Suillus bovinus

Ectomycorrhizal fungi: exploring themycelial frontierIan C. Anderson1 & John W.G. Cairney2

1The Macaulay Institute, Craigiebuckler, Aberdeen, UK; and 2Centre for Plant and Food Science, University of Western Sydney, NSW, Australia

Correspondence: Ian C. Anderson, The

Macaulay Institute, Craigiebuckler, Aberdeen

AB15 8QH, UK. Tel.: 144 1224 498200;

ext. 2357; fax: 144 1224 498207; e-mail:

[email protected]

Received 1 December 2006; revised 12

February 2007; accepted 5 March 2007.

First published online 28 April 2007.

DOI:10.1111/j.1574-6976.2007.00073.x

Editor: Ramon Diaz Orejas

Keywords

ectomycorrhizal fungi; soil-borne mycelia;

mycelial biomass; ectomycorrhizal

communities; elevated atmospheric CO2.

Abstract

Ectomycorrhizal (ECM) fungi form mutualistic symbioses with many tree species

and are regarded as key organisms in nutrient and carbon cycles in forest

ecosystems. Our appreciation of their roles in these processes is hampered by a

lack of understanding of their soil-borne mycelial systems. These mycelia represent

the vegetative thalli of ECM fungi that link carbon-yielding tree roots with soil

nutrients, yet we remain largely ignorant of their distribution, dynamics and

activities in forest soils. In this review we consider information derived from

investigations of fruiting bodies, ECM root tips and laboratory-based microcosm

studies, and conclude that these provide only limited insights into soil-borne ECM

mycelial communities. Recent advances in understanding soil-borne mycelia of

ECM fungi have arisen from the combined use of molecular technologies and

novel field experimentation. These approaches have the potential to provide

unprecedented insights into the functioning of ECM mycelia at the ecosystem

level, particularly in the context of land-use changes and global climate change.

Introduction

Many tree species in forest habitats worldwide rely upon

mutualistic ectomycorrhizal (ECM) fungi to fulfil their

nutrient requirements (Smith & Read, 1997). These fungi

contribute to tree nutrition by means of mineral weathering

(Landeweert et al., 2001) and mobilization of nutrients from

organic complexes (Read & Perez-Moreno, 2003). They are

also an important avenue for the delivery of carbon to soil

and are responsible for a substantial component of forest-

soil carbon fluxes (Soderstrom, 1992; Hogberg et al., 2001;

Hogberg & Hogberg, 2002; Godbold et al., 2006; Hobbie,

2006). ECM fungi are thus regarded as key elements of forest

nutrient cycles and as strong drivers of forest ecosystem

processes (Read et al., 2004).

The fungi that form ECM associations comprise a tax-

onomically broad suite of basidiomycetes and, to a lesser

extent, ascomycetes (Smith & Read, 1997). Typically, the

fungi form a mycelial mantle around short lateral roots of

their hosts and penetrate between epidermal and cortical

cells, surrounding them with a highly branched structure,

the Hartig net (Peterson et al., 2004). Significant variation

exists in the morphology of ECM root tips that are infected

by different fungal taxa, and analysis of macroscopic and

microscopic characteristics of ECM roots is used widely for

identification of the ECM fungi (Agerer, 1987–2002). The

fungus–plant interface formed by the Hartig net in the ECM

root is functionally critical to the mutualism, as it represents

the interface across which nutrients and carbon are trans-

ferred between the partners (Smith & Read, 1997).

In addition to the structures formed at the host root,

ECM fungi produce mycelia that extend from the mantle

into the surrounding soil (Fig. 1), although the extent and

structure of this extramatrical mycelium is thought to differ

between ECM fungal taxa (Agerer, 2001). The soil-borne

mycelia of ECM fungi are regarded as functionally impor-

tant to the mutualism in foraging for, and translocation of,

nutrients and water. They also infect short lateral roots

(Smith & Read, 1997) and potentially form a common

mycelial network that might facilitate carbon and/or nutri-

ent movement between individual tree hosts (Simard &

Durall, 2004; Selosse et al., 2006). At the ecosystem level

these mycelia are of further significance in the mobilization

of nutrients from organic and recalcitrant inorganic sources,

both in competition and in cooperation with soil-dwelling

saprotrophs, in the relocation of nutrients within the

soil–plant continuum, and in the delivery and distribution

of carbon belowground (Leake et al., 2002; Wallander,

2006). Despite the importance of soil-borne mycelia of

ECM fungi in forest ecosystems, along with their likely

importance in the global carbon budget (Hobbie, 2006)

and sensitivity to global change factors such as rising atmo-

spheric CO2 (Rillig et al., 2002), we know relatively little

regarding their extent, diversity and spatial location in forest

FEMS Microbiol Rev 31 (2007) 388–406c� 2007 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

soils. In particular, the current lack of fundamental informa-

tion regarding the physical and physiological continuity of

belowground mycelia limits our understanding of the im-

portance of ECM mycelial networks in nutrient cycling and

in the distribution of carbon and nutrients within forest-soil

systems (Cairney, 2005). Furthermore, although many de-

tailed investigations of communities of ECM fungi coloniz-

ing root tips have been undertaken (see below), the relative

importance of particular taxa in root-tip communities gives

no insight into the extent of their mycelial systems in soil

(Genney et al., 2006), nor into their contributions to

nutrient and carbon cycling.

To date, investigation of ECM mycelia in soil has been

constrained by the difficulty in identifying ECM mycelia and

in separating these from mycelia of non-ECM soil fungi.

Most of our current understanding has thus been derived via

inference from the distribution of fruiting bodies and ECM

root tips in the field and/or from observations of mycelia

Fig. 1. Mycelial systems of ectomycorrhizal fungi. (a) A typical microcosm system demonstrating the mycelium of Suillus variegatus growing from a

Scots pine seedling (photo taken by P.M.A. Fransson). (b) Russula sp. fruiting body and associated mycelium. (c) A close-up of (b) showing the base of

the fruiting-body stipe, the associated white mycelium, and ECM root tips (arrows) colonized by this fungus. (d) A dense mycelial mat that has been

lifted from a rock surface in a native Scots pine forest. Patches of brown, pink, white and yellow mycelium of different fungal species are visible (photo

taken by C.D. Campbell).

FEMS Microbiol Rev 31 (2007) 388–406 c� 2007 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

389Ectomycorrhizal fungal mycelia

produced in microcosms in the laboratory (Fig. 1). As we

will outline in this review, many of the assumptions upon

which these inferences are based are somewhat tenuous, and

current understanding of complex ECM mycelial systems in

forest soils remains limited. Recent years have, however,

witnessed the development and application of a suite of

molecular methods that are yielding unprecedented insights

into ECM mycelia in forest soils. In this review we highlight

some of the limitations of our current knowledge, along

with the most significant recent advances in our apprecia-

tion of the distribution, dynamics and activities of soil-

borne ECM mycelial systems along with their responses to

elevated atmospheric CO2 concentrations.

