Exploring structural definitions of mycorrhizas,with emphasis on nutrient-exchange interfaces1

R. Larry Peterson and Hugues B. Massicotte

Abstract: The roots or other subterranean organs of most plants develop symbioses, mycorrhizas, with fungal symbi-onts. Historically, mycorrhizas have been placed into seven categories based primarily on structural characteristics. Anew category has been proposed for symbiotic associations of some leafy liverworts. An important feature ofmycorrhizas is the interface involved in nutrient exchange between the symbionts. With the exception ofectomycorrhizas, in which fungal hyphae remain external to plant cell walls, all mycorrhizas are characterized by fun-gal hyphae breaching cell walls but remaining separated from the cell cytoplasm by a plant-derived membrane and aninterfacial matrix that forms an apoplastic compartment. The chemical composition of the interfacial matrix varies incomplexity. In arbuscular mycorrhizas (both Arum-type and Paris-type), molecules typical of plant primary cell walls(i.e., cellulose, pectins, β-1,3-glucans, hydroxyproline-rich glycoproteins) are present. In ericoid mycorrhizas, onlyrhamnogalacturonans occur in the interfacial matrix surrounding intracellular hyphal complexes. The matrix aroundintracellular hyphal complexes in orchid mycorrhizas lacks plant cell wall compounds until hyphae begin to senesce,then molecules similar to those found in primary cell walls are deposited. The interfacial matrix has not been studiedin arbutoid mycorrhizas and ectendomycorrhizas. In ectomycorrhizas, the apoplastic interface consists of plant cell walland fungal cell wall; alterations in these may enhance nutrient transfer. In all mycorrhizas, nutrients must pass into thesymplast of both partners at some point, and therefore current research is exploring the nature of the opposing mem-branes, particularly in relation to phosphorus and sugar transporters.

Key words: interface, apoplastic compartment, Hartig net, arbuscule, intracellular complex, nutrient exchange.

Résumé : Les racines et autres organes souterrains de la plupart des plantes développent des symbioses avec deschampignons, les mycorhizes. Historiquement, on a placé les mycorhizes dans sept catégories, basées surtout sur descaractéristiques structurales. Une nouvelle catégorie a été proposée pour les associations symbiotiques de certaines hé-patiques foliacées. Une importante caractéristique des mycorhizes est l’interface impliqué dans les échanges de nutri-ments entre les symbiotes. À l’exception des ectomycorhizes, dans lesquelles les champignons demeurent à l’extérieurdes parois cellulaires des plantes, toutes les mycorhizes se caractérisent par des hyphes fongiques traversant les paroiscellulaires, mais demeurant séparés du cytoplasme par une membrane dérivée de la plante et une matrice interfacialeformant un compartiment apoplastique. La composition chimique de la matrice interfaciale varie en complexité. Chezles mycorhizes arbusculaires (de type Arum aussi bien que Paris), on retrouve des molécules typiques des parois cellu-laires primaires (c.-à-d., cellulose, pectines, β-1,3-glucans, glycoprotéines riches en hydroxyproline). Chez les mycorhi-zes éricoïdes, il n’y a que des rhamnogalacturones dans la matrice interfaciale entourant les complexes d’hyphesintracellulaires. La matrice entourant les complexes intracellulaires ne comporte pas de matériel pariétal de la plantechez les mycorhizes des orchidées, jusqu’à ce que les hyphes deviennent sénescentes; on retrouve alors des moléculessimilaires à celles des parois primaires. La matrice interfaciale n’a pas été étudiée chez les mycorhizes arbutoïdes et lesectendomycorhizes. Chez les ectomycorhizes, l’interface apoplastique est constituée de paroi cellulaire végétale et deparoi cellulaire fongique; une altération de ces parois peut augmenter le transfert de nutriments. Chez toutes les mycor-hizes, les nutriments doivent passer par le symplaste des deux partenaires à un point donné, et c’est pourquoi les re-cherches actuelles portent sur la nature des membranes opposées, surtout en relation avec les transporteurs de sucre etde phosphore.

Mots clés : interface, compartiment apoplastique, réseau de Hartig, arbuscule, complexe intracellulaire, échange de nu-triments.

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Can. J. Bot. 82: 1074–1088 (2004) doi: 10.1139/B04-071 © 2004 NRC Canada

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Received 29 September 2003. Published on the NRC Research Press Web site at http://canjbot.nrc.ca on 18 August 2004.

R.L. Peterson. Department of Botany, University of Guelph, Guelph, ON N1G 2W1, Canada.H.B. Massicotte.2 Ecosystem Science & Management Program, College of Science and Management, University of Northern BritishColumbia, 3333 University Way, Prince George, B.C. Canada.

1This article is one of a selection of papers published in the Special Issue on Mycorrhizae and was presented at the FourthInternational Conference on Mycorrhizae.

2Corresponding author (e-mail: hugues@unbc.ca).

Introduction

Mycorrhizas are usually defined as mutualistic symbiosesbetween fungi and plants in which both partners can benefitfrom the association (Smith and Read 1997). It is recog-nized, however, that it is often difficult to assess the benefitsderived by each of the symbionts in mutualistic associations,since these must be considered in terms of the costs to eachpartner (Ahmadjian and Paracer 1986). There is considerablediscussion as to what constitutes a benefit to each of thepartners in mycorrhizas as environmental conditions change.In mycorrhizas, the main benefit to the plant is largely nutri-tional in nature, but protection from root pathogens and im-proved water relations may also accrue.

The term “balanced mycorrhizas” has been proposed todenote situations in which both organisms receive essentialmaterials through reciprocal exchange (Brundrett 2002,2004). An important step in the evolution of balancedmycorrhizas was, therefore, the development of a specializedinterface zone for bidirectional nutrient exchange (Brundrett2002).

The term “exploitive mycorrhizal associations” has beensuggested for those situations in which there is a unidirec-tional flow of nutrients, with the main benefit usuallygoing to the plant partner (Brundrett 2002, 2004). Myco-heterotrophic plant species without photosynthetic ability,and green plants in situations where the fungus seems togain little from the association, could be considered as ex-amples of exploitive mycorrhizas (Brundrett 2002, 2004). Itshould be emphasized, however, that whether balanced orexploitive, mycorrhizal associations are the norm for almostall vascular plants and a few nonvascular plants (Smith andRead 1997; Read et al. 2000). Mycorrhizal symbioses can,therefore, be distinguished from interactions between patho-genic fungi and roots, in which nutrient flow is always to thefungi.

