In vitro shiro formation between the ectomycorrhizal basidiomycete Tricholoma matsutake and Cedrela herrerae in the Mahogany family (Meliaceae)

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In vitro shiro formation between the ectomycorrhizal basidiomycete Tricholoma matsutake and Cedrela herrerae in the Mahogany family (Meliaceae) Hitoshi Murata a,*, Akiyoshi Yamada b, Tsuyoshi Maruyama c, Naoki Endo b, Kohei Yamamoto b, Norio Hayakawa b, Hitoshi Neda a a

Department of Applied Microbiology and Mushroom Science, Forestry & Forest Products Research Institute, Matsunosato 1, Tsukuba 305-8687, Japan b Department of Bioscience and Biotechnology, Faculty of Agriculture, Shinshu University, Minami-minowa, Nagano 399-4598, Japan c Department of Molecular and Cell Biology, Forestry and Forest Products Research Institute, Tsukuba, Ibaraki 305-8687, Japan

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abstract

Article history:

Tricholoma matsutake is an ectomycorrhizal basidiomycete that associates with Pinaceae

Received 14 August 2013

plants, forming a rhizospheric mycelial aggregate called “shiro” from which the prized

Received in revised form

“matsutake” mushrooms form. Here we document that T. matsutake associates in vitro

24 October 2013

with Andean Cedrela herrerae (Meliaceae) via root endophyte interactions and efficiently

Accepted 24 October 2013

forms shiro. C. herrerae produces many branches, leaves, and lateral roots in association

Available online 7 January 2014

with T. matsutake, unlike C. odorata, which grows in the tropics and produces few leaves and branches in association with the symbiont. This symbiosis may be a unique approach

Keywords: Endangered plant

to culturing matsutake as well as to cultivating endangered plant species in vitro. ª 2013 The Mycological Society of Japan. Published by Elsevier B.V. All rights reserved.

Meliaceae Mushroom cultivation Root endophyte Symbiosis

Tricholoma matsutake is an ectomycorrhizal basidiomycete that associates in subalpine and temperate regions with Pinaceae, such as Pinus densiflora, Tsuga diversifolia, and Picea glehnii. With these trees it establishes rhizospheric mycelial aggregates called “shiro”, from which the prized but currently uncultivable mushrooms “matsutake” are produced in natural

habitats (Ogawa 1978; Hosford et al. 1997; Yamada et al. 2010, 2014). We previously reported that root endophytic symbioses were axenically synthesized in vitro between T. matsutake and somatic plants of Cedrela odorata, which is an endangered tropical tree in the mahogany family (Meliaceae) and naturally harbors arbuscular-mycorrhizal fungi (Shi et al. 2006; Murata

* Corresponding author. Tel.: þ81 29 829 8279; fax: þ81 29 874 3720. E-mail address: [email protected] (H. Murata). 1340-3540/$ e see front matter ª 2013 The Mycological Society of Japan. Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.myc.2013.10.005

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et al. 2013b). In fact, C. odorata allowed T. matsutake to form shiro that gave off an aroma similar to that in natural P. densiflora forests, and the fungus conferred plant vigor as well, features that may help to develop new methods for culturing T. matsutake for fruiting, as well as for cultivating endangered plant species in vitro (Murata et al. 2013b). However, C. odorata produced only a few branches and leaves in association with T. matsutake (Murata et al. 2013b), and withered at the low temperatures (<20  C) required for matsutake fruiting (Pennigton et al. 1981; Pennington and Muellner 2010). In the present study, we examined whether Cedrela herrerae, another endangered Cedrela species that is native to the Andes mountain range (800e3500 m in elevation) and that tolerates a wide range of temperatures (Pennigton et al. 1981; Pennington and Muellner 2010), including those required for matsutake fruiting, establishes root endophytic symbiosis with T. matsutake and allows shiro production in vitro. Tricholoma matsutake Y1 (¼ATCC MYA-915, NBRC 33136) is an isolate from a P. densiflora forest in Ibaraki Prefecture that has been used for genetic, systematic and physiological analyses of matsutake (Murata et al. 1999, 2013ab; Ota et al. 2012). Mycelia were grown at 25  C on Modified Melin-Norkran’s (MMN) agar plates containing 1.5% V8 juice (¼MMN þ V8; Murata et al. 1999). Somatic plants of C. herrerae Ch-3 were regenerated through shoot cultures initiated from shoot-tip explants originating from 20-year-old micropropagated plants, which were siblings of a tree from Cuzco, Peru. The method for culturing somatic plants was the same as that described for C. odorata Co-Lf03 (Maruyama et al. 1989). For axenic dual cultivation, we used a granite-based soil substrate (hereafter “substrate”) that had allowed shiro formation and root endophytic symbiosis between T. matsutake and C. odorata (Murata et al. 2013b). The substrate was made by adding 129 ml of 1/4 strength MS medium

