Exploring structural definitions of mycorrhizas, with emphasis on ...

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<strong>Exploring</strong> <strong>structural</strong> <strong>definitions</strong> <strong>of</strong> <strong>mycorrhizas</strong>,
<strong>with</strong> <strong>emphasis</strong> on nutrient-exchange interfaces 1
R. Larry Peterson and Hugues B. Massicotte
Abstract: The roots or other subterranean organs <strong>of</strong> most plants develop symbioses, <strong>mycorrhizas</strong>, <strong>with</strong> fungal symbionts.
Historically, <strong>mycorrhizas</strong> have been placed into seven categories based primarily on <strong>structural</strong> characteristics. A
new category has been proposed for symbiotic associations <strong>of</strong> some leafy liverworts. An important feature <strong>of</strong>
<strong>mycorrhizas</strong> is the interface involved in nutrient exchange between the symbionts. With the exception <strong>of</strong>
ecto<strong>mycorrhizas</strong>, in which fungal hyphae remain external to plant cell walls, all <strong>mycorrhizas</strong> are characterized by fungal
hyphae breaching cell walls but remaining separated from the cell cytoplasm by a plant-derived membrane and an
interfacial matrix that forms an apoplastic compartment. The chemical composition <strong>of</strong> the interfacial matrix varies in
complexity. In arbuscular <strong>mycorrhizas</strong> (both Arum-type and Paris-type), molecules typical <strong>of</strong> plant primary cell walls
(i.e., cellulose, pectins, β-1,3-glucans, hydroxyproline-rich glycoproteins) are present. In ericoid <strong>mycorrhizas</strong>, only
rhamnogalacturonans occur in the interfacial matrix surrounding intracellular hyphal complexes. The matrix around
intracellular hyphal complexes in orchid <strong>mycorrhizas</strong> 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 studied
in arbutoid <strong>mycorrhizas</strong> and ectendo<strong>mycorrhizas</strong>. In ecto<strong>mycorrhizas</strong>, the apoplastic interface consists <strong>of</strong> plant cell wall
and fungal cell wall; alterations in these may enhance nutrient transfer. In all <strong>mycorrhizas</strong>, nutrients must pass into the
symplast <strong>of</strong> both partners at some point, and therefore current research is exploring the nature <strong>of</strong> the opposing membranes,
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 des
champignons, les mycorhizes. Historiquement, on a placé les mycorhizes dans sept catégories, basées surtout sur des
caractéristiques <strong>structural</strong>es. Une nouvelle catégorie a été proposée pour les associations symbiotiques de certaines hépatiques
foliacées. Une importante caractéristique des mycorhizes est l’interface impliqué dans les échanges de nutriments
entre les symbiotes. À l’exception des ectomycorhizes, dans lesquelles les champignons demeurent à l’extérieur
des parois cellulaires des plantes, toutes les mycorhizes se caractérisent par des hyphes fongiques traversant les parois
cellulaires, mais demeurant séparés du cytoplasme par une membrane dérivée de la plante et une matrice interfaciale
formant un compartiment apoplastique. La composition chimique de la matrice interfaciale varie en complexité. Chez
les mycorhizes arbusculaires (de type Arum aussi bien que Paris), on retrouve des molécules typiques des parois cellulaires
primaires (c.-à-d., cellulose, pectines, β-1,3-glucans, glycoprotéines riches en hydroxyproline). Chez les mycorhizes
éricoïdes, il n’y a que des rhamnogalacturones dans la matrice interfaciale entourant les complexes d’hyphes
intracellulaires. La matrice entourant les complexes intracellulaires ne comporte pas de matériel pariétal de la plante
chez les mycorhizes des orchidées, jusqu’à ce que les hyphes deviennent sénescentes; on retrouve alors des molécules
similaires à celles des parois primaires. La matrice interfaciale n’a pas été étudiée chez les mycorhizes arbutoïdes et les
ectendomycorhizes. Chez les ectomycorhizes, l’interface apoplastique est constituée de paroi cellulaire végétale et de
paroi cellulaire fongique; une altération de ces parois peut augmenter le transfert de nutriments. Chez toutes les mycorhizes,
les nutriments doivent passer par le symplaste des deux partenaires à un point donné, et c’est pourquoi les recherches
actuelles portent sur la nature des membranes opposées, surtout en relation avec les transporteurs de sucre et
de phosphore.
Mots clés : interface, compartiment apoplastique, réseau de Hartig, arbuscule, complexe intracellulaire, échange de nutriments.
[Traduit par la Rédaction] Peterson and Massicotte 1088
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 <strong>of</strong> Botany, University <strong>of</strong> Guelph, Guelph, ON N1G 2W1, Canada.
H.B. Massicotte. 2 Ecosystem Science & Management Program, College <strong>of</strong> Science and Management, University <strong>of</strong> Northern British
Columbia, 3333 University Way, Prince George, B.C. Canada.
1 This article is one <strong>of</strong> a selection <strong>of</strong> papers published in the Special Issue on Mycorrhizae and was presented at the Fourth
International Conference on Mycorrhizae.
2 Corresponding author (e-mail: hugues@unbc.ca).
Can. J. Bot. 82: 1074–1088 (2004) doi: 10.1139/B04-071 © 2004 NRC Canada

Peterson and Massicotte 1075
Introduction
Mycorrhizas are usually defined as mutualistic symbioses
between fungi and plants in which both partners can benefit
from the association (Smith and Read 1997). It is recognized,
however, that it is <strong>of</strong>ten difficult to assess the benefits
derived by each <strong>of</strong> the symbionts in mutualistic associations,
since these must be considered in terms <strong>of</strong> the costs to each
partner (Ahmadjian and Paracer 1986). There is considerable
discussion as to what constitutes a benefit to each <strong>of</strong> the
partners in <strong>mycorrhizas</strong> as environmental conditions change.
In <strong>mycorrhizas</strong>, the main benefit to the plant is largely nutritional
in nature, but protection from root pathogens and improved
water relations may also accrue.
The term “balanced <strong>mycorrhizas</strong>” has been proposed to
denote situations in which both organisms receive essential
materials through reciprocal exchange (Brundrett 2002,
2004). An important step in the evolution <strong>of</strong> balanced
<strong>mycorrhizas</strong> was, therefore, the development <strong>of</strong> a specialized
interface zone for bidirectional nutrient exchange (Brundrett
2002).
The term “exploitive mycorrhizal associations” has been
suggested for those situations in which there is a unidirectional
flow <strong>of</strong> nutrients, <strong>with</strong> the main benefit usually
going to the plant partner (Brundrett 2002, 2004). Mycoheterotrophic
plant species <strong>with</strong>out photosynthetic ability,
and green plants in situations where the fungus seems to
gain little from the association, could be considered as examples
<strong>of</strong> exploitive <strong>mycorrhizas</strong> (Brundrett 2002, 2004). It
should be emphasized, however, that whether balanced or
exploitive, mycorrhizal associations are the norm for almost
all vascular plants and a few nonvascular plants (Smith and
Read 1997; Read et al. 2000). Mycorrhizal symbioses can,
therefore, be distinguished from interactions between pathogenic
fungi and roots, in which nutrient flow is always to the
fungi.