Brundrett (2004) suggests that the nutrient-exchange inter-face established between the symbionts is unlikely to func-tion in the same manner for balanced and exploitivemycorrhizal associations. To evaluate this suggestion, it isimportant to consider the structural aspects of interfaces forall categories of mycorrhizas and to assess the evidence thatthese sites function in nutrient exchange.

In parallel with the evolution of nutrient-exchange inter-faces, the extraradical mycelium of successful symbioses de-veloped as an efficient soil–fungal interface to allow forexploration and extraction of nutrients and water from thesubstrate. This topic is discussed by Leake et al. (2004) inthis Special Issue.

In this review, we will use the seven traditional categoriesof vascular plant mycorrhizas based on structural character-istics identified by light and transmission electron micros-copy (Scannerini and Bonfante-Fasolo 1983; Bonfante-Fasolo and Scannerini 1992; Peterson and Farquhar 1994;Smith and Read 1997; Peterson et al. 2004), recognizing thatthere is some discussion as to new ways of classifyingmycorrhiza types (Brundrett 2004). These categories arearbuscular mycorrhizas (AM), ectomycorrhizas, ectendomy-corrhizas, ericoid mycorrhizas, arbutoid mycorrhizas, mono-tropoid mycorrhizas, and orchid mycorrhizas. We will alsoinclude a brief discussion of mycorrhizas occurring with

nonvascular plants, since these are receiving more attentionin the literature and may provide insight into the evolutionof symbioses between plants and fungi (Read et al. 2000;Kottke et al. 2003).

The main objectives of this review are, therefore, to dis-cuss what is known about the interfaces in all categories ofmycorrhizas, to evaluate the evidence for nutrient transfer atthese sites, to suggest future research that is needed, andfinally to comment on possible modifications in definingcategories of mycorrhizas.

Categories of vascular plant mycorrhizas

Arbuscular mycorrhizasArbuscular mycorrhizas, the symbiotic associations be-

tween the majority of vascular plant species and fungi in thenew phylum Glomeromycota (Schüßler et al. 2001), can besubdivided into two main types, the Arum-type and theParis-type (Gallaud 1905; Smith and Smith 1997). In the on-togeny of interface development in the Arum-type (Fig. 1),usually one arbuscule develops through repeated branchingof a hypha (trunk hypha) that penetrates through the corticalcell wall (Bonfante and Perotto 1995). Paired arbuscules inadjacent cortical cells arising from the same intercellularhypha but developing at different rates have recently beenreported by Dickson et al. (2003) in Linum usitatissimum.Figure 1 also illustrates paired arbuscules in cortical cells ofa Pisum sativum root, illustrating that this feature may bemore common than reported. In the Paris-type, penetrationof the cortical cell wall by a single hypha is followed by ex-tensive coiling of this hypha (Fig. 2) from which lateralbranches are initiated to form arbusculate coils (Whitbreadet al. 1996; Cavagnaro et al. 2001; also see Fig. 3). In theArum-type nutrient-exchange interface, the fungus developsfine, thin-walled arbuscule branches within plant corticalcells, the walls of which are modified by becoming very thin(Bonfante-Fasolo and Grippiolo 1982; Bonfante-Fasolo et al.1990) and which have an amorphous chitin deposition(Bonfante-Fasolo 1982; Bonfante-Fasolo et al. 1990). In theParis-type, both hyphal coils and arbusculate coils may beinvolved in nutrient exchange, an idea suggested by calcula-tions that show that the surface area of hyphal coils may beequal to that of arbuscules in Arum-type mycorrhizas (Dick-son and Kolesik 1999) as well as by the presence of a mem-brane and interfacial matrix around hyphal coils andarbusculate coils (Armstrong and Peterson 2002). Arbus-cules and arbusculate coils are separated from the cortical-cell cytoplasm by a periarbuscular membrane and interfacialmatrix material, both derived from the plant symbiont (Bon-fante and Perotto 1995; Armstrong and Peterson 2002).

The interfacial matrix, situated between the fungal cellwall and the periarbuscular membrane, is an apoplastic com-partment composed of plant cell wall constituents, includingcellulose, pectins, β-1,3-glucans, and hydroxyproline-richglycoproteins, as demonstrated by the use of various affinityprobes (Bonfante and Perotto 1995; Armstrong and Peterson2002) and in situ mRNA probes (Blee and Anderson 2000).Bonfante and Perotto (1995) claim that the development ofthis apoplastic compartment between fungus and plant cell isthe most important event marking successful colonization.With the development of the interfacial matrix, any transfer

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of nutrients from the plant cell to the fungus would involvethe periarbuscular membrane, the interfacial matrix, the fun-gal wall, and the fungal plasma membrane (Fig. 4). The re-verse would also be true as nutrients pass from the fungus tothe plant cell (Smith and Smith 1990). Since there is a bidi-rectional exchange of nutrients, with phosphate (P), nitrate,and trace elements moving from the fungus to the plant andsugars moving to the fungus (Franken et al. 2002), thiswould be an example of a balanced relationship (Brundrett2002, 2004).

H+-ATPase activity has been demonstrated cytochemicallyin the fungal plasma membrane in the interface (Gianinazzi-Pearson et al. 1991, 2000), and Saito (2000) has provided adetailed account of the localization of various phosphatasesin host and fungal membranes of AM associations, evidencethat active transport is likely involved in nutrient transfer.Ferrol et al. (2002) summarize the evidence for the up-regulation of host plasma membrane ATPase genes inmycorrhizal roots of a number of plant species. Also, there

is a difference in plasma membrane proteins, as shown by2D-PAGE analysis, between mycorrhizal and nonmy-corrhizal tomato (Lycopersicon esculentum) roots (Benab-dellah et al. 2000), but whether these are related to thenutrient-exchange interface is not known.

