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Networks of power and influence: the role of
mycorrhizal mycelium in controlling plant
communities and agroecosystem functioning
1
Jonathan Leake, David Johnson, Damian Donnelly, Gemma Muckle, Lynne Boddy,
and David Read
Abstract: Extraradical mycelia of mycorrhizal fungi are normally the “hidden half” of the symbiosis, but they are
powerful underground influences upon biogeochemical cycling, the composition of plant communities, and
agroecosystem functioning. Mycorrhizal mycelial networks are the most dynamic and functionally diverse components
of the symbiosis, and recent estimates suggest they are empowered by receiving as much as 10% or more of the net
photosynthate of their host plants. They often constitute 20%–30% of total soil microbial biomass yet are undetected
by standard measures of biomass used by soil scientists and agromomists. Mycorrhizal mycelia provide extensive path
-
ways for carbon and nutrient fluxes through soil, often exceeding tens of metres per gram of soil. We consider the
amounts of photosynthate “power” allocated to these mycelial networks and how this is used in fungal respiration, bio
-
mass, and growth and in influencing soil, plant, and ecosystem processes. The costs and functional “benefits” to plants
linking to these networks are fungal specific and, because of variations in physiology and host specificity, are not
shared equally; some plants even depend exclusively on these networks for carbon. We briefly assess the potential con
-
tribution of extraradical mycorrhizal mycelium to sustainable agriculture and maintenance of biodiversity and highlight
technologies that promise new vistas and improved fine-scale resolution of the dynamic spatial and temporal function-
ing of these networks in soil.
Key words: arbuscular mycorrhiza, ectomycorrhiza, extraradical mycelium, hyphal networks.
Résumé : Sauf exception, les mycéliums extraracinaires des champignons mycorhiziens constituent la face cachée de la
symbiose, mais ils exercent de puissantes influences sur le cyclage biogéochimique, la composition des communautés
végétales et le fonctionnement des écosystèmes. Les réseaux mycéliens des mycorhizes constituent les composantes les
plus dynamiques et les plus fonctionnellement diverses de la symbiose, et les estimations récentes suggèrent que leur
puissance vient du fait qu’ils reçoivent jusqu’à 10 % ou plus du produit net de la photosynthèse des plantes hôtes. Ils
constituent souvent de 20%à30%delabiomasse microbienne totale du sol, mais demeurent tout de même non dé
-
tectés par les mesures standards de biomasse utilisées par les pédologues et les agronomes. Les mycéliums mycorhi
-
ziens fournissent des sentiers extensifs permettant les flux de carbone et de nutriments à travers le sol, dépassant
souvent des dizaines de kilomètres par gramme de sol. Les auteurs considèrent les quantités de « puissance » photo
-
synthétique allouées à ces réseaux mycéliens, comment elles sont utilisées pour leur respiration, leur biomasse et leur
croissance, et comment elles influencent les processus à l’échelle de la plante et des écosystèmes. Le coût et les « bé
-
néfices » fonctionnels pour les plantes reliées à ces réseaux sont spécifiques au champignon et, dû aux variations phy
-
siologiques et de spécificité de l’hôte, ne sont par répartis également; certaines plantes dépendent même exclusivement
de ces réseaux pour leur carbone. Les auteurs évaluent brièvement la contribution potentielle des mycéliums mycorhi
-
ziens extraracinaires pour l’agriculture durable et le maintien de la biodiversité, et mettent en lumière les technologies
prometteuses pour le développement de nouveaux concepts et l’amélioration de la fine résolution de la dynamique spa
-
tiale et du fonctionnement temporel de ces réseaux du sols.
Mots clés : mycorhizes arbusculaires, ectomycorhizes, mycélium extraracinaire, réseaux mycéliens.
[Traduit par la Rédaction] Leake et al. 1045
Can. J. Bot. 82: 1016–1045 (2004) doi: 10.1139/B04-060 © 2004 NRC Canada
1016
Received 4 September 2003. Published on the NRC Research Press Web site at http://canjbot.nrc.ca on 16 August 2004.
J.R. Leake,
2
D. Johnson, G.E. Muckle, and D.J. Read. The University of Sheffield, Department of Animal and Plant Sciences,
Sheffield S10 2TN, UK.
D.P. Donnelly and L. Boddy. Cardiff School of Biosciences, Cardiff University, CF10 3TL, UK.
1
This article is one of a selection of papers published in the Special Issue on Mycorrhizae and was presented at the
Fourth International Conference on Mycorrhizae.
2
Corresponding author (e-mail: j.r.leake@sheffield.ac.uk).
Introduction
Mycorrhizal mycelial networks: the hidden half of the
symbiosis
The extraradical mycorrhizal mycelium (ERMM) is the
part of the symbiosis most intimately connected to the soil
and most directly involved in uptaking nutrients and influ
-
encing soil properties. These mycelial networks provide
pathways for reciprocal transfer of carbon (C) received from
host-plant roots and nutrients taken up from the soil. They
also transfer nutrients and C between plants interlinked by
the same mycelial network (Simard et al. 2002). ERMM are
complex and dynamic components of the symbiosis but are
highly sensitive to disturbance and to alterations in soil pro
-
perties, such as pH and nutrient status, brought about by pol
-
lution, conventional agricultural, and forest management
(Wallenda and Kottke 1998; Brunner 2001; Erland and Tay
-
lor 2002; McGonigle and Miller 2000; Kabir and Koide
2002). Whilst the ERMM is increasingly recognized as the
main nutrient-absorbing interface of the plant–mycorrhiza
soil system (Smith and Read 1997), it remains the most
poorly understood and most difficult part of the symbiosis to
study (Staddon et al. 2003). Nonetheless, very significant
advances have recently been made in our understanding of
the structure and functioning of these networks and their po-
tential effects on major processes such as biogeochemical
cycles, soil aggregation, the composition and functioning of
plant communities, and agroecosystem functioning.
This paper reviews some of the recent experimental and
observational studies on ERMM of the two main types of
mycorrhizas, arbuscular (AM) and ectomycorrrhiza (EM),
and presents compelling evidence that confirms the central
importance of these networks as major pathways for C flux
through soil and powerful influences on plant community
composition and agroecosystem functioning. As understand-
ing of the importance of ERMM has increased, there is
growing evidence of a major role for these fungal systems in
both seminatural ecosystems and, increasingly, in sustain
-
able agriculture, such as organic and reduced-tillage man
-
agement systems, where mycorrhizal functioning appears to
be more important than in conventional intensive agriculture.
Progress in understanding the nature, extent, functioning,
and identity of mycorrhizal fungal networks has been seri
-
ously hampered by the difficulties inherent in observing and
studying mycelial systems without disturbing and destroying
them. These difficulties are particularly hard to overcome
outside the laboratory, and our knowledge of the functioning
of these networks has been strongly based on reductionist
studies, the relevance of which to the field situation is often
difficult to confirm. Routine studies of mycorrhizal occur
-
rence and functioning ignore ERMM, since the standard
methods of assessing mycorrhizal occurrence are based on
fungal colonization of roots (e.g., Dalpé 1993; Horton and
Bruns 2001), or in the case of AM fungi, sometimes based
on the occurrence of soilborne spores (Bever et al. 2001).
Although ERMM are often the most important sources of
inoculum in established plant communities with AM
(Merryweather and Fitter 1998), spore counts remain the
most widely used method of quantifying AM inoculum, even
though spore viability is often very low (e.g., McGonigle
and Miller 1996). As a consequence, the external mycelium,
which is the fungal structure of mycorrhiza that is most inti
-
mately associated with the soil and furthest from the roots,
and by implication the most critical for nutrient uptake, is
normally overlooked and has been rarely recorded. Only in
the past decade have studies started to focus specifically on
the extent and functioning of ERMM in the field.
Not only are ERMM obscured by the soil matrix, but they
are also intermingled with mycelium of saprotrophic fungi
from which they are often not readily distinguishable. The
diffuse mycelium of many EM fungi appear identical to
hyphae of saprotrophs to which many of them may be
closely related (Hibbett et al. 2000) and with which they
share many functional attributes (Leake et al. 2002). Where
-
as AM mycelia have some features such as angular projec
-
tions, thick walls, and infrequent septa that enable the
thicker parts, albeit with some difficulty, to be visually dif
-
ferentiated from saprotrophs, there remain uncertainties, par
-
ticularly when examining the finer distal parts of their
hyphal networks.
The measurement of ERMM length, especially of AM
fungi, has played a central role in establishing the impor
-
tance of extraradical mycelium and its functional signifi
-
cance as an entity distinct from the root infecting hyphae.
Whilst the aqueous membrane filtration technique for extrac-
tion and quantification of hyphal lengths is long established,
in recent years it has undergone various refinements, such as
agitation with sodium hexametaphosphate to disperse clays
(Schweiger et al. 1999), which have improved hyphal clean-
ing and the increasing use of vital stains to distinguish active
and dead hyphae (Kabir et al. 1998a). The difficulty in dis-
tinguishing EM mycelia from saprotrophs remains a major
barrier to assessment of EM hyphal lengths, although a few
studies have attempted these measurements in simplified
systems using sand-grown plants, where fungal populations
are likely to be dominated by mycorrhizal mycelium
(Querejeta et al. 2003).
Developments in methods used to study mycorrhizal
mycelia
As awareness of the importance of ERMM has increased,
efforts to develop techniques to study it have redoubled, and
some of the most important recent developments have arisen
from innovative combinations of methods to address for
-
merly intractable aspects of structure and function of
ERMM (Table 1). Of particular importance has been the
development of methods such as root-free hyphal compart
-
ments, combined with isotope tracers and molecular analy
-
ses, which have allowed the effects of ERMM to be
distinguished from those of roots both in the laboratory, and
increasingly, in the field. This has enabled most of the major
functions of intact mycelial networks to be investigated:
transport of C, weathering of minerals, production of extra
-
cellular enzymes, mineralization of nitrogen (N) and phos
-
phorus (P), uptake of nutrients, transport of nutrients, and
interactions with other organisms. As a result of these meth
-
odological advances, substantial progress has recently been
made in our understanding of the lengths of mycorrhizal
hyphae in soil, the C and nutrient fluxes through them, their
contributions to the global C, P, and N cycles, and their in
-
teractions with other organisms.
© 2004 NRC Canada
Leake et al. 1017
Biochemical markers for biomass estimation and species
identification
Fluorescent antibody assays have been developed that can
selectively detect AM hyphae in soil. Assays based on anti
-
bodies that bind to the hydrophobic glycoprotein glomalin,
which is produced by all isolates of AM fungi that have
been tested to date (Wright 2000), are not well suited to sim
-
ple quantification of AM hyphae, because glomalin is often
stabilized in soil after the mycorrhizal mycelium has de
-
cayed. However, antibodies with AM fungal lineage speci
-
ficity have been reported (Allen et al. 1999) and may be
used in soil. Indeed, apart from these, there are no suitable
biochemical markers for estimating the biomass of mixed
species of AM fungi in the field. Other approaches, such as
relating root-length colonization by AM to ergosterol con
-
centrations (e.g., Hart and Reader 2002a, 2002b), have been
called into question since ergosterol appears to be absent
from all AM fungi tested to date both in axenic culture and
in spores and hyphae from soil (Olsson et al. 2003). The
other widely used AM marker, the phospholipid fatty acid
16:1
ω
5, must be calibrated to biomass for each species
(Olsson et al. 2003), so in natural mixed communities of
AM hyphae it provides only a very crude estimate of bio
-
mass. This marker is almost certainly not specific to AM, al
-
though it does appear to be especially abundant in them.
