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Plants of the Ericales are common components of the understorey vegetation of boreal forest ecosystems. The roots of these plants harbour a number of ericoid mycorrhizal ascomycetes, some of which have been shown to improve plant nutrient uptake from the soil (Smith & Read, 1997). Fungal strains accommodated in the Hymenoscyphus ericae–Scytalidium vaccinii species complex (Helotiaceae, Helotiales, Ascomycota) are undoubtedly the most commonly reported and studied representatives of the ericoid mycorrhizal group of fungi (Read, 1991, 1996; Smith & Read, 1997; Sharples et al., 2000). In addition, species of Oidiodendron, anamorphs of the ascomycete family Myxothricaceae (Onygenales), and a broad range of sterile mycelia with divergent morphologies and unknown identities, are commonly isolated from ericoid fine roots (Stoyke et al., 1992; Perotto et al., 1996; Hambleton & Currah, 1997; Hambleton et al., 1998; Liu et al., 1998; McLean et al., 1999; Monreal et al., 1999; Bergero et al., 2000; Sharples et al., 2000).
More than 95% of fine roots of boreal forest trees are ectomycorrhizal (Taylor et al., 2000). The fungal species that form ectomycorrhizas have traditionally been associated with a number of basidiomycetes forming conspicuous epigeic or hypogeic sporocarps (Molina et al., 1992; Smith & Read, 1997). However, several corticoid basidiomycetes and teleomorphic, anamorphic and sterile ascomycetes have been shown to form ectomycorrhizal relationships (Mikola, 1965; Danielson, 1984; Yang & Wilcox, 1984; Wilcox & Wang, 1987a,b; Ursic & Peterson, 1997; Vrålstad et al., 1998, 2000; Bergero et al., 2000; Grogan et al., 2000; Kõljalg et al., 2000). In a recent study, Vrålstad et al. (2000) demonstrated that fungal strains derived from the ectomycorrhizal morphotype Piceirhiza bicolorata (Brand et al., 1992) constituted an assemblage of very close relatives of the ericoid mycorrhizal fungus H. ericae. An inferred ITS1-phylogeny gave a 100% supported evolutionary clade of 15 closely related P. bicolorata ITS1-genotypes that embraced the ITS-genotypes of the H. ericae and S. vaccinii ex-types. This clade, consisting of five subclades, was collectively referred to as the H. ericae aggregate. The hypothesis was put forward that ericoid and ectomycorrhizal plants might share mycobionts of this species complex. However, the lack of resynthesis studies, using both ericoid and ectomycorrhizal hosts, prevented a test of this hypothesis.
Support for this hypothesis was presented by Bergero et al. (2000), who collected ecto- and ericoid mycorrhizal samples from neighbouring plants of Quercus ilex and Erica arborea in a Mediterranean forest habitat. They demonstrated that ectomycorrhizal root tips of Q. ilex harboured fungi (Oidiodendron spp. and unidentified sterile mycelia) that, in vitro, formed ericoid mycorrhizas with E. arborea. No fully developed ectomycorrhizas were observed when Q. ilex seedlings were re-inoculated with these fungal isolates, but sections of swollen root tips revealed a poorly developed mantle and/or coils inside the epidermal cells. A rudimentary Hartig net was also observed. Their results indicate that ericoid mycorrhizal fungi may coexist in ericaceous and ectomycorrhizal plants. Further support comes from the study of the mitotic fungus Phialophora finlandia, commonly referred to as Mycelium radicis atrovirens (MRA) or dark septate endophytes (DSE) (Jumpponen & Trappe, 1998), that forms ectomycorrhizas and/or ectendomycorrhizas with hardwoods and conifers, respectively (Wilcox & Wang, 1987a,b). Monreal et al. (1999) found that the ectomycorrhizal strain of P. finlandia (FAG15 = UAMH7454) formed typical ericoid mycorrhizas with the shrub Gaultheria shallon (Ericales). Thus, reports of ectomycorrhizal, ectendomycorrhizal and ericoid mycorrhizal behaviour for this particular strain of P. finlandia are available (Wilcox & Wang, 1987a,b; Monreal et al., 1999). Monreal et al. (1999) also showed that P. finlandia clustered in the H. ericae group based on sequence similarity analysis of the internal transcribed spacer 2 (ITS2).
