Matsutake morphological evidence of ectomycorrhiza formation between Tricholoma matsutake and host roots in a pure Pinus densiflora forest stand

Transcription

1 RESEARCH New Phytol. (2000), 147, Matsutake morphological evidence of ectomycorrhiza formation between Tricholoma matsutake and host roots in a pure Pinus densiflora forest stand WARWICK M. GILL *, ALEXIS GUERIN-LAGUETTE, FRE DE RIC LAPEYRIE AND KAZUO SUZUKI Laboratory of Forest Botany, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Yayoi 1 1 1, Bunkyo-ku, Tokyo , Japan Equipe de Microbiologie Forestie re, Centre de Recherche de Nancy, Institut National de la Recherche Agronomique, Champenoux, France Received 3 December 1999; accepted 17 March 2000 SUMMARY There has been conflicting debate over many years regarding the trophic status of Matsutake. Here we examined the morphology, structure and ultrastructure of Pinus densiflora roots collected from a Tricholoma matsutake Shiro within a pure Japanese red pine stand. Molecular investigations (PCR RFLP analyses) indicated that T. matsutake was the highly dominant fungus within both the Shiro and the colonized root tips, suggesting that reported root morphology modifications can be attributed to T. matsutake infection. The external morphology of Matsutakecolonized roots is consistent with previous descriptions. The presence of extraradical mycelium, mantle, and intracortical Hartig net hyphae indicates clearly that T. matsutake forms an ectomycorrhizal association with P. densiflora in naturally occurring Shiros. The elucidation, for the first time, of the Hartig net ultrastructure at the host fungus interface provides further and convincing evidence of a conventional ectomycorrhizal association. The progressive blackening, observed from base to tip in dominant mycorrhizal types, due to increased deposition of polyphenol and subsequent necrosis, appears to be a result of infection. However, the presence of highly nucleated vascular tissue indicates the viability of the vascular cylinder in these roots bearing necrotic cortices. Such a preponderance of black necrotic cortical tissues among colonized roots may reflect some atypical behaviour of T. matsutake. Key words: ectomycorrhiza, Pinus densiflora (Japanese red pine, Akamatsu), Tricholoma matsutake (pine mushroom, Matsutake). INTRODUCTION The sporocarps of many ectomycorrhizal fungi are edible and a number of these are much sought after for their flavour, aroma and other attributes which they impart to cuisine. These prerequisites are fulfilled in Japan largely by a single mushroom, Tricholoma matsutake, and closely related Tricholoma species, included under the collective name Matsutake (Wang et al., 1997). The supply of this species, however, often falls short of Japanese *Author for correspondence (present address): Department of Plant and Microbial Sciences, The University of Canterbury, Private Bag 4800, Christchurch, New Zealand (fax ). domestic-consumer demand. Annual harvest of matsutake peaked in 1941 at c tonnes. However, over the ensuing 60 yr, the annual harvest has decreased markedly due to circumstances such as the reduction of wood-gathering and other human activities in forests (Ogawa, 1974; Iwase, 1997), natural forest ageing and the application of modern forestry management practices which are not conducive to matsutake development (Wang et al., 1997). Furthermore, the decline of P. densiflora forests due to nematode infection has reduced the availability of the primary host species (Iwase, 1997). At present, a good fruiting year will yield a mere 1000 tonnes. The balance to meet domestic demand, approx tonnes annually, is imported pre-

