycoscience 38:403–408
Ota Y, Yamanaka T, Murata H, Neda H, Ohta A, Kawai M, Yamada A, Konno M, Tanaka C (2012) Phylogenetic relationship and species delimitation of matsutake and allied species based on multilocus phylogeny and haplotype analyses. Mycologia 104:1369–1380
Parladé J, Hortal S, Pera J, Galipienso L (2007) Quantitative detection of Lactarius deliciosus extraradical soil mycelium by real-time PCR and its application in the study of fungal persistence and interspecific competition. J Biotechnol 128:14–23
Parladé J, De la Varga H, De Miguel AM, Sáez R, Pera J (2013) Quantification of extraradical mycelium of Tuber melanosporum in soils from truffle orchards in northern Spain. Mycorrhiza 23:99–106
Peabody RB, Peabody DC, Sicard KM (2000) A genetic mosaic in the fruiting stage of Armillaria gallica. Fungal Genet Biol 29:72–80
Raidl S, Bonfigli R, Agerer R (2005) Calibration of quantitative real-time TaqMan PCR by correlation with hyphal biomass and ITS copies in mycelia of Piloderma croceum. Plant Biol 7:713–717
Sawahata T, Narimatsu M (2006) The abundance of Collembola collected from ectomycorrhizal hyphal mats of Tricholoma matsutake. Eur J Soil Biol 42:S301–S304
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 28:2731–2739
Tellenbach C, Grünig CR, Sieber TN (2010) Suitability of quantitative real-time PCR to estimate the biomass of fungal root endophytes. Appl Environ Microbiol 76:5764–5772
Ugawa S, Yamaguchi M, Miura S, Kaneko S (2012) A method for obtaining the relationship between the amount of DNA and the fine root weight from mixtures of fine roots and soil particles. Soil Sci Plant Nutr 58:510–516
Vaario LM, Pennanen T, Sarjala T, Savonen EM, Heinonsalo J (2010) Ectomycorrhization of Tricholoma matsutake and two major conifers in Finland—an assessment of in vitro mycorrhiza formation. Mycorrhiza 20:511–518
van der Linde S, Alexander I, Anderson IC (2008) A PCR-based method for detecting the mycelia of stipitate hydnoid fungi in soil. J Microbiol Methods 75:40–46
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 Press, New York, pp. 315–322
Xu J, Cadorin M, Liang Y-J, Yang ZL (2010) DNA-based geographic typing of the gourmet mushroom Tricholoma matsutake traded in China. Mycoscience 51:248–251
Xu J, Sha T, Li Y-C, Zhao Z-W, Yang ZL (2008) Recombination and genetic differentiation among natural populations of the ectomycorrhizal mushroom Tricholoma matsutake from southwestern China. Mol Ecol 17:1238–1247
Yamada A, Katsuya K (1995) Mycorrhizal association of isolates from sporocarps and ectomycorrhizas with Pinus densiflora seedlings. Mycoscience 36:315–323
Yamada A, Maeda K, Kobayashi H, Murata H (2006) Ectomycorrhizal symbiosis in vitro between Tricholoma matsutake and Pinus densiflora seedlings that resembles naturally occurring ‘shiro’. Mycorrhiza 16:111–116
Yamada A, Kobayashi H, Ogura T, Fukuda M (2007) Sustainable fruit body formations of edible mycorrhizal Tricholoma species for three years in open pot culture with pine seedling hosts. Mycoscience 48:104–108
Yamada A, Kobayashi H, Murata H, Kalmiş E, Kalyoncu F, Fukuda M (2010) In vitro ectomycorrhizal specificity between the Asian red pine Pinus densiflora and Tricholoma matsutake and allied species from worldwide Pinaceae and Fagaceae forests. Mycorrhiza 20:333–339
Yamada A, Endo N, Murata H, Ohta A, Fukuda M (2014) Tricholoma matsutake Y1 strain associated with Pinus densiflora shows a gradient of in vitro ectomycorrhizal specificity with Pinaceae and oak hosts. Mycoscience 55:27–34
Yamaguchi M, Nakamura M, Takano M, Sekiya A (2009) Quantification of the mycelial mass of the white-rot fungus Pleurotus pulmonarius by real-time PCR. Bull FFPRI 8:133–141
Yang J, Ruegger PM, McKenry MV, Becker JO, Bomeman J (2012) Correlations between root-associated microorganisms and peach replant disease symptoms in a California soil. PLoS One 7:e46420
Acknowledgments
This study was conducted under a “Technology Development for Optimizing Forest Resource Utilization” grant from the Ministry of Agriculture, Forestry, and Fishery of Japan. We also acknowledge financial support from the Forestry and Forest Products Research Institute (FFPRI, Tsukuba, Japan) and the FFPRI Encouragement Model in Support of Researchers with Family Responsibilities.
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
Funding
This study was funded by the Ministry of Agriculture, Forestry, and Fishery of Japan. This study was also funded by the Forestry and Forest Products Research Institute (FFPRI, Tsukuba, Japan) and the FFPRI Encouragement Model in Support of Researchers with Family Responsibilities.
Conflict of interest
The authors declare that they have no conflict of interest.
Rights and permissions
About this article
Cite this article
Yamaguchi, M., Narimatsu, M., Fujita, T. et al. A qPCR assay that specifically quantifies Tricholoma matsutake biomass in natural soil. Mycorrhiza 26, 847–861 (2016). https://doi.org/10.1007/s00572-016-0718-z
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00572-016-0718-z