Fruiting bodies as indicators ofcommunity structure

Although frequently used in surveys of higher fungal

diversity, fruiting bodies provide little useful information

regarding the diversity of ECM fungal mycelia in soil.

Clearly, the presence of an epigeous fruiting body indicates

that mycelium of the fungus must be present in one form or

another in the underlying soil. Because many ECM taxa

produce no epigeous fruiting bodies, or may not have

fruited during the duration of the survey, it is impossible to

infer the importance of a fungus in the belowground

community on the basis of fruiting-body observations. In

fact, where fruiting-body data have been compared with

ECM root tips (see below), there is generally a very poor

correlation between the two, with the latter typically indi-

cating greater species richness (Erland & Taylor, 2002).

Furthermore, there is evidence that, for Laccaria spp. at

least, physiological and/or genetic differences may influence

fruiting at the intraspecific level (Selosse et al., 2001).

Estimation of mycelial distribution usingfruiting bodies

For those ECM fungi that produce epigeous fruiting bodies,

their presence has been used to infer the distribution of

mycelia of the fungi in forest soils. This method centres on

analysis of the mycelial genotype in the vegetative tissue of

the fruiting body and mapping the distribution of the

genotypes in a forest stand. Fruiting bodies that have the

same vegetative genotype must have developed from a

genetically identical mycelium that has grown through soil,

thus allowing estimation of the distribution of soil-borne

mycelia (Dahlberg & Stenlid, 1995). Initially this was

achieved by isolation of vegetative fruiting-body stipe my-

celium into axenic culture, followed by somatic incompat-

ibility testing to identify which isolates are genetically

identical (e.g. Dahlberg & Stenlid, 1990, 1994; Baar et al.,

1994). While useful for fungi that are readily cultured, many

ECM fungi are difficult or impossible to isolate into axenic

culture by conventional methods (Brundrett et al., 1996),

limiting the usefulness of somatic compatibility testing for

ECM fungi. Concerns have also been raised about the

resolution of the method for identifying genotypes of some

ECM taxa (Sen, 1990; Jacobson et al., 1993).

More recently, molecular methods for the identification

of genotypes using DNA extracted from vegetative fruiting-

body tissue have been used in this context. A range of

dominant molecular markers, including random amplified

polymorphic DNA (RAPD), simple sequence repeat (SSR),

amplified fragment length polymorphism (AFLP), single-

strand conformational polymorphism (SSCP) and inter-

retrotransposon amplified polymorphism (IRAP) markers

(e.g. De la Bastide et al., 1994; Anderson et al., 1998; Bonello

et al., 1998; Sawyer et al., 1999; Redecker et al., 2001; Murata

et al., 2005), along with codominant repeat SSR markers

(e.g. Zhou et al., 2001a; Dunham et al., 2003; Kretzer et al.,

2004; Wu et al., 2005; Bergemann et al., 2006), has now been

used to infer the genotype distributions of certain ECM

fungi in a variety of forest habitats. While some of these

molecular methods may not resolve all genotypes in a

population (Redecker et al., 2001; Dunham et al., 2003;

Bagley & Orlovich, 2004), they have been widely used to

estimate the spatial distribution of ECM fungal genotypes.

It is thus evident that genotypes of some ECM fungi are

spread over tens of metres in some forests, implying that

mycelia can extend for considerable distances, and persist

for several years, in the field (e.g. Dahlberg & Stenlid, 1990;

Baar et al., 1994; Dahlberg, 1997; Anderson et al., 1998;

Bonello et al., 1998; Sawyer et al., 1999). Estimates of

belowground mycelial growth rates (e.g. Dahlberg & Stenlid,

1990; Bonello et al., 1998; Sawyer et al., 1999; Lian et al.,

2006) and investigations of the survival of genotypes intro-

duced as ECM inoculum in forest plantations (e.g. De la

Bastide et al., 1994; Selosse et al., 1998; Sawyer et al., 2001)

provide strong supplementary evidence that mycelia of

certain ECM fungi can persist in forest soils for at least tens

of years. Whether these genotypes persist in soil as contin-

uous mycelia [ = genets (Dahlberg & Stenlid, 1995)], or have

fragmented into smaller mycelia [ = ramets (Dahlberg &

Stenlid, 1995)] as a result of, for example, feeding activities

of mycophagous soil arthropods (Schneider et al., 2005) or

other forms of disturbance, remains unclear.

Fruiting-body collections indicate that multiple geno-

types of some species, such as Laccaria spp., frequently

occur in an area of a few square metres, suggesting the

presence of many spatially restricted and ephemeral mycelia

in the underlying soil (e.g. Gryta et al., 1997; Gherbi et al.,

1999; Fiore-Donno & Martin, 2001; Dunham et al., 2003;

Murata et al., 2005). Within genera such as Amanita

(Redecker et al., 2001; Sawyer et al., 2001, 2003; Bagley &

Orlovich, 2004; Liang et al., 2005), Russula (Redecker et al.,


390 I.C. Anderson et al.

2001; Bergemann & Miller, 2002; Liang et al., 2004; Riviere

et al., 2006) or Tricholoma (Murata et al., 2005; Gryta et al.,

2006; Lian et al., 2006) there are varying reports of wide-

spread and spatially restricted genotypes. Similar observa-

tions have been made in population studies of individual

species such as Suillus bovinus (Dahlberg & Stenlid, 1990,

1994), Laccaria bicolor (Baar et al., 1994; Selosse et al., 1998)

and Pisolithus albus (Anderson et al., 1998, 2001), making it

difficult to predict genotype distribution, and so infer the

dimensions of belowground mycelia, strictly on the basis of

taxonomic affiliation.

The occurrence of multiple spatially restricted genotypes

of an ECM fungus is thought to reflect frequent mycelial

establishment from meiospores, while a local population

dominated by widespread genotypes is characteristic of the

sustained growth of persistent belowground mycelia (Dahl-

berg & Stenlid, 1995). There is some evidence that, for

certain ECM fungi, the former may prevail in recently

disturbed or younger forests, while the latter predominate

in mature undisturbed forests (Dahlberg & Stenlid, 1990,

1995). This is not always the case, however, because the

distribution of host tree species in a mixed forest may

influence the distribution of ECM genotypes (Zhou et al.,

2000). Furthermore, multiple spatially restricted genotypes

have been identified in populations of some ECM fungi in

mature forests (e.g. Gherbi et al., 1999; Fiore-Donno &

Martin, 2001; Redecker et al., 2001), and widespread geno-

types in relatively young plantations (Selosse, 2003). The

meaning of these observations in terms of the distribution of

soil-borne mycelia is questionable. At best, such information

based on fruiting-body distribution allows estimation of

possible mycelial dimensions in the underlying soil. It

provides no information on mycelial continuity, density or

vertical distribution in the soil profile.