Brundrett (2004) suggests that the nutrient-exchange interface
established between the symbionts is unlikely to function
in the same manner for balanced and exploitive
mycorrhizal associations. To evaluate this suggestion, it is
important to consider the <strong>structural</strong> aspects <strong>of</strong> interfaces for
all categories <strong>of</strong> <strong>mycorrhizas</strong> and to assess the evidence that
these sites function in nutrient exchange.
In parallel <strong>with</strong> the evolution <strong>of</strong> nutrient-exchange interfaces,
the extraradical mycelium <strong>of</strong> successful symbioses developed
as an efficient soil–fungal interface to allow for
exploration and extraction <strong>of</strong> nutrients and water from the
substrate. This topic is discussed by Leake et al. (2004) in
this Special Issue.
In this review, we will use the seven traditional categories
<strong>of</strong> vascular plant <strong>mycorrhizas</strong> based on <strong>structural</strong> characteristics
identified by light and transmission electron microscopy
(Scannerini and Bonfante-Fasolo 1983; Bonfante-
Fasolo and Scannerini 1992; Peterson and Farquhar 1994;
Smith and Read 1997; Peterson et al. 2004), recognizing that
there is some discussion as to new ways <strong>of</strong> classifying
mycorrhiza types (Brundrett 2004). These categories are
arbuscular <strong>mycorrhizas</strong> (AM), ecto<strong>mycorrhizas</strong>, ectendo<strong>mycorrhizas</strong>,
ericoid <strong>mycorrhizas</strong>, arbutoid <strong>mycorrhizas</strong>, monotropoid
<strong>mycorrhizas</strong>, and orchid <strong>mycorrhizas</strong>. We will also
include a brief discussion <strong>of</strong> <strong>mycorrhizas</strong> occurring <strong>with</strong>
nonvascular plants, since these are receiving more attention
in the literature and may provide insight into the evolution
<strong>of</strong> symbioses between plants and fungi (Read et al. 2000;
Kottke et al. 2003).
The main objectives <strong>of</strong> this review are, therefore, to discuss
what is known about the interfaces in all categories <strong>of</strong>
<strong>mycorrhizas</strong>, to evaluate the evidence for nutrient transfer at
these sites, to suggest future research that is needed, and
finally to comment on possible modifications in defining
categories <strong>of</strong> <strong>mycorrhizas</strong>.
Categories <strong>of</strong> vascular plant <strong>mycorrhizas</strong>
Arbuscular <strong>mycorrhizas</strong>
Arbuscular <strong>mycorrhizas</strong>, the symbiotic associations between
the majority <strong>of</strong> vascular plant species and fungi in the
new phylum Glomeromycota (Schüßler et al. 2001), can be
subdivided into two main types, the Arum-type and the
Paris-type (Gallaud 1905; Smith and Smith 1997). In the ontogeny
<strong>of</strong> interface development in the Arum-type (Fig. 1),
usually one arbuscule develops through repeated branching
<strong>of</strong> a hypha (trunk hypha) that penetrates through the cortical
cell wall (Bonfante and Perotto 1995). Paired arbuscules in
adjacent cortical cells arising from the same intercellular
hypha but developing at different rates have recently been
reported by Dickson et al. (2003) in Linum usitatissimum.
Figure 1 also illustrates paired arbuscules in cortical cells <strong>of</strong>
a Pisum sativum root, illustrating that this feature may be
more common than reported. In the Paris-type, penetration
<strong>of</strong> the cortical cell wall by a single hypha is followed by extensive
coiling <strong>of</strong> this hypha (Fig. 2) from which lateral
branches are initiated to form arbusculate coils (Whitbread
et al. 1996; Cavagnaro et al. 2001; also see Fig. 3). In the
Arum-type nutrient-exchange interface, the fungus develops
fine, thin-walled arbuscule branches <strong>with</strong>in plant cortical
cells, the walls <strong>of</strong> 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 the
Paris-type, both hyphal coils and arbusculate coils may be
involved in nutrient exchange, an idea suggested by calculations
that show that the surface area <strong>of</strong> hyphal coils may be
equal to that <strong>of</strong> arbuscules in Arum-type <strong>mycorrhizas</strong> (Dickson
and Kolesik 1999) as well as by the presence <strong>of</strong> a membrane
and interfacial matrix around hyphal coils and
arbusculate coils (Armstrong and Peterson 2002). Arbuscules
and arbusculate coils are separated from the corticalcell
cytoplasm by a periarbuscular membrane and interfacial
matrix material, both derived from the plant symbiont (Bonfante
and Perotto 1995; Armstrong and Peterson 2002).
The interfacial matrix, situated between the fungal cell
wall and the periarbuscular membrane, is an apoplastic compartment
composed <strong>of</strong> plant cell wall constituents, including
cellulose, pectins, β-1,3-glucans, and hydroxyproline-rich
glycoproteins, as demonstrated by the use <strong>of</strong> various affinity
probes (Bonfante and Perotto 1995; Armstrong and Peterson
2002) and in situ mRNA probes (Blee and Anderson 2000).
Bonfante and Perotto (1995) claim that the development <strong>of</strong>
this apoplastic compartment between fungus and plant cell is
the most important event marking successful colonization.
With the development <strong>of</strong> the interfacial matrix, any transfer
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1076 Can. J. Bot. Vol. 82, 2004
Figs. 1–4. Interfaces <strong>of</strong> arbuscular <strong>mycorrhizas</strong>. Fig. 1. Arbuscules <strong>of</strong> Glomus aggregatum in a Pisum sativum root. Material stained
<strong>with</strong> acid fuchsin and viewed <strong>with</strong> scanning laser confocal microscopy. Arbuscules are <strong>of</strong> the Arum type. Photo courtesy <strong>of</strong> Ryan Geil.
Scale bar = 25 µm. Fig. 2. Intracellular hyphal coils <strong>of</strong> Glomus intraradices in a Panax quinquefolius root. These coils are typical <strong>of</strong>
Paris-type arbuscular <strong>mycorrhizas</strong>. Material stained <strong>with</strong> acid fuchsin and viewed <strong>with</strong> scanning laser confocal microscopy. Scale bar =
25 µm. Fig. 3. Arbusculate coils <strong>of</strong> Glomus intraradices in a root <strong>of</strong> Panax quinquefolius. Arrowheads indicate fine branches formed
on hyphal coils. Material cleared, stained <strong>with</strong> chlorazol black E, and examined by light microscopy. Scale bar = 25 µm. Fig. 4. Diagram
<strong>of</strong> arbuscule illustrating the components <strong>of</strong> the interface. The periarbuscular membrane (arrowheads), interfacial matrix (*), fungal
cell wall (arrows), and fungal plasma membrane (double arrowheads) are evident. T, trunk hypha; I, intercellular hypha. Scale bar =
25 µm.
<strong>of</strong> nutrients from the plant cell to the fungus would involve
the periarbuscular membrane, the interfacial matrix, the fungal
wall, and the fungal plasma membrane (Fig. 4). The reverse
would also be true as nutrients pass from the fungus to
the plant cell (Smith and Smith 1990). Since there is a bidirectional
exchange <strong>of</strong> nutrients, <strong>with</strong> phosphate (P), nitrate,
and trace elements moving from the fungus to the plant and
sugars moving to the fungus (Franken et al. 2002), this
would be an example <strong>of</strong> a balanced relationship (Brundrett
2002, 2004).