2 382 RESEARCH W. M. Gill et al. bp Uncut M Cfr HaeIII HinfI C M traced back to the host tree, were either immersed immediately in 70% ethanol or 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer (ph 7.2), or transferred undisturbed in plastic boxes fresh to the laboratory. Matsutake mycorrhizal laterals, stored in 70% ethanol, were tentatively identified according to previously published external morphological descriptions (Ogawa, 1975), classified into four distinct types (I IV) based on external morphological criteria and enumerated using a stereomicroscope. Fig. 1. The amplified intergenic spacer (IGS1) and RFLP profiles of Tricholoma matsutake sporocarp (lane 1), mycorrhizal root tip (lane 2) and soil colonizing white hyphae (lane 3) samples collected from the same Tricholoma matsutake Shiro within a pure Pinus densiflora stand. The PCR products (uncut) were subsequently cleaved with Cfr13I, HaeIII or HinfI restriction endonucleases. Lane C, control (DNA template omitted); lane M, molecular weight marker in base pairs (bp). dominantly from Korea, China, North America and North Africa. Unfortunately, these imports are often of inferior species and the time delay from harvest to delivery results in post harvest spoilage and a reduction in quality (Wang et al., 1997). In order to satisfy year-round demand with top quality mushrooms, an artificial cultivation system is required. To establish such a system, the basic nutritional mechanisms of the fungus must first be fully understood. There has been conflicting debate over many years regarding the trophic status of T. matsutake. The degree of uncertainty is well illustrated by Wang et al. (1997) who, in conclusion, wrote it appears that T. matsutake occupies zones within the triangle defined by pathogen, saprobe and symbiont and gravitates towards one or another depending on the time of year. Current research is therefore directed at elucidating the interaction of T. matsutake with host P. densiflora root tissue and ascertaining the true trophic status of the fungus. Here the morphology, structure and ultrastructure of colonized roots is examined. MATERIALS AND METHODS Sample root collection and classification Putative Tricholoma matsutake (S. Ito et Imai) Sing. Pinus densiflora (Sieb. et Zucc.) mycorrhizal roots were collected from directly beneath mature T. matsutake sporocarps (Agerer, 1991) in a Shiro within a pure stand of Japanese red pine in Nagano Prefecture (Central Japan) from 1997 to Shiro refers to the white or pale grey soil between or around host trees composed of a compact mass of mycelia. Uninfected P. densiflora roots were collected from outside the Shiro. Shiro samples, composed of mycelium, soil and roots which were PCR RFLP characterization From the fresh Shiro fragment brought to the laboratory, two samples (each 1 mg f. wt) of white hyphae forming abundant soil aggregates and four putative type I or II T. matsutake ectomycorrhizal lateral root tips were taken. DNA was extracted using the DNeasy Plant kit (Qiagen, Germany) and the set 5SA CNL12 was used to amplify the intergenic spacer (IGS1) (Selosse et al., 1996). Aliquots of 5-fold-diluted DNA extracts, each of 25 µl were combined with 25 µl of PCR mixture. The final reaction mixture (50 µl) comprised: 50 µm each of datp, dctp, dgtp and dttp (Applied Biosystems, USA), 0.2 µm each of primer (Kurabo, Japan) and 1X PCR buffer with 1 unit of Taq polymerase (Perkin Elmer). The reactions were run on the GenAmp PCR system 9600 (Perkin Elmer, USA), including a progressive extension temperature cycling step (Kraigher et al., 1995). A 25-fold dilution of the DNA extracted from a sporocarp fragment (c. 4mm ) collected previously from the same Shiro and a negative control (no DNA template) were similarly processed. PCR products (c. 1.5 µg) were cleaved with 5 units of HinfI, MspI, HaeIII, DdeI, RsaI (Wako) and Cfr13I (Takara) restriction endonucleases according to the manufacturers instructions. The restriction fragments were separated by horizontal electrophoresis on 3% high resolution (Sigma) agarose gels in 0.5X TBE (2 h 20 min at 4 V cm ). The gels were stained with ethidium bromide and photographed under UV light using a video imager system (Biorad Multi- Analyst software version 2.03, Biorad, CA, USA). DNA molecular weight marker VIII (Biochemica, Boehringer, Mannheim, Germany) was used as a size standard. Microscopy Freehand sections of mycorrhizas from the fresh Shiro sample were prepared by freezing short lengths of mycorrhizal root (2 3 mm) in a drop of water in a Leica Jung CM1500 cryotome and sectioning by hand with a razor blade. Frozen sections were transferred to glass slides, mounted in water beneath a coverslip and examined unstained with an Olympus BH2 light microscope.