A combination of biochemical markers has been used to
determine EM biomass, including ergosterol (Ek 1997; Wal
-
lander et al. 2001) and the phospholipid fatty acid 18:2
ω
6,9
(Wallander, et al. 2001; Nilsson and Wallander 2003), but
neither of these is EM specific. It was only by growing
plants in the laboratory in a sandy soil of very low organic
matter content and by having nonmycorrhizal control plants
for comparison that Ek (1997) was able to subtract the er
-
gosterol content of saprotrophic fungi and convert ergosterol
measurements to EM biomass. Both Wallander et al. (2001)
and Nilsson and Wallander (2003) have extended this ap
-
proach to forest soils in the field by comparing the concen
-
tration of ergosterol and phospholipid biochemical markers
© 2004 NRC Canada
1018 Can. J. Bot. Vol. 82, 2004
Method Example Reference
Detection and quantification of extraradical mycelium of AM
Extraction and measurement of hyphae
Aqueous membrane filtration Boddington et al. 1999
Rotating wire frame Vilariño et al. 1993
Buried membrane Baláz and Vosátka 2001
In vitro observations
Root-organ cultures Fortin et al. 2002
Whole plant monoxenic mycorrhizal culture Giovannetti et al. 2001
Phospholipid fatty acid analysis Olsson et al. 2003
Glomalin immunofluorescent binding assay Wright 2000
PCR identification of AM hyphae in soil Hunt et al. 2004
Detection and quantification of extraradical mycelium of EM
Thin-layer soil microcosms Read 1992
Digital image analysis linked to autoradiography Leake et al. 2002
Phospholipid fatty acid analysis Nilsson and Wallander 2003
Ergosterol Wallander et al. 2001
Hyphal ingrowth bags Wallander et al. 2001
T-RFLP to study the spatial distribution of hyphae of different EM fungi down the soil profile Dickie et al. 2002
Competitive PCR to quantify EM mycelia of particular species in forest soil Guidot et al. 2002a
Transport of nutrients and carbon in extraradical mycelium of AM
Root-excluding hyphal ingrowth compartments in laboratory and field studies with
32
P Schweiger and Jakobsen 2000
31
P NMR of intact mycelia in soil Rasmussen et al. 2000
Movement of tubular vacuole system revealed by fluorescence tracer microscopy Uetake et al. 2002
Mobilization and uptake of nutrients by hyphae in monoxenic root-organ cultures Hawkins et al. 2000
Root-excluding hyphal ingrowth compartments in laboratory and field studies with
13
C and
14
C Johnson et al. 2002a, 2002b
Measurement of
14
C in handpicked AM hyphae after host-plant exposure to
14
CO
2
-free air Staddon et al. 2003
Transport of nutrients and carbon in extraradical mycelium of EM
31
P NMR of polyphosphate metabolism Suillus bovinus mycorrhizal with pine Gerlitz and Gerlitz 1997
Digital image analysis linked to autoradiography of
32
P transport in mycelial networks Lindahl et al. 1999
14
C and digital autoradiography of mycelial networks of seedlings in soil microcosms Leake et al. 2001
Movement of tubular vacuole system revealed by fluorescence tracer microscopy Ashford and Allaway 2002
Mobilization and uptake of nutrients by hyphae in mesh ingrowth bags in the field Nilsson and Wallander 2003
Combined DNA identification of hyphae with analysis of their uptake or binding of elements Wallander et al. 2003
Note: AM, arbuscular mycorrhiza; EM, ectomycorrhiza.
Table 1. Some methods used to study the structure and functioning of extraradical mycorrhizal mycelium.
in trenched plots, from which mycorrhizal roots were ex
-
cluded, and adjacent untrenched plots.
The application of DNA-based identification methods for
mycorrhizal mycelium in soil has lagged far behind the use
of this technology for the characterization of mycorrhizal
communities on ectomycorrhizal root tips (Horton and
Bruns 2001; Tedersoo et al. 2003) or in roots of AM plant
communities (Husband et al. 2002; Vandenkoornhuyse et al.
2002). To date there are only a few records of molecular
identification of mycorrhizal mycelium in the soil, but effec
-
tive methods now exist for both AM (Jacquot et al. 2000;
Hunt et al. 2004) and EM fungi (Dickie et al. 2002).
Molecular identification of mycorrhizal hyphae has
opened a new window on the diversity and spatial structur
-
ing of ERMM in soil. Terminal restriction fragment length
polymorphism (T-RFLP) has allowed the spatial distribution
of hyphae of different EM fungi down the soil profile under
pine forests to be determined (Dickie et al. 2002; Lande
-
weert et al. 2003). By combining PCR identification and ele
-
mental analysis of EM mycelia colonizing nutrient patches
of wood ash, Wallander et al. (2003) have been able to iden
-
tify the specific fungi foraging in these patches and the main
elements that they are accumulating in their mycelia. These
kinds of approaches will be invaluable to establish func-
tional differences between mycorrhizal fungi in the field. A
further exciting development is the ability to quantify
amounts of mycelia of specific EM fungi in natural soil by
competitive PCR (Guidot et al. 2002a). This technique holds
exceptional promise for future studies on the importance of
mycorrhizal mycelia in the field, opening the way for popu-
lation and community functioning studies of mycorrhizal
mycelial networks in situ.
AM hyphal networks: observation in vitro and in soil
AM hyphal lengths are determined by painstaking visual
discrimination from nonmycorrhizal hyphae on gridded
membranes or by comparing total hyphal length counts in
soil from mycorrhiza-inoculated and nonmycorrhizal control
plants grown in sterilized soil. An inevitable limitation of
hyphal extraction methods is that they fragment the network
in the cleaning process. Although it may be possible to dis
-
tinguish live and dead AM hyphae using vital stains (Kabir
et al. 1998a), the part of the network that is likely to be most
active in uptake of nutrients, the distal portion that is most
finely branched and intimately attached to the soil, is not
readily recovered and is rarely observed. As a consequence,
new in vitro techniques such as mycorrhizal root-organ cul
-
tures (Fortin et al. 2002) have been important in allowing the
occurrence, development, growth, and properties of ex
-
tremely delicate and relatively short-lived mycorrhizal myce
-
lia to be observed nondestructively in the laboratory. Such
approaches have highlighted the potential importance of
branched absorbing structures (BAS) described by Bago et
al. (1998). The main findings from mycorrhizal root-organ
cultures have been comprehensively reviewed by Fortin et
al. (2002), who reported that three species of Acaulospora,
four of Gigaspora, three of Scutellospora, one of Sclero
-
cystis, and 16 of Glomus have now been grown mono
-
xenically by this method. This approach has enabled detailed
studies of AM nutrient transport, their abilities to assimilate
certain organic nutrients (Koide and Kabir 2000; Hawkins et
al. 2000), C metabolism and transport (Bago et al. 2002),
and various aspects of their growth, spore production, and
physiology (Fortin et al. 2002). Such studies have been con
-
ducted monoxenically and in some cases in dual cultures
with other soil microorganisms to enable interactions
between AM mycelia and mycoparasites, saprotrophs, and
nutrient-solubilizing bacteria to be investigated (Fortin et al.
2002).
Whilst these in vitro methods have made a major contri
-
bution to our understanding of AM mycelia, their artificial
-
ity requires that results be corroborated, wherever possible,
by studies in soil and in association with whole plants. Be
-
cause root-organ cultures lack shoots they cannot supply
sugars to their fungal partners in the same way as intact
plants with their shoots experiencing diurnal photoperiods.
Also, since shoots are the major sink for nutrients, uptake
rates may be lower in root-organ cultures than in whole
plants. There are indications that the short-term exposure to
normal laboratory light levels, as happens during routine
maintenance of root-organ cultures, can cause unusual
growth responses and stimulate the hyphal branching in
Gigaspora gigantea, Gigaspora rosea, and Glomus intra
-
radices (Nagahashi et al. 2000).
Whole plants with monoxenic AM cultures can also be
used to study mycelial functioning, and this has the advan-
tages of maintaining the normal pathways of photosynthate
supply to AM fungi and the shoot demand for nutrients and
avoids the requirement to supply exogenous sugar to roots
(Giovannetti et al. 2001).
To visualize the fine structure and rates of spread of AM
hyphae in soil, including in the field, the burial of mem-
branes on which the hyphae grow reveals much about the
mycelial branching patterns, hyphal anastomoses, and the
finer absorptive parts of the network (Baláz and Vosátka
2001). Many of these fine structures are normally broken up
or obscured when hyphae are extracted from soil by wet
sieving. The buried membrane method can be used in natural
soil and does not suffer restriction to a limited range of AM
species that applies to root-organ culture systems.
EM hyphal networks: observation in vitro and in soil
Whilst the measurement of AM hyphal lengths in soil has
become increasingly common and the methods have become
well established and tested (see e.g., Boddington et al.
1999), progress in the routine quantification of EM hyphae
in soil has lagged far behind. The lack of visual distinction
between many saprotrophic and EM fungi is highly prob
-
lematic, so reliable estimates of EM hyphal lengths can be
made only in plants grown in microcosms or pots (for exam
-
ple, containing sand) with poorly developed saprotroph pop
-
ulations.
Major insights into the structure and functioning of EM
mycelia in soil have been gained through a combination of
careful microscopic observations of ectomycorrhizal roots
with adhering soil collected from forests (Agerer 2001) and
the growth, normally of selected species of mycorrhizas, in
thin-layer soil microcosms (Fig. 1). In this approach, most of
the extraradical mycelium grows on the soil surface and
therefore can be studied nondestructively (Read 1984).
When combined with radioactive tracers, isotope imaging,
time-lapse photography, and image analysis, the transport of
© 2004 NRC Canada
Leake et al. 1019
C and nutrients, growth, and spatial and temporal foraging
activities of ERMM have been determined (see e.g., Read
1992; Bending and Read 1995; Leake et al. 2002). Real-time
digital radioisotope imaging has recently enabled nondestruc
-
tive multiple time-sequence quantification of
14
C transfer
from plants to ERMM, without requiring compartmental
-
ization of roots and hyphae (Leake et al. 2001). Whilst much
has been revealed about the behaviour and physiological ecol
-
ogy of genera such as Suillus, Rhizopogon, Paxillus,
Laccaria, Pisolithus, and Cenoccoccum (Horton and Bruns
2001), these represent a distinct subset of EM fungi that are
relatively easy to culture and, in most cases, produce fairly
extensive extraradical mycelium. Most EM fungi are not
in culture; many genera such as Inocybe, Cortinarius, and
Russula have proved generally intractable to routine labora
-
tory culturing. Furthermore, the molecular analysis of EM
communities on roots is revealing enormous diversity of
these symbionts in established forests, and the species that
are well represented in fruit bodies, which includes the ma
-
jority of cultured species, are often relatively minor compo
-
nents of the root-tip populations (Taylor 2002; Horton and
Bruns 2001).