Community structure and spatialdistribution based on ectomycorrhizalroot tips

Root tip-based assessment of belowground ECMfungal diversity

With the realization that analysis of ECM fungal commu-

nities on the basis of fruiting-body data is a poor indicator

of belowground communities of the fungi (Horton & Bruns,

2001; Erland & Taylor, 2002) came acceptance that commu-

nities of ECM fungi are best analysed on the basis of

identification and enumeration of short lateral roots in-

fected by ECM fungal taxa ( = ECM roots). Initially achieved

by morphological identification of ECM roots (e.g. Taylor &

Alexander, 1989; Massicotte et al., 1999), PCR-based mole-

cular approaches have become de rigueur for these investi-

gations in recent years (Horton & Bruns, 2001; Anderson,

2006), and a great deal of thought has been invested in

designing appropriate sampling strategies (e.g. Horton &

Bruns, 2001; Taylor, 2002). Although some specific habitats

appear to be characterized by low species diversity of ECM

roots (Chambers et al., 2005), numerous such investigations

have revealed that communities of ECM roots in multi-

farious forest habitats are generally extremely species-rich

(reviewed Horton & Bruns, 2001). It is also evident that

communities of ECM roots vary with vegetation/environ-

mental gradients and chronosequences (e.g. Kernaghan &

Harper, 2001; Wurzenburger et al., 2004; Dickie & Reich,

2005; Palfner et al., 2005; Robertson et al., 2006).

ECM root species richness and/or community structure

are influenced by a range of factors, including soil type

(Gehring et al., 1998; Moser et al., 2005), soil moisture (e.g.

Taylor & Alexander, 1989; Karen et al., 1996; Shi et al., 2002;

Walker et al., 2005), season (Giachini et al., 2004), natural

nutrient gradients (Toljander et al., 2006), fire (e.g. Visser,

1995; Stendell et al., 1999; Grogan et al., 2000; Smith et al.,

2005; and see the review by Cairney & Bastias, 2006),

activities of herbivores and plant parasites (Brown et al.,

2001; Cullings et al., 2005; Mueller & Gehring, 2006), and

the quality and quantity of organic matter (e.g. Conn &

Dighton, 2000; Cullings et al., 2003). ECM root-tip com-

munities can also be strongly influenced by a range of forest

management practices (reviewed Jones et al., 2003) and by

gradients of nitrogen deposition (Taylor et al., 2000; Lilles-

kov et al., 2002; Dighton et al., 2004). Further anthropogenic

factors that have been shown to influence ECM root com-

munities include localized nitrogen addition (e.g. Karen &

Nylund, 1997; Fransson et al., 2000; Peter et al., 2001; Avis

et al., 2003; Berch et al., 2006; Carfrae et al., 2006), acid

precipitation (e.g. Rapp & Jentschke, 1994; Qian et al., 1998;

Roth & Fahey, 1998), elevated atmospheric CO2 concentra-

tions (Rey & Jarvis, 1997; Fransson et al., 2001; Kasurinen

et al., 2005), toxic metal contamination (e.g. Markkola et al.,

2002), and other forms of anthropogenic pollution (re-

viewed Cairney & Meharg, 1999).

Diversity and distribution of ECM root tips atdifferent scales

Although belowground root-tip analyses have been instru-

mental in furthering our understanding of ECM fungal

communities, it is without doubt that such studies are

confounded both by sampling intensity and by the scale at

which the study was conducted (Horton & Bruns, 2001;

Taylor, 2002). By constructing species� area curves for data

published in four previous studies, Horton & Bruns (2001)

demonstrated that, in all but one, insufficient samples were

analysed to have covered the diversity of ECM taxa present.

This demonstrates that a potentially inaccurate or partial



picture of diversity and community composition can be

generated if the sampling intensity is insufficient. While this

alone has consequences for data interpretation, sampling

intensity cannot be considered independently from scale

(Lilleskov et al., 2004). This is particularly important

because in many field studies the desirable scale for the

analysis is the stand or forest level, but, in reality, pattern in

most ECM communities occurs at much finer scales. For

example, analysis of ECM root-tip community data from

eight studies suggested that it is necessary to take cores at

least 3 m apart in order to achieve the greatest sampling

efficiency for stand-level community analysis (Lilleskov

et al., 2004). However, ECM root-tip abundance and simi-

larity of community composition have been shown to be

highly variable at much finer scales (5–20 cm) (Tedersoo

et al., 2003; Izzo et al., 2005), with a complete change in

ECM species composition being observed at a scale of 50 cm

in a mixed forest (Tedersoo et al., 2003).

There is an obvious trade-off between scale and sampling

intensity in root tip-based investigations of ECM commu-

nities, and alternative sampling approaches, including the

selection of random root tips from soil cores (Horton &

Bruns, 2001; Tedersoo et al., 2003), down to as few as three

ECM root-tips per sample (Koide et al., 2005b), have been

suggested as a possible way of tackling this problem. In most

ECM communities, a small number of ECM species are

highly abundant and dominant (Taylor, 2002). In these

situations it is difficult to see how this would not confound

the ecological interpretation of the data when so few root

tips are analysed.

Vertical stratification in ECM root-tipcommunities

A major advance in our understanding of the belowground

ecology of ECM fungi has been the observation that ECM

root-tip communities are vertically stratified in the soil

profile, with different fungi typically occupying different

horizons (Goodman & Trofymow, 1998; Rosling et al., 2003;

Tedersoo et al., 2003; Izzo et al., 2005; Baier et al., 2006;

Genney et al., 2006). While this might signal a degree of

niche differentiation between ECM taxa, caution is required

in interpretation because the vertical distribution of ECM

roots does not necessarily reflect the distribution of mycelia

of the respective taxa (Genney et al., 2006; and see below).

Despite the extensive and often painstaking effort that has

been devoted to investigating communities of ECM roots, our

understanding of the belowground ecology of ECM fungi in

forest ecosystems remains limited. The ECM root-based

studies have provided unprecedented insights into the diver-

sity of fungal taxa that form ECM associations, along with

aspects of their dynamics under a range of conditions, yet they

have failed to consider the components of ECM fungi that are

functionally most important in forest soil nutrient and carbon

cycling processes � the soil-borne mycelial systems.