In the present study, 12 fungal strains (12 ITS1-genotypes) of the H. ericae aggregate were inoculated in vitro onto sterile ectomycorrhizal and ericoid mycorrhizal hosts. The syntheses were conducted in order to determine: if putative ectomycorrhizal strains of the H. ericae aggregate were capable of reforming the black-and-hyaline mantles characteristic of the Piceirhiza bicolorata morphotype, from which they were originally isolated; and if single strains could form a mycorrhizal association with both ericoid and ectomycorrhizal hosts. We also provide complete ITS1-5.8S-ITS2 rDNA sequences of the 12 test isolates, and report on their phylogenetic relatedness to Phialophora finlandia.
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The outcome of the inoculation trials is shown in Table 2. The H. ericae isolates of ericoid origin (UAMH5828, UAMH6735, ARON2888.S) all formed numerous intracellular infections within the cortical cells of the hair roots of V. vitis-idaea (Fig. 1a). None of these three isolates formed any ecto- or endomycorrhizal associations with the ectomycorrhizal hosts. However, in some instances, superficial hyphal associations with the roots were observed (Table 2). Three out of nine H. ericae-like isolates of ectomycorrhizal origin (ARON2810.S, ARON2893.S, ARON2917.S) formed ectomycorrhizas with all three ectomycorrhizal host plant species (Table 2). In the P. sylvestris inoculations, none of the isolate-host combinations developed visible ectomycorrhizal tips after three months, but after an additional 4 months, brown to black ectomycorrhizal morphotypes were observed. Another isolate of ectomycorrhizal origin, ARON2906.S, formed typical P. bicolorata mycorrhizas with spruce and birch, but failed to produce any mycorrhizas with pine. The ARON2948.S isolate of ectomycorrhizal origin failed to produce ECM with spruce and pine, but two mycorrhizal tips were found on one birch plant. This isolate also formed a superficial hyphal association with roots of V. vitis-idaea. None of the ECM-forming isolates produced any structures that resembled ericoid mycorrhizal infections when inoculated onto V. vitis-idaea. However, three of the isolates (ARON2810.S, ARON2906.S, ARON2917.S) penetrated the cortical cells of the older parts of the root systems, with no obvious reaction by the root cells to the hyphal penetration. In addition, the hyphae of these isolates that were associated with the root surface changed from a purely linear growth form to a more cellular, monilioid growth pattern. Three isolates of ectomycorrhizal origin (ARON2879.S, ARON2894.S, ARON2903.S) did not associate with the roots of any of the four host plant species, while another isolate of ectomycorrhizal origin (ARON2916.S) formed a superficial hyphal association with roots of B. pubescens and V. vitis-idaea. All control plants remained non-mycorrhizal.