Root-free hyphal compartmentation in the laboratory and
field
Our understanding of the functioning of ERMM in soil
has been greatly increased through the use of mesh barriers
to provide root-free compartments into which mycorrhizal
mycelium can grow (Schüepp et al. 1987; Jakobsen et al.
2001). Hyphal in-growth compartments made with root-
excluding nylon mesh have been invaluable for determining
the distance to which AM hyphae grow from roots
(Jakobsen et al. 1992a) and their effective distance of nutri
-
ent foraging using radioisotope tracers (Jakobsen et al.
1992b; Smith et al. 2000). They have also established direct
effects of AM networks on host and nonhost plants (Francis
and Read 1995). Mesh bags buried in the field that contain
soil mixed with radioactive P, with and without addition of
fungicide, have permitted the quantification of the extent of
P uptake by native populations of AM fungi (Schweiger et
al. 1999; Schweiger and Jakobsen 2000). This approach was
further refined by Johnson et al. (2001, 2002a, 2002b) who
developed mesh-walled, soil-filled cores in which mycor
-
rhizal mycelial development can be controlled by mechani
-
cal disruption (Fig. 2). The centre of the core is within 1 cm
of the nearest roots outside, and at this distance Jakobsen
et al. (1992a) found that for isolates of Acaulospora,
Scutellospora, and Glomus the concentration of AM hyphal
length per gram of soil was 60%–110% of values found at
the root surface. By rotating the cores in soil, mycorrhizal
mycelial connections to the plant roots are broken without
the need for addition of fungicides or soil sterilization (John-
son et al. 2001). The hyphal in-growth cores have allowed
in situ measurements of both the uptake of phosphate by
ERMM and transfer of C to the mycelium from the plants by
using radioactive and stable isotope tracers (Johnson et al.
2001, 2002a, 2002b).
The hyphal compartment method has also been used with
EM in laboratory microcosms to study the biomass and res
-
piration responses of ERMM supplied with nutrient patches
(Ek 1997; Bidartondo et al. 2001) and in the field to esti
-
mate biomass production of EM mycelia (Wallander et al.
2001; Nilsson and Wallander 2003). The main findings of
many of these studies are discussed in detail in the following
sections.
Networks of power: C allocation to
extraradical mycorrhizal mycelia
The hidden pathway of C flux from plant roots
through mycorrhizal mycelia
Central to the functional importance of mycorrhizal
mycelial networks is their energy supply from plants. Sapro
-
trophic soil microorganisms are typically C limited, their C
sources being spatially and temporally heterogeneous. In
contrast, mycorrhizal fungi, by gaining direct access to
plant-supplied sugars, are energized by a quality and quan
-
tity of available carbohydrate supply that is unparalleled
amongst soil microbial populations. Despite the many indi
-
cations that ERMM is a major component of soil biota, its
contribution to soil microbial biomass is difficult to quantify
(see previous section), and the pathway it provides for C
movement from roots to other soil organisms is largely un
-
recognized and ignored. Until recently, scant attention has
© 2004 NRC Canada
1020 Can. J. Bot. Vol. 82, 2004
Fig. 1. The extensive and dense cover of ectomycorrhizal
mycelial network of Suillus bovinus in association with Pinus
sylvestris in a thin-layer (2 mm depth) microcosm of nonsterile
peaty soil on a 20 cm × 20 cm sheet of Perspex. The seedling
was inoculated with the mycorrhizal fungus in Petri dishes of
peat–vermiculite, and this was transferred with the seedling into
the upper part of the microcosm and allowed to grow for
4 weeks. (From Leake et al. 2001, reproduced with permission
of Tree Physiol., Vol. 21, p. 74, © 2001 Heron Publishing.)
been paid to the importance of ERMM as conduits for C
movement from plants to soils. For example, De Ruiter et al.
(1994) present food-web models for agroecosystems that do
not recognize the existence of mycorrhizas, but place con
-
siderable emphasis on saprotrophic fungi and fungal-feeding
animals. Even studies that have specifically investigated C
fluxes from grassland plants to soil microbiota (e.g., Stewart
and Metherell 1999; Saggar et al. 2001; Kuzyakov et al.
2001) have entirely ignored the fact that the majority of
grassland plants have mycorrhizas and their mycelial sys
-
tems draw directly on host photosynthate. Indeed, the most
recent models of C fluxes between herbaceous plants and soil
have been based upon the assumption that root exudation,
sloughed cells, and dead roots provide the only significant
pathways for the supply of plant-fixed C to the free-living mi
-
crobial populations in soils (e.g., Toal et al. 2000; Kuzyakov
et al. 2001).
This situation has arisen in part because the standard mea
-
sures of soil microbial biomass discriminate against detec
-
tion of mycorrhizal mycelial biomass. The widely used
substrate-induced respiration technique (Anderson and
Domsch 1978) is applied to sieved soil samples in which
ERMM are fragmented and their vital connections to their
life-supporting carbohydrate supplies from plants are de
-
stroyed. Since ERMM of AM fungi appear to be unable
to assimilate exogenous sugar (Pfeffer et al. 1999), the
substrate-induced respiration method discriminates against
the detection of an AM contribution to microbial biomass.
Even methods such as fumigation–extraction (Voroney and
Winter 1993), unless applied to samples within a few hours
of collection, will fail to detect the full contribution of the
living mycorrhizal networks to microbial biomass and C
fluxes. This very serious limitation in these techniques is
rarely acknowledged. The insensitivity of these measures to
intact mycorrhizal mycelial biomass raises doubts as to their
suitability for assessment of microbial biomass.
However, recent estimates suggest that AM fungi comn
-
stitute over 50% of the fungal length in some soils (Rillig et
al. 2002). They can account for more than 20% of the total
soil microbial biomass in pasture and prairie grasslands
(Miller and Kling 2000), rising to over 30% of the microbial
biomass in sandy soils (Olsson and Wilhelmsson 2000). In
addition to their contribution to the live fraction, AM hyphae
secrete the protein glomalin that accumulates in soil, con
-
tributing a substantial amount of the more stable soil organic
C. For example in grassland, Miller and Kling (2000)
suggest that as much 15% of the soil organic C pool is con
-
tributed by AM fungi, and it makes a similarly large contri-
bution in tropical rain-forest soils where the dominant trees
form AM (Rillig et al. 2001).
Recent work has shown that EM mycelia makes a similar
or even larger contribution to soil C pools. By combined use
of sand-filled hyphal in-growth bags inserted into forest
plots and trenching to exclude living mycorrhizal roots from
control plots, Wallander et al. (2001) used PLFA and
δ
13
C
signatures to estimate the EM biomass in a Swedish Pinus
sylvestris and Picea abies forest. They estimated that the to-
tal biomass of EM mycelia and roots was 700–900 kg·ha
–1
,
and on the basis of previous estimates of the biomass of EM
sheath on roots (Kårén and Nylund 1997), concluded that
approximately 80% of the EM biomass was extraradical my
-
celium. A conservative estimate of the contribution of
ectomycorrhizas to soil microbial biomass in forest soil is
32%, based on field observations following a large-scale gir
-
dling experiment (Högberg and Högberg 2002).
Despite the substantial biomass and associated C drain on
their hosts, the actual “cost” of mycorrhiza to plants may be
negligible because mycorrhizal colonization can increase the
rate of photosynthesis (Wright et al. 1998), alleviate shoot N
and P limitation, and cause a substantial increase in leaf area
arising from improved nutrition (Read and Perez-Moreno
2003).
C allocation to mycorrhiza and extraradical mycelium
Although both AM and EM fungi clearly have an impor
-
tant role in the terrestrial C cycle, the quantities of C allo
-
cated to them by plants have rarely been determined. Only a
handful of studies have specifically attempted to measure the
C costs of ERMM.
Estimates (from pot based studies) of the net total C allo
-
cation from plants to AM range between about 2% and 20%
of current assimilate (Jakobsen and Rosendahl 1990;
Pearson and Jakobsen 1993; Smith and Read 1997). Esti
-
mates of the total C costs of EM, obtained from calculations
© 2004 NRC Canada
Leake et al. 1021
Fig. 2. Diagram of hyphal ingrowth cores developed to study
mycorrhizal functioning in the field. The cores (2 cm diameter
and 8 cm depth) are filled with sieved (2 mm) natural soil and
inserted into grassland to allow study of the functioning of
extraradical mycorrhizal mycelium in situ. Hyphae, but not roots,
grow through the 10–35-mm pore-sized nylon mesh windows
(mesh is shown partially cut away for clarity). Control cores (on
the right) are rotated to sever the hyphal connections with roots
and thus avoid the use of fungicides, which can have effects on
saproptrophic as well as mycorrhizal fungal hyphae (Johnson et
al. 2001).
based on field observations of mycorrhizal biomass (Vogt et
al. 1982) and from laboratory studies using
14
C pulse label
-
ling of tree seedlings or C budgeting studies, suggest that
7%–30% of net fixation is allocated to EM, of which 16%–
71% is lost as respiration, according to laboratory studies
(Finlay and Söderström 1992; Ek 1997; Bidartondo et al.
2001).
C allocation to AM extraradical mycelium
It was estimated (using mesh-compartmentalized pots)
that 0.7%–0.8% of net C fixation by cucumber was allocated
to the ERMM of its AM partner (out of a total of 20% allo
-
cation to mycorrhiza) in 4 d following
14
C pulse-labelling
of the plants (Jakobsen and Rosendahl 1990; Pearson and
Jakobsen 1993). These pioneering measurements were a ma
-
jor advance, but the values appear to have underestimated
the true C cost of AM mycelia, in part because maximum C
allocation to external mycelium occurs within 24 h after
pulse labelling of shoots (Johnson et al. 2002a; Staddon et
al. 2003). The amounts of C allocated to support ERMM in
the field has now been estimated for the first time, over a
shorter time course, in a permanent grassland using hyphal
in-growth cores and
13
CO
2
pulse labelling of the surrounding
turf with CO
2
supplied at atmospheric CO
2
concentrations
(Johnson et al. 2002a, 2002b). Control cores were briefly
twisted immediately prior to commencing labelling to sever
AM hyphae passing through the mesh. Respiratory fluxes of
C from the AM networks within 21 h of pulse labelling ac-
counted for 3.9%–6.2% of the net fixation, with peak release
of
13
CO
2
from the hyphal compartments occurring only 9–
14 h after the C was fixed in the shoots, whereas maximum
13
C allocation to fine roots peaked later, after 21 h (Johnson
et al. 2002a).