Currently there is considerable interest in characterizingtransporters in both the periarbuscular membrane and thefungal membrane, since these would be involved in the ac-tive transport across this interface (Harrison 1999a, 1999b;Smith et al. 2001; Ferrol et al. 2002). The recent review byFerrol et al. (2002) summarizes the information on P andcarbon transporters in AMs and points out the lack of infor-mation concerning the presence and activity of these in thenutrient-exchange interface.

Other changes that occur in cortical cells containingarbuscules include alterations in expression of genes encod-ing enzymes involved in sucrose metabolism (Blee and An-derson 2002), leading to the establishment of a sink forsucrose transport from the phloem to the arbuscules.

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Figs. 1–4. Interfaces of arbuscular mycorrhizas. Fig. 1. Arbuscules of Glomus aggregatum in a Pisum sativum root. Material stainedwith acid fuchsin and viewed with scanning laser confocal microscopy. Arbuscules are of the Arum type. Photo courtesy of Ryan Geil.Scale bar = 25 µm. Fig. 2. Intracellular hyphal coils of Glomus intraradices in a Panax quinquefolius root. These coils are typical ofParis-type arbuscular mycorrhizas. Material stained with acid fuchsin and viewed with scanning laser confocal microscopy. Scale bar =25 µm. Fig. 3. Arbusculate coils of Glomus intraradices in a root of Panax quinquefolius. Arrowheads indicate fine branches formedon hyphal coils. Material cleared, stained with chlorazol black E, and examined by light microscopy. Scale bar = 25 µm. Fig. 4. Dia-gram of arbuscule illustrating the components of the interface. The periarbuscular membrane (arrowheads), interfacial matrix (*), fungalcell wall (arrows), and fungal plasma membrane (double arrowheads) are evident. T, trunk hypha; I, intercellular hypha. Scale bar =25 µm.

The deposition of the interfacial matrix and the formationof the periarbuscular membrane must involve considerablecellular activity, and it has been suggested that alterationsobserved in the plant-cell cytoskeleton during arbuscule de-velopment may play a role in these processes (Genre andBonfante 1997, 1998; Peterson et al. 2000; Armstrong andPeterson 2002; Timonen and Peterson 2002). Methods com-bining GFP (green fluorescent protein) tagging of cell com-ponents such as endoplasmic reticulum and Golgi bodies, inaddition to cytoskeletal elements, and scanning laser confo-cal microscopy (Takemoto et al. 2003) could add additionalinformation as to how interfaces are formed between symbi-onts.

Intercellular hyphae, although not surrounded by eitherplant-derived membrane or interfacial matrix, interface withthe apoplast (intercellular space system) of roots (Ferrol etal. 2002). They have the potential to be involved in the trans-fer of nutrients, and it has been suggested that this might bea pathway for carbon acquisition by the fungus (Smith andSmith 1990). In this case, only the fungal plasma membranewould be involved in the uptake of carbon compounds.

EctomycorrhizasEctomycorrhizas, symbioses formed between both gym-

nosperm and angiosperm species and many basidiomycetefungi and fewer ascomycete fungi, are characterized primar-ily by the presence of a mantle (sheath) and an Hartig netconsisting of modified fungal hyphae that develop betweenroot cells (Smith and Read 1997). Generally, species belong-ing to the angiosperms have a paraepidermal Hartig net (i.e.,confined to the root epidermis, as shown in Fig. 5), whereasspecies belonging to the gymnosperms have an Hartig netthat develops around epidermal and cortical cells (Fig. 6).There are exceptions to this in that a few angiosperm genera(e.g., Dryas) possess an Hartig net that surrounds epidermaland cortical cells (Melville et al. 1988). In some species theHartig net may reach the endodermis, although this is vari-able. Because of the intimate association of Hartig nethyphae with root cell walls (Fig. 7), the labyrinthine branch-ing (Fig. 8) of these hyphae (Kottke and Oberwinkler 1986,1987), and the presence of numerous mitochondria and otherorganelles in Hartig net hyphae (Massicotte et al. 1986,1990), it has been concluded that this is the main interfaceinvolved in bidirectional transfer of nutrients (Kottke andOberwinkler 1986; Massicotte et al. 1987, 1989; Smith andRead 1997). Inner mantle hyphae that are not part of theHartig net but are positioned immediately adjacent to theouter tangential wall of root epidermal cells provide a sec-ond possible interface for nutrient exchange in ectomy-corrhizas (Dexheimer and Gérard 1994) (Figs. 9, 10). Thesehyphae may also become highly branched, and in the fewcases in which an Hartig net does not develop (e.g., inPisonia grandis), it is the only interface for nutrient ex-change (Ashford and Allaway 1982).

More direct evidence that the Hartig net is involved in thetransfer of P and carbon compounds comes from the use ofradioactive tracers followed by microautoradiography of rap-idly frozen and freeze-substituted poplar (Populus tremu-loides × Populus alba) ectomycorrhizas (Bücking andHeyser 2001). In this study, 33Pi accumulated rapidly in themantle and later was localized in the Hartig net and cortical

cells; the authors interpret the results to support the conceptthat P moves across the mantle through the symplast and notthe apoplast. Also, mantle hyphae and Hartig net hyphaein the median region of ectomycorrhizal roots were morehighly labelled than in either the apical or basal region, indi-cating that there are some spatial differences in uptake andtranslocation of P along the root axis. Similar results wereobtained for the distribution of labelled carbohydrates. Thissupports the observation that there is a gradient of host-cellresponse to fungi forming the Hartig net along the long axisof a root (Massicotte et al. 1986). It is known, also, thatectomycorrhizas of different ages affect the absorption andtranslocation of P (Cairney and Alexander 1992a) and car-bon compounds (Cairney and Alexander 1992b).

In some mycorrhizas, such as those formed in P. grandis(Ashford and Allaway 1982) and between Alnus crispa andAlpova diplophloeus (Massicotte et al. 1986), epidermalcells contiguous with mantle hyphae and Hartig net hyphae,if present, develop wall ingrowths (Fig. 10) that are envel-oped by plasma membrane and assume the structure oftransfer cells. Transfer-cell development is also found in sev-eral other ectomycorrhizas (Dexheimer and Gérard 1994).The increase in surface area of wall and membrane of trans-fer cells in P. grandis, although less than provided by the de-velopment of an Hartig net in other ectomycorrhizas, maybe adequate for transfer of nutrients in the environment inwhich this species is found (Allaway et al. 1985).

