RESEARCH New Phytol. (2000), 147, 389 400 Comparative anatomy of ectomycorrhizas synthesized on Douglas fir by Rhizopogon spp. and the hypogeous relative Truncocolumella citrina HUGUES B. MASSICOTTE *, LEWIS H. MELVILLE, R. LARRY PETERSON AND RANDY MOLINA University of Northern British Columbia, College of Science and Management, Faculty of Natural Resources and Environmental Studies, 3333 University Way, Prince George, BC, Canada V2N 4Z9 Department of Botany, University of Guelph, Guelph, Ontario, Canada N1G 2W1 USDA Forest Service, Pacific Northwest Research Station, Forestry Sciences Laboratory, 3200 Jefferson Way, Corvallis, Oregon 97331, USA Received 13 September 1999; accepted 20 March 2000 SUMMARY The morphology and anatomy of ectomycorrhizas of Rhizopogon parksii, Rhizopogon vinicolor and Rhizopogon subcaerulescens, and a hypogeous relative, Truncocolumella citrina, synthesized on Douglas fir in glasshouse conditions using spore slurries as inoculum, are described and compared. Mycorrhizas formed with R. parksii and R. vinicolor did not exhibit their characteristic subtuberculate morphology in these tests, but rather had a pinnate form. All species had diagnostic features of ectomycorrhizas: a well-developed Hartig net and a fungal mantle. In addition, several species exhibited crystal inclusions in the outer mantle, usually at the interface between the mantle and soil. Truncocolumella citrina had crystal-like inclusions within the mantle but external to fungal hyphae, a feature rarely described in ectomycorrhizas. Key words: Rhizopogon, Truncocolumella, Pseudotsuga menziesii, anatomy, scanning electron microscopy. INTRODUCTION Rhizopogon is a species-rich genus of hypogeous ectomycorrhizal basidiomycetes, and several species show strong host specificity with particular genera of Pinaceae (Massicotte et al., 1994; Molina & Trappe, 1994). A previous paper in this series (Massicotte et al., 1999) compared detailed anatomical structures of Pinus ponderosa (ponderosa pine) ectomycorrhizas synthesized with eight Rhizopogon spp. belonging to sections Amylopogon, Fulviglebae and Rhizopogon. This paper emphasizes anatomical features of Douglas-fir (Pseudotsuga menziesii) ectomycorrhizas formed by species of the sections Villosuli and Amylopogon. Field associations of sporocarps of section Villosuli spp. with particular hosts, and laboratory syntheses, show the strong specificity of section Villosuli for *Author for correspondence (fax 1 250 960 5538; e-mail hugues unbc.ca). Douglas fir (Massicotte et al., 1994; Molina & Trappe, 1994; Molina et al., 1999). This specificity is also borne out by the common association of section Villosuli spp. with Douglas fir in exotic plantations in Europe (Gross et al., 1980; Jansen & de Vries, 1989; Parlade &A lvarez, 1993; Parlade et al., 1996) and New Zealand (Chu-Chou & Grace, 1981, 1983a,b). In native Douglas-fir forests of Western North America, section Villosuli ectomycorrhizal morphotypes are common and dominant mycorrhizal components on both young seedlings and mature trees (Castellano & Trappe, 1985; Molina & Trappe, 1994; Simard et al., 1997a,b; Goodman & Trofymow, 1998; Molina et al., 1999), an indication of their ecological importance as multistage fungi (Visser, 1995). Rhizopogon spp. also typically form abundant rhizomorphs that might function in water uptake; R. vinicolor ectomycorrhizas have improved drought resistance for Douglas fir (Parke et al., 1983; Dosskey et al., 1990).