In the studies of Johnson et al. (2002a), the amount of C
allocated to biomass of ERMM could not be determined by
the
13
C pulse-labelling approach because the high organic
matter content of the soil diluted the
13
C tracer. However, in
a parallel analysis in which blocks of turf were removed
from the same field site and in which identical hyphal in-
growth cores were then established,
14
CO
2
pulse labelling of
the shoots was used to quantify C allocation to AM biomass
and AM-derived soil C (Johnson et al. 2002b). This revealed
that AM mycelia in the unrotated hyphal compartments ac
-
counted for about 3% of net fixation 72 h after pulse label
-
ling. There was no evidence that the chosen 72-h harvest
interval coincided with the maximum
14
C content in the
hyphal compartment, so the 3% allocation value should be
regarded as a conservative estimate. Taken together with the
estimated respiratory fluxes measured through the AM my
-
celia, the total C allocation to ERMM in the grassland in
early autumn was 9% of net C fixation. These figures are re
-
markably similar to the results of Domanski et al. (2001),
who found that 11% of the
14
C fixed by pot-grown Lolium
perenne was in the soil 6 h later. They found that about 10%
of the total C fixed was respired from below ground and 7%
remained in the soil, but in this study the contribution of
ERMM to these components was not considered.
The C allocation to ERMM in the grassland study (John
-
son et al. 2002a, 2002b) is about an order of magnitude
higher than estimated net fixation allocated to ERMM in
the pot studies of cucumber (Jakobsen and Rosendhal 1990;
Pearson and Jakobsen 1993). However, the latter did not in
-
clude respiratory fluxes through mycorrhiza, although they
noted that the peak in
14
CO
2
respiration from roots plus my
-
celium occurred during the 16-h labelling period. Their mea
-
surements of
14
C allocation to hyphae required extraction of
the fungal material from soil by a sieving procedure that
may have had incomplete recovery, and fragmentation of the
mycelia may have caused leakage and loss of labelled C.
Their measurements on C allocation to hyphal compartments
were taken 70–96 h after labelling, which is likely to be con
-
siderably past the peak
14
C concentration in the hyphae.
While the respiratory release of labelled C from AM
hyphae is now known to be maximal from 12 to 16 h after
pulse labelling of the plants, the optimal time for sampling
ERMM for total labelled C remains uncertain, but appears to
be soon after labelling, probably within the first 24 h (see
Staddon et al. 2003). It will be governed by the balance be
-
tween the rates of C transfer, the rates of respiration, and the
rates of C incorporation into hyphal C storage and growth.
Many pulse-labelling studies have either used relatively long
fixation periods, which make it difficult to establish the
chronology of C fluxes, or have taken the first samples many
days after labelling (e.g., Stewart and Metherell 1999), and
so may miss the importance of rapid C transfers and fluxes
that happen sooner than this, such as those through AM and
roots (Domanski et al. 2001). A fuller knowledge of the
rates, routes, and fate of C fluxes through the ERMM of AM
is required to avoid these difficulties in future.
For the first few hours after the hyphal in-growth cores
were removed from the soil in the field and the hyphal con-
nections to plant roots severed,
13
CO
2
release from the cores
remained fairly constant (Johnson et al. 2002a), but within
24 h it rapidly declined (Fig. 3), suggesting that the contri-
bution of AM mycelia to soil respiration is quickly lost
when all connections to host roots are severed. These obser-
vations corroborate the conclusions of laboratory studies that
have indicated that AM primarily use current plant assimi
-
late (Wright et al. 1998) and that most of the C flux from
plants to their mycorrhizal associates occurs rapidly, often
within 24 h of fixation. The dependence on recent assimilate
has also been shown in respiration from EM mycelia, which
decreased by 60%–95% within 24 h of detachment from
host roots (Söderström and Read 1987).
Pulse labelling of turf with
13
CO
2
combined with root-
excluding hyphal in-growth cores (Johnson et al. 2002a,
2002b)is a powerful tool for the study of in situ AM func
-
tioning. Further studies of these kinds are now clearly re
-
quired to establish the typical quantities of C allocated to
ERMM in a range of grassland, arable fields, and woodlands
using pulse-labelling studies of appropriate duration and
sampling frequencies. It is now established that the amounts
of C allocated from plants to AM vary depending upon both
the plant and fungal species (Lerat et al. 2003), and it is to
be expected that as AM fungi differ in the extent and bio
-
mass of ERMM, such differences will also occur in C allo
-
cation to the extraradical mycelium.
C allocation to EM extraradical mycelium
As with AM, there are very few estimates of the propor
-
tion of plant C fixation that is allocated to extraradical EM
mycelium. In mycorrhizal Pinus ponderosa seedlings, extra
-
© 2004 NRC Canada
1022 Can. J. Bot. Vol. 82, 2004
radical hyphae contributed 4% of the total respiration
(Rygiewicz and Anderson 1994), while 8%–19% of net fixa-
tion of Betula pendula in ectomycorrhizal association with
Paxillus involutus in a sandy forest soil was respired by
ERMM in a root-free hyphal compartment, and these myce-
lia received 20%–29% of the net fixation (Ek 1997). In these
studies, respiration from compartments containing both roots
and extraradical mycelium were not included in the esti
-
mates of C flux through ERMM, since root and mycelial
respiration cannot be distinguished in these compartments.
Consequently, the total C allocation and respiration from
mycorrhizal mycelium will have been even higher than the
reported values. Using a similar microcosm system, in
which half the soil volume was occupied by roots and my
-
corrhiza and the other half was a root-free hyphal compart
-
ment, the proportion of net fixation by Pinus muricata that
was allocated to ERMM in the hyphal compartments ranged
from 2.8%–10% with three species of Rhizopogon, 8.4%
with Suillus pungens, and 21% with Paxillus involutus
(Bidartondo et al. 2001). Since ERMM in the root compart
-
ments ranged from 18%–120% of the biomass in the hyphal
compartments, these remain very conservative estimates of
the total C allocation to extraradical mycelium.
In soil-based microcosms the transfer of
14
C label from
14
CO
2
fixed by shoots of Pinus sylvestris seedlings to Suillus
bovinus mycelium was first detected by digital auto
-
radiography within 8 h after photosynthesis and reached
maximum concentration in the mycelium by 30 h (Fig. 4),
whereas the
14
C activity in the roots peaked 18 h later
(Leake et al. 2001). These results confirm the importance of
recent fixed photosynthate allocated to EM mycorrhiza and
are corroborated by field studies showing that daily varia
-
tions in the
δ
13
C isotopic signature of recently fixed C in
trees is closely followed, within a few days, by the isotope
signature of root and rhizosphere respiration, which must in
-
clude EM mycelium (Ekblad and Högberg 2001). Measuring
© 2004 NRC Canada
Leake et al. 1023
Fig. 3. Isotopic signature of (
δ
13
C‰) of CO
2
release from static (solid circles) and rotated (open circles) mycorrhizal hyphal ingrowth
cores in field plots in which the turf was pulse labelled for5hwith
13
CO
2
as well as from pooled static and rotated cores from unla
-
belled plots (triangles). The cores were all removed from the ground 9 h after the pulse labelling, and the release of
13
CO
2
was moni
-
tored 80 min, 140 min, 24 h, and 7 d later. Bars indicate SE of the mean (n = 4). Data are replotted from Johnson et al. (2002a).
Fig. 4. The mean amount of supplied, pulse-labelled
14
C detected
by digital autoradiography in surface roots and mycorrhizal my-
celium up to 2 mm from roots (open circles) and in external
mycorrhizal mycelium >2 mm from roots (solid circles) over 0–
124 h after exposure of Pinus sylvestris shoots to
14
CO
2
for3h.
Bars indicate SE of the mean (n = 4 replicate microcosms).
total
14
C allocation into the ERMM of the EM fungus in the
microcosms revealed that it contained 9% of the total
14
Cin
the plants (including ectomycorrhizal roots) 5 d after label
-
ling (Leake et al. 2001). At its peak concentration in the
extraradical mycelium, 30 h after labelling, the
14
C percent
allocation is estimated to be approximately 14% of net fixa
-
tion. These estimates are close to the 17% net
14
C pulse-
label photosynthate allocated to extraradical EM mycelia of
an unknown fungus associated with Pinus densiflora seed
-
lings grown on black cotton cloth overlain onto soil, using a
similar digital autoradiographic method (Wu et al. 2002).
Most of this
14
C transfer to ERMM also occurred rapidly,
within 2–3 d after pulse labelling (Wu et al. 2002).
C pools and C turnover in mycorrhizal networks
In both the studies by Leake et al. (2001) and Wu et al.
(2002) there was a steady decline in
14
C concentration in
mycorrhizal mycelium once the peak was reached (Fig. 4).
This decline was most marked just behind the young, ac
-
tively growing margins of the network. These temporal vari
-
ations in
14
C content suggest that there are two distinct fates
of C within ERMM. A major part is rapidly translocated
throughout the network and quickly respired, whereas some
of the C enters more stable, longer term pools, for example,
in hyphal walls and structural components of mycelium. The
portion that has short residence time in the hyphae will in-
clude some C that is transferred back to host plants, for ex-
ample, as amino acids generated from the hyphal uptake of
inorganic N. The amounts of C returning to the plant by this
route will be equivalent to 5 C atoms for each atom of
NH
4
+
-N assimilated, but not all N assimilated requires return
of recent host assimilate, since considerable amounts of N
may be acquired by direct uptake of amino acids from soil.
The energy demands for mineral nutrient assimilation and
transport to the host appear to be quite considerable, for ex-
ample, respiration from ectomycorrhizal ERMM growing in
root-free soil compartments increased within 24 h after addi
-
tion of ammonium or NO
3
–
-N and continued to increase for
several days afterwards (Ek 1997). The mean respiration rate
of ERMM increased by 54%–180% over the 4 d after addi
-
tion of mineral N, and the mycelial respiratory output was
equivalent to 4–13 atoms of C for each atom of N gained by
the plants.
Evidence for two distinct C pools in ERMM of AM, one
fast and one slow turnover, has also been provided by
Staddon et al. (2003), who have used a novel isotope tracer
technique to study C flux through mycorrhizal mycelia.
They supplied Plantago lanceolata for 5 h during the day
with CO
2
derived from fossil fuel that was free of
14
C and
then extracted and analysed the
14
C concentration in extra
-
radical AM mycelia over a period of 1 month. Maximum de
-
pletion of
14
C concentration in AM mycelia occurred within
a day or so of C fixation; up to 16% of the C content of the
mycelia was replaced by new assimilate from the 5-h label
-
ling. This confirms the extremely rapid transfer of current
assimilates from plants to mycorrhizal mycelium. Over a
subsequent period of 6 d the
14
C concentration in the myce
-
lia showed a linear increase with time following the return to
fixation of atmospheric
14
CO
2
by the plants. From 6 to 30 d,
the
14
C concentration in ERMM changed very little with
time, but remained about 2.5% depleted.