Regardless of whether root cells differentiate as transfercells, or whether Hartig net or inner mantle hyphae are in-volved, the interface between symbionts consists of contigu-ous root cell walls and fungal hyphal walls with material(sometimes called cement) deposited between them, creatingan apoplastic compartment. Plasma membranes of each sym-biont would be involved during the bidirectional transfer ofnutrients (see Figs. 9, 10). It is important to ascertain, there-fore, whether changes that would enhance nutrient exchangeoccur in root cell walls and hyphal walls as an ectomy-corrhiza is formed. Features of root-cell plasma membranesand fungal plasma membranes in the interface region mayalso enhance nutrient exchange.

Both plant cell walls and fungal cell walls play an integralrole during all stages in the establishment and functioning ofectomycorrhizas, and considerable progress has been madein determining changes in both walls at the molecular level(Duplessis et al. 2002; Tagu et al. 2002). However, onlyinformation related to the walls of the symbionts in thenutrient-exchange interface will be considered here.

One change in host cell walls observed at the ultra-structural level is the apparent continuity between the walland the intercellular matrix adjacent to Hartig net hyphae,suggesting that the cell wall, in particular, is modified in theinterface (Dexheimer and Gérard 1994). Also, in two gym-nosperm ectomycorrhizal associations, large amounts of acidpolysaccharides (pectins) are present in cortical cell walls aswell as in the matrix material separating these walls andHartig net hyphae (Nylund 1987). This author concludes thatthis supports the concept that there is a “mycorrhizal infec-tion zone” in roots, in which changes to host walls enablethe development of the intercellular Hartig net; no commentwas made in terms of nutrient exchange in this zone. Using avariety of affinity techniques, Balestrini et al. (1996) showed

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that in ectomycorrhizas formed between Corylus avellanaand Tuber magnatum, there were no qualitative changes inthe chemical nature of cortical cell walls of colonized rootscompared with uncolonized roots. However, there was anapparent swelling of some cortical cell walls and the appear-ance of electron-lucent areas in these walls in the region ofthe Hartig net. They concluded that their observations sup-port the premise that ectomycorrhizal fungi secrete hydro-lytic enzymes to facilitate penetration of hyphae betweenroot cells (Cairney and Burke 1994). What needs to be de-termined is whether modifications to host cell walls that arecontiguous with Hartig net and inner mantle hyphae result inincreased permeability to inorganic ions and organic mole-cules compared with cell walls not adjacent to fungalhyphae.

Modifications of Hartig net fungal walls have been dem-onstrated using the Gomori–Swift cytochemical method forcystein-containing proteins (Paris et al. 1993). In these hy-phal walls, only one poorly reactive layer was present, com-pared with walls of mantle hyphae, in which both a highlyreactive and a poorly reactive layer were present. These au-thors suggest that the simpler organization of the Hartig nethyphal walls may enhance nutrient exchange, but no physio-logical evidence for this was provided. In ectomycorrhizasformed between Eucalyptus globulus and Pisolithustinctorius, a group of symbiosis-related acidic polypeptidesare enhanced during the colonization process (Burgess et al.1995), and these have been immunolocalized to the hyphalcell wall, including the walls of mantle and Hartig nethyphae (Laurent et al. 1999; Tagu et al. 2000, 2002). Sincesymbiosis-related acidic polypeptides are present in hyphalwalls at all stages of mycorrhiza development, their signifi-cance in enhancing nutrient exchange is uncertain.

One characteristic of host cell walls in the Hartig net re-gion is the presence of acid invertases that convert sucrose tofructose and glucose in the apoplastic compartment; thesesugars can then be taken up by Hartig net hyphae (Salzerand Hager 1993; Nehls et al. 2000, 2001b).

There has been and continues to be considerable interestin determining the involvement of the host and fungalplasma membranes in nutrient exchange in ectomycorrhizas.Earlier research demonstrated cytochemically that acid phos-phatase (Dexheimer et al. 1986) and H+-ATPase activity (Leiand Dexheimer 1988) are localized along the plasma mem-branes of both symbionts in the Hartig net region, indicatingthat a bidirectional, active transport mechanism is likely in-volved in nutrient exchange.

Although the movement of P from Hartig net hyphae intothe apoplastic interface is likely driven by a concentrationgradient (Bücking and Heyser 2000), the involvement of Ptransporters in the fungal plasma membrane in active trans-port has not been determined (Chalot et al. 2002). Likewise,the existence of P transporters in host-cell plasma mem-branes for P uptake from the apoplastic compartment in theexchange interface zone has not been documented (Chalot etal. 2002).

Although the movement of carbon compounds in the formof sucrose is usually from root cells to the apoplastic com-partment, where, as noted above, acid invertase converts su-crose to glucose and fructose that can be taken up by thefungal hyphae, there is no direct evidence that hexose trans-porters are involved. However, Nehls et al. (2001a) haveshown that, in Amanita–Populus ectomycorrhizas, expres-sion of the gene encoding a monosaccharide transporter(AmMst1) was enhanced six-fold in Hartig net hyphae com-pared with mantle hyphae.

Ectomycorrhizal fungal hyphae do possess various trans-porters, including Pi transporters and hexose transporters(see Chalot et al. 2002); it is important now to show their in-volvement in the nutrient-exchange interface.

In gymnosperm and those angiosperm ectomycorrhizas inwhich most of the cortical cells are interfaced with Hartignet hyphae, there is a question as to whether these cellsmaintain plasmodesmata connections subsequent to the for-mation of the Hartig net. In two systems, one a gymnosperm(Nylund 1980) and the other an angiosperm (Melville et al.1988), cortical cells do retain plasmodesmata, potentiallyproviding a symplastic continuity from the phloem to all lay-ers of cortical cells. Research is needed, however, to deter-mine the extent of symplastic continuity from the phloem tothe epidermis in ectomycorrhizas.