Table 2. Results of inoculation trials between isolates of the Hymenoscyphus ericae aggregate and potential ectomycorrhizal and ericoid hosts
|Isolate number – taxon or genotype||Mycorrhizal statusa|
|Betula pubescens||Picea abies||Pinus sylvestris||Vaccinium vitis-idaea|
|UAMH5828–S. vaccinii||− −s||− −||− −||+ + +|
|UAMH6735–H. ericae||− −||− −s||− −||+ + +|
|ARON2888.S–H. ericae||− −||− −||− −||+ + +|
|ARON2916.S – G1||− −s||− −||− −||− − −s|
|ARON2917.S – G2||+ +||+ +||− +||− − −i|
|ARON2903.S – G3||− −||− −||− −||− − −|
|ARON2894.S – G4||− −||− −||− −||− − −|
|ARON2879.S – G5||− −||− −||− −||− − −|
|ARON2948.S – G8||+ −||− −||− −||− − −s|
|ARON2893.S – G11||+ +||+ +||− +||− − −|
|ARON2810.S – G12||+ +||+ +||− +||− − −i|
|ARON2906.S – G13||+ +||+ −||− −||− − −i|
Figure 1. Ericoid and ectomycorrhizal structures formed between the Hymenoscyphus ericae aggregate and host plant roots: (a) light micrograph (400×) of Vaccinium vitis-idaea root colonized by Hymenoscyphus ericae isolate ARON2888.S, with intracellular hyphal coils (arrowhead) and emanating hyphae (arrow); (b) typical field-collected Piceirhiza bicolorata morphotype on Picea abies: black with bright hyaline tips. Notice the single light brown ectomycorrhiza (ECM) morphotype (arrowhead) that probably represents the morphotype of group I in vivo; (c–h) synthesized ectomycorrhizas between ARON2917.S +Betula pubescens (c), ARON2893.S + P. abies (d–e), and ARON2810.S + B. pubescens (f–h). Morphotype group I (c–e) – light brown to brown, some with translucent tips (c–d), and sparse (c,e) to extensive (d) growth of extramatrical hyphae (emh). Morphotype group II (f) – dark brown to black with whitish tip (classic P. bicolorata morphotype). Morphotype group III (g–h) – charcoal black with extensive growth of emh. Bars, 50 µm (a), 0.5 mm (c–g) and 5 mm (b,h).
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Ectomycorrhizal morphology and anatomy
While the five ECM-forming isolates of this study (ARON2810.S, ARON2893.S, ARON2906.S, ARON2917.S, ARON2948.S) had been isolated from field collected ectomycorrhizas of the distinctive black-and-hyaline Piceirhiza bicolorata morphotype (Fig. 1b), the isolates formed in vitro a series of morphotypes ranging in colour from light brown to charcoal black. For presentation purposes, this series has been divided roughly into three major groups: (I) light brown to brown morphotypes (outer mantle hyphae hyaline, subhyaline to brown; Fig. 1c–e); (II) dark brown to black morphotypes with hyaline tips =P. bicolorata (outer mantle hyphae melanized except where enclosing the root apex; Fig. 1f); and (III) charcoal black, Cenococcum-like morphotypes (outer mantle hyphae melanized continuously around the root apex; Fig. 1g,h). In the ARON2948.S-birch combination, only two brown ectomycorrhizal roots were observed. After 3 months, the isolates ARON2810.S, ARON2893.S and ARON2906.S had formed ECM that fit into all morphotype categories (Fig. 1c–h and 2a,b,h), while at this stage, isolate ARON2917.S had only formed morphotypes of group I (Fig. 2c). After 7 months, the latter isolate had developed fully melanized mycorrhizal tips with pine. In general, ectomycorrhizal tips were 1–3 mm in length (cf. Fig. 1h), and possessed sparse to extensive amounts of narrow (1.8–2.4 µm) and regularly septate extramatrical hyphae (Fig. 1c–g). Extramatrical hyphae were initially hyaline and smooth, later darkly pigmented, smooth to finely verrucose. Fig. 2 details a number of longitudinal sections of LR-white resin embedded ECM roots of isolate-host combinations representing the three morphotype groups. In all cases, the sections showed dense, continuous mantles (5–70 µm in thickness) and narrow, well developed uniseriate Hartig nets (Fig. 2). No intracellular penetrations were observed in the ECM roots in any of the isolate-host combinations. Fungal cytoplasm within active hyphae of mantles and Hartig nets was coloured bright turquoise by the Fast-Green F.C.F.-solution, and appeared as bright turquoise ‘granulae’ within hyphal cross sections (Fig. 2d–g,i). In the partial and completely melanized morphotypes, the mantles were consistently two-layered, with an outer melanized mantle layer (D1) and an inner hyaline layer (D2) from where the hyaline Hartig net extended between the root cells (Fig. 2a,d,i). In ECM roots of birch, the Hartig net was restricted to the epidermis, which consisted of a single layer of radially elongated cells (cf. Fig. 2a,c,d,f). In ECM roots of spruce and pine, the Hartig net always extended to the first layer of epidermal cells, and one to three cell layers further into the cortex (cf. Fig. 2b,e,h–i). Hyphae were never observed in the endodermis. The epidermal cells were often filled with tannins (Fig. 2e,i), and cortical cells were sometimes internally delineated with discrete, brown droplets (Fig. 2e) previously ascribed to polyphenolic deposits (Pichéet al., 1983). In all sections of apparently vital, intact ECM roots, the hyphae of the D2 mantle layer and Hartig net were consistently hyaline. In some sections of moribund ECM roots of pine, the plant cells had started to disintegrate while the still intact Hartig net was darkly pigmented. A darkly pigmented Hartig net was occasionally observed in the cortex of some long roots.