The fast-turnover C pool in the ERMM, revealed by the
magnitude and rate of
14
C concentration increase in the first
6 d following
14
C depletion, was interpreted by Staddon et
al. (2003) as evidence that most of the hyphae have a life
-
span of 5–6 d. However, this incorrectly presumes that the C
allocated to the mycelium is exclusively used in biomass
production, whereas it is more likely that the fast-turnover C
pool is the main source of the C used in respiration. The
studies by Johnson et al. (2002a, 2002b) suggest that respi
-
ration from ERMM of AM fungi may account for more than
two thirds of the C transferred to them from plants in the
first 3 d after pulse labelling. In addition, they found that the
13
CO
2
release from the hyphal compartments fell rapidly
soon after dawn on the day after labelling and remained low
during the day, but rose again at night. This suggests that
much of the hyphal respiration is of C fixed in the few
hours immediately preceding, except at night when it must
depend on sugars mobilized from stored plant carbohydrate
pools such as starch. The longer term C pool in the hyphae,
represented by the 2.5%
14
C depletion value found by
Staddon et al. (2003) from 6–30 d postlabelling, is likely to
more closely reflect biomass production and hyphal turn
-
over.
Another recent study that attempted to calculate the lon
-
gevity and turnover time of ERMM in grassland concluded
that only one quarter to one third of the hyphal network is
turned over annually (Miller and Kling 2000). Such esti-
mates are clearly prone to many sources of error, and it is
difficult to reconcile these calculated turnover rates with lab-
oratory observations of ERMM. Observations on AM net-
works in undisturbed monoxenic cultures have shown that
the very narrow thin-walled hyphae that form terminal
branches have a functional lifespan similar to that of arbus-
cules: they develop in 7 d, and by 5 weeks the cells are
empty and have septa separating them from the living myce-
lia (Bago 2000). In addition to natural senescence in the
field, disturbance of the hyphal networks by various soil ani
-
mals, such as worms and fungivores, by seasonal extremes
of temperature and water availability, and by fungal patho
-
gens is likely to ensure higher rates of turnover than nor
-
mally seen in the laboratory.
Knowledge of the C pools in ERMM of AM fungi and the
nature of the substances transported from the intraradical to
extraradical mycelium has advanced considerably. Bago et
al. (2002) indicate that lipid and glycogen are the main C
compounds transported into the extraradical mycelium, and
they suggest that N is transported back to the plants as
amino acids, possibly associated with polyphosphate. The
incorporation of mineral N into amino acids by the fungus
and their transfer to the plant would return some of the C
skeletons originally supplied by the plant. These postulated
metabolic and transport pathways match those that are
known to occur in EM mycelium (Martin et al. 1998), and
interdependence between N, P, K, and Mg transport has been
shown in EM mycelia (Jentschke et al. 2001).
Clearly, more work needs to be done to establish the turn
-
over rates of C in different ERMM networks and the nature
of their internal C pools. Undoubtedly, isotope tracer tools
have the potential to unlock the secrets of mycorrhizal func
-
tioning in laboratory and the field, and the use of labelling
with fossil
14
C-free CO
2
in the field has enormous potential,
© 2004 NRC Canada
1024 Can. J. Bot. Vol. 82, 2004
avoiding the high cost of both
13
C and
14
C and the restric
-
tions on use of the latter radioisotope.
Networks of power: the lengths, absorptive
areas, growth rates, and interconnections
of extraradical mycorrhizal mycelia
Lengths of mycorrhizal hyphae
In an increasing number of studies, the lengths of ERMM
have been measured over time courses in both the laboratory
and the field, revealing the nature and extent of these hyphal
networks and the rapidity of their production. Such studies
have confirmed the importance of mycorrhizal networks as
major components of soil microbial biomass and have shown
the importance of the length and absorptive area of mycor
-
rhizal networks for nutrient acquisition.
Lengths of arbuscular mycorrhizal hyphae in soil
ERMM length measurements are normally expressed per
unit mass or volume of soil or per unit length of mycorrhiza-
infected root (Table 2). The latter expression is affected by
the length of root present and by the proportion of it that is
infected by mycorrhiza. It is strongly affected by the nature
of the plant roots, how coarse or fine, and it is difficult to re-
late values obtained from different species or from studies
with very different rates of root colonization. Plants with
AM have widely different root types, and this has large ef-
fects on the length of hyphae per unit length of infected root.
For example, Lolium perenne gave 14 m AM hyphae·g soil
–1
butonly1mhyphae·m infected root
–1
, since this grass has
an extensive fibrous root system with moderate to high lev-
els of mycorrhizal colonization (Tisdall and Oades 1979). In
contrast, the much coarser rooted Trifolium repens supported
hyphal lengths in soil of only 3 m·g
–1
, but this provided
46 m hyphae·m infected root
–1
(Table 2).
The lengths of ERMM of AM fungi are typically one
to two orders of magnitude longer than the lengths of
mycorrhiza-colonized roots and typically range from 3–
30 m·g soil
–1
(Table 2). Considering that the studies of
hyphal lengths have been carried out on a wide range of
fungi and under various laboratory and field conditions on a
variety of soil types, the reported values are remarkably sim
-
ilar. In the pot studies (Table 2) the range of AM hyphal
lengths (2–29 m·g soil
–1
) are almost identical to those
reported from root-free hyphal compartments of pots (2–
36 m·g soil
–1
) where lower hyphal densities might have been
expected due to the greater distances from roots. This raises
the interesting possibility that AM hyphae grow more inten
-
sively in root-free soil, possibly because of the higher avail
-
ability of nutrients outside the rhizosphere depletion zones.
The highest recorded hyphal lengths in AM communities
have been reported in Acer saccharum woodland (Table 2),
but in this case AM fungi were not distinguished from
saprotrophs, although the mycorrhizal mycelium is likely to
make a considerable contribution to the total fungal biomass
(Klironomos et al. 1993). In the absence of woodland stud
-
ies, the highest densities of AM hyphal lengths have, un
-
surprisingly, been reported in grasslands, where there is
minimal soil disturbance and permanent plant cover. Here,
they ranged from 68–101 m·g soil
–1
in a prairie and from
45–74 m·g soil
–1
in a pasture, with maximum lengths in No
-
vember and minimum in June (Miller et al. 1995, Table 2).
These lengths of mycorrhizal hyphae are vast: assuming a
similar density of AM mycelia throughout the top 10 cm of
the soil profile alone, there would be sufficient length of
AM mycelial to stretch all the way around the equator of the
earth in just 4 m
2
of grassland. Further studies are required
to confirm that these observations are typical. Ploughing and
disturbance is known to reduce the extent of AM mycelial
networks (McGonigle and Miller 2000; Kabir et al. 1998a,
1998b), and in the field, AM hyphal lengths in annual crops
are typically in the range 2–8 m·g soil
–1
and tend to be lower
than in many of the pot studies. These differences are likely
to be due to higher planting and rooting densities of pot-
grown plants than in the field.
There remain very serious gaps in our knowledge of the
amounts of ERMM produced by many plant communities.
With the exception of measurements from maize grown in
an 8-month-old agroforestry plot (Boddington et al. 1999),
studies of AM hyphal lengths to date have focused almost
exclusively on herbaceous plants, and the laboratory studies
have been restricted to a very limited range of species, more
than half of which appear to have included a single plant
species, Trifolium subterraneum (Table 2). As a conse
-
quence, with the exception of records of AM hyphal lengths
of outdoor-grown Populus tremoloides saplings in open-top
chambers, where AM hyphal lengths significantly increased
with elevated CO
2
(Klironomos et al. 1997), the biomass and
extent of AM hyphae in temperate deciduous forests and
tropical rain forests dominated by plants with AM fungi
is unknown. The abundance of total hyphae in the Acer
saccharum woodland studied by (Klironomos et al. 1993),
which far exceeds the total AM and saprotophic fungi in
grassland and pot-plant studies, suggests that there may be
orders of magnitude longer AM hyphal lengths in woodlands
with AM host trees. ERMM are expected to be particularly
important in their biomass and functioning in woodland,
since trees have a high C-fixing potential and depend upon
efficient nutrient cycling to maintain productivity. In the fu
-
ture, in addition to studies of AM hyphal lengths in wood
-
land and seminatural vegetation, a much wider range of host
species should be studied, as AM hyphal production in field
plots has been shown to depend upon the host-plant species
(Rillig et al. 2002), and more fungi need to be studied, too,
since AM fungi differ greatly in the extent of their ERMM
(Jakobsen et al. 1992b).
Lengths of ectomycorrhizal hyphae in soil
As a consequence of lack of visual distinction between
hyphae of EM and saprotrophic fungi (see Introduction),
there are few estimates of lengths of hyphae produced by
EM (Table 3) and enormous uncertainty about their typical
densities in soil in the field. Current estimates range from
30–8000 m hyphae·m root
–1
(Smith and Read 1997) and 3–
600 m·g soil
–1
(Table 3). Local hyphal proliferation may
give much higher values than these. EM fungi differ greatly
in the extent to which they produce extraradical mycelium,
some species producing little mycelium, apart from the
mycorrhizal mantle, whereas others produce extensive
mycelial networks that extend decimetres from the roots and
have considerable biomass (see Agerer 2001).
© 2004 NRC Canada
Leake et al. 1025
© 2004 NRC Canada
1026 Can. J. Bot. Vol. 82, 2004
Culture conditions and plant species AM fungus
Hyphal length
per unit soil
mass (m·g
–1
)
Hyphal length
per metre
colonized root
length (m) Reference
Pot
Trifolium subterraneum,42d Acaulospora laevis 29 1055 Abbott and Robson 1985
Trifolium subterraneum,42d Glomus tenue 26 1422 Abbott and Robson 1985
Trifolium subterraneum Scutellospora calospora 2–25 nd Sanders et al. 1977
Trifolium subterraneum,42d Gigaspora calospora 17 1232 Abbott and Robson 1985
Lolium perenne Glomus sp. 8 49 Tisdall and Oades 1979
Pisum sativum, 33–57 d, mixed inoculum Glomus (three species) 4–6 470–600* Gavito et al. 2002
Trifolium subterraneum,42d Glomus fasciculatum 5 250 Abbott and Robson 1985
Allium cepa Glomus mosseae nd 79–250 Sanders 1975
Allium cepa Glomus mosseae nd 71 Sanders et al. 1977
Allium cepa Glomus macrocarpon nd 71 Sanders et al. 1977
Allium cepa Glomus microcarpon nd 71 Sanders et al. 1977
Pot, hyphal compartment
Linum usitatissimum Glomus mosseae (two isolates) 26–36 nd Schweiger and Jakobsen 2000
Trifolium subterraneum Glomus mosseae 6–29* 22–103 Drew et al. 2003
Trifolium subterraneum Glomus intraradices 4–27* 8–57 Drew et al. 2003
Cucumis sativus Glomus fasciculatum 27 113 Jakobsen and Rosendahl 1991
Trifolium subterraneum Acaulospora laevis 15–25 nd Jakobsen et al. 1992b
Trifolium subterraneum Glomus sp. 10–25 nd Jakobsen et al. 1992b
Linum usitatissimum Glomus intraradices 19 nd Schweiger and Jakobsen 2000
Linum usitatissimum Glomus caledonium 16 nd Schweiger and Jakobsen 2000
Trifolium subterraneum Scutellospora calospora 2–15 nd Jakobsen et al. 1992b
Trifolium subterraneum Native populations 4–11 nd Schweiger et al. 1999
Linum usitatissimum, 32 d Native populations 10 nd Thingstrup et al. 2000
Linum usitatissimum Glomus claroideum 5 nd Schweiger and Jakobsen 2000
Linum usitatissimum Glomus geosporum 5 nd Schweiger and Jakobsen 2000
Trifolium repens Glomus mosseae 2–5* nd Li et al. 1991
Pisum sativum, 33–57 d, mixed inoculum Glomus (three species) 2–3 210–220* Gavito et al. 2002
Linum usitatissimum Scutellospora callospora 2 nd Schweiger and Jakobsen 2000
Field
Acer saccharum forest, length of all fungi Native populations (640–4200) nd Klironomos et al. 1993
Prairie, 11 years old Native populations 68–101
†
800–1030 Miller et al. 1995
Ungrazed pasture, 17 years old Native populations 45–74
†
440–1240 Miller et al. 1995
Zea mays, preceded by fallow or cover crop Native populations 10–35 nd Kabir and Koide 2002
Sown Lolium perenne Native populations 14 1 Tisdall and Oades 1979
Populus tremuloides saplings in soil–sand, 14 months Native populations 2–8 nd Klironomos et al. 1997
Table 2. Length of arbuscular mycorrhizal (AM) extraradical mycorrhizal mycelium in soil, expressed per unit mass of soil or per metre of infected roots.