Similar to aboveground and belowground comparisons

of ECM communities, numerous studies have reported a

lack of congruence between ECM species colonizing root

tips and those detected using molecular methods to be

present as mycelia in the same soil volume (Koide et al.,

2005a; Kj�ller, 2006). Therefore, the true diversity of ECM

species may remain uncovered if those species that are only

present as mycelia in a given soil volume, and which are

likely to be of some functional importance to their host

partners, are not considered. In addition, even where intense

sampling protocols have been adopted, the spatial distribu-

tion patterns of root-tip communities may not necessarily

reflect the patterns of the mycelial distribution in soil of the

constituent species (Genney et al., 2006).

Because ECM fungi are ‘nonresource-unit-restricted’

fungi (sensu Boddy, 1999), their mycelia forage through soil

to greater or lesser extents for nutrients, water and new

short-lateral roots to colonize (Read, 1992). In doing so,

they form indeterminate, structurally and physiologically

heterogeneous interconnected networks of which the colo-

nized roots of the plant host form a part (Rayner, 1991;

Cairney & Burke, 1996). There is a growing awareness not

only that investigation of ECM mycelia in forest soils is

imperative to a functional understanding of ECM commu-

nities, but also that communities of ECM fungi when

considered from the perspective of their mycelial systems in

soil may be quite different from those identified on the basis

of colonized root tips (e.g. Horton & Bruns, 2001; Cairney,

2005; Anderson, 2006; Genney et al., 2006; Wallander, 2006).

Estimation of mycelial distribution inectomycorrhizal populations using roottips

In addition to community analysis, ECM roots have been

used to estimate the distribution of ECM mycelial genotypes

belowground. This work has confirmed the observations

based on fruiting-body distribution (see above) that geno-

types of some taxa are spatially restricted belowground and

that the distribution of others can be widespread (Guidot

et al., 2001; Hirose et al., 2004; Kretzer et al., 2004; Lian

et al., 2006). It is also clear that genotypes of some ECM

fungal taxa can infect multiple hosts in the field simulta-

neously (Lian et al., 2006), providing circumstantial support

for the notion of a common mycelial network in forest soil

(Peter, 2006). Importantly, it has also become evident that

while estimates of genotype distribution based on fruiting-

body distribution sometimes correlate roughly with esti-

mates based on root tips (Guidot et al., 2001; Zhou et al.,

2001b; Lian et al., 2006), in other instances root tip-based

estimates indicate more widespread genotype distribution



(Hirose et al., 2004). Furthermore, there is strong evidence

that belowground genotypes of at least some taxa, can differ

in temporal fruiting patterns (Guidot et al., 2001; Zhou

et al., 2001b; Hirose et al., 2004), meaning that, unless

repeated sampling is conducted, fruiting-body analysis may

only unveil part of the population that is present. Although,

with an appropriate sampling strategy, root-tip studies may

provide some information on the vertical distribution of

mycelial genotypes in ECM roots (Zhou et al., 2001b), they

do not necessarily provide an accurate reflection of the

vertical distribution of the equivalent soil-borne mycelia

(Genney et al., 2006).

Direct observation of ectomycorrhizalmycelia in the field

The indeterminate filamentous nature of fungal hyphae,

coupled with the complex nature of soil fungal communities

(Fig. 1) and difficulties in identifying fungal mycelia in the

absence of fruiting bodies, makes direct observation and

identification of fungal mycelia in soil virtually impossible,

and ECM fungi are no exception in this regard (Bridge &

Spooner, 2001; Cairney, 2005). Some higher fungi produce

macroscopic linear mycelial aggregates (= rhizomorphs sensu

Cairney et al., 1991) during growth through soil. In the case

of certain saprotrophic fungi, robust rhizomorphs are

produced within the soil� litter interface, and this has

facilitated excavation, direct measurement and mapping of

more-or-less intact mycelial systems (see Thompson, 1984).

Although many ECM fungi form rhizomorphs, these are

typically diminutive, and thus direct observations of ECM

mycelia in soil have been largely confined to those produced

within a few centimetres of ECM root tips or fruiting bodies.

From investigations of this nature, it is evident that different

ECM fungi produce different amounts of soil-borne myce-

lium, and that the propensity for mycelia to differentiate to

form rhizomorphs also varies considerably (reviewed by

Agerer, 2001). Mycelia that remain nonrhizomorphic are

thought to reflect a limited ability to explore surrounding

soil, while mycelia that comprise highly differentiated rhi-

zomorphs are regarded as more adapted to long-distance

exploration (Agerer, 2001). Between the two extremes are a

range of more-or-less differentiated mycelial types that

facilitate medium-distance exploration in soil (Agerer,

2001). According to Agerer (2001), the most highly differ-

entiated rhizomorphs can explore ‘up to several decimetres’

from the root surface in soil. Difficulties in excavating even

the most robust ECM rhizomorph systems in soil make it

difficult to determine the extent to which this underesti-

mates the exploration potential of ECM mycelia in soil, but

nonetheless this classification scheme represents a useful

way to categorize ECM fungi based on the potential

ecological relevance of their mycelial systems.

Some ECM fungi produce dense mycelial mats or shiro in

forest soil. Mat-forming fungi such as Gaultheria and

Hysterangium spp. produce persistent dense mycelia that

occupy the upper few centimetres in soil and occupy areas

that are typically o 1 m2 (Cromack et al., 1979; Griffiths

et al., 1991). While discrete mats produced by these fungi

can be identified and measured in soil, it is not clear whether

they comprise single or multiple mycelia of the fungi in

question. The shiro produced by Tricholoma matsutake

persist for many years and develop by outward growth in a

‘fairy ring’ fashion from a central point for up to several

metres (Ogawa, 1977). Recent analysis of vegetative mycelia

from fruiting bodies indicate that a single shiro of

T. matsutake can comprise multiple mycelia (Murata et al.,

2005); however, the distribution of these mycelia within the

shiro remains unclear.

Observations of mycelia in microcosms

Our understanding of the physiological activities of ECM

mycelia in soil has been built largely on microcosm studies.

The most widely used microcosm system comprises a thin

layer of milled peat or soil between two Perspex sheets

(Duddridge et al., 1980), or a similar construction (Fig. 1).