3 RESEARCH Matsutake mycorrhizas 383 (a) (b) (c) (d) (e) (f) Fig. 2. External morphology of Pinus densiflora roots colonized (a,c f) or not (b) by Tricholoma matsutake. (a) Dense, frequently branched root system collected from a Tricholoma matsutake Shiro. Note the varied morphologies of lateral mycorrhizal roots. Bar, 1.5 mm. (b) Uninfected P. densiflora lateral root with profuse root hairs and distinctive cream yellow root tip. Bar, 400 µm. (c f) Type I IV Matsutake mycorrhizal roots. Note the increasing blackening from the mycorrhizal root base and decrease in extraradical mycelium as development progresses from type I to type IV. Bars, 400 µm.

4 384 RESEARCH W. M. Gill et al. (a) (b) (c) (d) (e) (f) Fig. 3. For legend see opposite.

5 RESEARCH Matsutake mycorrhizas 385 Mycorrhizal roots from the Shiro sample stored in 70% ethanol were prepared for light microscopy. Primary fixation was carried out in 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer with 1% acrolein, for 2 h under vacuum at room temperature. They were then rinsed three times in distilled water (each for 20 min) and dehydrated in an ascending acetone series finishing with three changes (each for 15 min) of 100% propylene oxide. The mycorrhizal roots were finally embedded in Spurr s resin. Thin sections (2 4 µm) were cut with glass knives on a Super Nova Ultratome (Reichert Jung, Vienna, Austria) and gently heat-fixed to glass microscope slides. The sections were then stained in 0.1% toluidine blue O in 1% acetate buffer (Ling-Lee et al., 1977) for 10 min, destained in tap water for 15 min, air dried, then mounted in DPX (Fluka BioChemika, Tokyo, Japan) beneath a coverslip and examined as described previously. Matsutake mycorrhizas from the Shiro sample stored in 2.5% glutaraldehyde were prepared for transmission electron microscopy (TEM). The mycorrhizas underwent primary fixation as already described and after three buffer rinses (each 20 min in 0.1 M sodium cacodylate, ph 7.2), were postfixed in 2% osmium tetroxide in the same buffer for 90 min at room temperature. The mycorrhizas were then rinsed three times in distilled water and dehydrated and embedded as before. Gold sections were cut with a diamond knife on an LKB Ultratome (LKB, Stockholm, Sweden) and floated onto 200 mesh copper grids supported with Formvar (0.5% in chloroform). Sections were counterstained for 10 min in 2% uranyl acetate in 70% ethanol and for 5 min in basic lead citrate. Sections were examined with a Jeol 1200EX microscope (JEOL, Tokyo, Japan) at an acceleration voltage of 80 kv. RESULTS PCR RFLP characterization of colonizing hyphae DNA extracts from all the samples of white shiro mycelium, putative T. matsutake ectomycorrhizas and the T. matsutake sporocarp yielded a unique PCR product (Fig. 1). This product was determined to be 456 bp in length by cloning and sequencing (data not shown). Each of the six endonucleases tested, MspI, DdeI, RsaI (data not shown) and HinfI, HaeIII and Cfr13I (Fig. 1) generated identical RFLP patterns from all three samples. Colonized root morphology The dense, frequently branched P. densiflora root systems (Fig. 2a) beneath T. matsutake sporocarps consisted of monopodial colonized roots (Fig. 2c f) lacking root hairs as opposed to noncolonized roots (Fig. 2b). Roots, which were confirmed to be colonized by T. matsutake based on PCR RFLP analyses, were classified into four distinct types according to external morphological criteria (Fig. 2c f). Type I lateral root tips (Fig. 2c) were often straight and more or less clavate, with abundant extraradical mycelium. The cream yellow root tip was distinguishable from the light-brown axis, which became darker toward the base. A loose hyphal sheath was observed on the root surface. Type II colonized root axes (Fig. 2d) became progressively blackened from the base. The black proximal portion was characterized by a wrinkled and pitted surface, loss of ensheathing hyphae and reduced extraradical mycelium. The root tip retained a turgid cream yellow appearance. Type III root axes (Fig. 2e) were Fig. 3. Light micrographs of both fresh freehand sections and resin-embedded sections of Pinus densiflora roots colonized by Tricholoma matsutake. (a) Transverse freehand section through the distal region of a type I mycorrhizal root. The substantial mantle (m) overlies the epidermis (e), which is impregnated with brown tannins (t). The intercellular Hartig net (hn) colonizes both the epidermis and cortex (c). Bar, 30 µm. (b) Transverse freehand section through the distal region of a type I mycorrhizal root. Host cortical cells (c) are often lined