To date, most of the estimates of EM hyphal lengths have
been carried out with seedlings inoculated with single EM
species in laboratory studies, and the extent of mycelial pro
-
duction is difficult to extrapolate to natural forests and plan
-
tations with established trees and diverse assemblages of
mycorrhizal symbionts. The only attempt to determine EM
hyphal lengths in forest soil is based on an indirect approach
in which regression relationships between mycelial respira
-
tion and biomass were used to estimate mycorrhizal mycelia
length in a Swedish pine forest (Finlay and Söderström
1989), and this gave values of 200 m·g soil
–1
(Table 3).
Given that both the quantity and proportion of net C fixation
allocated to mycorrhiza is likely to normally be higher in
forests than in grassland, this seems a reasonable estimate
and not dissimilar to those indicated by recent soil micro
-
cosm studies with tree seedlings (cf. Tables 2 and 3).
In the species that produce the most extensive mycelial
networks, these are often highly differentiated structures
with hyphal cords or rhizomorphs comprising hydrophobic
hyphal aggregates, used for long-distance transport, whereas
the distal absorptive mycelium is typically hydrophilic to en
-
able the uptake of nutrients from soil solution (Olsson et
al. 2002). Some species, such as Hydnellum, Hysterangium,
Suillus, Paxillus, Amanita, and Boletus, produce extensive
hydrophobic conducting mycelium, whereas others, such as
Laccaria, Hebeloma, and Thelephora, produce mainly hy-
drophilic mycelial systems that can be sparse or extensive
depending on the individual species (Olsson et al. 2002).
Whilst many species produce extensive extraradical myce-
lium, some species such as Lactarius subdulcis are almost
completely confined to the sheath (Agerer 2001). These dif-
ferences must strongly influence the nature of the nutrient
pools that different EM fungi use.
Absorptive area of extraradical mycorrhizal mycelium
The finest absorptive AM hyphae in soil are typically
2
µ
m in diameter, compared with root-hair diameters of 10–
20
µ
m and fine-root diameters of 100–500
µ
m. Read (1999)
noted that assuming an equal dry mass per unit volume of
these different structures, in geometric terms the C cost per
unit absorptive area of fine mycorrhizal hyphae is approxi
-
mately 10 times more efficient than that of root hairs and
about 100 times more efficient than that of roots. Because of
the enormous length of mycorrhizal hyphae in soil they can
provide a surface area for absorption that is similar to, or
even greater than, that of roots. If we assume, for the sake of
argument, that mycorrhizal hyphae are 3
µ
m in diameter and
that the average root diameter is 500
µ
m, it would require
170 m of hyphae to provide the same surface area for ab
-
sorption as1mofroot lacking root hairs.
The typical lengths of AM hyphae of between 3 and
35 m·g soil
–1
, reported in Table 2, have an external surface
area of 0.3–2.9 cm
2
·g soil
–1
, if the mean hyphal diameter is
2.6
µ
m (as found by Jakobsen and Rosendhal (1990)), or
0.4–4.5 cm
2
·g soil
–1
, if the mean hyphal diameter is 4
µ
m (as
found by Miller et al. (1995)). However, external surface
area may vary even more widely than this, as AM hyphal di
-
ameters ranged between 1.2 and 18
µ
m (mean 3.7–7.9
µ
m)
in five species from three genera (Acaulospora, Glomus, and
Scutellospora) (Dodd et al. 2000). In a typical grassland soil
with bulk density close to 1 g·cm
–3
, the absorptive area of
© 2004 NRC Canada
Leake et al. 1027
Culture conditions and plant species AM fungus
Hyphal length
per unit soil
mass (m·g
–1
)
Hyphal length
per metre
colonized root
length (m) Reference
Wheat–rape–barley rotation Native populations 2–8 nd Boddington et al.1999
Native grassland, Serengeti Native populations 0–7 nd McNaughton and Oestenheld 1990
Avena barbata, 2 years Native populations 6 nd Rillig et al. 2002
Taeniatherum caput-medusae, 2 years Native populations 5 nd Rillig et al. 2002
Aegilops triuncialis, 2 years Native populations 4 nd Rillig et al. 2002
Trifolium microcephalum, 2 years Native populations 4 nd Rillig et al. 2002
Amsinckia douglasiana 2 years Native populations 3 nd Rillig et al. 2002
Tropical legume tree and Zea mays Native populations 3–4 nd Boddington et al.1999
Zea mays, conventional, reduced, and zero tillage Native populations 2–4* nd Kabir et al. 1998a
Trifolium repens Native populations 3 46 Tisdall and Oades 1979
Note: Values are rounded to the nearest whole number. nd, no data.
*Where original values were expressed per cubic centimetre of soil, these were expressed per gram of soil, assuming a typical bulk density of 1.32 g·cm
–3
for cultivated mineral soils (Brady 1984).
†
Where original values were expressed per cubic centimetre of soil, these were expressed per gram of soil, assuming a typical bulk density of 1.10 g·cm
–3
for permanent grassland (Brady 1984).
Table 2. (concluded).
© 2004 NRC Canada
1028 Can. J. Bot. Vol. 82, 2004
Culture conditions and plant species Ectomycorrhizal fungus
Hyphal length
per unit soil
mass (m·g
–1
)
Hyphal
length per
metre root
length (m) Reference
Pot or soil microcosm
Pinus sylvestris in nonsterile peat nd 1000–8000 Read and Boyd 1986
Pinus taeda in pots of fertilized sand Pisolithus tinctorius 7 504 Rousseau et al. 1994
Pinus taeda in pots of fertilized sand Cenococcum geophilum 3 nd Rousseau et al. 1994
Salix viminalis in pots of fertilized sand Thelephora terrestris nd 308 Jones et al. 1990
Salix viminalis in pots of fertilized sand Laccaria proxima nd 289 Jones et al. 1990
Pinus sylvestris and Alnus incana grown together in pots of sand Paxillus involutus 0.8 nd Ekblad et al. 1995
Betula pendula in sandy forest soil, EM colonized part of hyphal compartment Paxillus involutus 115–607* nd Ek 1997
Betula pendula in sandy forest soil–root compartment Paxillus involutus 14–29* nd Ek 1997
Pinus muricata in sandy forest soil, root-free hyphal compartment Rhizopogon (three spp.) 150–343* nd Bidartondo et al. 2001
Pinus muricata in sandy forest soil, root-free hyphal compartment Suillus pungens 383* nd Bidartondo et al. 2001
Pinus muricata in sandy forest soil, root-free hyphal compartment Paxillus involutus 570* nd Bidartondo et al. 2001
Quercus agrifolia in sandy soil mixture, root-free hyphal compartment Cortinarius collinitus 1.5 nd Querejeta et al. 2003
Quercus agrifolia in sandy soil mixture, root-free hyphal compartment Cenococcum geophilum 2.5 nd Querejeta et al. 2003
Field
Swedish Pinus sylvestris forest 200 nd Finlay and Söderström 1989
*Indicates values that are converted from biomass estimates, assuming that there are 0.83 m of hyphae per milligram of fungal biomass, which is the average of the values for Pisolithus tintorius and
Cenoccoccum geophilum, given by Rousseau et al. (1994). This will overestimate hyphal lengths of species that produce appreciable amounts of multicellular hyphal cords or rhizomorphs.
Table 3. Length of ectomycorrhizal (EM) extraradical mycorrhizal mycelium in soil, expressed per unit mass of soil or per metre of infected roots.
© 2004 NRC Canada
Leake et al. 1029
mycorrhizal hyphae in the top 10 cm of the profile could
therefore be expected to range from 3 to 90 m
2
·(m
2
turf)
–1
.
The ratio of absorptive area of ERMM to root area is typi-
cally much higher in EM than in AM plants, not only be-
cause EM plants have relatively short and course roots and
generally lack root hairs, but because the mycelial networks
of their mycorrhizas are normally much more extensive.
With sand-grown Pinus taeda inoculated with Pisolithus
tinctorius, the ectomycorrhizal mycelium constituted 75% of
the absorptive area, only 25% being provided by roots, but
the EM mycelium was only 5% of the biomass of root plus
mycorrhiza (Rousseau et al. 1994). The potential absorptive
area of EM hyphae per gram of soil in this study was only
0.47 cm
2
·g
–1
with P. tinctorius and 0.28 cm
2
·g
–1
when the
trees were grown with Cenococcum geophilum. Much higher
values than these would be expected in forest soils, as
growth and biomass production of EM fungi, and C alloca
-
tion to them, is greatly stimulated in the presence of forest
floor litter (Bending and Read 1995; Leake et al. 2001; Read
and Perez-Moreno 2003). Indeed, the mean area of soil cov
-
ered by hyphae of Paxillus involutus in association with
Pinus sylvestris in 20 cm × 20 cm peat microcosms
increased from 110 cm
2
in control microcosms to over
260 cm
2
within 10 d of adding a patch of 0.5 g of air-dried,
partially decayed pine litter (Fig. 5; Donnelly et al. 2003). In
this 10-d period the ERMM increased its occupancy of the
available soil surface area from 15% to 36% in response
to the single litter patch. Observations of these kinds have
demonstrated the exceptional speed, precision, intensity, and
effectiveness of nutrient foraging by mycorrhizal mycelium.