Such microcosms have been used to elegantly demonstrate

the roles of ECM mycelia in the absorption and transloca-

tion of water, phosphorus and nitrogen from soil to the

plant host (Duddridge et al., 1980; Finlay & Read, 1986a, b;

Finlay et al., 1988, 1989; Ek et al., 1994; Andersson et al.,

1996; Timonen et al., 1996; Ek, 1997), along with establish-

ing the mycelia as important conduits for carbon movement

from tree hosts into soil (Finlay & Read, 1986a, b; Ek, 1997;

Leake et al., 2001; Mahmood et al., 2001; Wu et al., 2002;

Heinonsalo et al., 2004; Rosling et al., 2004). The micro-

cosms have also been pivotal in identifying potential pat-

terns and processes involved in the growth, development

and differentiation of ECM mycelial systems (Read, 1992;

Donnelly et al., 2004), along with the likely influence of

edaphic changes such as pH (Erland et al., 1990; Ek et al.,

1994; Mahmood et al., 2001). An important observation

here has been the foraging behaviour of ECM mycelia in soil

and, in particular, their abilities to densely colonize discrete

patches of various organic substrates or necromass from

which they can effect nutrient mobilization (Finlay & Read,

1986a, b; Carleton & Read, 1991; Bending & Read, 1995a, b;

Perez-Moreno & Read, 2000, 2001a, b; Leake et al., 2001;

Lilleskov & Bruns, 2003; Donnelly et al., 2004; Wallander &

Pallon, 2005). Microcosms have also been used to investigate

factors such as ECM mycelial longevity and temporal

changes in elemental composition (Downes et al., 1992;

Wallander & Pallon, 2005), interactions with soil minerals

(Wallander et al., 2002), responses of mycelia to elevated

atmospheric CO2 concentrations (e.g. Rouhier & Read,



1998, 1999; Fransson et al., 2005), spatial heterogeneity in

enzyme and gene expression in mycelial systems (Timonen

& Sen, 1998; Wright et al., 2005), and to quantify ECM

myelial area, density, biomass and elemental content in soil

(Ek, 1997; Schubert et al., 2003; Agerer & Raidl, 2004;

Donnelly et al., 2004; Hagerberg et al., 2005).

Although microcosm-based studies have facilitated sig-

nificant advances in our appreciation of the activities of

ECM mycelia in soil, there is a danger that they may have

painted a somewhat unrealistic picture of the extent of ECM

mycelial system development in the field. Images of ECM

plants in microcosms generally portray a seedling with a

small root system and an ECM mycelial system that radiates

throughout much of the microcosm soil, exploring an area

of soil that is several times that occupied by the root system

(Fig. 1). The ‘extensive soil-colonizing vegetative mycelium’

produced in microcosms is viewed as representing an ideal

model of ECM systems in forest soil (Sen, 2000); however,

such extrapolation needs to be undertaken with caution for

several reasons. First, the extent of mycelial development in

more-or-less homogenous milled or sieved microcosm sub-

strates is unlikely to reflect that through the heterogeneous

matrix of natural forest soil. Indeed, as highlighted by Smith

& Read (1997), the intensive hyphal colonization observed

in patches of introduced organic matter (e.g. Finlay & Read,

1986a, b; Bending & Read, 1995b; Perez-Moreno & Read,

2000; Leake et al., 2001) may be more representative of

mycelial growth in forest soil than the extensive mycelial

exploration that occurs through the homogeneous micro-

cosm substrates. The largely two-dimensional nature of the

microcosms means that exploration of the substrate is

effectively on a single plane, and thus the distance that

mycelium grows from the root system is likely to be greater

than would be the case for the equivalent mycelial biomass

in a three-dimensional soil mass. Even where three-dimen-

sional microcosms have been used (e.g. Coutts & Nicholl,

1990), however, observations of mycelia rely on growth at

the interface between the soil and the transparent micro-

cosm window, and, as such, are unlikely to reflect growth of

the mycelia within the soil column. Add to this the lack of

vertical stratification of soil (see below) in the microcosms

(although the rudimentary soil profile reconstruction of

Heinonsalo et al. (2004) partially addressed this), along with

the small size of seedlings, for which the carbon balance and

patterns of carbon allocation are likely to be dissimilar to

that of mature trees in the field (Ericsson et al., 1996), and

the relative growth of mycelia in microcosms may not be

much of a guide to mycelia in the field.

Despite these limitations, rates of growth of ECM mycelia

in microcosms (Read, 1992) have been used to estimate rates

of mycelial expansion in the field (e.g. Bonello et al., 1998;

Sawyer et al., 1999). With the above in mind, and the fact

that the nature of the substrate and edaphic conditions can

strongly affect ECM mycelial growth (Erland et al., 1990;

Smith & Read, 1997), such estimation must be viewed with a

degree of caution. Attention must also be drawn to the fact

that, in most cases, microcosms reflect mycelial growth of a

single ECM fungus in the absence of competing ECM or

other macro fungi. Where competing ECM fungi have been

coinoculated in microcosms, it is clear that mycelial devel-

opment can be altered significantly (Wu et al., 1999). While

the outcomes of interactions appear to vary, largely accord-

ing to relative carbon availability to the individual fungi

(Lindahl, 2000; Lindahl et al., 2001), it is further evident that

the presence of saprotrophic basidiomycete mycelia can

influence the growth and development of ECM mycelia in

microcosms (Lindahl et al., 1999, 2001; Leake et al., 2001,

2002). Because mycelia of different taxa may preferentially

grow at different depths in the soil (Lindahl et al., 1999), the

largely two-dimensional nature of the microcosms will

reduce the potential for different mycelia to avoid competi-

tion by vertical stratification of growth (Cairney, 2005). Care

must therefore be taken when attempting to extrapolate

outcomes of competitive interactions between mycelia in

microcosms to the field.

Estimation of mycelial biomass in the field

Given the apparent importance of ECM mycelia in forest

carbon and nutrient cycles, an understanding of their

belowground biomass is pivotal to fully appreciating their

contributions to biogeochemical processes. While estimates

of mycelial biomass in simple nonsoil substrates have been

obtained in the laboratory (e.g. Colpaert et al., 1992;

Rousseau et al., 1994), determining ECM mycelial biomass

in field soil has, until recently, remained a vexed issue.

Hogberg & Hogberg (2002) adopted an indirect method

that involved tree girdling, soil fumigation and measure-

ments of dissolved organic carbon to estimate that ECM

mycelium accounts for 4 32% of the total microbial

biomass in a boreal forest, while estimates have also been

derived by long-term incubation of soil in the laboratory.

Incubation in the absence of a host plant means that ECM

mycelia should degrade during the incubation period, and

thus the difference between fungal biomass, estimated by

ergosterol or phospholipid fatty acid (PLFA) analysis, before

and after incubation, is used as an estimate of ECM fungal

biomass (Wallander et al., 2004). Such studies indicate that

ECM mycelial biomass decreases with soil depth (Baath

et al., 2004; Wallander et al., 2004).