390 RESEARCH H. B. Massicotte et al. Ectomycorrhizal syntheses with Douglas fir have been reported for several section Villosuli spp. (see Molina & Trappe, 1994 for a complete listing). Most ectomycorrhizal descriptions are from pure culture syntheses which might contain artefacts particularly if glucose is present in the rooting substrate (Duddridge, 1986a,b); most descriptions are only rudimentary and note mantle colour, texture, presence of rhizomorphs and extent of Hartig net development. Molina & Trappe (1994) noted similarity in overall appearance in ectomycorrhizas synthesized between Douglas fir and section Villosuli spp. Descriptions of field-collected Douglas fir associated with Rhizopogon spp. in the section Villosuli are fewer than for those synthesized in laboratory culture, but a common feature in all descriptions is the presence of dense patches of dark pigmented surface hyphae overlaying typically white or pale interior mantles (Molina & Trappe, 1982, 1994; Massicotte et al., 1994). The density and coverage of the darker surface hyphae are most pronounced on older ectomycorrhizas and described for Douglas fir R. parksii as a moderate reddish brown to greyish reddish brown fibrillose epicutis (Massicotte et al., 1994). Rhizopogon vinicolor Douglas-fir ectomycorrhizas have received the most detailed anatomical attention. They often develop as dense coralloid or tuberculate clusters and the tubercles are encased in a rind of thick, darkly pigmented hyphae (Trappe, 1965; Zak, 1971; Massicotte et al., 1992; Goodman, 1996). Trappe (1965) believed this rind to be of phycomycetous origin, but Zak (1971) later showed the rind to be differentiated aseptate, amber, thickwalled hyphae belonging to R. vinicolor. Ectomycorrhizas synthesized between R. vinicolor and Douglas fir by Zak (1971) did not fully develop into tubercles but did show a patchy structure of the differentiated, pigmented surface hyphae as noted later by Molina & Trappe (1994) for several section Villosuli spp. on Douglas fir. The presence of this unique epicutis of dark hyphae is a diagnostic feature used to distinguish section Villosuli Douglas-fir ectomycorrhizas in the field or on seedlings used in soil bioassays. Massicotte et al. (1992) also reported the presence of calcium oxalate crystals within R. vinicolor ectomycorrhizas as well as bacterial associates along the hyphae within the outer rind or on the surface of the tubercle. Li et al. (1992) isolated a nitrogenfixing, spore-forming Bacillus sp. from R. vinicolor tubercles. The morphology of Truncocolumella citrina Douglas-fir ectomycorrhizas has been described briefly (Massicotte et al., 1994; Eberhart & Luoma, 1996), but the anatomy of these mycorrhizas has received little attention. The purpose of this study was to compare detailed Douglas-fir ectomycorrhizal anatomies of two section Villosuli spp. (R. vinicolor and R. parksii) with R. subcaerulescens (section Amylopogon) and a close hypogeous relative, T. citrina. Bacterial associations and crystal formations were also examined. Ectomycorrhizas were synthesized in a previously reported spore inoculation study (Massicotte et al., 1994). MATERIALS AND METHODS Seedling preparation, growth conditions and inoculation Ectomycorrhizas (EM) were synthesized in the glasshouse using spore suspensions as part of a larger experiment on the specificity of the genus Rhizopogon (Massicotte et al., 1994). Details of seedling preparation, growth conditions, source of hypogeous sporocarps and inoculation were provided previously (Massicotte et al., 1994). Briefly, seedlings of Douglas fir (Pseudotsuga menziesii) (Mirb.) Franco were grown from seeds in the glasshouse in Pine cells (60 ml capacity) or Super cells (160 ml capacity) for 18 wk. At that time, inoculation with spore slurries of Rhizopogon parksii, R. vinicolor, R. subcaerulescens, and Truncocolumella citrina was performed with a repeat inoculation 3 wk later. Several