Figure 2. Longitudinal sections of LR-white embedded ectomycorrhizal (ECM) roots of Group I (b,c,e,f,g), Group II (a,d) and Group III (h,i) morphotypes. The following isolate-host combinations are presented: ARON2906.S +Betula pubescens (a,d), ARON2906.S +Picea abies (b,e), ARON2917.S + B. pubescens (c,f,g), ARON2893.S + P. abies (h–i). (a) Piceirhiza bicolorata ECM morphotype. Mantle (m) two-layered: inner layer hyaline and continuous over the root apex (double arrow), outer layer melanized except at the root apex (arrow). (b–c) Light brown to brown morphotypes – mantles hyaline to subhyaline with some melanized hyphae (arrowhead) in the outer part. (d) Enlarged view of (a), showing the two-layered mantle (m1 and m2), Hartig net (hn) uniseriate and restricted to a layer of radially elongated epidermal cells (Eec). Bright turquoise ‘granulae’ (g): fungal cytoplasm within hyphae in cross sections. (e) Enlarged view of (b), Hartig net uniseriate extending along the epidermal cells (ec) and one to three cell layers into the cortex (cc). Tannin-filled epidermal cells (t) and cortical cells with discrete, brown droplets (d). (f–g) Enlarged view of (c). (f) Hartig net (hn) restricted to Eec. (g) Paradermal section through epidermis showing a uniseriate Hartig net. (h) Charcoal black morphotype. Outer mantle melanized continuously around root apex. Extramatrical hyphae (emh). (i) Enlarged view of (h). Mantle two-layered and Hartig net extending two–three cell layers into the cortex. Bars, 20 µm.
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ITS-phylogeny of ericoid, ecto- and nonmycorrhizal strains of the H. ericae aggregate
Parsimony analyses of the included ITS1-5.8S-ITS2 rDNA sequences yielded 16 most parsimonious trees (MPTs). Fig. 3 shows the bootstrap consensus tree with clades that yielded > 70% bootstrap support. The bootstrap consensus tree retained one 100% supported main clade of the ingroup (the H. ericae aggregate), which was subdivided into five major subclades. Subclade 1 (100% supported) includes strictly ericoid mycorrhizal isolates of ‘true’H. ericae. Subclade 2 (99% supported) includes P. finlandia and four of the confirmed ectomycorrhizal test isolates. Subclade 3 (100% supported) includes the three isolates that did not associate with roots of any of the host plant species. Subclade 4 includes one single ectomycorrhizal isolate, and subclade 5 includes the isolate that only formed a superficial hyphal association with some of the host roots (Fig. 3, Table 2).
Figure 3. The bootstrap consensus tree yielded with the branch and bound search algorithm in PAUP* (Swofford, 1999) (2000 replications) based on complete ITS1-5.8S-ITS2 rDNA sequences of the test isolates of this study, Phialophora finlandia (isolate FAG15 = AF011327), and with the outgroups Botrytis cinerea (Z99664) and Hyaloscypha aureliella (U57495). Numbers above lines indicate percentage bootstrap support for each branch. Only clades with bootstrap support > 70% are retained. The 100% bootstrap supported main clade of the ingroup has previously been referred to as the Hymenoscyphus ericae aggregate (cf. Vrålstad et al., 2000). The H. ericae aggregate is subdivided into the subclades 1–5. The results of the inoculation trials are indicated for each isolate: += confirmed ectomycorrhizal (ECM) (blue) or ericoid mycorrhizal (ERM) (purple), − = no confirmed mycorrhizal formation. Subclade 1 (purple) includes confirmed ERM isolates, subclades 2 and 4 (blue) include confirmed ECM isolates, and subclades 3 and 5 (black) include isolates with no observed mycorrhizal abilities. Diamond, Phialophora finlandia (isolate FAG15) has previously been reported to form ECM (Wilcox & Wang, 1987a), and there is also limited evidence that this isolate form ERM (Monreal et al., 1999).