H + -ATPase activity has been demonstrated cytochemically
in the fungal plasma membrane in the interface (Gianinazzi-
Pearson et al. 1991, 2000), and Saito (2000) has provided a
detailed account <strong>of</strong> the localization <strong>of</strong> various phosphatases
in host and fungal membranes <strong>of</strong> AM associations, evidence
that active transport is likely involved in nutrient transfer.
Ferrol et al. (2002) summarize the evidence for the upregulation
<strong>of</strong> host plasma membrane ATPase genes in
mycorrhizal roots <strong>of</strong> a number <strong>of</strong> plant species. Also, there
is a difference in plasma membrane proteins, as shown by
2D-PAGE analysis, between mycorrhizal and nonmycorrhizal
tomato (Lycopersicon esculentum) roots (Benabdellah
et al. 2000), but whether these are related to the
nutrient-exchange interface is not known.
Currently there is considerable interest in characterizing
transporters in both the periarbuscular membrane and the
fungal membrane, since these would be involved in the active
transport across this interface (Harrison 1999a, 1999b;
Smith et al. 2001; Ferrol et al. 2002). The recent review by
Ferrol et al. (2002) summarizes the information on P and
carbon transporters in AMs and points out the lack <strong>of</strong> information
concerning the presence and activity <strong>of</strong> these in the
nutrient-exchange interface.
Other changes that occur in cortical cells containing
arbuscules include alterations in expression <strong>of</strong> genes encoding
enzymes involved in sucrose metabolism (Blee and Anderson
2002), leading to the establishment <strong>of</strong> a sink for
sucrose transport from the phloem to the arbuscules.
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Peterson and Massicotte 1077
The deposition <strong>of</strong> the interfacial matrix and the formation
<strong>of</strong> the periarbuscular membrane must involve considerable
cellular activity, and it has been suggested that alterations
observed in the plant-cell cytoskeleton during arbuscule development
may play a role in these processes (Genre and
Bonfante 1997, 1998; Peterson et al. 2000; Armstrong and
Peterson 2002; Timonen and Peterson 2002). Methods combining
GFP (green fluorescent protein) tagging <strong>of</strong> cell components
such as endoplasmic reticulum and Golgi bodies, in
addition to cytoskeletal elements, and scanning laser confocal
microscopy (Takemoto et al. 2003) could add additional
information as to how interfaces are formed between symbionts.
Intercellular hyphae, although not surrounded by either
plant-derived membrane or interfacial matrix, interface <strong>with</strong>
the apoplast (intercellular space system) <strong>of</strong> roots (Ferrol et
al. 2002). They have the potential to be involved in the transfer
<strong>of</strong> nutrients, and it has been suggested that this might be
a pathway for carbon acquisition by the fungus (Smith and
Smith 1990). In this case, only the fungal plasma membrane
would be involved in the uptake <strong>of</strong> carbon compounds.
Ecto<strong>mycorrhizas</strong>
Ecto<strong>mycorrhizas</strong>, symbioses formed between both gymnosperm
and angiosperm species and many basidiomycete
fungi and fewer ascomycete fungi, are characterized primarily
by the presence <strong>of</strong> a mantle (sheath) and an Hartig net
consisting <strong>of</strong> modified fungal hyphae that develop between
root cells (Smith and Read 1997). Generally, species belonging
to the angiosperms have a paraepidermal Hartig net (i.e.,
confined to the root epidermis, as shown in Fig. 5), whereas
species belonging to the gymnosperms have an Hartig net
that 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 epidermal
and cortical cells (Melville et al. 1988). In some species the
Hartig net may reach the endodermis, although this is variable.
Because <strong>of</strong> the intimate association <strong>of</strong> Hartig net
hyphae <strong>with</strong> root cell walls (Fig. 7), the labyrinthine branching
(Fig. 8) <strong>of</strong> these hyphae (Kottke and Oberwinkler 1986,
1987), and the presence <strong>of</strong> numerous mitochondria and other
organelles in Hartig net hyphae (Massicotte et al. 1986,
1990), it has been concluded that this is the main interface
involved in bidirectional transfer <strong>of</strong> nutrients (Kottke and
Oberwinkler 1986; Massicotte et al. 1987, 1989; Smith and
Read 1997). Inner mantle hyphae that are not part <strong>of</strong> the
Hartig net but are positioned immediately adjacent to the
outer tangential wall <strong>of</strong> root epidermal cells provide a second
possible interface for nutrient exchange in ecto<strong>mycorrhizas</strong>
(Dexheimer and Gérard 1994) (Figs. 9, 10). These
hyphae may also become highly branched, and in the few
cases in which an Hartig net does not develop (e.g., in
Pisonia grandis), it is the only interface for nutrient exchange
(Ashford and Allaway 1982).
More direct evidence that the Hartig net is involved in the
transfer <strong>of</strong> P and carbon compounds comes from the use <strong>of</strong>
radioactive tracers followed by microautoradiography <strong>of</strong> rapidly
frozen and freeze-substituted poplar (Populus tremuloides
× Populus alba) ecto<strong>mycorrhizas</strong> (Bücking and
Heyser 2001). In this study, 33 P i accumulated rapidly in the
mantle and later was localized in the Hartig net and cortical
cells; the authors interpret the results to support the concept
that P moves across the mantle through the symplast and not
the apoplast. Also, mantle hyphae and Hartig net hyphae
in the median region <strong>of</strong> ectomycorrhizal roots were more
highly labelled than in either the apical or basal region, indicating
that there are some spatial differences in uptake and
translocation <strong>of</strong> P along the root axis. Similar results were
obtained for the distribution <strong>of</strong> labelled carbohydrates. This
supports the observation that there is a gradient <strong>of</strong> host-cell
response to fungi forming the Hartig net along the long axis
<strong>of</strong> a root (Massicotte et al. 1986). It is known, also, that
ecto<strong>mycorrhizas</strong> <strong>of</strong> different ages affect the absorption and
translocation <strong>of</strong> P (Cairney and Alexander 1992a) and carbon
compounds (Cairney and Alexander 1992b).
In some <strong>mycorrhizas</strong>, such as those formed in P. grandis
(Ashford and Allaway 1982) and between Alnus crispa and
Alpova diplophloeus (Massicotte et al. 1986), epidermal
cells contiguous <strong>with</strong> mantle hyphae and Hartig net hyphae,
if present, develop wall ingrowths (Fig. 10) that are enveloped
by plasma membrane and assume the structure <strong>of</strong>
transfer cells. Transfer-cell development is also found in several
other ecto<strong>mycorrhizas</strong> (Dexheimer and Gérard 1994).
The increase in surface area <strong>of</strong> wall and membrane <strong>of</strong> transfer
cells in P. grandis, although less than provided by the development
<strong>of</strong> an Hartig net in other ecto<strong>mycorrhizas</strong>, may
be adequate for transfer <strong>of</strong> nutrients in the environment in
which this species is found (Allaway et al. 1985).