internally with discrete brown droplets (d). In this section, the mantle (m) is very thin and discontinuous. Note the Hartig net (hn) penetrating between cortical cells. Bar, 12 µm. (c) Longitudinal section through the distal cortex of a type I mycorrhizal root (stained resin-embedded material). In plan view, the distinctive, rarely septate, multibranched pseudoparenchymatous structure (mh) of the Hartig net is apparent. The intercellular Hartig net mycelium, seen also in transverse view (hn), appears as a single file of hyphae between the host cortical cells (c). Bar, 12 µm. (d) Transverse section through the epidermis in the distal region of a type I mycorrhizal root (Toluidine blue O-stained resin-embedded material). The thin mantle (m), containing green coloured polyphenolic inclusions (pi) overlies the epidermis (e), which contains similar polyphenolic material (p). Note the multibranched Hartig net (mh) within the outer cortex (c). Bar, 12 µm. (e) Transverse section through the necrotic region of a Matsutake mycorrhizal root (Toluidine blue O-stained resin-embedded material). The convoluted outline of the section reflects the wrinkled and grooved surface of the necrotic regions seen in external morphology. The outer cortex is impregnated with green coloured polyphenolics (p) and other dark staining inclusions (arrow). The cortex (c) has collapsed, but the intracortical Hartig net (hn) remains visible. The central vascular cylinder (vc) appears undisturbed. Note the absence of ensheathing mycelium. Bar, 60 µm. (f) Longitudinal section through the distal region of a type IV mycorrhizal root (Toluidine blue O-stained resin-embedded material). The collapsed, necrotic cortex (c) contains remnants of extracellular Hartig net (hn). The vascular cylinder (vc), however, is highly nucleate and undisturbed. Bar, 60 µm.

6 386 RESEARCH W. M. Gill et al. (b) (a) (c) Fig. 4. Transmission electron micrographs of a transverse section of the Tricholoma matsutake mycorrhiza Hartig net within the distal region of a type I mycorrhizal root of Pinus densiflora. (a) The multibranched pseudoparenchymatous Hartig net is apparent (mh). Separate branches are created by hyphal invaginations (arrows) which form adjacent to host cortical cells (c). Often, polyphenolics (p) are deposited within the host cortical cell. Adjacent fungal hyphae (fh) are often visible and might be derived from the same hyphal system. Bar, 1 µm. (b) At the interface between the host cortex (c) and the multibranched Hartig net (mh), the host cell wall (hw) and fungal cell wall (fw) might fuse and become indistinguishable, forming a characteristic involving layer (il). The host wall in places loses integrity and merges with the embedding matrix (arrow). Bar, 0.5 µm. (c) In other places, both the host wall (hw) and fungal wall (fw) merge, but remain distinct from each other (*). When separated, the symbiont walls retain their integrity and are distinguishable from the embedding matrix (em). Bar, 0.5 µm. often bent and the black, wrinkled, pitted surface was devoid of almost all extraradical and ensheathing mycelia. The blackening encroached upon the root tip, which became darker. The tip of type IV roots (Fig. 2f) became concolourous with the black axis and was no longer distinguishable. Neither extraradical nor ensheathing hyphae were observed. From examination of 200 tips, the relative abundance of each type was determined. Type II was dominant (51%) followed by type III (31%). Type I and IV represented 9 and 5% of sampled root tips, respectively. Examination of colonized roots by light microscopy Transverse freehand sections through the distal region of a type I putative mycorrhizal root (Fig. 3a) showed evidence of a mantle, c. 6 cells in thickness, overlying the epidermis which was impregnated with brown tannins. An intercellular Hartig net colonizing both the epidermis and cortex was discernable. Within the cortex of similar material (Fig. 3b), cells were often lined internally with discrete brown droplets previously described as polyphenolic deposits (Piche et al., 1983). Here the mantle was thin, not exceeding 2 cell layers, and discontinuous (Fig. 3b). The uniseriate Hartig net penetrated between cortical cells. The distinctive, rarely septate, multibranched pseudoparenchymatous Hartig net structures, termed palmetti by Mangin in 1910 (Smith & Read, 1997), were apparent in longitudinal section (Fig. 3c). In such toluidine blue O-stained resin-embedded type I mycorrhizas, the mantle contained green coloured polyphenolic inclusions and the underlying epidermis was impregnated with similar material (Fig.