other Rhizopogon spp. belonging to the different sub-sections were tested also for synthesis with Douglas fir, but most failed (Massicotte et al., 1994). However, R. subcaerulescens formed ectomycorrhizas with Douglas fir but usually in dual culture with ponderosa pine. These Douglas-fir R. subcaerulescens ectomycorrhizas were used for the present study. Source of material Several EM clusters, selected on the basis of turgidity (wrinkled clusters were not considered) and colour, were harvested when the plants were 10 14 months old. The EM clusters were fixed immediately and either processed for light microscopy using LR White embedding medium or for scanning electron microscopy (SEM) using standard protocols (Massicotte et al., 1992). Scanning electron microscopy Intact clusters or portions of small root systems, freshy washed from their supporting matrix, were sectioned with a razor blade and fixed immediately in 2.5% glutaraldehyde in 0.10 M Hepes (N-2- hydroxyethylpiperazine-n1 2-ethane sulfonic acid) buffer, ph 6.8 at ambient temperature for at least 24 h, rinsed in the same buffer and post-fixed in 2% aqueous osmium tetroxide for 2 h at 4 C. Tissue was then rinsed with buffer, treated with thiocarbohydrazide, rinsed thoroughly in water and fixed again with 1% aqueous osmium tetroxide. Tissue was then dehydrated in an ascending series of
RESEARCH Biology of Rhizopogon 391 ethanol, critical-point dried, mounted on aluminum stubs, coated with gold-palladium and photographed in a JEOL JSM-35C scanning electron microscope (Japan Electron Optics, USA). Some specimens were fractured using a razor blade for observations by SEM. When available, at least 2 3 clusters of each species were observed. Light microscopy Small fresh specimens, usually with 2 4 root tips present, were sectioned with a razor blade 3 4 mm from the tip and immediately fixed in glutaraldehyde as for SEM. Tissue was then dehydrated in an ethanol series and embedded in LR white resin (London Resin Co., Basingstoke, UK). Sections (1.0 1.5 µm) for light microscopy (LM) were cut with glass knives and stained with 0.05% toluidine blue O in 1% (w v) aqueous sodium borate. Several sections were also observed under crossed polarizers to reveal birefringent deposits in the mantle. RESULTS Ectomycorrhizas of Rhizopogon parksii Ectomycorrhizas were single to small pinnate (Fig. 1a) to large pinnate (Fig. 1b) normally with several rhizomorphs of different size present. Clusters with tuberculate or subtuberculate morphology were not observed. In some cases, colonization by R. parksii replaced what was most likely a Thelephora spp. as indicated by the change in mantle morphology (Fig. 1a). Scanning electron microscopy of a fully colonized apex revealed the intricate and profuse fungal growth (Fig. 1c) and at higher magnification, the mantle showed proliferation of hyphae, sometimes branched, that appeared to emanate from the outer mantle layer (Fig. 1d). Rhizomorphs were numerous, appearing compact and of variable width, with some emanating hyphae (Fig. 1e) and at higher magnification, they appeared delicately woven with clamped hyphae (Fig. 1f). Mycorrhizal tips, fractured in a transverse plane, revealed classical ectomycorrhiza features with a very thick mantle and distinct Hartig net hyphae between root cells (Fig. 2a). At higher magnification, the multi-layered mantle was obvious and the Hartig net hyphae surrounded epidermal and cortical cell layers (Fig. 2b). Light microscopy of median longitudinal sections of young colonized roots revealed an apex devoid of colonization and a mantle which, during its formation, showed close contact between hyphae and root hairs (Fig. 2c e). In more proximal regions, the fungal mantle was well developed, fairly loose, and with hyphae concentrated around root hairs (Fig. 2c e). Some root hairs had a thickened wall that was continuous with a similarly thickened wall in the subtending epidermal cell (Fig. 2d). Hartig net hyphae developed in the epidermal layer at this stage (Fig. 2d,e). Ectomycorrhizas of Rhizopogon vinicolor Ectomycorrhizas were single (Fig. 3a) to pinnate. Clusters with tuberculate or subtuberculate morphology were not observed. Some mycorrhizal roots were elongated, sinuous, and had a moderately thick mantle, except at the apex where colonization was minimal (Fig. 3a). The Hartig net was well developed, had a labyrinthic morphology and occurred close to the root apical meristem (Fig. 3a) and reached almost up to the endodermis (Fig. 3b). The mantle was dimorphic, showing a typical loose outer region with some rod-shaped bacteria present and a more compact inner region (Fig. 3c). The same section examined under crossed polarizers revealed numerous birefringent deposits at the interface between the compact inner mantle and the loose outer mantle (Fig. 3d). Ectomycorrhizas of Rhizopogon subcaerulescens Only single monopodial ectomycorrhizas covered by a prosenchymatous mantle were present (Fig. 4a,b). Numerous short, smooth emanating hyphae protruded beyond the layers of hyphae (Fig. 4c,d) that were coated with large, crystalline deposits (Fig. 4e). A moderately thick mantle with short emanating hyphae, as well as an obvious Hartig net were evident in mycorrhizas fractured in a transverse plane (Fig. 4f). Ectomycorrhizas of Truncocolumella citrina Ectomycorrhizas were single to small pinnate in morphology. Young monopodial roots had abundant mucigel masking colonizing hyphae in the apical region (Fig. 5a). Older ectomycorrhizas had abundant hyphae covering the apex (Fig. 5b) and intricate intermingling of hyphae, some of which had encrustations along their walls (Fig. 5c). Sections of well colonized ectomycorrhizas showed a moderately thick mantle and obvious Hartig net hyphae colonizing root tissues (Fig. 5d). The mantle showed a gradation in thickness with the thickest portion being in the most proximal region (Fig. 6a). The presence of numerous mitotic stages (Fig. 6b) indicated that, in spite of the well developed mantle and Hartig net (Fig. 6a), the root was in an active growth phase. Hyphae penetrated between the root cap cells that retained their nuclei (Fig. 6b). An enlargement of a proximal region showed a thick and mostly compact mantle and Hartig net hyphae that penetrated between cortical cells, the walls of which appeared sinuous in nature (Fig. 6c). Observations of the mantle in a similar
392 RESEARCH H. B. Massicotte et al. (e) (f) Fig. 1. Scanning electron microscopy of Rhizopogon parksii Pseudotsuga menziesii ectomycorrhizas. A portion of a small pinnate mycorrhiza partially colonized at the apex (arrowhead) by R. parksii. Rhizomorphs of R. parksii are obvious at the tip of the cluster (double arrowhead). The remainder of the root is colonized by a Thelephora sp. (arrow), with a smooth mantle. A large pinnate cluster entirely colonized by R. parksii. Rhizomorphs are numerous and in some cases, hold the peat moss leaves (arrowheads) from the substrate to the root. An enlargement of the fully colonized apex in showing the intricate and profuse fungal growth. An enlargement of the mantle surface in showing the proliferation of hyphae radiating out from the mantle, some of them branching and swollen in places (arrowheads). (e) An enlargement of a group of rhizomorphs seen in. Most rhizomorphs appear compact, of variable widths and with some emanating hyphae. (f) An enlargement of one rhizomorph, showing the compact and delicately interwoven nature of these hyphae. Some knee-shape appendages (arrowhead) can be seen. Some hyphae appear clamped (double arrowheads). Bars: 100 µm (a,b,e); 10 µm (c,d,f). region of the root showed that cytoplasmic material was present within the outer and inner mantle hyphae (Fig. 6d). An enlargement of the mantle in the most proximal region of the root where the mantle was thickest showed the dimorphic nature of the mantle; the outer mantle hyphae appeared to be
