Ectomycorrhization of Tricholoma Matsutake with Abies Veitchii and Tsuga Diversifolia in the Subalpine Forests of Japan
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mycoscience 56 (2015) 402e412 Available online at www.sciencedirect.com journal homepage: www.elsevier.com/locate/myc Full paper Ectomycorrhization of Tricholoma matsutake with Abies veitchii and Tsuga diversifolia in the subalpine forests of Japan Naoki Endo a, Preeyaporn Dokmai b,c, Nuttika Suwannasai b, Cherdchai Phosri d, Yuka Horimai c, Nobuhiro Hirai e, Masaki Fukuda a,c, * Akiyoshi Yamada a,c,f, a Department of Bioscience and Food Production Science, Interdisciplinary Graduate School of Science and Technology, Shinshu University, 8304, Minami-minowa, Nagano, 399-4598, Japan b Faculty of Science, Srinakharinwirot University, 114, Sukhumvit 23, Bangkok, 10110, Thailand c Department of Bioscience and Biotechnology, Faculty of Agriculture, Shinshu University, 8304, Minami-minowa, Nagano, 399-4598, Japan d Division of Science, Faculty of Liberal Arts and Science, Nakhon Phanom University, 167 Naratchakouy Sub- District, Muang District, Nakhon Phanom, 48000, Thailand e Graduate School of Agriculture, Kyoto University, Kitashirakawa-Oiwake-cho, Sakyo-ku, Kyoto, 606-8502, Japan f Division of Terrestrial Ecosystem, Institute of Mountain Science, Shinshu University, 8304, Minami-minowa, Nagano, 399-4598, Japan article info abstract Article history: Tricholoma matsutake is one of the most highly valued ectomycorrhizal mushrooms, pri- Received 15 July 2014 marily associated with conifers. Here, we examined the association of T. matsutake with Received in revised form hemlock and fir species native to the subalpine forests of Japan. Basidiomata of T. matsu- 12 December 2014 take were harvested from the forests of Honshu Island, Japan, along with two soil samples Accepted 15 December 2014 directly beneath the basidiomata; ectomycorrhizal root tips were also collected, and used Available online 28 January 2015 for fungal isolation and molecular analyses. Mycorrhizal fungi were isolated from the roots of Abies veitchii (Veitch's fir) and Tsuga diversifolia (northern Japanese hemlock). These fungi Keywords: were identified as T. matsutake based upon their production of whitish mycelial colonies on Ectomycorrhizal morphology nutrient agar media, and confirmed using molecular analyses; the ectomycorrhizal asso- Fir ectomycorrhiza ciation between T. matsutake and A. veitchii was also confirmed by mycorrhizal synthesis Matsutake mushroom in vitro. This work represents the first description of T. matsutake ectomycorrhizations with Mycorrhizal isolation Abies and Tsuga species, as well as a detailed morphological description of these Nuclear ribosomal RNA gene (rDNA) associations. IGS1 region © 2014 The Mycological Society of Japan. Published by Elsevier B.V. All rights reserved. * Corresponding author. Department of Bioscience and Biotechnology, Faculty of Agriculture, Shinshu University, 8304, Minami-minowa, Nagano, 399-4598, Japan. Tel.: þ81 265 77 1631; fax: þ81 265 77 1629. E-mail address: [email protected] (A. Yamada). http://dx.doi.org/10.1016/j.myc.2014.12.004 1340-3540/© 2014 The Mycological Society of Japan. Published by Elsevier B.V. All rights reserved. mycoscience 56 (2015) 402e412 403 2008; Yamanaka et al. 2011), P. yunnanensis Franch., P. densata 1. Introduction Masters, P. wallichiana A.B. Jacks., P. armandii Franch., Casta- nopsis orthacantha Franch., Q. aquifolioides Rehder & E.H. Wil- & Tricholoma matsutake (S. Ito S. Imai) Singer [synonym: T. son, Q. pannosa Hand.-Mazz., Q. guyavifolia H. Lev., Q. € “ nauseosum (A. Blytt) Kytov.], commonly known as matsu- semecarpifolia Sm., Lithocarpus spp., and Pasania spp. in ” take , is one of the most prized species of ectomycorrhizal southwestern China and Bhutan (Matsushita et al. 2005; mushrooms. These fungi are commonly found throughout Yamanaka et al. 2011), and P. taiwanensis Hayata in Taiwan Japan and China, along with parts of central and northern (Kobayasi 1939; Ogawa 1978; Zhou and Zhang 2005). Europe (Imazeki and Hongo 1987; Bergius and Danell 2000; Other conifers found within T. matsutake-endemic regions, Matsushita et al. 2005; Murata et al. 2008; Yamada et al. including P. parviflora var. pentaphylla (Mayr) A. Henry, P. kor- & 2010). The fungus belongs to section Caligata Konrad aiensis Siebold & Zucc., P. jezoensis var. hondoensis (Mayr) Maubl. ex Bon (Bon 1991), which includes other matsutake Rehder, A. mariesii Mast., A. homolepis Siebold & Zucc., A. firma mushroom species such as North American T. magnivelare Siebold & Zucc., Larix kaempferi (Lamb.) Carriere and Pseu- & (Peck) Redhead, Mediterranean T. anatolicum H.H. Dogan dotsuga japonica (Shiras.) Beissn., are either negative for T. Intini, European T. caligatum (Viv.) Ricken, and Mexican matsutake growth, or have yet