7 RESEARCH Matsutake mycorrhizas 387 3d). In transverse section through the necrotic region of a Matsutake mycorrhizal root (Fig. 3e) the convoluted outline of the section reflects the wrinkled and grooved surface of the necrotic regions as seen in external morphology. The outer cortex was impregnated with green coloured toluidine blue O-stained polyphenolics as well as with other dark staining inclusions (Fig. 3e). Although ensheathing mycelium was absent, the intracortical Hartig net hyphae were still visible within the collapsed cortex and the central vascular cylinder appeared intact (Fig. 3e). In longitudinal section through the distal region of type IV mycorrhizal roots in which the root tip was indistinguishable from the black axis, the vascular cylinder appeared highly nucleated and undisturbed (Fig. 3f). Here again the collapsed, polyphenolic-impregnated necrotic cortex contained remnants of Hartig net cells (Fig. 3f). Examination of the Hartig net by transmission electron microscopy Observation of the Hartig net region by TEM confirmed the multibranched Hartig net palmetti structure (Fig. 4a) within the distal cortex of a type I mycorrhizal root. Typical hyphal invaginations were observed (Fig. 4a). The chattered face of sectioned polyphenolic deposits within cortical cells conformed to previous descriptions (Pargney & Brimont, 1995). At the interface between the host cortex and the multibranched Hartig net (Fig. 4b), the host cell wall and fungal cell wall were fused and became indistinguishable forming a characteristic involving layer (Duddridge & Read, 1984). The host wall in places had lost integrity and merged with the embedding matrix (Fig. 4b, arrow). In other places, although both the host wall and fungal wall merged, they remained distinguishable (Fig. 4c, asterisk). When separated by embedding matrix, the symbiont walls often retained their integrity (Fig. 4c). DISCUSSION For many years, the relationship between the Matsutake Shiro mycelium, the root-colonizing hyphae and the T. matsutake sporocarp has been contentious and a source of concern regarding previous descriptions. Here we present molecular evidence linking the Shiro mycelium, type I and II mycorrhizal roots and the T. matsutake sporocarp. Similar profiles were obtained after restriction of all PCR products with six endonucleases. The absence of extraneous bands observed from the mycorrhizal root and Shiro samples, which were not present in the sporocarp samples, indicates that T. matsutake is the highly dominant fungus in the natural Shiro environment. The external morphology of Matsutake mycorrhizal roots reported here is consistent with previous descriptions (Masui, 1927; Ogawa, 1975; Yamada et al., 1999). With regard to the internal morphology, although Yamada et al. (1999) provided evidence of Hartig net development in P. densiflora mycorrhizal roots collected within a Shiro, Ogawa (1985) reported that such colonized roots lack both a mantle and Hartig net, although hyphae were seen to invade the intercellular spaces of the host outer cortex. The presence, in the mycorrhizal roots investigated here, of extraradical mycelium (Fig. 2c,d), mantle (Fig. 3a c) and intracortical Hartig net hyphae (Fig. 3a f) indicates clearly that T. matsutake forms an ectomycorrhizal association with P. densiflora within naturally occurring Shiros. Furthermore, the elucidation, for the first time, of the Hartig net ultrastructure at the host fungus interface (Fig. 4) provides further convincing evidence of a conventional ectomycorrhizal association. Fungal DNA from type III and IV mycorrhizas could not be amplified, probably because of the very small amount of colonizing