EctendomycorrhizasThere is some debate as to whether ectendomycorrhizas

should be placed in a separate category, or considered eitheras a modified ectomycorrhiza (Egger and Fortin 1988) or afungal morphotype (Brundrett 2004). For this discussion,however, a separate category is maintained to emphasize thefact that there appears to be only a few species of ascomy-cete fungi that colonize mostly Pinus spp. and Larixspp. roots to form a unique structural relationship (Yu et al.2001). Most of the structural work on ectendomycorrhizashas involved pine species associated with one member of thePezizales, Wilcoxina mikolae; hence the full range of struc-

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Figs. 5–10. Features of interfaces in ectomycorrhizas. Fig. 5. Longitudinal section of a root tip of Alnus crispa colonized by Alpovadiplophloeus. A thick mantle (M) and a paraepidermal Hartig net (arrowheads) are evident. Material embedded in resin, stained withtoluidine blue O, and examined with light microscopy. Scale bar = 50 µm. Fig. 6. Longitudinal section of Pinus strobus root colonizedby Pisolithus tinctorius showing dichomtomy of apex, a thin mantle (M), and Hartig net hyphae (arrowheads) interfaced with epider-mal and cortical cells. Material prepared as in Fig. 5. Scale bar = 50 µm. Fig. 7. Higher magnification of Hartig net region from mate-rial similar to that in Fig. 5 showing Hartig net hyphae (arrowheads) interfaced with epidermal (E) cells. M, mantle. Scale bar =50 µm. Fig. 8. Inner mantle (M) and branched Hartig net hyphae (arrowheads) in a Betula alleghaniensis – Laccaria bicolor mycor-rhiza. Material prepared as in Fig. 5. Bar = 50 µm. Fig. 9. Diagram illustrating the interface between fungus and root cells inectomycorrhizas. The root cell wall (CW) abuts on the cell wall of branched inner mantle (arrowhead) and branched Hartig net (doublearrowheads) hyphae. Nutrient exchange would also involve the plasma membrane of root cells (large arrows) and the plasma membraneof fungal hyphae (small arrows). Scale bar = 50 µm. Fig. 10. The interface region of some ectomycorrhizas may involve wall in-growths (arrowheads) in root cells, which increases the surface area for nutrient exchange. Scale bar = 50 µm.

tural characteristics has yet to be determined. In those ecten-domycorrhizas studied, a mantle and Hartig net forms as inectomycorrhizas, but in addition, intracellular hyphal com-plexes develop within epidermal and cortical cells (Figs. 11,12). As a result, three potential sites for nutrient exchangeare formed: the inner mantle – epidermal cell interface, theHartig net epidermal – cortical cell interface, and the intra-cellular hyphal complex – root-cell cytoplasm. Hartignet hyphae are branched, a perifungal membrane develops

around the intracellular hyphal complex (Fig. 13), and rootcells remain alive, as judged by the presence of a nucleusand other organelles (Scales and Peterson 1991). When sec-tions are viewed with transmission electron microscopy aninterfacial matrix is apparent between the perifungal mem-brane and the hyphal wall; however, its composition has notbeen determined (Scales and Peterson 1991). There has beenno experimental work to determine if there is an exchange ofnutrients between the symbionts at any interface in this my-

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corrhiza category, and nothing is known about the molecularaspects of root colonization and the development of thenutrient-exchange interface.

Ericoid mycorrhizasThere is considerable specificity shown in the formation

of ericoid mycorrhizas in that few fungal species, primarilyascomycetes, form symbioses with members of the familiesEricaceae and Epacridaceae (Smith and Read 1997). Al-though relatively few plant species in the Ericaceae andEpacridaceae have been investigated intensively in terms oftheir mycorrhizas, the colonization process is very similaramong those examined, regardless of the particular fungalspecies involved (Perotto et al. 1995). Fungal hyphae contactthe thickened epidermal cell walls of the very fine roots(hair roots), penetrate through these walls, and form intra-cellular hyphal complexes within epidermal cells (Figs. 14–16). Although, in most instances, each epidermal cell iscolonized by a separate “unit”, we have observed some ex-ceptions to this in that fine hyphae can pass from epidermalcell to epidermal cell (Fig. 15). In any event, the intracellularhyphal coil is separated from the host cytoplasm by a peri-fungal membrane and interfacial matrix material (Bonfante-Fasolo and Gianinazzi-Pearson 1979; Bonfante-Fasolo andPerotto 1988; Perotto et al. 1995). This is shown in Fig. 16.The interfacial matrix differs in composition from that inAM in that immunolabelling shows the presence of onlyrhamnogalacturonan as a wall constituent and a low level oflabelling for the enzyme polygalacturonase (Perotto et al.1995). As in other mycorrhizas, an apoplastic compartmentis present between the symbionts, and bidirectional transportwould have to occur across this interface (Fig. 16). Littleresearch has been conducted on this interface, althoughATPase activity has been localized on the perifungal mem-brane (Gianinazzi-Pearson et al. 1984). There is consider-able scope in extending observations made with ericoidmycorrhizas to mycorrhizas formed with epacrid plant spe-cies, since there is limited structural work with this mycor-rhiza category (Cairney and Ashford 2002).

Arbutoid mycorrhizasArbutoid mycorrhizas occur in a group of species belong-

ing to the Ericales (Smith and Read 1997). Structurally, theyresemble ectendomycorrhizas in that a mantle, Hartig net,

and intracellular hyphal complexes form (Fusconi andBonfante-Fasolo 1984; Münzenberger et al. 1992; Mas-sicotte et al. 1993), but they differ in that the intracellularhyphal complexes are confined to the epidermis (Figs. 17,19). In Arbutus menziesii, the outer row of cortical cells de-velops suberin lamellae in their walls; these may limit thedevelopment of the Hartig net to the epidermis (Massicotteet al. 1993). The intracellular hyphal complexes in Arbutusunedo mycorrhizas are surrounded by epidermal-cell plasmamembrane and an interfacial matrix material (Fusconi andBonfante-Fasolo 1984; Münzenberger et al. 1992; Filippi etal. 1995). As in ectendomycorrhizas, there are three possiblesites of nutrient exchange: the interface between inner man-tle hyphae and the tangential wall of epidermal cells, the in-terface between Hartig net hyphae and epidermal cells, andthe interface between hyphal complexes and epidermal cellcytoplasm (Figs. 18, 19). Although Münzenberger et al.(1992) have shown that vesicles containing osmiophilic in-clusions fuse with the membrane surrounding intracellularhyphae, experimental evidence is lacking as to the transferof nutrients between symbionts.