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The resynthesis experiments carried out in this study confirm that some genetically close relatives of the ericoid mycorrhizal fungus H. ericae are true ectomycorrhizal partners with coniferous and angiosperm trees. In the birch ectomycorrhizas, the Hartig net was restricted to radially elongated cells of epidermis (Fig. 2a,c,d,f), which is characteristic for woody angiosperm ectomycorrhizas (Wilcox & Wang, 1987a; Perumalla et al., 1990; Massicotte et al., 1998). The spruce and pine ectomycorrhizas had a Hartig net that extended between the epidermal, often tannin-filled cells, and further into the cortex (Fig. 2b,e,h,i), which is characteristic for gymnosperm ectomycorrhizas (Pichéet al., 1983; Wilcox & Wang, 1987a; Ursic & Peterson, 1997; Gill et al., 2000).
Sequence similarity analysis of the internal transcribed spacer 2 (ITS2) has previously shown a link between the ectomycorrhizal fungus P. finlandia and H. ericae (Monreal et al., 1999), and the parsimony analysis of the complete ITS-region in the present study showed that P. finlandia grouped within subclade 2 of the H. ericae aggregate (Fig. 3). Hence, the isolates of subclade 2 are closer related to P. finlandia than to H. ericae. In the present study, only ‘true’H. ericae isolates of ericoid mycorrhizal origin (i.e. subclade 1, Fig. 3) formed classic ericoid mycorrhizal associations, while ectomycorrhizal formation was restricted to P. finlandia-like strains of ectomycorrhizal origin (subclade 2) as well as the single isolate of subclade 4. These results suggest that both ericoid and ectomycorrhizal symbioses have evolved within the H. ericae aggregate (Fig. 3), but do not support the hypothesis that single isolates have the ability to form both kinds of mycorrhizal symbioses. Hence, the H. ericae aggregate seems to contain a number of closely related taxa with wide ecological attributes.
We found that four of the five confirmed ectomycorrhizal strains of the H. ericae aggregate formed black-and-hyaline morphotypes that shared anatomical features with the original description of P. bicolorata (Brand et al., 1992) from which they originated. This morphotype appeared to represent an intermediate developmental stage in a sequence from light brown, dark brown, black-and-hyaline to completely black morphotypes, caused by a gradual acropetal melanization of the outer (D1) mantle layer. P. finlandia has been reported to produce ectomycorrhizal morphotypes ranging from light amber, brown, bicoloured to black (Wilcox & Wang, 1987a; Ursic & Peterson, 1997), which correspond to the observed morphotypes of our study. A progressive blackening of mycorrhizas formed by the ectomycorrhizal basidiomycete Tricholoma matsutake, from light brown, bicoloured to black morphotypes, was recently reported (Gill et al., 2000). The blackening was due to increased deposition of plant polyphenols and subsequent necrosis, with the totally black morphotypes being devoid of an intact mantle (Gill et al., 2000). By contrast, the darkening of the morphotypes in the present study did not appear to affect ectomycorrhizal structures of the ECM roots.