Regardless <strong>of</strong> whether root cells differentiate as transfer
cells, or whether Hartig net or inner mantle hyphae are involved,
the interface between symbionts consists <strong>of</strong> contiguous
root cell walls and fungal hyphal walls <strong>with</strong> material
(sometimes called cement) deposited between them, creating
an apoplastic compartment. Plasma membranes <strong>of</strong> each symbiont
would be involved during the bidirectional transfer <strong>of</strong>
nutrients (see Figs. 9, 10). It is important to ascertain, therefore,
whether changes that would enhance nutrient exchange
occur in root cell walls and hyphal walls as an ectomycorrhiza
is formed. Features <strong>of</strong> root-cell plasma membranes
and fungal plasma membranes in the interface region may
also enhance nutrient exchange.
Both plant cell walls and fungal cell walls play an integral
role during all stages in the establishment and functioning <strong>of</strong>
ecto<strong>mycorrhizas</strong>, and considerable progress has been made
in determining changes in both walls at the molecular level
(Duplessis et al. 2002; Tagu et al. 2002). However, only
information related to the walls <strong>of</strong> the symbionts in the
nutrient-exchange interface will be considered here.
One change in host cell walls observed at the ultra<strong>structural</strong>
level is the apparent continuity between the wall
and the intercellular matrix adjacent to Hartig net hyphae,
suggesting that the cell wall, in particular, is modified in the
interface (Dexheimer and Gérard 1994). Also, in two gymnosperm
ectomycorrhizal associations, large amounts <strong>of</strong> acid
polysaccharides (pectins) are present in cortical cell walls as
well as in the matrix material separating these walls and
Hartig net hyphae (Nylund 1987). This author concludes that
this supports the concept that there is a “mycorrhizal infection
zone” in roots, in which changes to host walls enable
the development <strong>of</strong> the intercellular Hartig net; no comment
was made in terms <strong>of</strong> nutrient exchange in this zone. Using a
variety <strong>of</strong> affinity techniques, Balestrini et al. (1996) showed
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Peterson and Massicotte 1079
Figs. 5–10. Features <strong>of</strong> interfaces in ecto<strong>mycorrhizas</strong>. Fig. 5. Longitudinal section <strong>of</strong> a root tip <strong>of</strong> Alnus crispa colonized by Alpova
diplophloeus. A thick mantle (M) and a paraepidermal Hartig net (arrowheads) are evident. Material embedded in resin, stained <strong>with</strong>
toluidine blue O, and examined <strong>with</strong> light microscopy. Scale bar = 50 µm. Fig. 6. Longitudinal section <strong>of</strong> Pinus strobus root colonized
by Pisolithus tinctorius showing dichomtomy <strong>of</strong> apex, a thin mantle (M), and Hartig net hyphae (arrowheads) interfaced <strong>with</strong> epidermal
and cortical cells. Material prepared as in Fig. 5. Scale bar = 50 µm. Fig. 7. Higher magnification <strong>of</strong> Hartig net region from material
similar to that in Fig. 5 showing Hartig net hyphae (arrowheads) interfaced <strong>with</strong> 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 mycorrhiza.
Material prepared as in Fig. 5. Bar = 50 µm. Fig. 9. Diagram illustrating the interface between fungus and root cells in
ecto<strong>mycorrhizas</strong>. The root cell wall (CW) abuts on the cell wall <strong>of</strong> branched inner mantle (arrowhead) and branched Hartig net (double
arrowheads) hyphae. Nutrient exchange would also involve the plasma membrane <strong>of</strong> root cells (large arrows) and the plasma membrane
<strong>of</strong> fungal hyphae (small arrows). Scale bar = 50 µm. Fig. 10. The interface region <strong>of</strong> some ecto<strong>mycorrhizas</strong> may involve wall ingrowths
(arrowheads) in root cells, which increases the surface area for nutrient exchange. Scale bar = 50 µm.
that in ecto<strong>mycorrhizas</strong> formed between Corylus avellana
and Tuber magnatum, there were no qualitative changes in
the chemical nature <strong>of</strong> cortical cell walls <strong>of</strong> colonized roots
compared <strong>with</strong> uncolonized roots. However, there was an
apparent swelling <strong>of</strong> some cortical cell walls and the appearance
<strong>of</strong> electron-lucent areas in these walls in the region <strong>of</strong>
the Hartig net. They concluded that their observations support
the premise that ectomycorrhizal fungi secrete hydrolytic
enzymes to facilitate penetration <strong>of</strong> hyphae between
root cells (Cairney and Burke 1994). What needs to be determined
is whether modifications to host cell walls that are
contiguous <strong>with</strong> Hartig net and inner mantle hyphae result in
increased permeability to inorganic ions and organic molecules
compared <strong>with</strong> cell walls not adjacent to fungal
hyphae.
Modifications <strong>of</strong> Hartig net fungal walls have been demonstrated
using the Gomori–Swift cytochemical method for
cystein-containing proteins (Paris et al. 1993). In these hyphal
walls, only one poorly reactive layer was present, compared
<strong>with</strong> walls <strong>of</strong> mantle hyphae, in which both a highly
reactive and a poorly reactive layer were present. These authors
suggest that the simpler organization <strong>of</strong> the Hartig net
hyphal walls may enhance nutrient exchange, but no physiological
evidence for this was provided. In ecto<strong>mycorrhizas</strong>
formed between Eucalyptus globulus and Pisolithus
tinctorius, a group <strong>of</strong> symbiosis-related acidic polypeptides
are enhanced during the colonization process (Burgess et al.
1995), and these have been immunolocalized to the hyphal
cell wall, including the walls <strong>of</strong> mantle and Hartig net
hyphae (Laurent et al. 1999; Tagu et al. 2000, 2002). Since
symbiosis-related acidic polypeptides are present in hyphal
walls at all stages <strong>of</strong> mycorrhiza development, their significance
in enhancing nutrient exchange is uncertain.
One characteristic <strong>of</strong> host cell walls in the Hartig net region
is the presence <strong>of</strong> acid invertases that convert sucrose to
fructose and glucose in the apoplastic compartment; these
sugars can then be taken up by Hartig net hyphae (Salzer
and Hager 1993; Nehls et al. 2000, 2001b).
There has been and continues to be considerable interest
in determining the involvement <strong>of</strong> the host and fungal
plasma membranes in nutrient exchange in ecto<strong>mycorrhizas</strong>.
Earlier research demonstrated cytochemically that acid phosphatase
(Dexheimer et al. 1986) and H + -ATPase activity (Lei
and Dexheimer 1988) are localized along the plasma membranes
<strong>of</strong> both symbionts in the Hartig net region, indicating
that a bidirectional, active transport mechanism is likely involved
in nutrient exchange.
Although the movement <strong>of</strong> P from Hartig net hyphae into
the apoplastic interface is likely driven by a concentration
gradient (Bücking and Heyser 2000), the involvement <strong>of</strong> P
transporters in the fungal plasma membrane in active transport
has not been determined (Chalot et al. 2002). Likewise,
the existence <strong>of</strong> P transporters in host-cell plasma membranes
for P uptake from the apoplastic compartment in the
exchange interface zone has not been documented (Chalot et
al. 2002).
Although the movement <strong>of</strong> carbon compounds in the form
<strong>of</strong> sucrose is usually from root cells to the apoplastic compartment,
where, as noted above, acid invertase converts sucrose
to glucose and fructose that can be taken up by the
fungal hyphae, there is no direct evidence that hexose transporters
are involved. However, Nehls et al. (2001a) have
shown that, in Amanita–Populus ecto<strong>mycorrhizas</strong>, expression
<strong>of</strong> the gene encoding a monosaccharide transporter
(AmMst1) was enhanced six-fold in Hartig net hyphae compared
<strong>with</strong> mantle hyphae.