Monotropoid mycorrhizasMonotropoid mycorrhizas occur in several genera belong-

ing to the subfamily Monotropoideae (family Ericaceae). Allplant hosts in this category are myco-heterotrophic and,therefore, depend on adjacent autotrophic plant species fortheir source of carbon (Leake 1994). Plants within theMonotropoideae form close associations with specific fungalsymbionts (Bidartondo and Bruns 2001, 2002; Young et al.2002). These fungi form hyphal links between the rootsof heterotrophic plants and the roots of autotrophic plants,through which carbon compounds can be transported. Cur-rently, there is no evidence that the fungus is receiving nutri-ents from the heterotrophic host, therefore, according toBrundrett (2002, 2004), this would be an example of anexploitive mycorrhiza.

Structural features of monotropoid mycorrhizas areunique (Figs. 20–22). A mantle and paraepidermal Hartignet form but, in addition, fungal hyphae penetrate epidermalcells to form “fungal pegs”, one per epidermal cell (Figs. 20,21). In Monotropa species, fungal pegs originate from innermantle hyphae that penetrate the outer tangential wall of epi-dermal cells (Lutz and Sjolund 1973; Duddridge and Read

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Figs. 11–13. Interfaces in ectendomycorrhizas. Fig. 11. Longitudinal section of Pinus banksiana root colonized by Wilcoxina mikolaevar. mikolae. A thin mantle (arrows), Hartig net hyphae (arrowheads), and intracellular hyphae (double arrowhead) are present. Mate-rial processed as in Fig. 5. Scale bar = 50 µm. Fig. 12. Higher magnification of portion of root shown in Fig. 11 showing Hartig nethyphae (arrowheads) and intracellular hyphal complexes (double arrowheads) surrounding cortical cell nuclei (N). Scale bar = 50 µm.Fig. 13. Diagram showing the interface between Hartig net hyphae (HN) and the intracellular hyphal complex (*) with root cells. Thelatter interface involves the perifungal membrane (arrowheads), interfacial material (double arrowheads), fungal cell wall (small ar-rows), and the fungal plasma membrane (large arrow). P, plasmodesmata between cells. Scale bar = 50 µm. Figs. 14–16. Interface inericoid and epacrid mycorrhizas. Fig. 14. An intracellular hyphal complex within an epidermal cell of a hair root of Kalmiaangustifolia. Material cleared, stained with acid fuchsin, and examined by differential interference contrast microscopy. Scale bar =25 µm. Fig. 15. Epidermal cells of Gaultheria procumbens with intracellular hyphal complexes. Narrow hyphae (arrowheads) joinhyphal complexes (*) of adjacent epidermal cells. Material cleared, stained with acid fuchsin, and examined with scanning laser confo-cal microscopy. Scale bar = 25 µm. Fig. 16. Diagram of intracellular hyphal complexes in epidermal cells of typical ericoid andepacrid mycorrhizas. The outer tangential wall (*) of epidermal cells is thickened. The interface between the epidermal cell cytoplasmand fungus is similar to that illustrated in Fig. 13, with perifungal membrane (arrowheads) and interfacial matrix material (double ar-rowheads) separating the fungus from the epidermal cell cytoplasm. Scale bar = 25 µm.

1982; Snetselaar and Whitney 1990). Details of this areshown in Figs. 20 and 22. In Pterospora andromedea andSarcodes sanguinea, however, fungal pegs have their originfrom Hartig net hyphae that penetrate radial walls of epider-mal cells (Robertson and Robertson 1982; Massicotte et al.2004; and see Fig. 21). Each fungal peg becomes encased ina complex of finger-like projections of wall material sur-rounded by a membrane (Fig. 22). This interface consists offungal plasma membrane, fungal wall, a plant-derived wall,and a plant-derived membrane (Fig. 22). The amplificationof wall and surrounding plasma membrane surface area issimilar to what occurs in transfer cells that are found where

there is rapid flux of materials (Gunning and Pate 1969).However, there is no experimental evidence for bidirectionaltransfer or, indeed, unidirectional transfer of nutrients at thisinterface.

A complicating feature of the “fungal peg” is that theplant and fungal wall at the tip apparently break down, re-sulting in a membranous sac extending into the epidermalcell (Duddridge and Read 1982; Robertson and Robertson1982; Dexheimer and Gérard 1993). Although it has beensuggested that nutrients might be transferred to the epider-mal cell at this point (Francke 1934), there is no evidencefor this. As pointed out by Robertson and Robertson (1982),

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the contents of this sac do not resemble those of the fungalpeg. The function of this structure remains enigmatic.

Other interfaces that are potentially involved in bidi-rectional transfer of nutrients include inner mantle andHartig net hyphae that are contiguous with epidermal cells,but, as yet, this has not been shown.

Orchid mycorrhizasAll orchid species have minute seeds that usually lack an

endosperm and have limited storage of reserves in the undif-ferentiated embryo (Rasmussen 1995). Subsequent develop-ment of the embryo into a protocorm is dependent on thecolonization of the germinating seed by soilborne fungi thatare able to convert complex carbon compounds in the sub-strate into simple sugars, some of which are transported tothe developing protocorm (Peterson et al. 1998). There maybe some specificity in the fungal species that associate withparticular orchid species at the seed germination stage. Forexample, the successful colonization of germinating seedsof Neottia nidus-avis in nature is dependent on a specificSebacina-like fungus (McKendrick et al. 2002). To sustaingrowth, the developing protocorm and seedling receive car-bon compounds via fungal hyphae (McKendrick et al. 2000;Alexander and Hadley 1985; Smith and Read 1997), butthere is no evidence that the fungal symbiont receives nutri-ents from the plant in exchange. At this early stage of orchidplant development, therefore, this is an example of anexploitive association (Brundrett 2002, 2004).