Growth rates of extraradical mycorrhizal mycelium
The production of ERMM is very dynamic, and the
lengths can increase very quickly when plants are establish-
ing in bare soil. For example, Thingstrup et al. (2000) have
shown that over a 32-d growth period the length of AM
hyphae in pots of flax increased in the final 9 d from less
than 2 m·g soil
–1
to over 10 m·g soil
–1
, and there was no in-
dication that it had stopped increasing at that point. With
Trifolium subterraneum grown in pots inoculated with either
Glomus fasciculatum or Gigaspora calospora, AM hyphal
lengths increased rapidly from 10 m·g soil
–1
at 4 weeks, to
20 m·g soil
–1
at 5 weeks, and 25 m·g soil
–1
at 7 weeks
(Abbott and Robson 1985). At the same time the length of
hyphae per unit root length infected also changed markedly,
but in this case peak values were attained at 4 or 5 weeks,
and the values fell sharply by week 7, as the length of in
-
fected root increased more rapidly than hyphal lengths dur
-
ing the last 2 weeks. Consequently, in most pot studies the
duration of the experiment will have a critical effect on
hyphal length estimates, and where only single harvests are
taken the reported values may not be representative of ma
-
ture plants. Similarly, with annual field crops, large seasonal
variations in AM hyphal lengths are to be expected and have
recently been confirmed for maize (Kabir et al. 1998a), in
which the maximum AM hyphal densities increased up to
flowering but declined thereafter.
AM hyphal extension rates have frequently been reported
in the range 0.3–3.3 mm·d
–1
(Jakobsen et al. 1992a), but
much higher rates occur in some EM fungi. In peat micro
-
cosms, two strains of Paxillus involutus and two of Suillus
bovinus, all grown in association with Pinus sylvestris, had
radial extension rates of 7–8 and 5–6 mm·d
–1
, respectively
(Donnelly et al. 2003). At a forest ecosystem scale,
Wallander et al. (2001) have estimated that the equivalent of
between 125–200 kg ERMM·ha
–1
·year
–1
grew into sand-
Fig. 5. The mean (±SE) hyphal cover of Paxillus involutus in mycorrhizal association with Pinus sylvestris in peat microcosms (see
Fig. 1), which received additions of 0.5 g of air-dried, partially decayed Pinus litter from a forest floor (solid symbols) or (open sym
-
bols) received no litter (n = 3 replicate microcosms). The litter was added 32 d after the mycorrhizal seedlings were transplanted into
the peat. An arrow indicates the time of litter addition. Letters indicate significant differences between mean values for treatments with
and without litter (P < 0.05) for comparisons on the same date. Note the doubling of hyphal cover in less than 10 d.
filled mesh bags buried in Swedish pine–spruce forest soil,
and most of this biomass production occurred in a relatively
short time from July to September. Assuming a mean hyphal
length to mass ratio of 0.83 m·
µ
g
–1
, (Rousseau et al. 1994;
see Table 3) the ERMM in the forest annually produces an
absorptive area of 70–112 m
2
·(m
2
of forest floor)
–1
, and the
total length of new mycorrhizal hyphae produced in 2.5 m
2
of forest floor would be long enough to stretch from pole to
pole around the world each year.
Vegetative spread of ectomycorrhizal networks and
spatial distribution of genets
The extensive growth and biomass of ectomycorrhizal
mycelial systems allows many of these fungi to spread vege
-
tatively from root to root below ground and for a single
genotyope to colonize extensive areas. By somatic compati
-
bility testing, Dahlberg and Stenlid (1990) showed that the
sizes of clones of the ectomycorrhizal fungus Suillus
bovinus in a Swedish pine forest increased with age of the
forest. More recently, DNA sequence studies (Sawyer et al.
2003; Guidot et al. 2004) or microsatellite markers (Dunham
et al. 2003; Kretzer et al. 2004) have been used to study the
extent of genets of a range of ectomycorrhizal fungi. Such
methods avoid the tedious requirement to isolate and culture
the fungus from each sample location to test its genet iden-
tity. The DNA-based approaches have greatly facilitated
studies of genet sizes, including in species that are not
readily culturable. Genet sizes vary widely between species
and may be strongly affected by the age of the forest and by
factors such as disturbance (Guidot et al. 2002b). Individual
genets may extend over large contiguous areas, with a num-
ber of reports showing individual genets occupying over
50 m
2
(Anderson et al. 2001; Sawyer, et al. 2003), but it is
most unlikely that the mycelial network remains completely
interlinked over this scale. The development of large genets
confirms long-term genet persistence of representatives of
some genera like Suillus, but in other cases, like Hebeloma
cylindrosporum, it has been suggested that most genets turn
over rapidly and may be replaced annually through new
spore germination (Guidot et al. 2004).
Interconnections of mycorrhizal networks
Amongst the key features of mycorrhizal mycelial net
-
works that make them functionally distinctive from roots is
their ability to form unique pathways for nutrient and C
transport interconnecting plants below ground (Simard et al.
2002). Further, EM mycelia, particularly in those species
that produce extensive mycelia, are often topologically com
-
plex, with frequent anastomoses that provide multiple alter
-
native transport pathways between foraging mycelium and
their C sources, the roots. In the older portions of EM net
-
works produced by species that develop multicellular hyphal
cords, such as Suillus bovinus, a tangentially interconnected
net-like structure is often well developed.
The structural complexity of internal transport pathways
within mycelial networks has only recently been discovered.
Tubular vacuole systems that allow simultaneous bi
-
directional transport to be maintained in the hyphae have
been observed in both EM (Ashford and Allaway 2002) and
AM (Uetake et al. 2002) fungi. In view of their continuity
over long distances, the tubular vacuole system in the largely
aseptate arterial hyphae of AM fungi (Uetake et al. 2002)
and in septate EM mycelia (Allaway and Ashford 2001) pro
-
vide the most likely means of simultaneous rapid transport
in opposite directions of C obtained from the host and nutri
-
ents acquired from the soil. Motile pleomorphic tubule–
vacuole systems have been shown to move in opposite
directions in the same cell, but experimental proof of nutri
-
ent and C transport are required (Ashford and Allaway
2002). Regulation and control of the internal transport path
-
ways is likely to be complex, since the directions of C and
nutrient flows may alter when parts of the network colonize
new roots or access new nutrient sources.
The extent of mycelial interconnection between plants
must be controlled by host–fungus specificity. At an ecosys
-
tem level, plants that share one particular type of mycor
-
rhiza, for example, EM or AM, form guilds (Perry 1998)
within which plants may join common mycelial networks.
The low global diversity of AM fungi, which may number
a few hundred species in contrast with the high diversity
of plants (approx. 250 000 species; Wilson 1992), most of
which have this type of mycorrhiza, has engendered low
host specificity. However, molecular studies have revealed
diverse assemblages of 20–30 AM fungal species in estab
-
lished plant communities (Vandenkoornhuyse et al. 2002;
Husband et al. 2002) and uncovered hitherto hidden patterns
of host specificity (Vandenkoornhuyse et al. 2002). Together
with results from pot studies of specificity in host–fungus
interactions affecting plant growth and AM spore production
(Bever 2002), the established view of very low host specific-
ity in AM (Sanders 2002) is now being seriously challenged
(Sanders 2003). It seems likely that in diverse plant commu-
nities virtually all mycorrhiza-compatible plants will join
common mycelial networks but that not all plant species will
share the same fungal partners (species or individuals of the
same genet), so that a complex community of overlapping
host–fungus species interactions occurs.
Networks of influence: the effect of ERMM
on biogeochemical cycles, plant community
composition, and agroecosystem
functioning
In parallel with the recent increasing awareness of the bio
-
mass, extent, interconnections, and C fluxes through
ERMM, there has been a growing recognition of the multi
-
functional importance of these networks in biogeochemical
cycles, plant community composition, and agroecosystem
functioning.
Mycorrhizal mycelial networks: a major
force in biogeochemical cycling
The empowerment of mycorrhizal networks with substan
-
tial amounts of host-derived C allows them to play central
roles in major biogeochemical cycles. The ERMM of EM
fungi show considerable interspecific and interstrain differ
-
ences in the structure and functioning of their ERMM
(Colpaert et al. 1992; Ek 1997; Leake et al. 2001; Donnelly
et al. 2003).
Whilst there is less structural complexity and variation in
AM hyphal networks, there are, nonetheless, clearly emerg
-
© 2004 NRC Canada
1030 Can. J. Bot. Vol. 82, 2004
ing functional differences between AM fungi in the extent
and efficiency with which they forage for P from roots
(Smith et al. 2000), and in part this may be explained by dif
-
ferences in the lengths and extension distances of AM
hyphae (Schweiger and Jakobsen 2000). There also appear
to be differences between AM fungal species and strains in
their abilities to assimilate organic N sources (Hawkins et al.
2000).
P and N uptake by mycorrhizal mycelium
The most important function of AM for plant growth is
normally to increase uptake of P, but AM fungi, as has long
been recognized for EM, are involved in the uptake of many
different nutrient elements (N, Cu, Fe, K, and Zn), including
some organic sources of N and P (Smith and Read 1997). It
is only in the past decade that there has been strong evidence
that AM mycelia play an important role in mineralization
and uptake of organic P (Tarafdar and Marschner 1994), and
using monoxenic cultures it has now been possible to un
-
equivocally demonstrate this process (Koide and Kabir
2000). AM hyphal networks possess wall-bound extra
-
cellular phosphatase enzymes (Joner et al. 2000a), and their
narrow diameters and rapid linear extension rates should en
-
able them to place these enzymes in soil pores that are too
small and too far from the root to be directly accessed by
root hairs. The extent to which the potential of AM fungi to
use organic P sources is realized in the field is, however,
unclear (see Joner et al. 2000a), although recent studies sug-
gest it may be significant in some soil types (Feng et al.
2003).
It is well established that many EM fungi are active pro-
ducers of phytase and phosphatase enzymes (Leake and
Read 1997), and some can obtain both P and N from a range
of organic sources, including partially decayed tree litter,
pollen, and nematodes (Read and Perez-Moreno 2003). In
soil microcosms, between 35% and 40% of the total P con
-
tent of partially decayed tree litter was removed by coloniz
-
ing EM mycelium, the majority of this P being mobilized
from organic compounds. In the absence of EM mycelium,
moist and nonsterile, partially decayed tree litter only very
slowly releases inorganic P (Bending and Read 1995).
Taking the efficiency of nutrient recovery from pollen and
nematodes by mycorrhizal tree seedlings in soil microcosms
and estimates of the annual production of these nutrient
sources in boreal forests, Read and Perez-Moreno (2003)
suggest that 15% of P and 12% of N supplied to trees in
these forest ecosystems may come just from nutrient uptake
from these sources by EM mycelium. Furthermore, there is
evidence that some EM fungi are toxic to fungal-feeding
microarthropods such as collembola and that significant
amounts of N can be obtained by mycorrhizal fungi digest
-
ing dead collembola (Klironomos and Hart 2001).