Hyphal ingrowth bags offer a more direct method for

analysis of ECM mycelial growth and biomass in soil. These

are nylon mesh (50 mm) bags containing acid-washed sand

that are buried in soil and later retrieved for analysis of total

fungal content by ergosterol or PLFA analysis (Wallander

et al., 2001). The absence of organic matter in the sand



substrate is thought to select for mycelia of ECM fungi over

saprotrophs, because ECM fungi have a carbon source from

their tree hosts (Wallander et al., 2001). Comparison of

mycelial biomass in hyphal ingrowth bags from trenched

plots (designed to sever ECM roots from their tree hosts,

and so exclude ECM mycelia from the plots) with that from

untrenched plots suggests that some 85–90% of the myce-

lium that colonizes the sand-filled bags in Swedish forest

soils is ECM (Wallander et al., 2001; Hagerberg & Wallander,

2002; Nilsson & Wallander, 2003). This is supported by d13C

values derived from the bags (Wallander et al., 2001), along

with molecular analysis of the fungal taxa present as mycelia

in hyphal ingrowth bags from a range of forest habitats

(Wallander et al., 2003; Bastias et al., 2006; Kj�ller, 2006 and

see below). The method relies upon the assumptions that the

use of acid-washed sand provides a reasonable approxima-

tion of mycelial growth in native soil, and that there is no

turnover of mycelium during the period in which bags are

buried in soil (Wallander et al., 2001; Hendricks et al., 2006).

Recent data suggest that ECM mycelial colonization of bags

containing acid-washed sand can be considerably reduced

compared with that of native soil, indicating that the former

may underestimate ECM biomass in the surrounding soil, a

fact that may be exacerbated if mycelial turnover occurs

during the burial period (Hendricks et al., 2006). The use of

soil as a substrate in ingrowth bags, however, requires that

there is relatively low fungal biomass in the soil (Wallander,

2006), and further work on the influence of substrate is

clearly required. It has also been suggested that ingrowth

bags may select for certain ECM fungal taxa that are able to

colonize the substrate rapidly at the expense of slower-

growing taxa (Wallander et al., 2003).

Despite the various questions regarding the efficacy of

ingrowth bags for investigation of ECM mycelia, this

method remains the most direct for estimation of ECM

mycelial biomass in the field. Work of this nature indicates

that ECM fungal biomass in coniferous forests can be of the

order of 100–600 kg ha�1 and that it comprises a substantial

part of the soil fungal biomass (Wallander et al., 2001, 2004;

Hagerberg et al., 2003; Hendricks et al., 2006). Growth of

ECM mycelium appears to be seasonal, with greatest activity

in autumn and little or no growth during winter (Wallander

et al., 2001; Hagerberg & Wallander, 2002). ECM mycelial

biomass has also been shown to vary with soil depth and

host tree species, being generally greater in the upper part of

the soil profile than in the underlying soil and correlated

with the distribution of tree roots in the profile (Wallander

et al., 2004; Goransson et al., 2006). It is also evident that

ECM mycelial biomass in forest soil can be negatively

influenced by soil nutrient, particularly nitrogen, status

(Nilsson & Wallander, 2003; Nilsson, 2004; Nilsson et al.,

2005; Hendricks et al., 2006). In contrast, fertilization of

arctic tundra has recently been shown to significantly

increase ECM mycelial biomass (Clemmensen et al., 2006).

The extent to which these observations reflect altered

mycelium production per se, or a shift in the ECM fungal

community towards taxa that produce more/less soil-borne

mycelium, however, remains unclear. It is also evident that

ECM mycelial production can be influenced by temperature

(Clemmensen et al., 2006), and that mycelia can grow in

response to certain soil minerals (Hagerberg & Wallander,

2002; Hagerberg et al., 2003) and can contribute to the

mobilization of minerals such as apatite (Wallander et al.,

2002, 2003).

Analysis of ECM communities by directDNA extraction

In recent years methods have been developed to allow

analysis of microbial communities by direct nucleic acid

extraction from soil. These techniques include cloning,

denaturing gradient gel electrophoresis (DGGE), tempera-

ture gradient gel electrophoresis (TGGE) and terminal

restriction fragment length polymorphism (T-RFLP). The

technical considerations and limitations associated with

these approaches in the context of both general soil (Ander-

son & Cairney, 2004; Bidartondo & Gardes, 2005) and ECM

(Anderson, 2006) fungal communities have been reviewed

recently and thus will not be considered here.

Direct DNA extraction from soil

Recent investigations based on direct DNA extraction from

soil have yielded information on the diversity and distribu-

tion of ECM mycelia in soil (Table 1). Although they did not

target ECM fungi specifically, several studies identified

mycelia of ECM fungi as being present in the general fungal

assemblages of forest soils (Chen & Cairney, 2002; Anderson

et al., 2003; Landeweert et al., 2003a). Given that the

majority of ECM fungi are basidiomycetes (Smith & Read,

1997), basidiomycete-specific PCR primers have been used

to increase the detection of ECM mycelia in soil DNA

extracts. Using this method, Landeweert et al. (2003a)

reported vertical stratification of certain ECM fungi in the

soil profile, while Smit et al. (2003) demonstrated that

removal of litter and humus layers from a pine plantation

increased the diversity of ECM mycelia. However, the use

of basidiomycete-specific primers for the analysis of

ECM mycelial communities has been cautioned (Anderson,

2006), because the diversity (e.g. Vralstad et al., 2002;

Tedersoo et al., 2006) and abundance (e.g. Koide et al.,

2005a) of ascomycetes in ECM root-tip communities is

often high.

An alternative means of targeting ECM fungi in DNA

extracted directly from soil is T-RFLP analysis and inter-

rogation of a database containing reference terminal frag-

ments of known ECM fungi from the site. This has



facilitated confirmation of the vertical stratification of ECM

mycelial communities in the soil profile (Dickie et al., 2002;

Genney et al., 2006). More importantly, Koide et al. (2005b)

demonstrated that positive or negative interspecific interac-

tions can influence the distribution of ECM mycelia in soil

and that such interactions can vary according to soil

conditions.

T-RFLP analysis of DNA extracted from multiple adjacent

soil cores allowed Edwards et al. (2004) to estimate the

minimum width of mycelial patches of several ECM taxa in a

horizontal plane, the widest patch-size recorded being

160 cm for Thelephora terrestris. Moreover, Genney et al.