Both autotrophic and myco-heterotrophic species occur inthe family, Orchidaceae. Roots of autotrophic orchids be-come colonized by fungi, and these mainly supply the hostwith mineral nutrients such as nitrogen and P (Alexander etal. 1984; Smith and Read 1997), although there is some evi-dence that plants may also receive some carbon compoundsvia the fungal hyphae (Smith and Read 1997).

Myco-heterotrophic species are associated with fungi thatprovide hyphal links to neighbouring autotrophic plant spe-cies, through which they obtain photosynthates (Leake1994). There appears to be some specificity in fungi in-volved with particular myco-heterotrophic orchid species.For example, Cephalanthera austinae is associated withmycobionts belonging to the Thelephoraceae (Taylor andBruns 1997).

Regardless of whether fungi associate with developing

protocorms, roots of autotrophic species, or roots of myco-heterotrophic species, the colonization process is similar inthat hyphae within parenchyma cells of protocorms (Fig. 23)and roots (Fig. 24) form hyphal complexes called pelotons(Fig. 25). These are surrounded by a plant-derived mem-brane and an interfacial matrix (Fig. 26). These plant cell –fungus interfaces are assumed to be the sites of nutrientexchange in most orchid mycorrhizas. This mode of nutrientexchange has been termed tolypophagy (Burgeff 1909; Ras-mussen 2002). Pelotons undergo degradation (Fig. 23) and,subsequent to this, cells containing the remains of thepeloton can be recolonized. The interfacial matrix changesin composition, depending on the age of the peloton (Peter-son et al. 1996). Early in the colonization process, the inter-facial matrix lacks pectins, cellulose, and β-1,3-glucans, asdetermined by using various affinity probes; these wall com-ponents are, however, present following peloton senescence,and these encase the degenerating hyphae (Peterson et al.1996).

There is some evidence suggesting that the perifungalmembrane has different physiological characteristics fromthat of the peripheral plasma membrane. For example, inprotocorms of Spiranthes sinensis colonized by Cerato-basidium cornigerum, the perifungal membrane failed toreact for adenylate cyclase activity when cytochemical meth-ods were used, whereas the peripheral plasma membrane did(Uetake and Ishizaka 1995); the significance of this in termsof the functioning of the interface is not clear. Also, certainATPases are active on the perifungal membrane but not onthe peripheral plasma membrane (Serrigny and Dexheimer1985), and neutral and acid phosphatases have been local-ized on the perifungal membrane (Dexheimer et al. 1988).

As with AMs, the plant cytoskeleton undergoes consider-able reorganization in that both microtubules and actin fila-ments become closely associated with developing pelotons(Uetake et al. 1997; Uetake and Peterson 1997, 1998). Therole of these components of the cytoskeleton in establishingthe nutrient-exchange interface is unknown.

In some orchid species, there appears to be a unique modeof nutrient acquisition termed ptyophagy, first described byBurgeff (1909) and more recently by Wang et al. (1997),in the orchid Gastrodia elata. In this type, degradation ofhyphae within regions of roots containing “digestive cells”results in the release of nutrients that are then presumably

Figs. 17–19. Interfaces in arbutoid mycorrhizas. Fig. 17. Longitudinal section of Arbutus menziesii – Pisolithus tinctorius mycorrhiza.A thick mantle (M), Hartig net hyphae (arrowheads), and intracellular hyphae (double arrowheads) are present. Material embedded inLR White resin, stained with acriflavine HCl, and viewed with blue light using epifluorescence microscopy. Scale bar = 100 µm.Fig. 18. Portion of an Arbutus menziesii – Piloderma bicolor mycorrhiza showing a thin mantle (M), paraepidermal Hartig net hyphae(arrowheads), and sectioned intracellular hyphal complexes (double arrowheads) within epidermal cells. Material embedded in LRWhite resin, stained with toluidine blue O, and viewed with light microscopy. Scale bar = 25 µm. Fig. 19. Diagram of interfaces. Man-tle (M) and paraepidermal Hartig net (HN) hyphae contact the wall of epidermal cells. Intracellular hyphae are separated from the epi-dermal cell cytoplasm by a perifungal membrane (arrowheads) and interfacial matrix material (double arrowheads). Scale bar =25 µm. Figs. 20–22. Interfaces in monotropoid mycorrhizas. Fig. 20. Mantle (M), paraepidermal Hartig net (arrow), and fungal pegs(arrowheads) in portion of Monotropa uniflora root. Material prepared as in Fig. 5. Scale bar = 50 µm. Fig. 21. Portion of aPterospora andromedea root showing mantle (M), paraepidermal Hartig net (arrows), and fungal peg (arrowhead). Material prepared asin Fig. 5. Scale bar = 25 µm. Fig. 22. Diagram showing the interfaces in monotropoid mycorrhizas. Mantle (M) and Hartig net (ar-rows) hyphae contact epidermal cell walls. In addition, hyphal pegs (*) are enveloped by epidermal-cell-derived wall material (arrow-heads) and plasma membrane (double arrowheads). Scale bar = 25 µm.

taken up by intact pelotons in adjacent cells. As pointed outby Rasmussen (2002), this appears to be a novel mycorrhizatype and deserves further study.

Nonvascular plant mycorrhizasThe Bryophytes consist of three divisions: Hepatophyta

(liverworts), Anthocerophyta (hornworts), and Bryophyta(mosses). For reasons unknown, only the mosses have failedto establish associations with symbiotic fungi (Read et al.2000). The majority of experimental and structural work hasinvolved liverworts, perhaps because of the diversity in mor-phology of the dominant gametophyte phase and the factthat fungi involved in the symbioses include members of thethree groups of mycorrhizal fungi (Read et al. 2000). It isbeyond the scope of this review to consider in detail the re-search published on bryophytes, but examples are given toillustrate the variation in symbiotic associations and possiblenutrient-exchange interfaces that develop.

In associations with AM fungi, the thalli of the genusPellia develop typical arbuscules (Read et al. 2000).