Ectomycorrhizal fungal hyphae do possess various transporters,
including P i transporters and hexose transporters
(see Chalot et al. 2002); it is important now to show their involvement
in the nutrient-exchange interface.
In gymnosperm and those angiosperm ecto<strong>mycorrhizas</strong> in
which most <strong>of</strong> the cortical cells are interfaced <strong>with</strong> Hartig
net hyphae, there is a question as to whether these cells
maintain plasmodesmata connections subsequent to the formation
<strong>of</strong> 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, potentially
providing a symplastic continuity from the phloem to all layers
<strong>of</strong> cortical cells. Research is needed, however, to determine
the extent <strong>of</strong> symplastic continuity from the phloem to
the epidermis in ecto<strong>mycorrhizas</strong>.
Ectendo<strong>mycorrhizas</strong>
There is some debate as to whether ectendo<strong>mycorrhizas</strong>
should be placed in a separate category, or considered either
as a modified ectomycorrhiza (Egger and Fortin 1988) or a
fungal morphotype (Brundrett 2004). For this discussion,
however, a separate category is maintained to emphasize the
fact that there appears to be only a few species <strong>of</strong> ascomycete
fungi that colonize mostly Pinus spp. and Larix
spp. roots to form a unique <strong>structural</strong> relationship (Yu et al.
2001). Most <strong>of</strong> the <strong>structural</strong> work on ectendo<strong>mycorrhizas</strong>
has involved pine species associated <strong>with</strong> one member <strong>of</strong> the
Pezizales, Wilcoxina mikolae; hence the full range <strong>of</strong> struc-
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1080 Can. J. Bot. Vol. 82, 2004
tural characteristics has yet to be determined. In those ectendo<strong>mycorrhizas</strong>
studied, a mantle and Hartig net forms as in
ecto<strong>mycorrhizas</strong>, but in addition, intracellular hyphal complexes
develop <strong>with</strong>in epidermal and cortical cells (Figs. 11,
12). As a result, three potential sites for nutrient exchange
are formed: the inner mantle – epidermal cell interface, the
Hartig net epidermal – cortical cell interface, and the intracellular
hyphal complex – root-cell cytoplasm. Hartig
net hyphae are branched, a perifungal membrane develops
around the intracellular hyphal complex (Fig. 13), and root
cells remain alive, as judged by the presence <strong>of</strong> a nucleus
and other organelles (Scales and Peterson 1991). When sections
are viewed <strong>with</strong> transmission electron microscopy an
interfacial matrix is apparent between the perifungal membrane
and the hyphal wall; however, its composition has not
been determined (Scales and Peterson 1991). There has been
no experimental work to determine if there is an exchange <strong>of</strong>
nutrients between the symbionts at any interface in this my-
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Peterson and Massicotte 1081
Figs. 11–13. Interfaces in ectendo<strong>mycorrhizas</strong>. Fig. 11. Longitudinal section <strong>of</strong> Pinus banksiana root colonized by Wilcoxina mikolae
var. mikolae. A thin mantle (arrows), Hartig net hyphae (arrowheads), and intracellular hyphae (double arrowhead) are present. Material
processed as in Fig. 5. Scale bar = 50 µm. Fig. 12. Higher magnification <strong>of</strong> portion <strong>of</strong> root shown in Fig. 11 showing Hartig net
hyphae (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 (*) <strong>with</strong> root cells. The
latter interface involves the perifungal membrane (arrowheads), interfacial material (double arrowheads), fungal cell wall (small arrows),
and the fungal plasma membrane (large arrow). P, plasmodesmata between cells. Scale bar = 50 µm. Figs. 14–16. Interface in
ericoid and epacrid <strong>mycorrhizas</strong>. Fig. 14. An intracellular hyphal complex <strong>with</strong>in an epidermal cell <strong>of</strong> a hair root <strong>of</strong> Kalmia
angustifolia. Material cleared, stained <strong>with</strong> acid fuchsin, and examined by differential interference contrast microscopy. Scale bar =
25 µm. Fig. 15. Epidermal cells <strong>of</strong> Gaultheria procumbens <strong>with</strong> intracellular hyphal complexes. Narrow hyphae (arrowheads) join
hyphal complexes (*) <strong>of</strong> adjacent epidermal cells. Material cleared, stained <strong>with</strong> acid fuchsin, and examined <strong>with</strong> scanning laser confocal
microscopy. Scale bar = 25 µm. Fig. 16. Diagram <strong>of</strong> intracellular hyphal complexes in epidermal cells <strong>of</strong> typical ericoid and
epacrid <strong>mycorrhizas</strong>. The outer tangential wall (*) <strong>of</strong> epidermal cells is thickened. The interface between the epidermal cell cytoplasm
and fungus is similar to that illustrated in Fig. 13, <strong>with</strong> perifungal membrane (arrowheads) and interfacial matrix material (double arrowheads)
separating the fungus from the epidermal cell cytoplasm. Scale bar = 25 µm.
corrhiza category, and nothing is known about the molecular
aspects <strong>of</strong> root colonization and the development <strong>of</strong> the
nutrient-exchange interface.
Ericoid <strong>mycorrhizas</strong>
There is considerable specificity shown in the formation
<strong>of</strong> ericoid <strong>mycorrhizas</strong> in that few fungal species, primarily
ascomycetes, form symbioses <strong>with</strong> members <strong>of</strong> the families
Ericaceae and Epacridaceae (Smith and Read 1997). Although
relatively few plant species in the Ericaceae and
Epacridaceae have been investigated intensively in terms <strong>of</strong>
their <strong>mycorrhizas</strong>, the colonization process is very similar
among those examined, regardless <strong>of</strong> the particular fungal
species involved (Perotto et al. 1995). Fungal hyphae contact
the thickened epidermal cell walls <strong>of</strong> the very fine roots
(hair roots), penetrate through these walls, and form intracellular
hyphal complexes <strong>with</strong>in epidermal cells (Figs. 14–
16). Although, in most instances, each epidermal cell is
colonized by a separate “unit”, we have observed some exceptions
to this in that fine hyphae can pass from epidermal
cell to epidermal cell (Fig. 15). In any event, the intracellular
hyphal coil is separated from the host cytoplasm by a perifungal
membrane and interfacial matrix material (Bonfante-
Fasolo and Gianinazzi-Pearson 1979; Bonfante-Fasolo and
Perotto 1988; Perotto et al. 1995). This is shown in Fig. 16.
The interfacial matrix differs in composition from that in
AM in that immunolabelling shows the presence <strong>of</strong> only
rhamnogalacturonan as a wall constituent and a low level <strong>of</strong>
labelling for the enzyme polygalacturonase (Perotto et al.
1995). As in other <strong>mycorrhizas</strong>, an apoplastic compartment
is present between the symbionts, and bidirectional transport
would have to occur across this interface (Fig. 16). Little
research has been conducted on this interface, although
ATPase activity has been localized on the perifungal membrane
(Gianinazzi-Pearson et al. 1984). There is considerable
scope in extending observations made <strong>with</strong> ericoid
<strong>mycorrhizas</strong> to <strong>mycorrhizas</strong> formed <strong>with</strong> epacrid plant species,
since there is limited <strong>structural</strong> work <strong>with</strong> this mycorrhiza
category (Cairney and Ashford 2002).