The anamorph genus Phialophora Medlar represents a poorly defined assemblage of darkly pigmented and little differentiated hyphomycetes, which have their teleomorphs in a broad range of ascomycete orders (e.g. Helotiales, Caliciales, Sordariales and Diaporthales), and represent soil, wood and plant saprobes, specialized plant and human pathogens as well as mutualists (Gams, 2000). P. finlandia has been reported to be beneficial, promoting the growth and survival of their ectomycorrhizal hosts both in highly acidic environments (Wilcox & Wang, 1987b) and iron contaminated soils (Lobuglio & Wilcox, 1988). Wilcox & Wang (1987a) conducted a long-term study on the ectomycorrhizal development of P. finlandia on red pine (Pinus resinosa), red spruce (Picea rubens) and yellow birch (Betula alleghaiensis) and found that the mantle developed before and after the Hartig net in ECM roots of birch and conifers, respectively. P. finlandia occupied long roots through a continuum of root sizes forming a Hartig net in virtually all roots (Wilcox & Wang, 1987a), as was occasionally observed in our study. Such a Hartig net, running through cortex of the long roots, may serve as an internal source of inoculum for ectomycorrhizal infection of emerging short roots.
The isolates of subclade 2 melanized more quickly than the single isolate of subclade 4, corroborating observed axenic culture features (cf. Vrålstad et al., 2000). Axenic culture isolates from subclade 2 regularly turned charcoal black within a week of subculturing, while the isolate from subclade 4 remained hyaline up to 4 wk before melanization occurred (Vrålstad et al., 2000). The black colonies possessed whitish margins of hyaline, actively growing hyphae (Vrålstad et al., 2000), a ‘bicoloured’ morphological feature also expressed in the ectomycorrhizal structures.
The ecto- and ericoid mycorrhizal potentials of the isolates of subclade 3 and subclade 5 (Fig. 3) remain uncertain. These isolates were originally derived from P. bicolorata ectomycorrhizas, but it is possible that they represented nonmycorrhizal mantle-associated or root-endophytic fungi. The isolates of subclade 3 remained hyaline in axenic culture (Vrålstad et al., 2000), suggesting that these isolates were unlikely to be responsible for the black-and-hyaline ECM morphotype from which they originated. Sequences of the complete ITS-region show that these isolates are more divergent from H. ericae (Fig. 3) than previously suggested based on partial ITS1-data (cf. Figure 2 in Vrålstad et al., 2000).
The black morphotypes produced by ectomycorrhizal members of the H. ericae aggregate (group III, cf. Fig. 1g–h and 2h) resemble the ECM morphotype produced by Cenococcum geophilum (Agerer & Gronbach, 1988). Wilcox & Wang (1987a,b) concluded that microscopic examination was required in order to distinguish between C. geophilum, P. finlandia and Chloridium paucisporum ectomycorrhizas. It is highly probable that the prevalence of C. geophilum has been largely overestimated in ectomycorrhizal studies at the expense of H. ericae- and P. finlandia-like representatives.
Melanized cell walls are not common among ectomycorrhizal fungi, except in tomentelloid basidiomycetes (Kõljalg et al., 2000) and the ascomycetes C. geophilum, Chloridium paucisporum, P. finlandia (Wilcox & Wang, 1987a,b) as well as the ectomycorrhizal strains of the H. ericae aggregate. Fungal melanins may act as a boundary between fungal cells and their environments, and protect against physical, chemical and biological stresses (Pigott, 1982; Wheeler & Bell, 1988; Gadd, 1993; Butler et al., 2001). The P. bicolorata morphotype has been observed to dominate root systems of hardwood and conifer seedlings colonizing heavy metal contaminated Cu-mine spoils in central Norway (Vrålstad et al., 2000; Vrålstad, 2001). It was suggested that a high tolerance to certain heavy metals by the fungi of the H. ericae aggregate forming the P. bicolorata morphotype, was responsible for the high prevalence of these fungi. Furthermore, it was observed that the P. bicolorata morphotype was much more abundant in postfire habitats than in undisturbed forest habitats (Vrålstad et al., 2000; Vrålstad, 2001). Microscopic examinations of the partially and fully melanized morphotypes formed by the H. ericae-like relatives revealed an inner mantle layer and Hartig net that remained hyaline. The melanized hyphae of the outer mantle layer and extramatrical mycelium may function as protective barriers between the external environment and the active fungus-plant symbiosis inside the root. This could be a key-factor to their apparent successful colonization of burnt and metal polluted habitats.