Schüßler (2000), using two isolates of Glomus claroideum,has also demonstrated arbuscule formation in the thallus ofthe hornwort Anthoceros punctatus. Collections of the primi-tive liverwort Haplomitrium showed a symbiotic associationthat resembled an AM association, but hyphae were re-stricted to the outermost layer of cells in the organs colo-nized (Carafa et al. 2003). In the latter example, fine hyphaeresembled arbuscular branches in that they were surroundedby a periarbuscular membrane and interfacial matrix mate-rial, but collapsed fungal swellings were very unusual in thatthey were of wide diameter and were encased by very thickinterfacial matrix material containing crystalline deposits.As indicated by Read et al. (2000), the functional aspect ofthese associations has yet to be explored.

The rhizoids of some liverworts associate with Hymeno-scyphus ericae, an ascomycete that forms ericoid mycor-rhizas with some members of the Ericaceae (Duckett et al.1991; Duckett and Read 1995; Read et al. 2000). Rhizoidtips become swollen as they are colonized by fungal hyphaeand peg-like wall deposits occur around the penetrating

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Figs. 23–26. Interface in orchid mycorrhizas. Fig. 23. Section of Goodyera repens protocorm colonized by Ceratobasidium cereale.Sections of intracellular complexes (pelotons) (arrowheads), as well as collapsed pelotons (double arrowheads), are present. Materialprepared as in Fig. 17. Scale bar = 100 µm. Fig. 24. Transverse section of Cypripedium arietinum root showing sections of pelotons(arrowheads) and collapsed pelotons (double arrowheads). Material prepared as in Fig. 5. Photo courtesy of Carla Zelmer. Scale bar =100 µm. Fig. 25. Peloton in root of Paphiopedilum sanderianum. Material was embedded in LR White resin, stained with sulfarho-damine, and viewed with scanning laser confocal microscopy. Photo courtesy of Carla Zelmer. Scale bar = 50 µm. Fig. 26. Diagram ofpeloton showing the interface between fungus and root cell. The fungus is separated from the root cytoplasm by a perifungal mem-brane (arrowheads), and interfacial matrix material (double arrowheads). Scale bar = 50 µm.

hyphae (Duckett et al. 1991). These authors compare this tothe fungal pegs in monotropoid mycorrhizas, noting the ab-sence of the elaborated branched wall structure, and con-clude that this site is where nutrient exchange occurs.

In a combined structural and molecular study, Kottke et al.(2003) examined the fungal associations of species in 12families of leafy liverworts and found considerable variabil-ity in fungal associates. They concluded that both asco-mycete and basidiomycete hyphae could become surroundedby host-derived wall material and agreed with Read et al.(2000) that these peg-like structures are probable sites of nu-trient exchange. Kottke et al. (2003) have proposed the term“jungermannioid mycorrhiza” for symbiotic associationsbetween members in one clade of leafy liverworts, theJungermanniidae, and either basidiomycete or ascomycetefungal symbionts.

Interestingly, it has been demonstrated that some leafy liv-erworts can form symbiotic associations with basidiomycetefungi that can also form mycorrhizas with adjacent tree spe-cies (Read et al. 2000). These develop a typical mantle andHartig net in the tree roots but coiled hyphal complexes inthe liverwort. This suggests that the same fungus can formdivergent nutrient-exchange interfaces depending on the hostand can potentially establish fungal bridges between vascu-lar and nonvascular plants.

Future work with liverworts should include a more in-depth study of the nutrient-exchange interface between thesymbionts.

Discussion

As suggested by Brundrett (2004), any attempt to redefineand recategorize mycorrhizas must be based on understand-ing the variation that occurs in these associations. With re-spect to the symbionts in question, this is relevant at variouslevels, including the development of the interface that is es-tablished at the cellular level. From the literature and fromour own observations on the development and functioningof interfaces between symbionts (Peterson et al. 2004), itis clear that most information available is for AMs andectomycorrhizas. This reflects the fact that most plant spe-cies develop either one or the other of these mycorrhizas(Smith and Read 1997). It is also evident that, regardless ofthe mycorrhiza category, the interface between symbiontsincludes an apoplastic compartment and opposing plasmamembranes of the symbionts (Smith and Smith 1990;Dexheimer and Pargney 1991). With the exception ofectomycorrhizas, in which the exchange interface is externalto plant cell walls, interfaces of all other mycorrhizas in-volve the formation of an intracellular apoplastic compart-ment (Smith and Smith 1990), the chemical nature of whichhas only been studied for a few mycorrhiza categories.

The evidence for transfer of nutrients is mostly indirect,relying on structural features of the interface and membraneproperties of the symbionts forming this interface (Smithand Smith 1990). Characterization of the membranes interms of activation of various transporters and other featuresneeds to be extended beyond the studies that have primarilyinvolved AMs (Harrison 1999a, 1999b; Saito 2000) andectomycorrhizas (Chalot et al. 2002). More direct ap-proaches, such as microautoradiography, as used by Bücking

and Heyser (2001, 2003) in their study of transfer of labelledP and carbon compounds in ectomycorrhizas, could be usedto confirm nutrient transfer through the interface of othermycorrhizas.

Although it has been suggested that the interfaces between“balanced” mycorrhizas and “exploitive” mycorrhizas arelikely to differ, with lysis of fungal hyphae in exploitivemycorrhizas being more important in the release of nutrients(Brundrett 2004), further research such as that by Imhof(2003) on a range of mycorrhizal associations could verifythis.

Considerable interest exists in exploring the interfacecharacteristics of symbiotic associations of nonvascularplants, particularly the liverworts, because of the diversitythat is being discovered in this primitive group of plants(Read et al. 2000; Carafa et al. 2003; Kottke et al. 2003).These systems may provide valuable clues with respect tothe evolution of mycorrhizal symbioses (Kottke et al. 2003).

We suggest that the present categories of mycorrhizas beretained for symbiotic associations with vascular plants butthat consideration be given to erecting one or more catego-ries for symbiotic associations with nonvascular plants.Kottke et al. (2003) have initiated this discussion. We alsosuggest that there is scope for further structural studies ofmycorrhizal categories that have received limited attention todate.

Acknowledgements

We are indebted to Lewis Melville for the drawings,Linda Tackaberry and two anonymous reviewers for valuablecomments on the manuscript, and the Natural Sciences andEngineering Research Council of Canada (NSERC) for fi-nancial support.

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