Arbutoid <strong>mycorrhizas</strong>
Arbutoid <strong>mycorrhizas</strong> occur in a group <strong>of</strong> species belonging
to the Ericales (Smith and Read 1997). Structurally, they
resemble ectendo<strong>mycorrhizas</strong> in that a mantle, Hartig net,
and intracellular hyphal complexes form (Fusconi and
Bonfante-Fasolo 1984; Münzenberger et al. 1992; Massicotte
et al. 1993), but they differ in that the intracellular
hyphal complexes are confined to the epidermis (Figs. 17,
19). In Arbutus menziesii, the outer row <strong>of</strong> cortical cells develops
suberin lamellae in their walls; these may limit the
development <strong>of</strong> the Hartig net to the epidermis (Massicotte
et al. 1993). The intracellular hyphal complexes in Arbutus
unedo <strong>mycorrhizas</strong> are surrounded by epidermal-cell plasma
membrane and an interfacial matrix material (Fusconi and
Bonfante-Fasolo 1984; Münzenberger et al. 1992; Filippi et
al. 1995). As in ectendo<strong>mycorrhizas</strong>, there are three possible
sites <strong>of</strong> nutrient exchange: the interface between inner mantle
hyphae and the tangential wall <strong>of</strong> epidermal cells, the interface
between Hartig net hyphae and epidermal cells, and
the interface between hyphal complexes and epidermal cell
cytoplasm (Figs. 18, 19). Although Münzenberger et al.
(1992) have shown that vesicles containing osmiophilic inclusions
fuse <strong>with</strong> the membrane surrounding intracellular
hyphae, experimental evidence is lacking as to the transfer
<strong>of</strong> nutrients between symbionts.
Monotropoid <strong>mycorrhizas</strong>
Monotropoid <strong>mycorrhizas</strong> occur in several genera belonging
to the subfamily Monotropoideae (family Ericaceae). All
plant hosts in this category are myco-heterotrophic and,
therefore, depend on adjacent autotrophic plant species for
their source <strong>of</strong> carbon (Leake 1994). Plants <strong>with</strong>in the
Monotropoideae form close associations <strong>with</strong> specific fungal
symbionts (Bidartondo and Bruns 2001, 2002; Young et al.
2002). These fungi form hyphal links between the roots
<strong>of</strong> heterotrophic plants and the roots <strong>of</strong> autotrophic plants,
through which carbon compounds can be transported. Currently,
there is no evidence that the fungus is receiving nutrients
from the heterotrophic host, therefore, according to
Brundrett (2002, 2004), this would be an example <strong>of</strong> an
exploitive mycorrhiza.
Structural features <strong>of</strong> monotropoid <strong>mycorrhizas</strong> are
unique (Figs. 20–22). A mantle and paraepidermal Hartig
net form but, in addition, fungal hyphae penetrate epidermal
cells to form “fungal pegs”, one per epidermal cell (Figs. 20,
21). In Monotropa species, fungal pegs originate from inner
mantle hyphae that penetrate the outer tangential wall <strong>of</strong> epidermal
cells (Lutz and Sjolund 1973; Duddridge and Read
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1082 Can. J. Bot. Vol. 82, 2004
1982; Snetselaar and Whitney 1990). Details <strong>of</strong> this are
shown in Figs. 20 and 22. In Pterospora andromedea and
Sarcodes sanguinea, however, fungal pegs have their origin
from Hartig net hyphae that penetrate radial walls <strong>of</strong> epidermal
cells (Robertson and Robertson 1982; Massicotte et al.
2004; and see Fig. 21). Each fungal peg becomes encased in
a complex <strong>of</strong> finger-like projections <strong>of</strong> wall material surrounded
by a membrane (Fig. 22). This interface consists <strong>of</strong>
fungal plasma membrane, fungal wall, a plant-derived wall,
and a plant-derived membrane (Fig. 22). The amplification
<strong>of</strong> wall and surrounding plasma membrane surface area is
similar to what occurs in transfer cells that are found where
there is rapid flux <strong>of</strong> materials (Gunning and Pate 1969).
However, there is no experimental evidence for bidirectional
transfer or, indeed, unidirectional transfer <strong>of</strong> nutrients at this
interface.
A complicating feature <strong>of</strong> the “fungal peg” is that the
plant and fungal wall at the tip apparently break down, resulting
in a membranous sac extending into the epidermal
cell (Duddridge and Read 1982; Robertson and Robertson
1982; Dexheimer and Gérard 1993). Although it has been
suggested that nutrients might be transferred to the epidermal
cell at this point (Francke 1934), there is no evidence
for this. As pointed out by Robertson and Robertson (1982),
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Peterson and Massicotte 1083
Figs. 17–19. Interfaces in arbutoid <strong>mycorrhizas</strong>. Fig. 17. Longitudinal section <strong>of</strong> Arbutus menziesii – Pisolithus tinctorius mycorrhiza.
A thick mantle (M), Hartig net hyphae (arrowheads), and intracellular hyphae (double arrowheads) are present. Material embedded in
LR White resin, stained <strong>with</strong> acriflavine HCl, and viewed <strong>with</strong> blue light using epifluorescence microscopy. Scale bar = 100 µm.
Fig. 18. Portion <strong>of</strong> an Arbutus menziesii – Piloderma bicolor mycorrhiza showing a thin mantle (M), paraepidermal Hartig net hyphae
(arrowheads), and sectioned intracellular hyphal complexes (double arrowheads) <strong>with</strong>in epidermal cells. Material embedded in LR
White resin, stained <strong>with</strong> toluidine blue O, and viewed <strong>with</strong> light microscopy. Scale bar = 25 µm. Fig. 19. Diagram <strong>of</strong> interfaces. Mantle
(M) and paraepidermal Hartig net (HN) hyphae contact the wall <strong>of</strong> epidermal cells. Intracellular hyphae are separated from the epidermal
cell cytoplasm by a perifungal membrane (arrowheads) and interfacial matrix material (double arrowheads). Scale bar =
25 µm. Figs. 20–22. Interfaces in monotropoid <strong>mycorrhizas</strong>. Fig. 20. Mantle (M), paraepidermal Hartig net (arrow), and fungal pegs
(arrowheads) in portion <strong>of</strong> Monotropa uniflora root. Material prepared as in Fig. 5. Scale bar = 50 µm. Fig. 21. Portion <strong>of</strong> a
Pterospora andromedea root showing mantle (M), paraepidermal Hartig net (arrows), and fungal peg (arrowhead). Material prepared as
in Fig. 5. Scale bar = 25 µm. Fig. 22. Diagram showing the interfaces in monotropoid <strong>mycorrhizas</strong>. Mantle (M) and Hartig net (arrows)
hyphae contact epidermal cell walls. In addition, hyphal pegs (*) are enveloped by epidermal-cell-derived wall material (arrowheads)
and plasma membrane (double arrowheads). Scale bar = 25 µm.
the contents <strong>of</strong> this sac do not resemble those <strong>of</strong> the fungal
peg. The function <strong>of</strong> this structure remains enigmatic.