RESEARCH Biology of Rhizopogon 393 (e) Fig. 2. Scanning electron microscopy (a,b) and light microscopy (c e) of Rhizopogon parksii Pseudotsuga menziesii ectomycorrhizas. A fractured transverse section of a mycorrhizal tip in a well colonized portion of root. A very thick mantle surrounding the root (*), distinct Hartig net hyphae between root cells (arrowheads), and stelar tissues (S), are obvious. An enlargement of showing loose outer mantle (arrows) and a compact multi-layered inner mantle (*). Epidermal and cortical cell layers are surrounded by Hartig net hyphae (arrowheads). A median longitudinal section showing a young mycorrhizal root devoid of colonization at the apex. Several colonized root hairs (arrowheads) are obvious. An enlargement of a similar section of the root shown in. The loose mantle is well developed and fungal hyphae have concentrated around root hairs (arrows). The Hartig net (arrowheads) appears to have developed up to the first cortical layer (*). (e) An enlargement of a root hair similar to those in showing the peculiar cell wall structure of the root hair (arrowheads). Fungal hyphae in the mantle (arrows) and in the Hartig net (double arrowhead) are easily distinguished. Bars: 100 µm (a,c,d); 10 µm (b,e). coated with dark material (Fig. 6e). Often, the nuclei of fungal hyphae present in the inner mantle and Hartig net could be distinguished (Fig. 6e). Numerous birefringent deposits located mostly in the inner tissues of the mantle, but with some external to the mantle, were present (Fig. 7a). An
394 RESEARCH H. B. Massicotte et al. Fig. 3. Light microscopy of Rhizopogon vinicolor Pseudotsuga menziesii ectomycorrhizas. A median longitudinal section of a sinuous, long ectomycorrhizal root showing a moderately thick mantle (*) except at the apex and a well-developed Hartig net (arrowheads). A lateral root primordium (arrow) is present. An enlargement of in the young Hartig net zone showing a moderately thick mantle (*), labyrinthic Hartig net hyphae reaching inner cortical layers (arrowheads). E indicates the endodermis. An enlarged portion of the mantle in a root similar to showing the loose outer mantle (*) and the compact inner mantle (double arrowhead). Some rod-shaped bacteria (arrows) can be seen in the outer mantle. The same section as in examined under crossed polarizers revealing numerous birefringent deposits at the interface between the compact inner mantle and the loose outer mantle. Bars: 100 µm (a d). enlargement of a root showed the several locations of these inclusions (compare Fig. 7b with c). Under normal light microscopy, they could barely be distinguished in the inner mantle and Hartig net (Fig. 7b) whereas under crossed polarizers, they were obvious (Fig. 7c). These inclusions are multibranched, with needle-like patterns within them (Fig. 7d); they appeared to have been initiated at a locus at the periphery of a cell and then to have grown within the cell. In the inner portions of the root, where the Hartig net hyphae can be distinguished (Fig. 7e), these inclusions also appeared
RESEARCH Biology of Rhizopogon 395 (e) (f) Fig. 4. Scanning electron microscopy of Rhizopogon subcaerulescens Pseudotsuga menziesii ectomycorrhizas. A long, monopodial root completely colonized by fungal hyphae. An enlargement of in the region indicated by the arrow, showing a well developed prosenchymatous mantle with several emanating hyphae (arrowheads). Higher magnification of the mantle showing numerous short emanating hyphae (arrowheads). Higher magnification showing larger inner mantle hyphae coated with encrustations. (e) An enlargement of a section similar to showing the details of encrustations on hyphae. (f) A fractured section of a well colonized tip showing the moderately thick mantle with short emanating hyphae (arrowheads) and obvious Hartig net hyphae colonizing root tissues (arrows). Bars: 100 µm ; 10 µm (b d,f); 1 µm (e).