(2006) coupled T-RFLP analysis with a soil-slicing approach

to investigate the distribution of ECM mycelia in a volume

of soil (800 cm3) at a fine scale. Mycelial patches of indivi-

dual ECM taxa were variously shown to occupy different

volumes up to a maximum of 312 cm3 for Cadophora

finlandica. A combination of T-RFLP analysis and cloning

has also been used to demonstrate vertical separation of

ECM and saprotrophic mycelia in a forest-soil profile

(Lindahl et al., 2007). While each of these studies demon-

strated the potential distribution of mycelia of various ECM

species in forest soil, none considered the distribution

of individual mycelia ( = genet) of the species. Zhou et al.

(2001a, b), however, have demonstrated that simple se-

quence repeat (SSR) markers can be applied to investigate

the distribution of individual mycelia of Suillus grevillei

following direct DNA extraction from soil. With the increas-

ing development of SSR markers for ECM fungi (e.g.

Bergemann & Miller, 2002; Kretzer et al., 2004; Hitchcock

et al., 2006), information regarding the distribution of

individual mycelia within the soil volume should increase

markedly in the near future.

Even where individual mycelia of ECM fungal taxa can be

identified in soil, observations are restricted to determining

only the presence or absence of the mycelium in a sampled

soil volume, and the methods provide no information on

mycelial density. Competitive PCR, however, offers a means

of achieving this goal. Indeed, Guidot et al. (2002, 2004)

have used this approach successfully to show that soil-borne

mycelia of Hebeloma cylindrosporum are ephemeral, extend

for o 50 cm, and decrease in density with increasing

distance from the base of basidiomes.

A fundamental assumption in the interpretation of data

generated by direct DNA extraction from soil is that the taxa

identified are present as mycelia rather than as spores or

other resting propagules. Although approaches such as the

collection of soil samples after basidiome production has

ceased (Dickie et al., 2002) have been used to minimize the

likely detection of spore DNA, the fact that DNA from a

small number of H. cylindrosporum spores can be detected

in soil DNA extracts (Guidot et al., 2004) demonstrates

the potential for spore DNA to be amplified. Given that

dormant propagules are likely to contain little RNA relative

to active mycelia, RNA-based approaches (e.g. Anderson &

Parkin, 2006) may provide a more robust means to detect

active ECM mycelia in soil in the future.

The importance of analysing ECM fungal communities

by direct DNA extraction from soil is emphasized by the lack

of congruence between community structures inferred from

root-tip and soil-based analyses (Koide et al., 2005a; Land-

eweert et al., 2005). Furthermore, it is evident that spatial

segregation can exist between root tips and mycelia of

individual ECM taxa in the soil profile, with abundant

mycelium of some species occurring in the absence of

infected roots tips (Genney et al., 2006). Indeed, the same

Table 1. Investigations of soil ECM mycelial communities using direct DNA extraction and molecular analysis methods

Forest type DNA extracted from Analysis method Target fungal group Reference

Larix kaempferi forest Soil and ECM roots Simple sequence repeat analysis Suillus grevillei Zhou et al. (2001b)

Sclerophyll forest Soil Cloning/sequencing Soil fungi Chen & Cairney (2002)

Pinus resinosa plantation Soil T-RFLP ECM Dickie et al. (2002)

Pinus pinaster forest Soil Competitive PCR Hebeloma cylindrosporum Guidot et al. (2002)

Pinus sylvestris forest Soil DGGE/sequencing Soil fungi Anderson et al. (2003)

Coniferous forest Soil Cloning/sequencing Basidiomycetes Landeweert et al. (2003a)

Pinus sylvestris plantation Soil DGGE and cloning/sequencing Basidiomycetes Smit et al. (2003)

Pinus taeda plantation Soil T-RFLP Basidiomycetes Edwards et al. (2004)

Pinus pinaster forest Soil Competitive PCR Hebeloma cylindrosporum Guidot et al. (2004)

Pinus resinosa plantation Soil and ECM roots T-RFLP ECM Koide et al. (2005a)

Pinus resinosa plantation Soil and ECM roots T-RFLP ECM Koide et al. (2005b)

Pinus sylvestris forest Soil and ECM roots DGGE/sequencing Basidiomycetes Landeweert et al. (2005)

Wet sclerophyll forest Hyphal ingrowth bags DGGE and cloning/sequencing ECM Bastias et al. (2006)

Pinus sylvestris plantation Soil and ECM roots T-RFLP ECM Genney et al. (2006)

Fagus sylvatica forest Hyphal ingrowth bags Cloning/sequencing ECM Kj�ller (2006)

Picea abies plantation Hyphal ingrowth bags DGGE/sequencing ECM Korkama et al. (2007)

Pinus sylvestris forest Soil T-RFLP and cloning/sequencing ECM and saprotrophic fungi Lindahl et al. (2007)



appears to be true for individual mycelia of S. grevillei (Zhou

et al., 2001b).

DNA extraction from hyphal ingrowth bags

Hyphal ingrowth bags, in conjunction with ergosterol and/

or PLFA analysis, has been the main method used to

measure mycelial growth and biomass in forest soil (see

above). More recently, however, the approach has been

combined with direct DNA extraction from the ingrowth-

bag substrate and molecular methods to identify the mycelia

of ECM fungal taxa colonizing the bags. Regardless of the

molecular method used, these studies indicated that

83–91% of taxa colonizing hyphal ingrowth bags in a wet

sclerophyll forest (Bastias et al., 2006), a Fagus sylvatica

forest (Kj�ller, 2006) or a Picea abies plantation (Korkama

et al., 2007) were ECM fungi, which is broadly similar to

hyphal ingrowth bag-based estimates of ECM fungal bio-

mass in Swedish forest soils using d13C measurements and

trenching (Wallander et al., 2001; Hagerberg & Wallander,

2002; Nilsson & Wallander, 2003).

The hyphal ingrowth-bag approach has been successfully

used in conjunction with DGGE to investigate the effects of

plant host and forest management practices on ECM

mycelial communities. Thus, Korkama et al. (2007) demon-

strated that fast-growing Picea abies clones support different

ECM fungal mycelial communities from slower-growing

clones, with Atheliaceae taxa more abundant in the former.

Bastias et al. (2006) used this method to establish that long-

term repeated prescribed burning of wet sclerophyll forest

can significantly alter the composition of mycelial commu-

nities of ECM fungi in the upper 10 cm of the soil profile.

Cloning and sequencing of DNA extracted from the in-

growth bags further suggested that frequent burning had a

strong negative effect on Cortinariaceae and a positive effect

on Thelephoraceae taxa (Bastias et al., 2006). Comparison of

ECM fungi colonizing hyphal ingrowth bags with those

species colonizing root tips adjacent to the ingrowth bags

in a Fagus sylvatica forest showed that Boletoid species

occurred more frequently as mycelia than in root tips, in

contrast to Russuliod and Cortinarius spp., for which the

opposite was true (Kj�ller, 2006). It should be noted that, in

each of these investigations, only sand was used as substrate

in the ingrowth bags. Because the nature of the substrate can

influence mycelial colonization of hyphal ingrowth bags

(Hendricks et al., 2006), it may also bias community

composition. To date there is no published comparison

between mycelia of ECM fungi colonizing sand-filled hyphal

ingrowth bags with mycelia in the surrounding soil.