Other interfaces that are potentially involved in bidirectional
transfer <strong>of</strong> nutrients include inner mantle and
Hartig net hyphae that are contiguous <strong>with</strong> epidermal cells,
but, as yet, this has not been shown.
Orchid <strong>mycorrhizas</strong>
All orchid species have minute seeds that usually lack an
endosperm and have limited storage <strong>of</strong> reserves in the undifferentiated
embryo (Rasmussen 1995). Subsequent development
<strong>of</strong> the embryo into a protocorm is dependent on the
colonization <strong>of</strong> the germinating seed by soilborne fungi that
are able to convert complex carbon compounds in the substrate
into simple sugars, some <strong>of</strong> which are transported to
the developing protocorm (Peterson et al. 1998). There may
be some specificity in the fungal species that associate <strong>with</strong>
particular orchid species at the seed germination stage. For
example, the successful colonization <strong>of</strong> germinating seeds
<strong>of</strong> Neottia nidus-avis in nature is dependent on a specific
Sebacina-like fungus (McKendrick et al. 2002). To sustain
growth, the developing protocorm and seedling receive carbon
compounds via fungal hyphae (McKendrick et al. 2000;
Alexander and Hadley 1985; Smith and Read 1997), but
there is no evidence that the fungal symbiont receives nutrients
from the plant in exchange. At this early stage <strong>of</strong> orchid
plant development, therefore, this is an example <strong>of</strong> an
exploitive association (Brundrett 2002, 2004).
Both autotrophic and myco-heterotrophic species occur in
the family, Orchidaceae. Roots <strong>of</strong> autotrophic orchids become
colonized by fungi, and these mainly supply the host
<strong>with</strong> mineral nutrients such as nitrogen and P (Alexander et
al. 1984; Smith and Read 1997), although there is some evidence
that plants may also receive some carbon compounds
via the fungal hyphae (Smith and Read 1997).
Myco-heterotrophic species are associated <strong>with</strong> fungi that
provide hyphal links to neighbouring autotrophic plant species,
through which they obtain photosynthates (Leake
1994). There appears to be some specificity in fungi involved
<strong>with</strong> particular myco-heterotrophic orchid species.
For example, Cephalanthera austinae is associated <strong>with</strong>
mycobionts belonging to the Thelephoraceae (Taylor and
Bruns 1997).
Regardless <strong>of</strong> whether fungi associate <strong>with</strong> developing
protocorms, roots <strong>of</strong> autotrophic species, or roots <strong>of</strong> mycoheterotrophic
species, the colonization process is similar in
that hyphae <strong>with</strong>in parenchyma cells <strong>of</strong> protocorms (Fig. 23)
and roots (Fig. 24) form hyphal complexes called pelotons
(Fig. 25). These are surrounded by a plant-derived membrane
and an interfacial matrix (Fig. 26). These plant cell –
fungus interfaces are assumed to be the sites <strong>of</strong> nutrient
exchange in most orchid <strong>mycorrhizas</strong>. This mode <strong>of</strong> nutrient
exchange has been termed tolypophagy (Burgeff 1909; Rasmussen
2002). Pelotons undergo degradation (Fig. 23) and,
subsequent to this, cells containing the remains <strong>of</strong> the
peloton can be recolonized. The interfacial matrix changes
in composition, depending on the age <strong>of</strong> the peloton (Peterson
et al. 1996). Early in the colonization process, the interfacial
matrix lacks pectins, cellulose, and β-1,3-glucans, as
determined by using various affinity probes; these wall components
are, however, present following peloton senescence,
and these encase the degenerating hyphae (Peterson et al.
1996).
There is some evidence suggesting that the perifungal
membrane has different physiological characteristics from
that <strong>of</strong> the peripheral plasma membrane. For example, in
protocorms <strong>of</strong> Spiranthes sinensis colonized by Ceratobasidium
cornigerum, the perifungal membrane failed to
react for adenylate cyclase activity when cytochemical methods
were used, whereas the peripheral plasma membrane did
(Uetake and Ishizaka 1995); the significance <strong>of</strong> this in terms
<strong>of</strong> the functioning <strong>of</strong> the interface is not clear. Also, certain
ATPases are active on the perifungal membrane but not on
the peripheral plasma membrane (Serrigny and Dexheimer
1985), and neutral and acid phosphatases have been localized
on the perifungal membrane (Dexheimer et al. 1988).
As <strong>with</strong> AMs, the plant cytoskeleton undergoes considerable
reorganization in that both microtubules and actin filaments
become closely associated <strong>with</strong> developing pelotons
(Uetake et al. 1997; Uetake and Peterson 1997, 1998). The
role <strong>of</strong> these components <strong>of</strong> the cytoskeleton in establishing
the nutrient-exchange interface is unknown.
In some orchid species, there appears to be a unique mode
<strong>of</strong> nutrient acquisition termed ptyophagy, first described by
Burgeff (1909) and more recently by Wang et al. (1997),
in the orchid Gastrodia elata. In this type, degradation <strong>of</strong>
hyphae <strong>with</strong>in regions <strong>of</strong> roots containing “digestive cells”
results in the release <strong>of</strong> nutrients that are then presumably
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1084 Can. J. Bot. Vol. 82, 2004
Figs. 23–26. Interface in orchid <strong>mycorrhizas</strong>. Fig. 23. Section <strong>of</strong> Goodyera repens protocorm colonized by Ceratobasidium cereale.
Sections <strong>of</strong> intracellular complexes (pelotons) (arrowheads), as well as collapsed pelotons (double arrowheads), are present. Material
prepared as in Fig. 17. Scale bar = 100 µm. Fig. 24. Transverse section <strong>of</strong> Cypripedium arietinum root showing sections <strong>of</strong> pelotons
(arrowheads) and collapsed pelotons (double arrowheads). Material prepared as in Fig. 5. Photo courtesy <strong>of</strong> Carla Zelmer. Scale bar =
100 µm. Fig. 25. Peloton in root <strong>of</strong> Paphiopedilum sanderianum. Material was embedded in LR White resin, stained <strong>with</strong> sulfarhodamine,
and viewed <strong>with</strong> scanning laser confocal microscopy. Photo courtesy <strong>of</strong> Carla Zelmer. Scale bar = 50 µm. Fig. 26. Diagram <strong>of</strong>
peloton showing the interface between fungus and root cell. The fungus is separated from the root cytoplasm by a perifungal membrane
(arrowheads), and interfacial matrix material (double arrowheads). Scale bar = 50 µm.
taken up by intact pelotons in adjacent cells. As pointed out
by Rasmussen (2002), this appears to be a novel mycorrhiza
type and deserves further study.
Nonvascular plant <strong>mycorrhizas</strong>
The Bryophytes consist <strong>of</strong> three divisions: Hepatophyta
(liverworts), Anthocerophyta (hornworts), and Bryophyta
(mosses). For reasons unknown, only the mosses have failed
to establish associations <strong>with</strong> symbiotic fungi (Read et al.