to be confirmed in their natural unidentified Tricholoma species (Ota et al. 2012; Murata et al. environments (Hamada 1964; Ogawa 1978; Shindo 2009; 2013b). More distantly related species, such as the Asian T. Yamada et al. 2014). Of the symbiotic associations that have bakamatsutake Hongo and T. fulvocastaneum Hongo, and the been identified, only two include detailed descriptions of the € European T. dulciolens Kytov. also are occasionally grouped mycorrhizal structures in nature: P. densiflora (Masui 1927; with the matsutake mushrooms (Ota et al. 2012; Yamada et al. 1999) and P. pumila (Ogawa 1976a). Five others, Christensen and Helmann-Clausen 2013; Murata et al. P. glehnii (Ogawa 1976b), T. diversifolia (Ogawa 1977a), T. sieboldii 2013a). (Ogawa 1977b), A. sachalinensis (Murata and Minamide 1989), Annual sales of matsutake mushrooms are estimated at and Q. semecarpifolia (Yamanaka et al. 2011) have also been ¥ 30 billion yen in Japan alone (Suzuki 2005; Yamada 2005), described, however it remains unclear whether these conifer though the long-term viability of T. matsutake cultivation in plants possess true ectomycorrhizal structures, due to a lack Japan remains uncertain. Numerous attempts have been of sophisticated anatomical characteristics; i.e., Hartig net made to improve T. matsutake production in the Pinus densi- hyphae. & flora Siebold Zucc. forests, however the success of these Examinations of ectomycorrhization of T. matsutake with efforts has been limited due to extensive damage to forests various conifers in vitro suggest a potential gradient of intra- caused by the pine wilt disease, particularly those in the cellular colonization between plants at the roots cortex western part of Japan, which have been severely affected in (Yamada et al. 2014). While certain species, including P. den- recent decades (Kishi 1995; Suzuki 2004). Annual production siflora, P. parviflora, P. koraiensis, P. sylvestris, P. glehnii, P. abies, of matsutake in Japan peaked in 1941 at 12,000 tons (Ogawa and T. diversifolia, exhibited clear ectomycorrhizal structures, e 1978), but has since plummeted to as low as 50 100 tons in other conifers, such as P. thunbergii, P. jezoensis, L. kaempferi, recent years (Ministry of Agriculture, Fishery, and Forestry, and A. veitchii exhibited more equivocal mycorrhizal struc- Japan). As a result of these shortfalls, over 95% of matsutake tures; i.e., discontinuous Hartig net development; direct mushrooms sold in Japan must now be imported from observation of the root systems of these plants in their natural abroad. environment will be necessary to establish definitive matsu- The host specificity of ectomycorrhizal fungi is an takeeplant associations. important part of forest ecosystems, which can be used as a In this study, we focused on two subalpine conifers, A. method of managing forest resources (Marx et al. 2002; Smith veitchii and T. diversifolia, to determine their potential for ecto- and Read 2008). Targeted cultivation of edible ectomycor- mycorrhizal association with T. matsutake. In the subalpine rhizal fungi requires a thorough understanding of host forests of Honshu Island, Japan, T. diversifolia is well-known specificity and other environmental conditions (Hosford et al. among commercial pickers of wild edible mushrooms as a 1997; Pilz and Molina 2002; Pilz et al. 2002). After identifying a suitable host of T. matsutake (Hamada 1953; Ogawa 1978), suitable pairing, fungi can then be used to inoculate mycor- though the details of this association remain largely unchar- rhizal seedlings, allowing for the co-cultivation of trees and acterized, with little knowledge regarding the environmental fungi (Cairney and Chambers 1999; Hall et al. 2003; Guerin- conditions necessary to support ectomycorrhization. In Laguette et al. 2014). Natural hosts of T. matsutake in Japan contrast, A. veitchii is one of the most abundant conifers found include P. densiflora, P. pumila (Pall.) Regel, P. parviflora Siebold on Honshu Island, but is not generally regarded as a host of T. & Zucc., P. thunbergii Parl., Picea glehnii Mast., P. jezoensis matsutake. Although T. matsutake fruiting in pure A. sachalinensis Carriere, Tsuga sieboldii Carriere, T. diversifolia (Maxim.) Mast., forests has been described on Hokkaido Island, Japan (Murata Abies sachalinensis Mast. and A. veitchii Lindl. (Kobayasi 1939; and Minamide 1989; Murata et al. 2001), fir species are rarely Kawamura 1955; Imazeki and Hongo 1957, 1987; Hamada found to support T. matsutake growth anywhere on the Eurasian 1964; Ogawa 1978; Murata and Minamide 1989). Other hosts continent. It is uncertain whether this lack of an association is include P. sylvestris and P. abies in Scandinavia and the Alps indicative of a poor growth environment, or is simply limited by (Bergius and Danell 2000; Matsushita et al. 2005; Vaario et al. the