A combination of PLFA analysis and molecular ap-

proaches allows determination of both mycelial biomass

and ECM species identity in hyphal ingrowth bags (Korka-

ma et al., 2007), but it is difficult to correlate these two

independent measurements directly. Correlation of both

relative abundance (community evenness) and species iden-

tity (species richness) is important for determining the

mycelial biomass of individual ECM species and the re-

sponse of individual ECM taxa in manipulative field experi-

ments. This approach is common in root tip-based

community studies of ECM fungi, although the limitations

of root tip-based measurements of relative abundance have

been previously discussed (Taylor, 2002). While determina-

tion of the relative abundance of mycelia of individual ECM

species is yet to be achieved, this should be possible in the

future by combining quantitative molecular methods such

as competitive PCR (e.g. Guidot et al., 2002, 2004) or real-

time quantitative PCR (Landeweert et al., 2003b; Schubert

et al., 2003; Raidl et al., 2005) with the hyphal ingrowth-bag

approach. Given the known variation in the growth form of

mycelial systems of different ECM species (Agerer, 2001),

this will be an important future development in the use of

hyphal ingrowth bags.

ECMmycelia and climate change

The importance of understanding the diversity, extent and

dynamics of ECM mycelial systems in forest soils is empha-

sized by current interest in belowground responses to

elevated atmospheric CO2 in the context of global climate

change (Pendall et al., 2004) and the need to understand

both plant and ECM fungal responses (Alberton et al.,

2005). ECM fungal mycelia can comprise 80% of the total

fungal biomass and 30% of the microbial biomass in some

forest soils (Wallander et al., 2001, 2003; Hogberg &

Hogberg, 2002), with carbon allocation to ECM fungi

estimated to be as much as 22% of net primary production

(Hobbie, 2006). ECM fungi are thus an important compo-

nent of forest carbon cycles, and the effects of elevated

atmospheric CO2 on these fungi have deservedly received

recent attention. Much of this work has focused on interac-

tions between CO2 concentration and root colonization by

ECM fungi. With one exception (Rouhier & Read, 1999),

such investigations have reported increased percentage root

colonization by ECM fungi under elevated atmospheric CO2

conditions (Norby et al., 1987; Ineichen et al., 1995;

Berntson et al., 1997; Godbold et al., 1997; Tingey et al.,

1997; Rouhier & Read, 1998; Walker et al., 1998; Kasurinen

et al., 2005).

Elevated atmospheric CO2 has also been shown to alter

ECM fungal root-tip community structure in growth-

chamber experiments (Godbold & Berntson, 1997; Godbold

et al., 1997; Rey & Jarvis, 1997; Rygiewicz et al., 2000) and

in the field (Fransson et al., 2001; Kasurinen et al., 2005). In

one such experiment, a change in ECM root-tip community

composition in favour of morphotypes that appeared to

produce emanating hyphae and/or rhizomorphs was noted



under elevated atmospheric CO2 conditions (Godbold &

Bernston, 1997; Godbold et al., 1997). Because no attempt

was made to quantify soil-borne mycelia, however, this

provides no direct information on the mycelial response of

ECM fungi under these conditions. Several laboratory-based

microcosm experiments have been undertaken with a view

to determining the responses of ECM mycelia to elevated

atmospheric CO2 (Table 2). While Gorissen & Kuyper

(2000) observed no significant increase in mycelial produc-

tion, other studies have reported increases in mycelial

biomass or spread of up to 400% (Ineichen et al., 1995;

Rouhier & Read, 1998, 1999; Fransson et al., 2005). It should

be emphasized that these investigations were conducted in

simplified microcosm systems (see above) using seedlings

inoculated with a single ECM fungus. As such, the observed

responses may not reflect the responses of ECM mycelia to

elevated atmospheric CO2 in the field, highlighting the need

for field-based investigations. Although elevated atmo-

spheric CO2 was found to alter the structure of a basidio-

mycete mycelial community (that included ECM fungi) in a

scrub oak forest (Klamer et al., 2002), to date only one such

study has focused on ECM fungi. By analysing PLFAs from

hyphal ingrowth bags buried under Betula pendula trees in

open-top chambers, Kasurinen et al. (2005) established that

elevated atmospheric CO2 had no significant effect on

mycelial production by ECM fungi.

The inability to generalize based on this single field

experiment notwithstanding, these data relate only to the

response of the ECM mycelial community and there-

fore provide no information on responses of individual

ECM species to elevated atmospheric CO2 in the field.

Further studies on how mycelial systems of ECM fungi

respond to elevated atmospheric CO2 concentrations will

be crucial to modelling and understanding carbon dynamics

in complex forest ecosystems under future climate-change

scenarios.

Conclusions

Soil-borne mycelia of ECM fungi are a dominant and

important component of soil microbial communities in

forest ecosystems, where they play crucial roles in nutrient

and carbon cycling processes. Our appreciation of the roles

of ECM fungi in these processes is hampered by a funda-

mental lack of understanding of their mycelial systems in

soil. Much of the currently available information on the

diversity, distribution and dynamics of ECM fungi is derived

from investigations of fruiting bodies and ECM root tips,

and provides only limited insights into soil-borne ECM

mycelial communities. Information derived from micro-

cosm studies, while unarguably enhancing our understand-

ing of ECM mycelial functioning, cannot necessarily be

extrapolated directly to mycelia in the field, emphasizing

the necessity for direct analysis of soil-borne mycelia of

ECM fungi in situ. Molecular approaches, combined with

new experimental approaches, such as hyphal ingrowth

bags, facilitate analysis of ECM fungal communities from

the perspective of mycelia in soil. Furthermore, they have

the potential to increase our understanding of the distribu-

tion of individual mycelia and so enhance our appreciation

of the physical and physiological continuity of mycelial

systems in soil. Such information will give unprecedented

insights into the functioning of ECM mycelia at the ecosys-

tem level and will assume particular importance when

considering the responses of ECM fungi to future perturba-

tions, particularly in the context of land-use changes and

global climate change.

Acknowledgements

This work was supported by an Australian Research Council

Linkage International Awards grant (LX0455012) and a

Macaulay Development Trust collaboration grant. I.C.A.

receives funding from the Scottish Executive Environment

and Rural Affairs Department.

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