396 RESEARCH H. B. Massicotte et al. Fig. 5. Scanning electron microscopy of Truncocolumella citrina Pseudotsuga menziesii ectomycorrhizas. A portion of a monopodial single root newly colonized by fungal hyphae. The beaded appearance and mucigel (double arrowheads) is obvious and is masking hyphae colonizing the apex. Hyphae have clamps (arrowheads). A portion of a monopodial single mycorrhizal root, well colonized by fungal hyphae. An enlargement of showing intermingling tubular hyphae, some of which have encrustations (arrowheads). A fractured portion of a well colonized tip showing a thick mantle (*) and obvious Hartig net hyphae (arrowheads) colonizing several cortical layers. Bars: 100 µm (a,b); 10 µm (c,d). to occupy the cortical cell volume (Fig. 7e) and many had a dark matrix surrounding them (Fig. 7f,g). DISCUSSION Pseudotsuga menziesii (Douglas fir) is one of the most receptive ectomycorrhizal hosts, with over 2000 suggested fungal symbionts (Trappe, 1977; Molina et al., 1992). Rhizopogon is one of the major fungal symbionts with Douglas fir (Molina & Trappe, 1994) and a number of species can form ectomycorrhizas with seedlings, and young and mature trees (Molina et al., 1999). This should, theoretically, lead to a considerable variation in the appearance of Douglasfir ectomycorrhizas in the field and, indeed, many morphotypes attributable to Rhizopogon spp. have been documented on Douglas fir and other tree species (Molina et al., 1999). In the present study, a comparison of morphological and anatomical features of ectomycorrhizas formed by inoculating seedlings with basidiospore suspensions of two section Villosuli spp. (R. parksii and R. vinicolor), a section Amylopogon sp. (R. subcaerulescens), and a hypogeous relative (T. citrina) revealed distinctive characteristics, some of which have not been reported previously. Ectomycorrhizas synthesized using spore slurries conformed in overall appearance with others described for Douglas fir: the morphology varied from single monopodial types to more or less compact pinnate clusters (compare with Massicotte et al., 1994, 1999; Molina & Trappe, 1994). Rhizopogon vinicolor and R. parksii produced ectomycorrhizas with mostly single monopodial to pinnate morphology, but the tuberculate morphology, a type frequently seen on field material (Zak, 1971; Massicotte et al., 1992; Goodman, 1996) was absent. Rhizopogon subcaerulescens Douglas-fir ectomycorrhizas were of a monopodial morphology. The duration of the experiment might not have been long enough for either tuberculate or pinnate clusters to form. It should be noted that, with R. subcaerulescens, ectomycorrhizas only formed on Douglas fir when this species was grown with a companion species
RESEARCH Biology of Rhizopogon 397 (e) Fig. 6. Light microscopy of Truncocolumella citrina Pseudotsuga menziesii ectomycorrhizas. A median longitudinal section of a long ectomycorrhizal root showing a thick mantle (much thicker proximally) and a well developed Hartig net (arrowheads). An enlargement of the apex shown in detailing the cellular features of meristematic cells, some of which are in different phases of mitosis (arrows). The mantle is subcompact and moderately thick. Some hyphae are present between root cap cells (double arrowheads). An enlargement of the region marked A in. The mantle is thick and mostly compact with some loose outer mantle hyphae, and the Hartig net hyphae have penetrated between cortical cells (arrowheads). Cortical cells walls (double arrowheads) appear sinuous in nature. Wide diameter outer mantle hyphae with various cytoplasmic materials. (e) An enlargement of the region marked B in. The mantle is very compact and hyphae in the outer portion have deposited a black matrix, coating the hyphae. Nuclei (arrowheads) can be distinguished within hyphae of the inner mantle and the Hartig net. Hartig net hyphae show some branching (double arrowhead). Bars: 100 µm (a,b,e); 10 µm (c,d). such as ponderosa pine (Massicotte et al., 1994). This suggests that the simpler morphology might be a result of a lesser degree of compatibility between these symbionts even though a mantle and Hartig net had formed. Given the typical appearance of mantles and Hartig nets, however, we assume these ectomycorrhizas were fully functional. This assumption needs to be tested by physiological experiments.
398 RESEARCH H. B. Massicotte et al. (e) (f) (g) Fig. 7. Light microscopy of Truncocolumella citrina Pseudotsuga menziesii ectomycorrhizas. A median longitudinal section of a small ectomycorrhizal root examined under crossed polarizers revealing numerous birefringent deposits in the inner tissues of the cortex (arrowheads). An enlargement of a portion of the root shown in, revealing a very thick mantle (*), and a Hartig net (arrowheads) that has colonized cortical cells. The same section as in examined under crossed polarizers revealing large druse-like encrustations situated in the inner mantle and inner Hartig net layers (arrowheads). Smaller incrustations are present in the outer mantle hyphae. An enlargement of a portion of the root in showing a detail of one encrustation (arrowheads) in the outer portion of the root. This crystal appears to occupy a portion of the necrotic root cap cells. The compact inner mantle (*) is obvious. (e) An enlargement of a portion of the root in showing a detail of three large encrustations (*) in the inner portion of the root. The crystals (*) are occupying a portion of the cortical cell and appear coated with a dark matrix (arrows). (f) Another view of crystals within root tissues (arrowheads) showing the dark nature of matrix surrounding the encrustations. (g) The same section as in (f) examined under crossed polarizers revealing these large druse-like encrustations situated among cortical cells (arrowheads). Bars: 1 mm ; 100 µm (b,c); 10 µm (d g).
RESEARCH Biology of Rhizopogon 399 Ectomycorrhizas formed between Douglas fir and R. parksii showed root hair hyphal interactions similar to those reported for Betula alleghaniensis Laccaria bicolor mycorrhizas (Massicotte et al., 1989). In both systems, hyphal contact induces wall thickening in root hairs that might prevent hairs from collapsing during early stages of mantle formation. All fungal species tested resulted in the formation of fundamental characteristics typifying ectomycorrhizas: a mantle and Hartig net. This observation was made for the Rhizopogon spp. used as inoculum in a previous study with ponderosa pine (Massicotte et al., 1999). The only Rhizopogon spp. examined in detail for both Douglas fir and ponderosa pine was R. subcaerulescens. In a comparison of the results, the influence of the host genotype on ectomycorrhiza formation is evident. Although both host species had a similar prosenchymatous mantle with short emanating hyphae, crystalline deposits in the mantle, and a cortical Hartig net, prominent rhizomorphs and pinnate root morphology were present only with ponderosa pine. Rhizomorphs have also been reported from R. subcaerulescens mycorrhizas on Tsuga heterophylla (Agerer et al., 1996). Rhizomorphs were also present with R. parksii Douglas-fir ectomycorrhizas as well as with R. occidentalis ponderosa pine and R. truncatus ponderosa pine ectomycorrhizas (Massicotte et al., 1999). It is well documented that many Rhizopogon spp. form abundant rhizomorphs in soil and these might enhance nutrient uptake and translocation (Molina et al., 1999). Lack of rhizomorphs on the R. subcaerulescens Douglas fir ectomycorrhizas again leads us to question the compatibility and functionality of these ectomycorrhizas under the experimental conditions. The Hartig net encompasses the cortex up to the endodermis in all of the ectomycorrhizas synthesized with Douglas fir. The same was true with ponderosa pine and several Rhizopogon spp. synthesized using the same inoculation system (Massicotte et al., 1999). In field-collected Douglas-fir roots, however, the inner cortical cells at times appear to impede Hartig net development (Massicotte et al., 1992). The presence of crystalline structures in the mantle formed between Douglas fir and both R. vinicolor and T. citrina extends observations made with ponderosa pine (Massicotte et al., 1999). Although the chemical nature of these crystalline structures was not determined, it is likely that they are calcium oxalate as reported for other hypogeous fungal species (Cromack et al., 1979; Entry et al., 1992). Although the crystals are primarily located in the mantle, they could still indicate that these fungal species play a similar role in nutrient release from soils to the hyphal mats of ectomycorrhizas formed between Douglas fir and Hysterangium (Entry et al., 1992). The composition of the crystals, particularly that of the intracellular crystals in Douglas-fir T. citrina ectomycorrhizas, needs to be determined before a function can be assigned to them. The unusual presence of large crystalline structures within the root tissues is, to our knowledge, novel to mycorrhizal structures. ACKNOWLEDGEMENTS H. B. M. and R. L. P. thank the Natural Sciences and Engineering Research Council of Canada for continued support in the form of operating grants. Our sincere thanks to Linda Tackaberry for help with root processing, Doni McKay and Jane Smith for glasshouse assistance and Laurie Winn for typing the manuscript. REFERENCES Agerer R, Mu ller WR, Bahnweg G. 1996. 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