hyphae present (Figs 2e,f, 3e,f). However, intermediate developmental stages between types I and IV were observed suggesting a developmental continuum. We therefore considered that type III and IV morphologies were also due to colonization of host roots by T. matsutake. Type II and III mycorrhizas are highly predominant in the Shiro, together representing 80% of the total mycorrhizal root tips observed. These mycorrhizal types are reminiscent of the ageing, but active, mycorrhizas described on Picea abies roots (Al-Abras et al., 1988). Here the presence of highly nucleated vascular tissues (Fig. 3f) similarly indicates the viability of the vascular cylinder in such roots bearing necrotic cortices, some of which might retain the potential for elongation giving rise to new type I mycorrhizas. A year-round seasonal survey of Matsutake mycorrhizal roots would be required to elucidate the dynamics of this developmental process, which is probably influenced by a combination of factors, such as time, season, environment and both tree and mycelium activities. The mantle, host epidermal and outer cortical cells of Matsutake mycorrhizal roots contain a large number of darkly stained inclusions, consistent with Pinus ectomycorrhizas (Foster & Marks, 1966). The progressive blackening and subsequent necrosis observed from base to tip in type I through to type IV mycorrhizal roots, appears to be a result of infection and progressive root colonization. Axial blackening is due to increased deposition of polyphenol, identified by a green reaction with toluidine blue O (Feder & O Brien, 1968; Ling-Lee et al., 1977). While the deposition of polyphenols, frequently observed in both gymnosperm and angiosperm mycorrhizas (reviewed by Munzenberger et al., 1990), resembles a defence reaction, the role of these high-molecular-weight precipitated polyphenols in controlling mycorrhizal infection is

8 388 RESEARCH W. M. Gill et al. questionable. Indeed the hyphal colonization and subsequent degradation of such deposits (Pargney & Brimont, 1995) casts doubt on their role in limiting fungal infection. Furthermore, the soluble lowmolecular-weight phenolics, which exhibit higher antifungal activity, reportedly decrease rather than increase in mycorrhizas compared with uninfected roots (Munzenberger et al., 1990, 1995). However, in the present case, expansion of the necrotic zone correlates with the disappearance of mantle and extraradical mycelium, suggesting that phenolics might perform a regulatory function in relation to T. matsutake. The preponderance of black necrotic cortical tissues among colonized roots (Fig. 2a) might also reflect some atypical behaviour of T. matsutake. It has been suggested for many years that T. matsutake might exhibit some pathogenic characteristics. Fungal penetration of host cortical cells has been observed in both natural (Masui, 1927; Ogawa, 1985; Yamada et al., 1999) and artificially synthesized (Wang et al., 1997; W. M. Gill, unpublished) Matsutake mycorrhizas. Furthermore, some members of the genus Armillaria, near relatives of Tricholoma, are regarded as pathogenic (Shaw & Kile, 1991). We have demonstrated here that T. matsutake forms an ectomycorrhizal association with its host P. densiflora, in concurrence with Yamada et al. (1999) and Gill et al. (1999). The distinctive progressive blackening and necrotic morphology of developing Matsutake mycorrhizal roots illustrated here, is perhaps influenced by a shift in the trophic status of T. matsutake as the association matures (Wang et al., 1997). The complete characterization of the natural Matsutake mycorrhiza life cycle and its nutritional relationships remain important subjects