(Murashige and Skoog 1962) containing 0.1% glucose and 0.5% sucrose to 500 ml of dried, granite-based, natural soil obtained from the B-layer of a P. densiflora forest (Satomi, Ibaraki, Japan). The substrate was packed into 1.8-l mayonnaise jars and sealed using a transparent cap with a hole covered by Milliseal (EMD Millipore Co, Billerica, MA); the jars were then sterilized by autoclaving at 121  C for 40 min. Prior to transplantation, a hole large enough to hold plant roots and inocula was made in the center of the substrate using a sterilized metal spatula. Eight pieces of ca. 7.5  7.5  4.0 mm MMN þ V8 agar carrying the mycelia were placed in the soil with the root system of the plants and covered by soil. These spawns were incubated for 160 d. Plants and fungal mycelia were individually cultured in the substrate as negative controls. At least, three replicates were conducted in each inoculation set, and the entire system was repeated independently. A total of nine plants were inoculated with fungus and there were six controls each of plants and the fungus cultured independently. Anatomic analyses of shiro and symbiotic organs were carried out using microscopes (MS5, Leica, Wetzlar, Germany; Axioplan 2 Imaging, Zeiss, Jena, Germany) as previously described (Murata et al. 2013b). The root systems were thoroughly washed with water for microscopic examination. Aggregates of roots and mycelia that gave off a unique aroma and required considerable force to remove from the soil with forceps were regarded as shiro (Murata et al. 2013b). After anatomical analysis, the specimens were dried at 65  C for 48 h, and the dry weight of the above- and below-ground parts of each plant was measured to analyze plant growth (Yamada et al. 2006; Murata et al. 2013b). All numerical data were analyzed using Student’s t-test, and a significant difference (P < 0.05) was determined (KaleidaGraph ver. 4J, Hulinks Inc., Tokyo, Japan).

Fig. 1 e Somatic plants of Cedrela herrerae CH-3 colonized by mycelia of Tricholoma matsutake Y1 during a 160-d incubation period. A: No fungal inoculation. B: Inoculation with T. matsutake Y1.

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Shiro with a typical aromatic odor were formed between T. matsutake Y1 and somatic C. herrerae CH-3 by 160 d after inoculation (Figs. 1, 2A, B). Roots associated with fungi were brown without root hairs and developed a number of lateral roots, while those without the fungus were bluish-brown with root hairs and developed few lateral roots (Fig. 2B, C). Hyphae heavily covered the entirety of the main roots and lateral roots, and aerial fungal hyphae spread extensively over the rhizosphere forming shiro (Figs. 1, 2A, B). Plants with shiro grew markedly better than the non-inoculated plants (Fig. 1). The control fungal mycelia cultured without plants did not grow over the substrate, and inocula remained at the inoculation site. Sections of live lateral roots of C. herrerae CH-3 and those associated with T. matsutake Y1 were examined under a microscope. The exodermis, as well as other internal portions of the roots, of C. herrerae CH-3 without inocula had no fungal hyphae and developed root hairs. In contrast, C. herrerae CH-3 colonized by T. matsutake Y1 had thick fungal sheaths over the exodermis (Fig. 3A, B). The fungal hyphae penetrated the intra- and intercellular spaces (Fig. 3BeF), often forming hyphal bundles or a pseudoparenchymatous organization (Fig. 3E, F). In some areas, hyphae colonized inside the cortical cells (Fig. 3B). Fig. 3 shows prominent cases in lateral roots directly generated from main roots. A small number of continuous T. matsutake hyphae penetrated intercellular spaces, behaving like root endophytes (Fig. 3C, D). The Hartig net, an intercellular hyphal network characteristic of ectomycorrhizal associations was not observed (Fig. 3C, D). Rather, the hyphae penetrated parallel to the direction of root growth (Fig. 3CeF), as observed previously in C. odorata (Murata et al. 2013b). Tricholoma matsutake Y1 promoted the growth of C. herrerae CH-3 (Figs. 1, 4). A significant beneficial effect (P ¼ 0.0070) was noted underground, resulting in considerable increases in root biomass (Figs. 1, 2, 4). In addition, somatic plants of C. herrerae had several leaves and branches on robust shoots above a well-grown root system; however, a significant difference was not detected (P ¼ 0.0750; Fig. 4). Overall, the total biomass of inoculated plants was markedly greater than that for control plants (P ¼ 0.0080). These data demonstrated the establishment of mutualistic symbioses between these plants and fungi, which is more desirable than the symbiosis between T. matsutake and C. odorata (Murata et al. 2013b). “Ecological specificity”, a theory widely accepted in mycorrhizal symbiosis, implies that artificial mycorrhizal association in vitro do not reflect actual plantemicrobe interactions in nature because biotic and abiotic factors surrounding the organisms, such as competition with preexisting microbes, strongly influence mycorrhiza syntheses (Harley and Smith 1983; Molina et al. 1992; Smith and Read 2008). In other words, there could be a great potential in axenic in vitro co-culture for a number of unknown symbiosis that do not occur in nature. In addition to its tolerance of low temperatures, C. herrerae has an advantage as a host of T. matsutake in vitro. Somatic plants of C. herrerae in association with T. matsutake grew well both above and below ground, unlike those of C. odorata, which grew shoots and lateral roots, but fewer branches and leaves (Murata et al. 2013b). Thus, the in vitro shiro-nursery

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Fig. 2 e Dissecting micrograph of the root systems of Cedrela herrerae CH-3. A: External view of shiro formed between C. herrerae CH-3 and T. matsutake Y1. Note the solid aggregate of soil, roots and areal hyphae. B: Internal view of shiro formed between C. herrerae CH-3 and T. matsutake Y1. Note the brown colored roots covered by areal hyphae. C: Cedrela herrerae CH-3 without T. matsutake Y1. Note white fluffy hairy roots surrounding bluish-brown roots. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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Fig. 3 e Differential interference Nomarski micrographs of root tissues of somatic Cedrela herrerae CH-3 colonized by Tricholoma matsutake Y1 in shiro. A, B: Surface area of root exodermis. C, D: Cross-sections of somatic C. herrerae CH-3. E, F: Longitudinal sections of somatic C. herrerae CH-3. Arrows indicate a single hypha penetrating the intercellular space (C, D). co: Cortical cell. ex: Exodermis. hb: Hyphal bundle. sh: Fungal sheath. Bars: 10 mm.

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Fig. 4 e Plant growth conferred by Tricholoma matsutake Y1. Bars show mean value plus/minus standard errors. Asterisks indicate a significant difference (P < 0.05) in treatment between plants with (inoculated) and without inocula (control) on the basis of Student’s t-test. The number of specimens analyzed is given in parentheses. S/ R ratio: shoot/root ratio. system may be a unique approach for growing both somatic C. herrerae, which is an endangered plant species due to excessive harvesting for lumber, and shiro of T. matsutake for fruiting, although plant acclimatization and out-planting experiments must be performed to confirm this potential.

Acknowledgments This work was supported by a grant from the Forestry and Forest Products Research Institute and from the Institute of Fermentation, Osaka, Japan.

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