2000). The majority <strong>of</strong> experimental and <strong>structural</strong> work has
involved liverworts, perhaps because <strong>of</strong> the diversity in morphology
<strong>of</strong> the dominant gametophyte phase and the fact
that fungi involved in the symbioses include members <strong>of</strong> the
three groups <strong>of</strong> mycorrhizal fungi (Read et al. 2000). It is
beyond the scope <strong>of</strong> this review to consider in detail the research
published on bryophytes, but examples are given to
illustrate the variation in symbiotic associations and possible
nutrient-exchange interfaces that develop.
In associations <strong>with</strong> AM fungi, the thalli <strong>of</strong> the genus
Pellia develop typical arbuscules (Read et al. 2000).
Schüßler (2000), using two isolates <strong>of</strong> Glomus claroideum,
has also demonstrated arbuscule formation in the thallus <strong>of</strong>
the hornwort Anthoceros punctatus. Collections <strong>of</strong> the primitive
liverwort Haplomitrium showed a symbiotic association
that resembled an AM association, but hyphae were restricted
to the outermost layer <strong>of</strong> cells in the organs colonized
(Carafa et al. 2003). In the latter example, fine hyphae
resembled arbuscular branches in that they were surrounded
by a periarbuscular membrane and interfacial matrix material,
but collapsed fungal swellings were very unusual in that
they were <strong>of</strong> wide diameter and were encased by very thick
interfacial matrix material containing crystalline deposits.
As indicated by Read et al. (2000), the functional aspect <strong>of</strong>
these associations has yet to be explored.
The rhizoids <strong>of</strong> some liverworts associate <strong>with</strong> Hymenoscyphus
ericae, an ascomycete that forms ericoid <strong>mycorrhizas</strong>
<strong>with</strong> some members <strong>of</strong> the Ericaceae (Duckett et al.
1991; Duckett and Read 1995; Read et al. 2000). Rhizoid
tips become swollen as they are colonized by fungal hyphae
and peg-like wall deposits occur around the penetrating
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Peterson and Massicotte 1085
hyphae (Duckett et al. 1991). These authors compare this to
the fungal pegs in monotropoid <strong>mycorrhizas</strong>, noting the absence
<strong>of</strong> the elaborated branched wall structure, and conclude
that this site is where nutrient exchange occurs.
In a combined <strong>structural</strong> and molecular study, Kottke et al.
(2003) examined the fungal associations <strong>of</strong> species in 12
families <strong>of</strong> leafy liverworts and found considerable variability
in fungal associates. They concluded that both ascomycete
and basidiomycete hyphae could become surrounded
by host-derived wall material and agreed <strong>with</strong> Read et al.
(2000) that these peg-like structures are probable sites <strong>of</strong> nutrient
exchange. Kottke et al. (2003) have proposed the term
“jungermannioid mycorrhiza” for symbiotic associations
between members in one clade <strong>of</strong> leafy liverworts, the
Jungermanniidae, and either basidiomycete or ascomycete
fungal symbionts.
Interestingly, it has been demonstrated that some leafy liverworts
can form symbiotic associations <strong>with</strong> basidiomycete
fungi that can also form <strong>mycorrhizas</strong> <strong>with</strong> adjacent tree species
(Read et al. 2000). These develop a typical mantle and
Hartig net in the tree roots but coiled hyphal complexes in
the liverwort. This suggests that the same fungus can form
divergent nutrient-exchange interfaces depending on the host
and can potentially establish fungal bridges between vascular
and nonvascular plants.
Future work <strong>with</strong> liverworts should include a more indepth
study <strong>of</strong> the nutrient-exchange interface between the
symbionts.
Discussion
As suggested by Brundrett (2004), any attempt to redefine
and recategorize <strong>mycorrhizas</strong> must be based on understanding
the variation that occurs in these associations. With respect
to the symbionts in question, this is relevant at various
levels, including the development <strong>of</strong> the interface that is established
at the cellular level. From the literature and from
our own observations on the development and functioning
<strong>of</strong> interfaces between symbionts (Peterson et al. 2004), it
is clear that most information available is for AMs and
ecto<strong>mycorrhizas</strong>. This reflects the fact that most plant species
develop either one or the other <strong>of</strong> these <strong>mycorrhizas</strong>
(Smith and Read 1997). It is also evident that, regardless <strong>of</strong>
the mycorrhiza category, the interface between symbionts
includes an apoplastic compartment and opposing plasma
membranes <strong>of</strong> the symbionts (Smith and Smith 1990;
Dexheimer and Pargney 1991). With the exception <strong>of</strong>
ecto<strong>mycorrhizas</strong>, in which the exchange interface is external
to plant cell walls, interfaces <strong>of</strong> all other <strong>mycorrhizas</strong> involve
the formation <strong>of</strong> an intracellular apoplastic compartment
(Smith and Smith 1990), the chemical nature <strong>of</strong> which
has only been studied for a few mycorrhiza categories.
The evidence for transfer <strong>of</strong> nutrients is mostly indirect,
relying on <strong>structural</strong> features <strong>of</strong> the interface and membrane
properties <strong>of</strong> the symbionts forming this interface (Smith
and Smith 1990). Characterization <strong>of</strong> the membranes in
terms <strong>of</strong> activation <strong>of</strong> various transporters and other features
needs to be extended beyond the studies that have primarily
involved AMs (Harrison 1999a, 1999b; Saito 2000) and
ecto<strong>mycorrhizas</strong> (Chalot et al. 2002). More direct approaches,
such as microautoradiography, as used by Bücking
and Heyser (2001, 2003) in their study <strong>of</strong> transfer <strong>of</strong> labelled
P and carbon compounds in ecto<strong>mycorrhizas</strong>, could be used
to confirm nutrient transfer through the interface <strong>of</strong> other
<strong>mycorrhizas</strong>.
Although it has been suggested that the interfaces between
“balanced” <strong>mycorrhizas</strong> and “exploitive” <strong>mycorrhizas</strong> are
likely to differ, <strong>with</strong> lysis <strong>of</strong> fungal hyphae in exploitive
<strong>mycorrhizas</strong> being more important in the release <strong>of</strong> nutrients
(Brundrett 2004), further research such as that by Imh<strong>of</strong>
(2003) on a range <strong>of</strong> mycorrhizal associations could verify
this.
Considerable interest exists in exploring the interface
characteristics <strong>of</strong> symbiotic associations <strong>of</strong> nonvascular
plants, particularly the liverworts, because <strong>of</strong> the diversity
that is being discovered in this primitive group <strong>of</strong> plants
(Read et al. 2000; Carafa et al. 2003; Kottke et al. 2003).
These systems may provide valuable clues <strong>with</strong> respect to
the evolution <strong>of</strong> mycorrhizal symbioses (Kottke et al. 2003).
We suggest that the present categories <strong>of</strong> <strong>mycorrhizas</strong> be
retained for symbiotic associations <strong>with</strong> vascular plants but
that consideration be given to erecting one or more categories
for symbiotic associations <strong>with</strong> nonvascular plants.
Kottke et al. (2003) have initiated this discussion. We also
suggest that there is scope for further <strong>structural</strong> studies <strong>of</strong>
mycorrhizal categories that have received limited attention to
date.
Acknowledgements
We are indebted to Lewis Melville for the drawings,
Linda Tackaberry and two anonymous reviewers for valuable
comments on the manuscript, and the Natural Sciences and
Engineering Research Council <strong>of</strong> Canada (NSERC) for financial
support.
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