for further investigation. ACKNOWLEDGEMENTS This research was supported by a grant from the Biooriented Technology Research Advancement Institution (BRAIN). REFERENCES Agerer R Characterization of ectomycorrhiza. In: Norris JR, Read DJ, Varma AK, eds. Methods in microbiology, vol. 23. Techniques for the study of mycorrhiza. London, UK: Academic Press Ltd, Al-Abras K, Bilger I, Martin F, Le Tacon F, Lapeyrie F Morphological and physiological changes in ectomycorrhizas of spruce (Picea excelsa (Lam.) Link) associated with ageing. New Phytologist 110: Duddridge JA, Read DJ The development and ultrastructure of ectomycorrhizas I. Ectomycorrhizal development on pine in the field. New Phytologist 96: Feder N, O Brien TP Plant microtechnique: some principles and new methods. American Journal of Botany 55: Foster RC, Marks GC Observations on the mycorrhiza of forest trees. I. The fine structure of the mycorrhizas of Pinus radiata D. Don. Australian Journal of Biological Sciences 19: Gill WM, Lapeyrie F, Gomi T, Suzuki K Tricholoma matsutake an assesment of in situ and in vitro infection by observing cleared and stained whole roots. Mycorrhiza 9: Iwase K Cultivation of mycorrhizal mushrooms. Food Review International 13: Kraigher H, Agerer R, Javornik B Ectomycorrhizae of Lactarius lignyotus on Norway spruce, characterized by anatomical and molecular tools. Mycorrhiza 5: Ling-Lee M, Chilvers GA, Ashford AE A histochemical study of phenolic materials in mycorrhizal and uninfected roots of Eucalyptus fastigata Deanne and Maiden. New Phytologist 78: Masui K A study of the ectotrophic mycorrhizas of woody plants. Memoirs of the College of Science, Kyoto Imperial University, Series B, III, 2: Munzenberger M, Heilemann J, Strack D, Kottke I, Oberwinkler F Phenolics of mycorrhizas and nonmycorrhizal roots of Norway spruce. Planta 182: Munzenberger M, Kottke I, Oberwinkler F Reduction of phenolics in mycorrhizas of Larix decidua Mill. Tree Physiology 15: Ogawa M Ecology of Tricholoma matsutake (Ito et Imai) Sing., mycorrhizal fungus, in pine forest. In: Mori K, ed. Mushroom Science 9. Proceedings of the Ninth International Scientific Congress on the Cultivation of Edible Fungi, Tokyo, Japan: Japan Science Press, Ogawa M Microbial ecology of mycorrhizal fungus Tricholoma matsutake (Ito et Imai) Sing. in pine forest. II. Mycorrhiza formed by Tricholoma matsutake. Bulletin of the Government Forestry Experimental Station 278: Ogawa M Ecological characters of ectomycorrhizal fungi and their mycorrhizae: an introduction to the ecology of higher fungi. Japan Agricultural Research Quarterly 18: Pargney JC, Brimont A Production of concentrated polyphenols by the root cap cells of Corylus associated with Tuber: ultrastructural study and element localization using electron energy loss spectroscopy and imaging. Trees 9: Piche Y, Peterson RL, Howarth MJ, Fortin JA A structural study of the interaction between the ectomycorrhizal fungus Pisolithus tinctorius and Pinus strobus roots. Canadian Journal of Botany 61: Selosse M-A, Costa G, Di Battista C, La Tacon F, Martin F Meiotic segregation and recombination of the intergenic spacer of the ribosomal DNA in the ectomycorrhizal basidiomycete Laccaria bicolor. Current Genetics 30: Shaw CG, Kile GA Armillaria root disease. Washington DC, USA: United States Department of Agriculture. Smith SE, Read DJ Mycorrhizal symbiosis, 2nd edn. London, UK: Academic Press Ltd. Wang Y, Hall IR, Evans LA Ectomycorrhizal fungi with edible fruiting bodies. I. Tricholoma matsutake and related fungi. Economic Botany 51: Yamada A, Kanekawa S, Ohmasa M Ectomycorrhiza formation of Tricholoma matsutake on Pinus densiflora. Mycoscience 40: