Review
Blackwell Science Ltd
Tansley review no. 135
Tansley review no. 135
Biology of mycorrhizal associations of
epacrids (Ericaceae)
Author for correspondence:
John W. G. Cairney
Tel: +61 29685 9903
Fax: +61 29685 9915
Email: [email protected]
John W. G. Cairney1 and Anne E. Ashford2
1
Mycorrhiza Research Group, Centre for Horticulture and Plant Sciences, University of Western Sydney,
Parramatta Campus, Locked Bag 1797, PENRITH SOUTH DCL, NSW 1797, Australia; 2School of
Biological Science, University of New South Wales, Sydney 2052, Australia
Received: 20 August 2001
Accepted: 4 January 2002
Contents
Summary
305
I. Epacrid plant hosts
306
II. Evolution of ericoid mycorrhizas in epacrids
306
III. Epacrid hair roots and their mycorrhizal associations
307
IV. Seasonality and incidence of mycorrhizal infection
310
VI. Nature of the mycorrhizal fungal endophytes
VII. Community and population biology of mycorrhizal
endophytes
318
VIII. Functional aspects of mycorrhizas in epacrids
319
IX. Conclusions
V. Structure and development of mycorrhizal associations
315
322
Acknowledgements
322
References
322
311
Summary
Key words: Epacridaceae, Ericaceae,
ericoid mycorrhiza, hair roots, Australia.
Epacrids, a group of southern hemisphere plants formerly considered members of
the separate family Epacridaceae, are in fact most closely allied to the Vaccinioid tribe
(Ericaceae). Epacrids and other extant ericoid mycorrhiza-forming plants appear to
have a monophyletic origin. In common with many Ericaceae they form ericoid
mycorrhizas. ITS sequence data indicate that the fungi forming ericoid mycorrhizas
with epacrids and other extant Ericaceae are broadly similar, belonging to a
poorly defined group of ascomycetes with phylogenetic affinities to Helotiales. The
basic development and structure of ericoid mycorrhizal infections in epacrids is
similar to other Ericaceae. However, data are limited on the structure and physiology
of both hair roots and ericoid mycorrhizas for all Ericaceae. Relatively little is known
about the functional significance of ericoid mycorrhizas in epacrids in southern
hemisphere habitats that are often poor in organic matter accumulation. However
the abilities of fungal endophytes of epacrids to utilize organic N and P substrates
equal those of endophytes from northern hemisphere heathland plant hosts.
Investigations using 15N/13C-labelled organic N substrates suggest that mycorrhizal
endophytes are important, at least, to the N nutrition of their epacrid hosts in some
habitats.
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I. Epacrid plant hosts
Plants that are known to form ericoid mycorrhizas have a
global distribution and have been traditionally assigned to
three families of Ericales: the Ericaceae, Empetraceae and
Epacridaceae (Watson et al., 1967; Cronquist, 1981; Thorne,
1992). They are common components of heathland and some
forest ecosystems (Specht, 1979; Read, 1991) and are united
ecologically by their occurrence in a hydrologically diverse range
of extremely nutrient-poor sandy or humic acid soils (Read,
1996). Empetraceae have been regarded as morphologically
distinct from Ericaceae. While some morphological distinctions, including differences in leaf venation and patterns of
anther dehiscence have been shown to exist between many
Epacridaceae and Ericaceae, no consistent morphological differences have been identified between the two groups (Stevens,
1971; Kron, 1996; Powell et al., 1996). In fact, cladistic analyses based on morphological, phytochemical and embryological
features (Anderberg, 1992, 1993; Judd & Kron, 1993) have
suggested that both Empetraceae and Epacridaceae belong
within the Ericaeae. These observations are strongly supported
by the combined data from comparisons of DNA sequences
from several chloroplast and nuclear genes and intergenic
regions (Kron & Chase, 1993; Cullings, 1996; Kron et al.,
1996; Kron, 1996; Crayn & Quinn, 2000). The epacrids
are thus now regarded as a derived lineage within the family
Ericaceae, and are thought to comprise seven tribes that
encompass some 34 genera and > 450 species (Powell et al.,
1996; Crayn et al., 1998; Crayn & Quinn, 2000). Currently
supported epacrid tribes are: Archerieae, Cosmelieae, Epacrideae,
Richeae, Oligarrheneae, Prionoteae and Styphelieae (Crayn
et al., 1998; Crayn & Quinn, 2000), however, the boundaries
of some tribes and their interrelationships remain poorly
resolved (Crayn & Quinn, 2000). Phylogenetic analyses of
morphological characteristics and DNA sequences place
epacrids in a clade within Ericaceae that is most closely
related to a group of vaccinioid taxa. This clade includes
tropical blueberry species, Vaccinium macrocarpon Ait. and
Gaultheria spp. (Fig. 1) (Kron et al., 1999). In light of these
observations, recent taxonomic treatments of the angiosperms have placed epacrids within Ericaceae (Bremer et al.,
1998), and we have adopted this classification throughout
this review.
II. Evolution of ericoid mycorrhizas in epacrids
Phylogenetic data support the proposed evolution of the
ericoid mycorrhizal condition in plants ancestral to the expanded
Ericaceae (Cullings, 1996). Similarities between the fungi that
are known to form ericoid mycorrhizas with extant epacrids
and those that form the associations with other extant Ericaceae,
provide further advocacy for a monophyletic origin of plants
that form ericoid mycorrhizas (Cairney, 2000; see below).
The geological time frame within which ericoid mycorrhizas
Fig. 1 Schematic representation of relationships between selected
Ericaceae taxa, based on published parsimony analysis of chloroplast
matK gene sequences from Kron et al. (1999). Branch lengths are
arbitrary. Taxa included in the analysis by Kron et al. (1999) were:
epacrids – Andersonia, Archeria, Brachyloma, Cosmelia,
Dracophyllum, Epacris, Leucopogon, Lysinema, Monotoca,
Pentachondra, Prionotes, Rupicola, Sphenotoma, Sprengelia spp.;
vacciniids – Agapetes, Cavendishia, Costera, Dimorphanthera,
Disterigma, Macleania, Satyria, Sphyrospermum, Symphysia,
Vaccinium spp.; Gaultheria group – Chamaedaphne, Gaultheria spp.;
Lyonia group – Agarista, Craibiodendron, Lyonia, Pieris spp.;
rhododendroids – Calluna, Ceratiola, Erica, Daboecia,
Menziesia, Rhododendron, Rhodothamnus, Therorhodion,
Tsusiophyllum spp.
evolved is poorly resolved. Macrofossils of epacrids (in
Tasmania) date from the Eocene, some 30 million years ago
( Jordan & Hill, 1995, 1996). Microfossil records, however,
indicate that pollen, similar to that of some extant epacrids
and other Ericaceae, was present in Australia during the late
Cretaceous about 80 million years ago (Dettmann, 1992).
Macrofossils that resemble extant Ericaceae are also known
from North America from the Cretaceous (Nixon & Crepet,
1993). Based on available fossil records and molecular clock
estimates of ascomycete evolution, Cullings (1996) suggested
that the ancestral ericoid mycorrhizal condition had arisen
by the early Cretaceous c. 140 million years ago. This is in
keeping with the proposed origin of the epacrid lineage in
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Tansley review no. 135
southern Gondwana during the Campanian c. 80 million
years ago (Dettmann, 1992), along with the hypothesised
origin of ancestral Ericales in, and radiation from, Gondwana
during the Mesozoic (Specht, 1979). The geographic distribution of extant epacrid taxa further implies that epacrids
advanced northwards during their evolution (Copeland, 1954).
Evolution of ancestral Ericaceae in southern Gondwana
during the Cretaceous is compatible with the hypothesis of
the evolution of sclerophylly in southern Gondwana during
that period (Specht, 1979; Dettmann, 1992). Although
climatic patterns at this time are not known, biogeographical
evidence based on the extant flora seems to indicate that the
various groups of the expanded Ericaceae probably became
confined to discrete climate-influenced habitats as Gondwana
drifted apart (Specht, 1979; Kron et al., 1999). The extent
to which their mycorrhizal associations have diverged as a
result of habitat restriction, however, remains an engaging
question.
So, too, is the relationship between Ericaceae and leafy liverworts with respect to their abilities to form associations with
the same group of fungi. The ability of ericoid mycorrhizal
endophytes from northern hemisphere Ericaceae to form
mycorrhiza-like hyphal complexes in the rhizoids of certain
leafy liverwort families has been clearly demonstrated (Duckett
& Read, 1991, 1995). Similar infection has also been observed
in some liverworts from Antarctica (Williams et al., 1994)
and an eastern Australian sclerophyll forest habitat inhabited
by several epacrid taxa (Chambers et al., 1999). Although no
cross infection experiments have been performed between
epacrid endophytes and liverworts, endophytes from the latter
in Australia and Antarctica form part of the Hymenoscyphus
species complex (Chambers et al., 1999; Sharples et al., 2000;
see below), implying that a similar relationship exists in
the southern hemisphere. Liverworts are regarded as having
branched as a separate lineage at a very early stage of land
plant evolution (Kenrick & Crane, 1997; Duff & Nickrent,
1999), precluding a direct evolutionary relationship with
Ericaceae. The relationship between Ericaceae and liverworts
thus appears to be purely ecological, and it has been suggested
that the liverworts may act as centres for mycelia that will
subsequently infect Ericaceae seedlings (Duckett & Read,
1995). Whether or not endophytes form a physiologically
functional mycorrhiza with the liverworts is not known (Read
et al., 2000), but we may intuitively predict that the association must enhance the fitness of the host to some degree.
This notwithstanding, the association of ericoid mycorrhizal
endophytes with some liverworts in both the southern and
northern hemispheres indicates that the association is clearly
an ancient one that probably arose in Gondwana. Ericoid
mycorrhizal fungal endophytes are thought to have evolved
from ancestral saprotrophic ascomycetes (Cullings, 1996;
Cairney, 2000), but whether they evolved first as associates
of the liverworts or of ancestral Ericaceae has yet to be
resolved.
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Review
III. Epacrid hair roots and their mycorrhizal
associations
All epacrids so far examined have ericoid mycorrhizas (Reed,
1987, 1996). A sheathing mycorrhiza has also been found on
Astroloma humifusum (Cav.) R.Br. in the wild (McGee, 1986).
As in other Ericaceae, ericoid mycorrhizas in Epacridaceae are
always produced on hair roots. These can be viewed as short
roots of determinate growth borne on dimorphic root systems
which also have larger indeterminate roots that may become
secondarily thickened.
Bell et al. (1996) have described root systems of 92 species
in 15 epacrid genera from South-west Australia. They found
that 75% had a small root system with a single main root and
laterals, that did not spread beyond the shoot canopy. They
identified four types of root morphology correlated with fire
response. Species that survive fire as seeds tended to have
shallow, less well developed root systems with a single well
defined main root, whereas resprouting species had lignotubers
giving rise to several large diameter roots. The root system
of one of the reseeders, Lysinema ciliatum R.Br., has been
described in detail from pot-grown nonmycorrhizal cuttings
(Allaway & Ashford, 1996). It is dimorphic system and fits
fairly well into the concept of there being ‘framework roots’
and ‘fine nutrient gathering roots’ (McCully, 1999). The latter
are hair roots.
In L. ciliatum cuttings adventitious roots which are relatively large and of indeterminate growth give rise both to other
large indeterminate first-order branches (long roots) and to
the hair roots which are much smaller and of determinate
growth (short roots). Hair roots may themselves branch and
there may be as many as three orders of them, ranging from
c. 70 µm diameter in penultimate to < 50 µm in ultimate roots.
The L. ciliatum cuttings were not mycorrhizal, but nevertheless produced an apparently normal root system, which
compares well with mycorrhizal root systems of 3-yr-old
L. ciliatum plants collected from field sites in Western Australia.
In the latter, of course the first order root is a primary root.
This root system also fits the general concept of an ericaceous
root system based on earlier work on Calluna vulgaris (L.)
Hull by Read & Stribley (1975). It is not known whether
colonisation increases the total number of roots or promotes
growth of hair roots in epacrids, as has been shown by Berta
et al. (1988) for C. vulgaris and by Duclos et al. (1983) for
Erica carnea L. It is therefore not clear how widespread this
phenomenon is or how many endophytes promote the
response. It is, however, clear that mycorrhizal infection is not
necessary for a root system with hair roots to develop, an
observation also made much earlier on Epacris impressa Labill.
by McLennan (1935).
Hair roots from all species are basically similar in anatomy.
At maturity they consist of an epidermis, only two cortical cell
layers both of which are suberised, and a stele which may or
may not have a complete ring of pericycle cells, the number
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Fig. 2 Transverse section of Woollsia
pungens showing the arrangement of tissues
typical of the finest hair roots. Shown in the
section is an epidermis, a two-layered cortex
comprising exodermis and endodermis lying
on the same radius and a monarch stele
containing only one file of xylem elements
(tracheids), one file of sieve elements, one
companion cell and only two pericycle cell
profiles. The epidermal cells are thick walled
and one has collapsed. Remnants of root cap
mucilage are retained especially around
mycorrhizal hyphae on the root surface. All
epidermal cells except the collapsed one are
colonised and contain hyphal profiles: a
penetration point may be seen. All cells
contain hyphal profiles of viable hyphae with
well preserved contents and aggregates of
poorly preserved hyphae surrounded by
material. This indicates either more than one
penetration or a heterogeity in the
penetration structure. Micrograph by
Dr C.L. Briggs. Bar, 5 µm.
depending on root order and size (see Fig. 2). The identity of
the suberised cortical layers as an exodermis and endodermis
is determined by examining the distribution of suberin with
sudan black B and confirmation of the position of Casparian
bands and suberised lamellae by electron microscopy. Either
or both of these layers are frequently seen to be collapsed in
electron micrographs, leading to the proposal that cortical
cells collapse early in development. This is most likely due to
failure of electron microscopy reagents to penetrate the cells,
as they become more impermeable during their development,
as well as to poor fixation and embedding due to their high
content of phenolic compounds. This failure of the exodermis
and endodermis to fix well when mature is a problem common to all roots (though not all roots have an exodermis). In
nonfixed hair roots suberised exodermal cells are seen to
remain viable even after the epidermis is collapsed or shed (see
below). Elements of both xylem and phloem are individually
small and few in number. The finest ultimate hair roots are
monarch with a single file each of tracheids and sieve cells
(Fig. 2). These elements are very small. In L. ciliatum the
tracheid lumen is only 2.4 µm in diameter, indicating that
resistance to mass flow is likely to be high and water flows in
individual roots very low (Allaway & Ashford, 1996; Briggs &
Ashford, 2001). Even the larger penultimate hair roots have
only three files of tracheids the largest of these being only
3.8 µm in diameter. This paucity and small size of xylem elements can be seen in cleared roots of a wide range of species
(A. E. Ashford, unpublished). Mycorrhizal plants of L. ciliatum
(Ashford et al., 1996; A. E. Ashford & W. G. Allaway, unpublished) and also of other epacrids, notably Dracophyllum
secundum R.Br. (Allen et al., 1989), Leucopogon parviflorus
(Andr.) Lindl. (Steinke et al., 1996), Leucopogon ericoides
(Smith) R.Br. (Fig. 2a in Read, 1996) and Leucopogon juniperinus
R. Br. (A. E. Ashford unpublished) collected from the field all
show these features. This anatomy can be seen as a means of
conserving water in the vicinity of individual roots, thereby
prolonging the survival of hyphae in that region.
Similar observations were made on the structure of hair
roots for northern hemisphere Ericaceae by earlier workers.
Burgeff (1961) showed that the two-layered cortex comprises
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Tansley review no. 135
a suberised exodermis and an endodermis and traced their
origin to the apex in Calluna vulgaris. He stated that the fungus
colonises only the epidermal cells. Nieuwdorp (1969) also
noted the common origin of the two cortical layers but
called the outer a subepidermis. He showed that this layer
in V. macrocarpon contains a suberised lamella and develops
to tertiary state, as is typical for an exodermis. An exodermis
is commonly found in Angiosperms (Peterson, 1989;
Perumalla et al., 1990) and there is every reason to suppose
that it is universally present in hair roots. The development
of the epidermal layer and its common origin with the root
cap as distinct from the two cortical layers is shown by Berta
& Bonfante-Fasolo (1983), who also show that it is the
epidermal layer that becomes colonised. Notwithstanding the
earlier work, many subsequent workers have misinterpreted
the anatomy of the hair root and erroneously called the
mycorrhizally colonised surface layer ‘cortical cells’ (see
Fig. 12.2 in Smith & Read, 1997).
Hair roots lack root hairs but have a prominent layer of
mucilage over their surface at least in the tip region, regardless
of whether they are infected or not. This applies to epacrids
(Ashford et al., 1996; Steinke et al., 1996; Briggs & Ashford,
2001) as well as other Ericaceae (Leiser, 1968; Berta &
Bonfante-Fasolo, 1983; Peretto et al., 1990). The mucilage is
thinner and more patchy in differentiated regions. The composition of this mucilage has been examined in detail in only
two species, C. vulgaris, a nonepacrid (Peretto et al., 1990)
and W. pungens (C. L. Briggs & A. E. Ashford, unpublished).
In C. vulgaris it reacts with periodic acid Schiff and periodic
acid-thiocarbohydrazide-silver proteinate (PATAg) reagents
(general stains indicating carbohydrates), and with two lectins
Ricinus communis agglutinin 120 indicating β-galactose, and
Concanavalin A (ConA) indicating polymers containing
glucose and mannose residues. The soluble fraction was found
to contain glucose, galactose, mannose, xylose, rhamnose and
arabinose, but only traces of uronic acids and proteins and
no fucose. Staining reactions of the mucilage in W. pungens
(C. L. Briggs & A. E. Ashford, unpublished) and other epacrid
species are in general agreement with these observations.
As it becomes fully hydrated in fresh aqueous mounts the
mucilage of L. ciliatum expands to form a thick layer with soil
debris at the surface (Ashford et al., 1996). A similar expansion
is seen in maize where it is known that root cap mucilage can
take up 1000 times its weight in water (Guinel & McCully,
1986; McCully & Sealy, 1996). Mucilage is not well preserved
in chemically fixed embedded hair roots where it is either lost
or tends to become coagulated and patchy in distribution.
Detached root cap cells are seen to be embedded in this
mucilage in L. ciliatum (Fig. 3; Ashford et al., 1996). Figure 4
shows the apex of a whole cleared root. Most of the cap cells
have already been released and common origin between cap
cells and epidermal cells from the same initials is clear. These
cap cells have not been studied in detail in any hair root but
are seen in L. ciliatum to be living and, if they function like
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Fig. 3 Line diagram of cleared Leucopogon ericoides root showing
changes in the epidermal layer as well as the root cap with loosely
arranged cells. The distribution of living cells released from the
cap and embedded in the mucilage layer is shown (arrows).
Bar, 20 µm.
those in other roots, they will remain alive and will continue
to secrete mucilage for some time after their release from the
cap (McCully, 1999). They tend to be easily lost or damaged
and so are likely to be missed, especially in older parts of
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Tansley review no. 135
Fig. 4 Root cap-epidermis junction of cleared Leucopogon ericoides
root showing the common origin of cells of these two tissues. This
root cap has shed most of its cells and the epidermis cells are not so
columnar as in younger apices. Bar, 20 µm.
roots. In other species root cap cell release is programmed and
their number regulated, cell production being modulated by
a range of environmental and endogenous stimuli. They are
thought to play an important role in rhizosphere dynamics
since in some cases they attract and in others repel specific
fungi and bacteria (Hawes, 1990; Hawes et al., 1998), but not
in lubricating the passage of roots through the soil as was
formerly supposed. They are the first cells that would be
contacted by rhizosphere microorganisms, and must surely be
considered in any evaluation of fungal recognition reactions
in ericoid associations.
Hair root apices with the capacity for active growth have a
small root cap of a few cells overlying a small meristem. There
is a short region of columnar, densely cytoplasmic, epidermal
cells which are not usually invaded by fungus (Fig. 3; McNabb,
1961; Ashford et al., 1996; Briggs & Ashford, 2001). Within
a short distance of the apex, epidermal cells elongate and the
transition zone can be quite abrupt. Fully elongated epidermal
cells often appear inflated and balloon-like in surface view
with a tendency to pull apart at the radial walls, but some
become collapsed with distance from the apex. Hair roots are
determinate and of short length (mean length was 3.4 mm in
ultimate hair roots of L. ciliatum) and many apices show features
indicating cessation of growth. These apices have no root cap,
no distinct meristem, and all the tissues are differentiated
(Allaway & Ashford, 1996). The epidermis is enlarged and
vacuolated and the two suberised cortical layers are differentiated around the apex, so that the root is completely waterproofed, although it is clear that it is still alive. While actively
growing apices are rarely mycorrhizally colonised, differentiated
apices frequently contain hyphae in the enlarged epidermal
cells quite close to the tip. These features all agree well with
those of other Ericaceae indicating commonality in hair root
structure (Berta & Bonfante-Fasolo, 1983; Duclos et al., 1983;
Peretto et al., 1990; see also Fig. 12.1 in Smith & Read, 1997).
Hair roots, in fact, are not really all that unusual. Other fine
roots, for example those of maize, are only 70 µm in diameter
and have a reduced cortex comprising only an exodermis
and endodermis, as well as a very small stele with very small
xylem elements. They are also determinate, reaching their
final length in < 2.5 d. Similarly the root cap is lost and
tissues differentiate to the tip. However these determinate
roots persist for the life of the plant and gradually shorten as
the distal ends slowly die back (McCully, 1999), while hair
roots are usually considered to be ephemeral, though reliable
data from field situations are scant. Fine roots are known to
be lost when plant root systems are dug out from the soil
and washed; as much as 50% of the total root length may
be preferentially removed (Pallant et al., 1993). In view of
their extreme fragility and small size hair roots are likely to be
markedly under-represented in any analysis of root systems
from the soil.
IV. Seasonality and incidence of mycorrhizal
infection
Ericoid mycorrhizas are reported to be present in the roots of
all epacrid species examined in Australia, but not necessarily
all year round (Reed, 1987, 1996). Since hair roots have finite
growth, indicating that they are determinate in length
(Bonfante-Fasolo et al., 1981; Allaway & Ashford, 1996;
A. E. Ashford, unpublished) and may be short lived, the
question arises as to whether ericoid mycorrhizas are strongly
seasonal. Read (1996) has proposed that different moisture
regimes will result in distinctive patterns of seasonal hair root
development. This is particularly relevant for Australian
epacrids which grow in situations ranging from permanently
moist to seasonally droughted. All the data from Australia
point to loss of hair roots and presumably mycorrhizal
function in situations where there is a dry season and the
plants become droughted. In a broad survey Reed (1987,
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Tansley review no. 135
1996) did not find any relationship between infection
intensity and either latitude, altitude, edaphic or climatic
factors. However he did report that some species lacked any
fine roots in the dry season at sites in north Queensland and
south-west Australia (Reed, 1996). Seasonal variation in the
abundance of hair roots and mycorrhizal infection has been
examined in detail for several species in south-west Australia
(Hutton et al., 1994; Bell & Pate, 1996). These locations are
subject to a Mediterranean-type climate with cool wet winters
(May–July) and hot dry summers (Dec–March). In plants of
all species examined, total hair root length rapidly declined
to a low level in summer as the soil dried out. Hair roots
reappeared in autumn (April) and hair root length
progressively increased while the soil was moist. Hair roots
became mycorrhizal over the moist winter period, with the
highest level of infection per plant in spring (generally Sept–
Nov). In this climate mycorrhizal function is highly seasonal,
occurring in the winter period when sufficient moisture is
available for hair root survival. Most of Australia, however, is
not subject to a Mediterranean climate. For example, much of
eastern Australia has longer term rainfall variations associated
with the El Niño-Southern Oscillation and there may be
several moist years followed by years of drought. Reed (1989)
found hair root tips at all times of the year in L. juniperinus
collected at a site 12 km NW of Sydney but the incidence of
infection was much lower in January and November; he
attributed higher infection in winter to slow root growth.
A similar result was obtained for Woollsia pungens (Cav.)
F. Muell. at another site (Lane Cove) in the Sydney
Metropolitan area (E. Kemp et al., unpublished). Hair roots
persisted throughout the year, comprising at least approx.
50% of the root system at all times. Although the percentage
hair root length that was colonised by mycorrhizal fungi in
the winter months was almost double that in summer, there
was at least approx. 20% root length colonised all year round.
These measurements were taken in a La Niña year and the
soil was moist all year round. The difference in level of
mycorrhizal infection in W. pungens was not correlated with
moisture but was negatively correlated with temperature.
Persistence of infection all year round is reported for most
species studied in the northern hemisphere (Read & Kerley,
1995). Such findings indicate that, except in very dry conditions, ericoid mycorrhizas may function all year round. However
the presence of coils is not necessarily an indicator of their
viability (Bonfante-Fasolo et al., 1981) and degree of infection
cannot be translated directly into functional benefit. Furthermore
the number of sites examined using appropriate analyses is
very small especially if the high degree of plant variation to be
expected is taken into account. This illustrates how little we
know about the timing and level of the contribution of ericoid
mycorrhizal hair roots to plant nutrition in the wild, and how
difficult it is to provide definitive answers even for associations
where it is known that colonisation can enhance nitrogen and
phosphorus nutrition.
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V. Structure and development of mycorrhizal
associations
There are very few detailed investigations on the structure
and molecular cytology of epacrid infections. All are from
field collected material where neither the fungal partner nor
the length of time since initiation of infection is known. This
is attributable to the difficulties in establishing epacrid seedlings and initiating mycorrhizas on them under controlled
conditions (McLean et al., 1998), as well as the failure of hair
roots to fix well. What is known suggests that they are fundamentally similar to the ericoid mycorrhizas described in detail
in several ericaceous species from the Northern hemisphere
(C. vulgaris, V. macrocarpon, Vaccinium myrtillus L., Rhododendron
ponticum L., Rhododendron sp. and E. carnea) either from
material collected from the field, or synthesized under various
conditions (Nieuwdorp, 1969; Bonfante-Fasolo & GianinazziPearson, 1979; Bonfante-Fasolo, 1980; Peterson et al., 1980;
Bonfante-Fasolo et al., 1981; Duddridge & Read, 1982; Berta
& Bonfante-Fasolo, 1983; Duclos et al., 1983; Read, 1983).
In the following section both will be considered together
emphasizing similarities and differences.
Development of the symbiosis is initiated when a fungal
hypha contacts a compatible region of hair root. The apical
region of actively growing roots is usually not infected. Hair
roots of different order are reported to carry different infection
levels and infection in those epacrids studied appears somewhat less than that reported in other Ericaceae. Extra-radical
mycelium on the root surface is usually rather sparse in fieldcollected epacrid mycorrhizas (Fig. 5a) as in C. vulgaris and
V. myrtillus (Bonfante-Fasolo & Gianinazzi-Pearson, 1979;
Bonfante-Fasolo et al., 1981).
In field-collected epacrid mycorrhizas it is common to see
single hyphae oriented longitudinally along the surface of a
hair root with short perpendicular branches towards the
root surface (Fig. 5b). On root contact an appressorium-like
structure is formed in some species (Fig. 5c). There is no
information on what controls its formation as there is for
some pathogens and AM fungi, and though common it is
not always present. Appressorium formation is followed by
the development of a narrow penetration hypha. This usually
grows through the outer tangential wall (or occasionally the
outer part of the radial wall) and enters the periplasmic space of
an epidermal cell where it widens and forms a coil (Fig. 5d,e).
Typically there is a single penetration point in epacrids as in
other Ericaceae. The existence of a halo around penetrating
hyphae indicates that wall digestion has occurred. There is no
information on the extent of control exerted by the root as
there is for AM fungi where it is known that the penetration
step and form that the intracellular hyphae take are under a
complex genetic control by the plant (Harrison, 1999).
Before penetration, a hypha encounters the surface mucilage
and there are many images of hyphal profiles on the root
surface completely enveloped in this mucilage both for epacrids
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Tansley review no. 135
and other Ericaceae (Allen et al., 1989; Steinke et al., 1996;
Briggs & Ashford, 2001). It has been suggested that both the
cell wall and mucilage overlying hair roots are important in
reactions controlling the establishment of ericoid mycorrhizal
associations (Bonfante-Fasolo, 1988). A sheath of extracellular
mucilage also surrounds the fungus Hymenoscyphus ericae
(Read) Korf & Kernan. This binds to ConA and binding is
inhibited by addition of glucose and mannose, indicating that
it also contains glucose and mannose side chains. Mucilage
differs in abundance and ConA stainability in different strains,
and differences are correlated with infective capacity (GianinazziPearson & Bonfante-Fasolo, 1986; Bonfante-Fasolo et al., 1987;
Perotto et al., 1995). It is reported to be more abundant in
infective strains and also more abundant around hyphae
growing on the root surface than with the same fungus in pure
culture, and it is suggested that it may be involved in adhesion
and/or recognition reactions, though definitive evidence for
this is lacking.
Within the root, the fungal coil (also called peloton by
some) occurs inside the periplasmic space. It is therefore intracellular but outside the epidermal cell plasma membrane and
so is an apoplasmic structure as far as the root is concerned.
The establishment of coils should provide a large surface area
of contact between host and endophyte but this has never
actually been measured in any ericoid mycorrhiza as it has
been in arbuscular mycorrhizae (Dickson & Kolesik, 1999).
Only the epidermis is colonised. The identity of the colonised
surface cell layer as an epidermis is clear if it is traced to the
root apex either in longitudinal sections or whole roots (see
Fig. 12.1 in Smith & Read, 1997). Here it is seen to have a
common origin with the very small root cap, both of which
arise from a layer of meristematic cells quite distinct from that
giving rise to the cortex. The problem of identifying the
mycorrhizally colonised layer as epidermis has arisen because
the suberised cortex usually fixes badly and the cells collapse
so that it is not easy to distinguish cell layers clearly in whole
roots or freehand cross sections. Once within the root the
fungus remains in the first colonised cell and does not spread
to adjacent cells (Fig. 5c–e). The best descriptions of the next
stages are for other nonepacrid Ericaceae, but even for these it
is hard to obtain a precise chronological sequence since the
timing of infection varies from cell to cell. A description of
events in these species primarily from electron microscopy
of chemically fixed material is covered in detail in a number of
articles (Read, 1983; Smith & Read, 1997). Data of a similar
nature are not available for a single epacrid species. In the few
epacrids studied using electron microscopy there are only isolated
images of apparently mature relationships. They are of similar
Review
appearance to those of other Ericaceae and by extrapolation it
is assumed that they mature and function in a similar way.
Production of fungal mucilage is suppressed once hyphae are
inside the root. The fungus does, however, continue to secrete
wall material, as demonstrated by wheat germ agglutinin staining of N-acetylglucosamine residues of chitin (as well as other
wall polysaccharides) in the region around the hyphal profiles
of epidermal coils (Bonfante-Fasolo et al., 1987; Perotto et al.,
1995). An electron-lucent gap containing dispersed material
separates the fungal wall from the invaginated plant plasma
membrane as in most EM images of ericoid mycorrhizas
(Allen et al., 1989; Briggs & Ashford, 2001). This region has
been termed an ‘interfacial matrix’ (Smith & Read, 1997). It
clearly is the site across which any nutrient exchange will
occur and so it is important to know its structure and dimensions, but it is not clear to what extent the gap is an artifact of
specimen preparation.
The fungi forming coils inside the epidermal cells of epacrid
hair roots most usually have simple septa and Woronin
bodies, an indication that they are mostly ascomycetes or their
anamorphs (Allen et al., 1989; Steinke et al., 1996; Briggs &
Ashford, 2001). Ascomycetes also predominate in other
Ericaceae (Bonfante-Fasolo & Gianinazzi-Pearson, 1979;
Bonfante-Fasolo, 1980; Bonfante-Fasolo et al., 1981). There
is however, a report of hyphae with nonperforated parenthesomes characteristic of Tulasnellales (Basidiomycotina) in
D. secundum (Allen et al., 1989). Such reports are sufficiently
common in Ericaceae sensu lato for this to be taken seriously
(Bonfante-Fasolo, 1980; Peterson et al., 1980). These form
what appear to be identical infection structures to those of
ascomycetes although there is no evidence of mutual benefit
(Allen et al., 1989; Peterson et al., 1980). The findings of
Peterson et al. (1980) are of particular interest since theirs
appears to be the only report showing colonisation of what is
clearly a suberised cell as indicated by the relatively electrontransparent suberised lamella (see Fig. 17 in Peterson et al.,
1980) and is presumably exodermis. However the significance
of this is difficult to evaluate since no information is given on
the frequency of this observation, or on the status of either the
fungus or the roots.
To evaluate the function of ericoid mycorrhizas it is necessary to know how long individual coils survive in epidermal
cells. All reports indicate that they are relatively short lived
but there are no precise values. There have been a number
of descriptions of changes in the structure of the cytoplasm
following colonisation. These changes are described from a
number of other Ericaceae based on examination of roots by
electron microscopy at various times after planting in infected
Fig. 5 Various images from cleared whole Leucopogon root hair roots stained with chlorazol black E and viewed by differential interference
contrast (DIC) optics. (a) The typical sparse distribution of hyphae on the root surface, L. ericoides; (b) pioneer hyphae with a series of side
branches have penetrated individual epidermal cells, L. juniperinus; (c–e) images from a through focus series of a coil within an epidermal cell
showing the surface appressorium (arrow in (c)), the narrowing of the hypha as it has penetrated the wall (arrow in (d)) and features of the coil
which shows hyphae of varying diameter, some with obvious contents (arrow (e)). Bar, 5 µm.
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soil in pots, or after contact with a known symbiont, and
constructing a sequence of events from the images obtained.
All indicate that the root cytoplasm degenerates before that of
the hyphae. This contrasts with other mycorrhizal associations
such as those of orchids and arbuscular mycorrhizas where
individual plant cells outlive the colonising hyphae and may
be repeatedly colonised. It suggests that the root cytoplasm
may be utilized by the fungus, there being no evidence of
other microbial colonisation or general cell wall disruption.
Under these circumstances it is difficult to see why fungal
death should follow soon afterwards, as is reported. In a large
number of the images of plant cells containing degenerating
hyphae there are also profiles of nonmoribund hyphae (as
noted by most authors). Some are surrounded by an encasing
material (Duddridge & Read, 1982). Such images indicate
that with the death of the plant cytoplasm the coil may become
reorganized and parts of it may continue to survive. In these
difficult-to-fix roots it has always been a problem to separate
fixation damage and plasmolysis from degenerative changes.
Localized changes in permeability or deposition of phenolics
may alter preservation dramatically even in adjacent cells.
There is now a large body of literature cataloguing the artifacts
resulting from the continued membrane flow and post mortem
effects that occur during aldehyde fixation, even in relatively
well fixed cells (Hoch & Howard, 1981; Mims et al., 1988).
This whole area needs revisiting using techniques such as
high pressure freezing and freeze substitution which improve
preservation and avoid many of these artifacts.
In older roots of pot grown L. ciliatum the uninfected epidermal cells have either collapsed or been shed, laying bare
the exodermis (Ashford et al., 1996). Infected cells can also be
sloughed from roots but it is not known to what extent this
occurs in the wild. The exposed suberised exodermis is not
invaded by fungus though there are frequently hyphae running over its surface (except in Peterson et al., 1980; see above).
Roots in this condition are alive and cytoplasmic streaming
may be observed in both the endodermal and exodermal cells.
These roots have the capacity to generate new hair roots from
cell divisions of the endodermis and pericycle. Though they often
superficially look dead, they might be longer lived than is generally assumed. However data on root dynamics are lacking.
A similar phenomenon of epidermal cell collapse is seen in
unfixed whole, field collected roots of a range of epacrid species
as well as in Calluna vulgaris (collected Denmark & Skåne,
Sweden, June 2001). The surviving noncollapsed epidermal
cells, which often occur in clusters, are invariably mycorrhizally
infected. McNabb (1961) described a similar phenomenon,
but viewed the hyphae as devoid of contents and dead. It is
difficult to determine the status of the fungus in these cells
but some coils are clearly alive since they accumulate Oregon
green 488 carboxylic acid (Fig. 6). If coils retain any viable
hyphal segments they could act as a source of inoculum in the
field. Epidermal cells produced by macerating hair roots are
effective for isolating the endophyte and, when plated out on
water agar, hyphae readily grow out from them to develop
viable mycelium (see Figs 1–4 in Read, 1983; Fig. 1 in Reed,
1987 and Fig. 3 in Read, 1996). Cells containing any live
hyphae at the surface of existing roots would be strategically
placed to generate infections on newly developed roots as we
have suggested (Ashford et al., 1996). However Hutton et al.
(1997) stated that soil collected from depths where hair root
fragments are expected was not effective as a source of infection, and expressed the opinion that at their site sloughed cells
are not the primary aestivating structures. There are intriguing
inconsistencies in observations of mycorrhizal dynamics in
the field and this area needs further investigation.
Most of the ericoid mycorrhizas examined show some
thickening of the epidermal walls but an exceptional thickening of the outer tangential and radial epidermal walls has been
described for a number of epacrid species as well as two other
Ericaceae (Ashford et al., 1996). In L. ciliatum and W. pungens
the thickened walls are multilayered and show great complexity of structure, with regions that look spongy in electron
micrographs alternating with those of more normal appearance (Ashford et al., 1996; Briggs & Ashford, 2001). Surface
views of hair roots in L. ciliatum show a mosaic of thick and
thin-walled cells and the physiology of these thick walled cells
is different (for example they appear to be more permeable to
4′-6-diamidino-2′-phencplindole [DAPI]). The role of the
thick wall has not been definitively established but it is clear
that thick-walled cells are preferentially colonised by the
fungus. In unfixed roots of field-collected plants it is common
to find thick-walled epidermal cells containing coils with live
hyphae surrounded by collapsed thin-walled uncolonised
cells. The thick wall in W. pungens has been characterized by
in situ staining (Briggs & Ashford, 2001). It is a multilamellate
secondary wall containing typical helicoidal arrays of cellulose
microfibrils, as also found in slightly thickened outer tangential walls of epidermal cells in C. vulgaris (Peretto et al., 1990;
Perotto et al., 1995). Staining with various basic dyes at controlled pH and other tests indicate that all wall regions carry
net negative charge attributed to carboxyl groups. However
lack of reaction with JIM5 and a poor reaction with JIM7
antibodies indicates negligible amounts of unesterified pectin
and pectin with up to 50% esterification. This has also been
found for C. vulgaris epidermal cell walls (Peretto et al., 1990;
Perotto et al., 1995). In both cases this low level of staining
contrasts with high levels of staining in the cortical and stelar
cell walls with the same antibodies, a difference that is found
in other families as well (Knox et al., 1990). It is viewed by
Perotto et al. (1995) to be significant in endophyte infection
of hair roots, and they have suggested that low levels of pectin
might cause switching of the fungus from a saprotrophic
phase where it secretes pectinases to a more mycorrhizal phase
where they are turned off (Perotto et al., 1995). There is some
support for this in that C. vulgaris endophytes do secrete polygalacturonases into the medium but none are found on the
surface of hyphae inside root epidermal cells. Reaction of the
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Tansley review no. 135
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Fig. 6 (a,b) Accumulation of Oregon green 488 carboxylic acid in tubular and rounded vacuoles in some fungal coils in ericoid hair roots. Not
all visible coils were labelled and it is suspected that they were not accessible to the probe. (a) Hair root of Calluna vulgaris. (b) Hair root of
Woollsia pungens. Bar, 20 µm.
W. pungens thick walls with the two lectins, peanut agglutinin
and Bandeira simplicifolia I lectin, but not ConA, shows them
to be rich in galactose side chains, but not glucose and mannose side chains. Wall histochemistry is consistent with there
being a high level of galactomannans. These polysaccharides
are implicated in controlling water relations of some legume
seeds under drying conditions. Colonised cells retain their
integrity longer than noncolonised cells and it has been suggested that the thick wall may in some way protect the fungus
or prolong the relationship (Briggs & Ashford, 2001).
VI. Nature of the mycorrhizal fungal endophytes
Putative ericoid mycorrhizal endophytes have been isolated from
a number of epacrid hosts using modifications of the maceration
(Reed, 1989; Hutton et al., 1994; Hutton et al., 1996) or direct
plating (Liu et al., 1998) methods that were developed by
Pearson & Read (1973) for isolation of mycorrhizal endophytes
from other Ericaceae hosts. A method whereby surface-sterilised
© New Phytologist (2002) 154: 305 – 326 www.newphytologist.com
hair root pieces are incubated in a solution containing bovine
serum albumin (BSA) (Williams, 1990) has also been used successfully (Steinke et al., 1996; McLean et al., 1999). Regardless
of the method employed, a diverse morphological array of
slow-growing sterile putative endophyte mycelia have been
obtained. Structural observations (Reed, 1987; Hutton et al.,
1994; Steinke et al., 1996) and recent molecular analyses (see
below) indicate that all endophytes so far isolated are ascomycetes or their anamorphs. Many of these endophytes have
been shown to form typical ericoid mycorrhizal structures in
hair root epidermal cells of epacrids (Reed, 1987; McLean
et al., 1998; Anthony et al., 2000) or other Ericaceae (Reed, 1989;
Liu et al., 1998), suggesting a mycorrhiza-forming ability.
As mentioned in Section V, as in northern hemisphere
Ericaceae hosts (Seviour et al., 1973; Bonfante-Fasolo, 1980;
Peterson et al., 1980; Mueller et al., 1986), basidiomycete
mycelia have occasionally been observed in viable epidermal
hair root cells of some epacrid hosts (Allen et al., 1989). There
are even occasional reports of colonisation by arbuscular
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mycorrhiza-like fungi in some epacrids (Khan, 1978; McGee,
1986; Bellgard, 1991; McLean & Lawrie, 1996; Reed, 1996).
While the apparent arbuscular mycorrhizal infection seems
most likely to reflect opportunistic nonsymbiotic colonisation
of hyphae growing from nearby arbuscular mycorrhizal
host plant roots (Leake & Read, 1991; Reed, 1996) the status
of basidiomycete endophytes remains unclear (see above).
Englander & Hull (1980) demonstrated reciprocal transfer of
carbon and phosphorus between a basidiome of Clavaria and
roots of a Rhododendron sp., but there remains no evidence
of synthesis of ericoid mycorrhizas between basidiomycetes
and members of the Ericaceae under controlled laboratory
conditions. It has further been suggested recently that the isolation techniques currently employed for ericoid mycorrhizal
endophytes may be selective for only certain endophyte taxa,
and that many endophytes may have been overlooked in
studies conducted to date (Bergero et al., 2000). Further work,
in particular development of molecular methods for identification of endophytes in planta, is urgently required to resolve
these issues in both epacrids and other Ericaceae hosts.
Identification of the fungi forming coils inside the roots does
not definitively prove their mycorrhizal status or degree of
benefit to the plant (Read & Kerley, 1995) but nevertheless it
is a very important step in understanding which endophytes
occur in hair roots in the wild.
It has been known for some time that the Helotiaceae
ascomycete H. ericae and anamorphic Myxotrichaceae
Oidiodendron species are common mycorrhizal endophytes
of northern hemisphere Ericaceae (Read, 1974; Couture
et al., 1983). Acremonium strictum W. Gams, an anamorphic
Hypocraeales ascomycete, is also known to be a common
mycorrhizal endophyte of Gaultheria shallon Pursh in some
north American habitats (Xiao & Berch, 1996). The biology
of H. ericae, in particular, has received much attention (Smith
& Read, 1997; Cairney & Burke, 1998), and it has become
the model fungus in laboratory studies of ericoid mycorrhizal
associations. Such emphasis on this taxon in the laboratory
has perhaps resulted in a somewhat erroneous assumption of
its ubiquitous ecological importance. While studies of endophyte diversity in the field indicate that H. ericae is indeed the
most commonly isolated mycorrhizal endophyte of northern
hemisphere Ericaceae hosts at some sites (Sharples et al., 2000),
this is not always the case in all habitats (Perotto et al., 1996;
Xiao & Berch, 1996). Neither does it appear to be true for
Australian epacrids. Despite the presence of an isolate that
showed 97.9% internal transcribed spaces (ITS) sequence
identity with H. ericae as a rhizoid endophyte of the leafy
liverwort Cephaloziella exiliflora (Taylor) Stephani in eastern
Australia (Chambers et al., 1999), there is currently no convincing evidence for H. ericae as a mycorrhizal endophyte of
an epacrid host. Parry et al. (2000) found that a polyclonal
antiserum to H. ericae cross-reacted with mycorrhizal endophytes
of eastern Australian epacrids. As conceded by the authors,
however, this suggests only that serological similarities exist
between the fungi and does not necessarily imply a close taxonomic relationship. Similarly, although Hutton et al. (1994)
reported pectic zymogram profiles from sterile endophytes
from Western Australian epacrids to be more similar to H. ericae
than to Oidiodendron spp., taxonomic relatedness to H. ericae
cannot be inferred from such an observation.
The pectic zymogram analyses conducted by Hutton et al.
(1994, 1996) were an important step in establishing that
epacrids are naturally infected by a diverse array of ascomycete mycorrhizal endophytes. Subsequent molecular analysis has confirmed that, in common with Ericaceae from the
northern hemisphere (Perotto et al., 1996; Sharples et al., 2000),
root systems of individual epacrid plants typically house an
array of endophyte taxa (Chambers et al., 2000). As is the case
for ectomycorrhizal fungal communities (Dahlberg, 2001),
however, ericoid mycorrhizal endophyte communities within
the root system of single plants are dominated by a relatively
small number of common taxa (Liu et al., 1998; Chambers
et al., 2000; Midgley et al., 2001; see below).
The precise taxonomic status of the sterile ascomycete
mycorrhizal endophytes of epacrids remains largely elusive,
but ITS sequence comparisons indicate that one endophyte
(isolated from W. pungens in an eastern Australian sclerophyll
forest) is an Oidiodendron species (Chambers et al., 2000).
Phylogenetic analyses of ITS sequence data from sterile
endophytes of epacrids and known ascomycete taxa suggest
that the unidentified endophytes belong in the order Helotiales
(= Leotiales) (McLean et al., 1999; Chambers et al., 2000).
These observations parallel the conclusions from the molecular
data obtained by Hambleton et al. (1998) and Monreal et al.
(1999) from sterile isolates of mycorrhizal endophytes of
North American Ericaceae. More importantly, they suggest
that mycorrhizal endophytes from epacrids and other Ericaceae,
collected from locations in the southern and northern hemispheres, respectively, show broad taxonomic similarity.
Sharples et al. (2000) conducted phylogenetic analyses of
a large number of ITS sequences from mycorrhizal and
root-associated (mycorrhizal status unconfirmed) endophytes
from epacrids and other Ericaceae taxa, along with a number of
Helotiales ascomycetes. Both neighbour-joining and parsimony
analyses separated the fungi into two strongly supported
(100% bootstrap support) clades (Fig. 7). Most mycorrhizal
Fig. 7 Neighbour-joining tree based on internal transcribed spaces (ITS)-sequence data from ericoid mycorrhizal fungi (purple text), unidentified
endophytes from northern hemisphere Ericaceae ( green text), unidentified endophytes from Australian epacrids (red text) and selected
nonmycorrhizal ascomycetes (yellow text). The analysis was performed as described by Sharples et al. (2000), however, some sequences were
omitted from the present analysis. GenBank accession codes for all isolates are detailed in Sharples et al. (2000). Circles, strongly supported
(> 90% bootstrap percentiles) branches.
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endophytes from epacrids formed part of one clade, along with
mycorrhizal and root-associated endophytes from the northern
hemisphere Ericaceae hosts C. vulgaris and G. shallon, Oidiodendron
spp. and several Hyaloscyphaceae ascomycetes. The second
clade encompassed two epacrid endophytes, all Hymenoscyphus
spp. isolates and certain sterile mycorrhizal and root-associated
endophytes from non-epacrid Ericaceae. A similar pattern
representing a Hymenoscyphus-like clade and a separate unspecified Helotiales clade was observed by Monreal et al. (1999)
for ITS2 sequences for mycorrhizal endophytes from northern
hemisphere Ericaceae hosts. Taken together these clearly indicate that the unknown mycorrhizal endophytes from Ericaceae
worldwide are either part of a Hymenoscyphus-like group or
of a separate, poorly defined, group that has phylogenetic
affinities with Helotiales. The broad range of taxa included in
the analyses, along with the poor bootstrap support obtained
for subgroupings within the latter, requires that more detailed
molecular comparisons, using sequence data from further
(more conserved) loci, be made before taxonomic affinities
within this group can be resolved in further detail.
The Hymenoscyphus-like clade warrants further mention.
As more DNA sequence data are obtained, there is an increasing consensus that, rather than being a single genetically
well-defined taxon, H. ericae represents an aggregate (or
complex) of genetically related isolates (Monreal et al., 1999;
Read, 2000; Sharples et al., 2000; Vrålstad et al., 2000). This
aggregate encompasses isolates that form ericoid mycorrhizas
with Ericaceae, but also isolates found in association with
ectomycorrhizal roots of trees (Bergero et al., 2000; Vrålstad
et al., 2000) and rhizoids of liverworts (Chambers et al., 1999).
The abilities of the isolates from ectomycorrhizas to form
ericoid mycorrhizas remains the subject of speculation, but
H. ericae isolated from ericoid mycorrhizal roots of C. vulgaris is
known to form infection coils in liverwort rhizoids (Duckett
& Read, 1995). ITS sequence data obtained recently in one
of our laboratories indicate that a mycorrhizal endophyte
from W. pungens in New South Wales, Australia is part of the
H. ericae complex (Midgley et al., 2001). The fungus has
> 99% ITS sequence identity with an unidentified mycorrhizal
endophyte (isolate E1-9) from the epacrid Epacris impressa in
Victoria, Australia (McLean et al., 1998), suggesting that the
isolates are probably conspecific (D. J. Midgley et al., unpublished). It further suggests that members of the Hymenoscyphus
aggregate are, in fact, widespread as endophytes of at least
some Epacridaceae in eastern Australia. The two broad groupings of endophytes outlined above thus appear to form ericoid
mycorrhiza with both Ericaceae hosts worldwide.
We are mindful that, to date, molecular analyses have been
conducted only on endophytes from epacrids collected in
the temperate zones of south-eastern Australia. No analyses
have been undertaken on material from other habitats such
as Western Australian sandplain heathlands, subtropical wet
heathland, feldmark-like communites in alpine zones or from
epacrids other than those from continental Australia. Given
the clear similarities between the epacrid endophytes so far
investigated and those from northern hemisphere Ericaceae,
however, it seems unlikely that different taxa have evolved as
endophytes of epacrids in these habitats. Further investigation
thus seems unlikely to alter our current perspective on the
broad taxonomic boundaries of mycorrhizal endophytes of
epacrids. Evolution of particular ecological traits in endophytes
from the different habitats with the potential of parallel evolution or coevolution with their respective epacrid hosts, however,
remains a possibility.
Based on phylogenetic analysis of 28 s rRNA gene sequences,
Cullings (1996) proposed that extant plant taxa with the
ability to form ericoid mycorrhizas share a common ancestral
origin. Given the global distribution of the Ericaceae, and the
fact that their centre of diversity is in Australia, Cullings
suggested that such ancestral plants evolved in the united
Gondwana, some 140 million years ago. Subsequent radiation
northwards is suggested to have led to the spread of ancestral
Ericales to the northern hemisphere (Cullings, 1996). The
extant distribution of the Hymenoscyphus aggregate and undefined Helotiales group as endophytes of both Epacridaceae
and Ericaceae in the southern and northern hemispheres,
respectively, is in keeping with the suggested monophyletic
origin of ericoid mycorrhizas. Indeed, although it has only
been tested for a few endophytes, endophytes from Ericaceae
and Epacridaceae appear to be intercompatible with either
host, at least in terms of the ability to form typical ericoid
mycorrhiza-like coils in epidermal cells (Reed, 1989; Read,
1996; Liu et al., 1998; McLean et al., 1998). Such apparent
intercompatibility implies that evolution of recognition systems
between the fungi and their hosts is likely to have occurred
in a common ancestral host plant taxon (Cullings, 1996).
The information currently available thus points strongly
to evolution of ericoid mycorrhizal associations during the
Cretaceous. This is in keeping with the fossil evidence for
appearance of Ericales-like plants (see above) and molecular
clock estimates for a major evolutionary radiation of ascomycetes
(Berbee & Taylor, 1993).
VII. Community and population biology of
mycorrhizal endophytes
Liu et al. (1998) isolated endophytes from randomly selected
2–3 mm long hair root pieces from four juvenile W. pungens
plants in a 10-m2 plot in a dry sclerophyll forest in eastern
Australia. ITS sequence analysis revealed that at least six
endophyte taxa (including an Oidiodendron sp.) were present
in the total assemblage of endophytes isolated from the plants.
However those from three of the plants were largely of a single
taxon (Liu et al., 1998; Chambers et al., 2000). This suggests
that root systems of individual W. pungens plants at the site
were dominated by a single ericoid mycorrhizal endophyte
taxon, that is now known to fall within the unspecified
Helotiales clade of Sharples et al. (2000).
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Tansley review no. 135
Inter-simple sequence repeat PCR (ISSR-PCR) revealed
that most of the isolates of this taxon obtained from an individual plant were of a single genotype, implying that epacrid
root systems may be extensively colonised by individual
mycelia (Liu et al., 1998). Furthermore, two endophyte genotypes were found to be common to hair roots of two plants
that were collected in juxtaposition, raising the possibility that
epacrid plants can be joined by common endophyte mycelia
where their roots come into contact or are in close proximity
(Liu et al., 1998). Similar symbiont-mediated interconnections
between arbuscular mycorrhizal and ectomycorrhizal plants
have been widely reported and, while it remains the subject
of considerable debate (Fitter et al., 1999; Robinson & Fitter,
1999), this may facilitate some degree of solute movement
between individual plants (Newman, 1988; Simard et al.,
1997). Although informative, the data derived from the work
of Liu et al. (1998) left many unanswered questions regarding
the spatial distribution of the various endophyte taxa and
genotypes within root systems.
By carefully recording positions of root segments from
which isolates were obtained on a digital image of the root
system of a W. pungens seedling from a different sclerophyll
forest site, Midgley et al. (2001) were able to map their spatial
distribution. While five putative taxa were identified in the
isolate assemblage, c. 75% of these were of a single taxon,
confirming the observations of Liu et al. (1998). This taxon was
widely distributed within the root system, but the remaining
four were confined to small, discrete patches of hair root.
ISSR-PCR discriminated six genotypes of the dominant
taxon, but most isolates were of a single genotype which was
widespread within the hair roots of the plant (Midgley et al.,
2001). H. ericae is known to display considerable intraspecific
physiological heterogeneity, at least in terms of nitrogen
utilisation, and it has been suggested that the presence of multiple genotypes of the fungus in a root system might serve
to maximise nitrogen acquisition from the complex soil pool
(Cairney et al., 2000). In the case of W. pungens, data from this
single plant suggest a low degree of functional diversity in
the endophyte community, but more extensive analysis of
replicate plants is clearly required to ascertain if this pattern
is common to other epacrids and at other sites. The most
frequently isolated endophyte taxon was identified by ITS
sequence comparison to fall within the Hymenoscyphus clade
(Midgley et al., 2001), which contrasts with the unspecified
Helotiales affinity of the dominant taxon observed by Liu et al.
(1998). This may reflect site-specific edaphic conditions, but
again, further analyses are required.
VIII. Functional aspects of mycorrhizas
in epacrids
The mutualistic basis of ericoid mycorrhizal symbiosis was
established by demonstration of carbon movement from host
to endophyte (Stribley & Read, 1974) and fungus-mediated
© New Phytologist (2002) 154: 305 – 326 www.newphytologist.com
Review
enhancement of plant nitrogen and phosphorus nutrition via
utilisation of organic sources of the nutrients in soil (Stribley
& Read, 1980; Bajwa & Read, 1985; Myers & Leake, 1996).
This work has been conducted largely using H. ericae and in
the context of the northern hemisphere mor-humus C. vulgaris
heathland habitat, and as such, does not necessarily serve as a
model for ericoid mycorrhiza functioning in epacrids (Straker,
1996; Whittaker & Cairney, 2001). Recent work on North
American G. shallon has broadened our perspective on ericoid
mycorrhizal functioning, and indicates that Oidiodendron maius
Barron, A. strictum and unidentified Helotiales endophytes
can facilitate host access to nitrogen in organic forms (Xiao &
Berch, 1999). To date, however, no in planta physiological
work has been conducted on axenic mycorrhizal epacrid plants.
The mycorrhizal status of endophytes isolated from epacrid
roots has been confirmed simply by formation of mycorrhizalike structures between the fungi and Ericaceae plant hosts
when grown in dual axenic culture (Reed, 1989; Liu et al.,
1998; McLean et al., 1998; Anthony et al., 2000). The limitations of this approach (Leake & Read, 1991) are obvious, and
it is possible that some of the fungi identified as mycorrhizal
only by their abilities to infect potential host plants, may not
serve to enhance host fitness. It also means that our current
functional understanding of mycorrhizas in epacrids relies on
information derived from gross measures of host nutrition in
the glasshouse and field, axenic culture work in isolated endophytes and, perhaps misplaced, inferences from knowledge
of the northern hemisphere systems. Although similarities
exist between northern and southern hemisphere heathlands
in terms of the extremely low availability of nitrogen and phosphorus (Groves, 1983; Straker, 1996), the southern hemisphere
systems are, as a result of relatively scant litter production and
the influence of fire, characteristically low in organic matter
content (Straker, 1996).
The lack of research activity on functional aspects of mycorrhizal associations of epacrids can be explained, in part, by the
acknowledged difficulties in germinating seeds of many taxa
and propagating seedlings under axenic conditions (Willams,
1986; Fox et al., 1987; Bunn et al., 1989; Reed, 1989; McLean
et al., 1994). Even when seedlings are obtained, initiation
and persistence of hair roots under axenic conditions can
constitute a further problem (McLennan, 1935; A. E. Ashford
& J. W. G. Cairney, unpublished). As is the case for many
South African Ericaceae (Ojeda, 1998), it is now clear that
seed germination in many epacrid species can be increased
significantly by treatment with smoke (or aqueous smoke
derivatives) and/or heat shock (Dixon et al., 1995; Keith, 1997),
making the task of obtaining seedlings considerably less tiresome. Furthermore, Leucopogon objectus Benth., E. impressa and
Richea scoparia Hook. f. have been successfully micropropagated
under axenic conditions (Bunn et al., 1989; Anthony et al.,
2000), so that large numbers of sterile plants of at least these
taxa can now be obtained with relative ease. Importantly,
micropropagated E. impressa produced hair roots that were
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Tansley review no. 135
successfully infected by mycorrhizal endophytes (McLean et al.,
1998; Anthony et al., 2000), suggesting that this system may
have considerable potential for use in future physiological
studies. Although persistence of hair root systems beyond a
few weeks has not been demonstrated, variation of the composition of the rooting medium may enhance this (Anthony
et al., 2000). Similarly, transferring plantlets to more complex
media containing, for example, a solid substrate such as sterile
sand (McLennan, 1935), may lead to more sustained hair root
production and increase the applicability of the technique for
use in physiological studies.
Australian heathland and forest soils that are inhabited by
epacrids are characteristically low in phosphorus and nitrogen
(Groves, 1983). Several studies that have used mycorrhizal
plants from the field have provided much information on
some of the likely benefits bestowed upon epacrids by ericoid
mycorrhizal association in such soils. Bell et al. (1994) transferred intact soil cores (from a Western Australian open Banksia
spp. woodland) containing mycorrhizal seedlings of Andersonia
gracilis DC., Astroloma xerophyllum (DC.) Sond., Leucopogon
conostephioides DC. or Leucopogon kingeanus (F. Muell.) C.A.
Gardner to the glasshouse. The cores contained an acid sandy
soil, and were experimentally supplemented with a range of
nutrients. Although none of the epacrids responded to phosphorus addition, all but L. kingeanus showed significantly
increased growth in response to nitrogen addition. Seedlings
grown in unamended soil cores contained significantly less
nitrogen than equivalent seedlings left in the field for the
duration of the experiment (6 months), suggesting that nitrogen depletion had occurred in the limited volume (c. 0.8 l) of
the soil core. Together, these important observations suggest
that in the Western Australian sandplain habitat, growth of
most epacrids is limited largely by nitrogen deficiency, rather
than phosphorus deficiency as might have been predicted for
Australian soils (Bell et al., 1994). Analysis of root xylem sap
of mycorrhizal epacrids from dune heath, kwongan sandplain, swamp, Banksia woodland and Eucalyptus forests in
Western Australia has shown that they vary markedly in their
nitrogen and phosphorus content, although no definitive
habitat-related pattern was observed (Bell & Pate, 1996). The
significance, if any, of mycorrhizal endophytes in this variation is unclear.
The relative availability of the various organic and inorganic nitrogen fractions in soils that support native Australian
vegetation varies seasonally, and is strongly influenced by
edaphic factors such as fire and water availability (Pate et al.,
1993; Erskine et al., 1996; Schmidt & Stewart, 1997). Soluble
organic nitrogen, in forms that include protein and amino
acids, occurs in soils of a broad range of Australian habitats
(Schmidt & Stewart, 1999). In at least one Australian epacrid
habitat, the subtropical wet (wallum) heathland, soluble protein
is the most abundant nitrogen source, and amino acids can
constitute a further major nitrogen fraction, particularly
after waterlogging (Schmidt & Stewart, 1997, 1999). As is the
case for northern hemisphere Ericaceae (Abuarghub & Read,
1988a,b), organic nitrogen is thus a potentially important
nitrogen source for mycorrhizal epacrids in their native
habitats. 15N from ground, dead wheat root material was
assimilated by certain epacrids when applied to soil (Bell &
Pate, 1996). Furthermore, when supplied in simple solution,
15N-labelled glycine was metabolised by nonsterile, presumably mycorrhizal, roots of Epacris pulchella Cav. (Schmidt &
Stewart, 1997, 1999) implying an ability to utilise amino
acids in the soil solution. Bell & Pate (1996) found that 15N
incorporation from labelled plant debris by epacrids did not
differ significantly from accumulation by nonmycorrhizal
Banksia taxa, suggesting that the material may have been
mineralised by the general soil microflora prior to acquisition
by the plants. Indeed, when the epacrids were supplied with
double (13C, 15N)-labelled plant debris, no evidence for 13Cenrichment of the shoots was observed, further suggesting
that mineralisation of nitrogen to inorganic forms probably
occurred before uptake by the plant. While is not possible
from these data to elucidate the relative contributions of
mycorrhizal endophytes and nonsymbiotic rhizosphere microorganisms to this process, results of physiological investigations
of isolated mycorrhizal endophytes suggest that they play a
central role in organic nitrogen utilisation by epacrids.
Unidentified Helotiales mycorrhizal endophytes and an
Oidiodendron sp., isolated from the epacrid W. pungens at a
dry sclerophyll forest site in eastern Australia have been
shown to use NO3− and NH4+ for growth (Chen et al., 1999;
Whittaker & Cairney, 2001). Significantly, they were also
shown readily to utilise certain amino acids and simple protein
(BSA) as sole nitrogen sources (Chen et al., 1999; Whittaker
& Cairney, 2001). Data for the W. pungens endophytes were
broadly similar to those obtained for a strain of H. ericae
isolated from a Calluna heathland, suggesting that their abilities to access nitrogen from simple organic forms in soil are
comparable (Chen et al., 1999; Whittaker & Cairney, 2001).
In common with H. ericae, the epacrid endophytes utilised
acidic, neutral and basic amino acids. Despite the reported
variation in amino acid preference, there was no consistent
pattern in amino acid preference that separated the epacrid
endophytes from H. ericae (Whittaker & Cairney, 2001). By
contrast to the W. pungens endophytes, isolates of the ectomycorrhizal genus Pisolithus from a similar dry sclerophyll
forest habitat were found by Anderson et al. (1999) to use the
basic amino acids arginine and histidine very poorly. Such
differential use of amino acids may indicate that a degree of
niche separation exists between the epacrid and ectomycorrhizal plants in terms of nitrogen use in their native habitat
(Whittaker & Cairney, 2001).
Use of BSA as a sole nitrogen source by endophytes from
W. pungens (Chen et al., 1999) implies that they produce extracellular proteolytic activity as has been clearly demonstrated
for H. ericae (Leake & Read, 1989), further emphasising the
parallelism in the nitrogen nutrition of these fungi. In H. ericae,
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Tansley review no. 135
extracellular proteolysis does not initiate mineralisation of
protein nitrogen. Rather, the resulting amino acids are thought
to be directly transported into hyphae (Read et al., 1989). The
abilities of W. pungens endophytes to utilise amino acids and
BSA as sole nitrogen and carbon sources for growth (Chen
et al., 1999) suggest a similar scenario in epacrid mycorrhizal
endophytes. A degree of physiological congruence between
H. ericae and epacrid mycorrhizal endophytes is further
emphasised by the fact that utilisation of the sulphur-containing
amino acid cysteine was enhanced in both by sulphur deficiency
(Bajwa & Read, 1986; Whittaker & Cairney, 2001). Both
groups of endophytes, may thus benefit their hosts under
conditions of sulphur deficiency by accessing the element
from the soil organic pool.
The form in which nitrogen is transferred to the host remains
unclear. Based upon their failure to observe 13C-enrichment
in shoots of mycorrhizal epacrids exposed to labelled substrate, Bell et al. (1994) suggested that, following absorption
of inorganic nitrogen or catabolism of amino acids in the
mycorrhizal endophytes, NH4+ may be the form of nitrogen
that is transferred. The fact that endophytes from W. pungens
can utilise amino acids as a source of both nitrogen and
carbon (Chen et al., 1999) certainly indicates that the fungi
can catabolise amino acids, but provides no indication of
the chemical form in which nitrogen transfer to the host is
effected. Discrimination between carbon isotopes can occur
during metabolic processing of carbon compounds by fungi
(Henn & Chapela, 2000), raising the possibility that isotope
fractionation may occur before transfer from mycorrhizal fungi
to their plant hosts. This certainly appears to the case for
nitrogen (Schmidt & Stewart, 1997; Kohzu et al., 2000), and
such an effect might mask the transfer of amino acid carbon
in 13C-labelling experiments. In fact, there is good evidence
from investigations of nitrogen metabolising enzymes that
amino acids are transferred to the host via the ectomycorrhizal
interface, regardless of whether inorganic or organic forms of
nitrogen are absorbed by the fungi (Smith & Read, 1997).
Similar enzymological studies of nitrogen metabolism in ericoid
mycorrhizal endophytes are clearly required, however, it seems
reasonable to hypothesise that the compounds transferred
will be broadly similar to those transferred in ectomycorrhizal
associations.
Mycorrhizal epacrids in Western Australian Banksia woodlands were found by Bell et al. (1994) to be phosphorus sufficient, indicating that the plant–fungus system can efficiently
extract phosphorus from naturally occurring sources. Despite
the equivocal nature of some of their data, Chen et al. (1999)
demonstrated that, at least, some mycorrhizal endophytes from
W. pungens in eastern Australia can utilise a phosphomonoester
(inositol hexaphosphate) and a phosphodiester (DNA) as
phosphorus sources. In this way, their abilities to capture phosphorus from organic sources in soil seem likely to approximate
those of H. ericae (Leake & Miles, 1996; Myers & Leake, 1996).
W. pungens endophytes (along with H. ericae) have further been
© New Phytologist (2002) 154: 305 – 326 www.newphytologist.com
Review
shown to solubilise sparingly soluble inorganic phosphorus
supplied in the form of hydroxylapatite [Ca5(PO4)3OH]
(Van Leerdam et al., 2001). Solubilisation occurred in the
presence of NH4+, but not NO3−, as the nitrogen source,
suggesting that it was a function of acidification of the medium
via H+ secretion from hyphae during NH4+ transport. In common with other soil fungi (Lapeyrie et al., 1991; Whitelaw
et al., 1999), neither the epacrid endophytes nor H. ericae
were able to solubilise the more recalcitrant fluorapatite
[Ca5(PO4)3F] (Van Leerdam et al., 2001). As is apparently the
case for nitrogen nutrition, current knowledge suggests no
obvious differences between epacrid mycorrhizal fungi and
endophytes from northern hemisphere Ericaceae with regard
to their contribution to the phosphorus nutrition of their
hosts.
Much of the nitrogen and phosphorus in Calluna heathland
soils is chemically or physically complexed with either plant
wall material or soluble/insoluble polyphenolic compounds
(including quinones and humic/fulvic acids). The latter are
of particular significance because they can precipitate and
inhibit decomposition of organic compounds like proteins,
amino acids and nucleic acids (Bending & Read, 1996).
Much has been made of the fact that H. ericae produces a
range of hydrolytic and oxidative enzyme activities that are
thought to facilitate access to such bound nutrients in soil
(Cairney & Burke, 1998). Furthermore, many of the phenolic
acids in mor-humus, and the organic acids derived from these
by microbial activity, are known to be phytotoxic ( Jalal &
Read, 1983) and H. ericae appears to be capable of reducing
their toxicity toward C. vulgaris (Leake & Read, 1991). Tannic
acid, for example, may be polymerised by catechol oxidase
to reactive quinones that are subsequently polymerised to
more complex quinones (Bending & Read, 1996; Cairney &
Burke, 1998). This is thought to reduce toxicity, decrease
protein binding and so facilitate fungal access to otherwise
unavailable tannin-bound protein (Bending & Read, 1996).
As discussed by Straker (1996), the phytotoxic nature of the
Calluna heathland soils and detoxification of the soil environment by H. ericae may contribute to the ability of C. vulgaris
to form pure stands in these habitats. By contrast, soils in
the drier habitats inhabited by epacrids have a relatively low
organic matter content, and phenolic compounds accumulate
to a much lesser extent, this being due to limited litter production as well as both fire and decomposer activities (Groves,
1983; Read & Mitchell, 1983; Straker, 1996). In these
habitats epacrids characteristically form a component of more
complex plant communities. Aside from the demonstration
that epacrid mycorrhizal endophytes produce extracellular
β-1– 4-endoxylanase activity (Cairney et al., 1996), there have
been no investigations of hydrolytic or oxidative enzyme
production by these fungi. While they appear to share with
their northern hemisphere counterparts an ability to utilise
simple organic forms of nitrogen and phosphorus, those
from the drier habitats, at least, may be less well adapted for
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Tansley review no. 135
dealing with complexed forms of these nutrients. Testing this
hypothesis should be relatively straightforward and would
potentially reveal much information regarding the evolution
of ericoid mycorrhizas and their roles in structuring plant
communties in northern and southern hemisphere habitats.
Ericoid mycorrhizal fungal partners of northern hemisphere
Ericaceae are regarded as resistant to a range of potentially
toxic metals including Al, Cu, Fe, Pb and Zn (Bradley et al.,
1981, 1982; Hashem, 1990, 1995; Burt et al., 1986). For Cu
and Zn, at least, there is convincing evidence that mycorrhizal infection can confer a degree of metal resistance on an
otherwise metal sensitive Ericaceae host (Bradley et al., 1981,
1982; Hashem, 1990, 1995). In this way, ericoid mycorrhizal
infection is regarded as important in colonisation of metal
contaminated sites by certain Ericaceae taxa (Meharg &
Cairney, 2000). The apparently constitutive resistance to
certain metals is thought to reflect an evolved response to
extreme low pH and anaerobic conditions (and consequent
solubilisation of toxic metals such as Al, and redox-active
metals such as Fe and Mn) that characterise the mor humus
soils (Meharg & Cairney, 2000). Recent data indicate that
mycorrhizal endophytes from W. pungens are broadly similar
to H. ericae strains in terms of their resistance to the potentially toxic metals Cd, Cu and Zn (Cairney et al., 2001). This
further emphasises the apparent physiological uniformity of
ericoid mycorrhizal fungi, but also raises questions regarding
the evolution of metal insensitivity. The soils from which the
W. pungens endophytes were obtained contain low levels of
potentially toxic metals and are generally sandy with moderately acidic pH (Cairney et al., 2001). Bioavailability of toxic
metals in these soils is thus likely to be considerably lower than
in mor-humus soils and it is difficult to envisage that such an
environment would select for metal resistance in mycorrhizal
fungal populations.
IX. Conclusions
Although the biology of mycorrhizas in epacrids has received
considerably less attention than certain northern hemisphere
Ericaceae, this should not be taken to indicate their relative
ecological importance. Epacrids form an important component
of some heathland and open forest communities in the
southern hemisphere, and it is likely that their ericoid
mycorrhizal status is important in their success in these
habitats. Clear patterns exist in the geographical distribution
of epacrids and other ericoid mycorrhiza-forming Ericaceae
taxa. The habitats in which they thrive, however, characteristically display extremely low nutrient availability and are
subject to considerable edaphic stress. From what is known at
present, the fungi forming ericoid mycorrhizas with epacrids
appear to be broadly similar to those from other Ericaceae
taxa. Penetration of hair root cells by ericoid mycorrhizal fungi
and the subsequent development of the symbiosis also appear
to follow similar patterns. By contrast to certain northern
hemisphere Ericaceae, relatively little is known regarding
symbiotic functioning of ericoid mycorrhizas in epacrids.
Investigations using naturally mycorrhized plants and of the
activities of isolated endophytes in axenic culture, however,
suggest that the benefits bestowed upon epacrid hosts may be
broadly similar to those identified for northern hemisphere
Ericaceae.
Acknowledgements
JWGC thanks the University of Western Sydney for provision
of a period of Academic Study Leave and AEA gratefully
acknowledges Professor Bengt Söderström, Department of
Microbial Ecology, Lund University and the Swedish Research
Council for supporting a guest Professorship, during which
time this review was written. We thank Dr Candy Briggs for
her useful comments on the original manuscript.
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