In addition to their roles in P nutrition, both AM and es
-
pecially EM fungi play a major role in the uptake of N by
plants. Evidence of the involvement of AM mycelium in the
uptake of N by plants has also been strengthened by studies
of monoxenic fungal cultures that have demonstrated uptake
of ammonium, nitrate, glycine, and glutamine. Uptake of
15
N-labelled amino acids by AM hyphae and transport of N
into roots has been measured for the first time (Hawkins et
al. 2000). This lends support to the observation that some
AM fungi increase decomposition and subsequent capture of
inorganic N from complex organic materials such as plant
litter and proliferate their hyphae in organic-resource
patches (Hodge et al. 2001). These kinds of responses have
been considered characteristic of EM but not AM fungi, and
the mechanisms involved in N mobilization by AM are un
-
clear, since there appears to be little evidence that AM fungi
produce the range of macromolecule hydrolysing and oxi
-
dizing enzymes of the former (Leake and Read 1997). How
-
ever, few studies have examined N use by AM fungi
specifically isolated from organic-rich soils, and it is notable
that the AM fungi that were most effective at taking up or
-
ganic N came from an environment with low nutrient input
(Hawkins et al. 2000).
In pot studies, particularly in relatively dry soil where
mass flow of nutrients is restricted, AM hyphae have been
found to provide an effective uptake pathway for 7%–49%
of the plant N uptake from nitrate and ammonium sources
(Hawkins et al. 2000; Johansen et al. 1994). Whilst the con
-
tribution of AM to plant N uptake is unusually high under
these rather extreme water-stress conditions, the potential of
these symbionts to assist plant N nutrition is confirmed.
Once mineral N is taken up by AM hyphae, it is most likely
to be transported through the hyphae to the plant in amino
acids (Bago et al. 2002), as has been shown to occur in
ectomycorrhizas. The incorporation of mineral N into amino
acids has high metabolic costs: in plants, 20 mol ATP are re-
quired for each mole of glutamate formed from nitrate, and
5 mol ATP are required per mole glutamate formed from
ammonium (Salsac et al. 1987). Mineral N assimilation con-
sequently has a high C cost to the mycelium, and if trans-
ported as amino acids, a significant amount of this C may be
transferred back to the host.
EM mycelia are particularly effective in the uptake of am-
monium from soil. Ek (1997) found that 9% of the nitrate
and 18% of the ammonium supplied to the mycelia of the
fungus Paxillus involutus was transferred to its host plant,
birch, in 4 d, and the mycelium incurred a significant C cost
of this nutrient uptake as its respiration increased by 55%–
180%. As a consequence of such rapid and effective uptake
by EM, forest soils naturally contain very low concentra
-
tions of mineral N. For example, Nilsson and Wallander
(2003) found only 4.6
µ
gNH
4
+
·g soil
–1
and 0.2
µ
gNO
3
·g
soil
–1
in April in a Swedish conifer forest with active mycor
-
rhizal networks, but in plots with plastic tubes inserted a
year earlier to exclude EM hyphae, this increased to 68.7
µ
g
NH
4
+
·g soil
–1
and 0.8
µ
gNO
3
·g soil
–1
.
Ectomycorrhizal mycelia can short-circuit the N cycle
In addition to their effective scavenging and assimilation
of inorganic N, especially ammonium, ectomycorrhizal fungi
have high-affinity amino acid uptake systems (Wallenda et
al. 2000), and some of these fungi also have highly devel
-
oped proteolytic capabilities enabling them to directly access
macromolecular N (Abuzinadah and Read 1989). This pro
-
cess, which is particularly important in boreal forests, has
necessitated a revision of the N cycle (Fig. 6a) and a reap
-
praisal of the nature of competition for N between plants in
these ecosystems (Näsholm et al. 1998). Utilization of or
-
ganic N by EM, which is particularly important in N-limited
forests (Taylor et al. 2000), not only short-circuits the con
-
© 2004 NRC Canada
Leake et al. 1031
ventional N cycle and greatly reduces the energetic costs of
N assimilation by the fungi, but in bypassing the normal
mineralization pathway it restricts the supply of mineral N
to plants and microorganisms that depend upon it (Fig. 6a).
By gaining a monopoly on the N, ectomycorrhizal fungi
may assist their host plants in suppressing other guilds
of plants, such as AM herbs and some hardwood saplings,
whose AM fungal partners are less effective at N capture,
and may strongly select for subsets of forest plants that have
at least some ability to use organic N (Nåsholm et al. 1998).
The reduced flow of N through the ammonification pathway,
together with effective scavenging of ammonium by EM,
also serves to minimize the residence time of mineral N in
soil solution. In turn, this will virtually eliminate nitrifica-
tion so that losses of N from the ecosystem through ammo-
nium leaching, nitrate leaching, and denitrification are
minimized and N is conserved in the forest ecosystem.
The situation is very significantly altered under enrich-
ment by anthropogenic N deposition (Fig. 6b), where the
proportion of EM fungi that have well-developed proteolytic
capabilities decreases (Taylor et al. 2000; Lilleskov et al.
2002) and the accumulation of ammonium in the soil
enhances mineralization and nitrification, culminating with
major leaching losses of nitrate together with base cations
(Schulze et al. 2000), often leading to serious acidification
(Brunner 2001). Enrichment with mineral N provides high N
availability to all guilds of plants. Whilst these effects are
clearly not driven solely by the responses of the mycorrhizal
fungi, their role is especially important, as they provide the
major N-absorbing system for the trees.
Laboratory studies have revealed a clear mechanistic link
between C supply to EM mycelia and their abilities to use
protein (Eaton and Ayres 2002). The C supply to EM from
their hosts has been shown to be reduced where N supply is
no longer limiting plants, and in these circumstances the ca
-
pacity of the fungi to take up and to immobilize N in their
own biomass and tissues is reduced. This is reflected
δ
15
N
signatures in pot-grown plants (Hobbie and Colpaert 2003).
Since EM trees commonly have well over 90% of their
root tips colonized by mycorrhiza, and the fungus com
-
pletely ensheaths the roots, it is fair to assume that virtually
all the nutrients and water taken up by the plants occurs
through their fungal partners. What is more difficult to as
-
certain is the proportion of the nutrients and water taken up
by established trees that could not have been obtained by the
plants if they lacked mycorrhizas. In a study by Read and
Perez-Moreno (2003), comparisons were made between N
and P uptake from pollen and nematode necromass by
mycorrhizal and nonmycorrhizal tree seedlings grown in
non-sterile soil. From their data, the proportion of the total
N and P supplied in a patch of pollen that was removed by
the action of ERMM was 33% and 62%, respectively. In the
case of the nematode necromass, 31% of the N removal and
41% of the P removal was attributable to foraging by EM
mycelium. By comparing the nutrient uptake by the
mycorrhizal and nonmycorrhizal plants the proportion of the
added N and P in the pollen that was only accessible to the
plants through mycorrhiza can be calculated and was found
to be 17% of the N and 18% of the total P.
Nutrient mobilization and mineral weathering by
mycorrhizal networks
AM hyphae appear to be able to acquire P from a range of
inorganic P sources, including some calcium and aluminium
phosphates that have extremely low solubility (Yao et al.
2001), but it is not known whether the fungi are directly in-
volved in their solubilization. Uptake of insoluble P sources
by AM may be facilitated by P-solubilizing bacteria, and
there may be mutualistic interactions between these two
groups of organisms (Villegas and Fortin 2001). EM mycelia
have also been shown to obtain P from a range of sparingly
soluble mineral sources such as aluminium phosphate
(Cumming and Weinstein 1990), and their production of or
-
ganic chelators such as citric and oxalic acids, together with
hydroxamate siderophores, are implicated in major mineral
weathering processes and podzolization (van Breemen et al.
2000). The sparingly soluble calcium phosphate apatite stim
-
ulates EM hyphal growth in N-fertilized forest plots (Nilsson
and Wallander 2003), and the enhanced weathering of apa
-
tite-P and biotite-K by some EM fungi has been demon
-
strated (Wallander 2000a, 2000b; Wallander et al. 2002).
These findings are of enormous significance for bio
-
geochemistry and processes of soil maturation. Whilst the
importance of some organisms, particularly lichens, has re
-
ceived considerable attention in studies of the weathering of
rocks and development of etched and fissured mineral sur
-
faces (see, e.g., Birkeland 1999), until very recently the in
-
volvement of mycorrhizal fungi in these processes in soils
was entirely unrecognized. However, there is simply no
comparison between the biomass, C flux, organic acid pro
-
duction, and surface area of contact with rock-forming min
-
erals achieved by lichens, which are restricted to a few
millimetres of bare rock surfaces or litter layers in open hab
-
itats and forest floors, and by mycorrhizal mycelia (see, e.g.,
© 2004 NRC Canada
1032 Can. J. Bot. Vol. 82, 2004
Fig.6.(a) The nitrogen (N) cycle for N-limited forest ecosystems dominated by ectomycorrhizal trees. The trees gain direct access to
organic N in plant and microbial litter through the activities of their fungal partners (pathway 1a; largest arrow), thereby short-
circuiting the normal mineralization pathways (pathways 3 and 4). There can be intense competition for labile organic N between
ectomycorrhizal fungi and microorganisms (pathways 1a and 1) and for the very small amounts of ammonium that are mineralized
(pathway 3). The main microbially driven pathways are (1) microbial depolymerisation and assimilation of organic N; (2) release of
microbial litter; (3) mineralization (ammonification); (4) nitrification; (6) microbial immobilization; (7) humification; and (8) N fixa
-
tion. Nitrate leaching and denitrification are negligible because nitrification is absent or occurs at insignificant rates. (b) The N cycle in
N-polluted forest ecosystems dominated by ectomycorrhizal trees. Note the importance of inorganic N as the main N sources used by
ectomycorrhizal plants and the great reduction in the utilization of organic N by ectomycorrhiza. There are increased amounts of N in
organic matter and especially in ammonium and nitrate pools. There are increased rates of mineralization, nitrification, and resultant N
losses through nitrate leaching and denitrification (5). The intensity of competition for N between mycorrhizal and saproptrophs is
much lower than in (a).
© 2004 NRC Canada
Leake et al. 1033
van Breemen et al. 2000; Wallander et al. 2002). Mycor
-
rhizal mycelium grows through decimetre depths of soil,
may receive as much as 10% or more of net C fixation, can
produce copious amounts of low molecular weight organic
acids (Wallander et al. 2002), extends to many metres per
gram of soil, and is abundant in virtually all terrestrial eco
-
systems.
The track etching of feldspar and other minerals with
hyphal-sized tubular pores at the estimated annual rate of
15 cm·cm
3
of soil particles·year
–1
(Jongmans et al. 1997) has
been attributed to EM fungi, which are now thought to be re
-
sponsible for greatly accelerated mineral weathering rates in
many forest soils (van Breemen et al. 2000). The high affin
-
ity of both AM and EM mycorrhizal hyphae for specific
nutrient ions such as P and K results in depletion of the
available pool of these ions (e.g., Li et al. 1991; Paris et al.
1995), and this, together with localized acidification caused
by respiration and proton release by hyphae, particularly as
-
sociated with ammonium uptake (Villegas and Fortin 2001),
will tend to increase the passive dissolution of minerals con
-
taining these ions. These processes are further accelerated by
organic acid chelators that are produced in abundance by
many EM fungi and can form extensive crystalline deposits
on their surfaces (Wallander et al. 2002).
Competition for nutrients between mycorrhizal mycelial
networks and saprotrophs and effects on the C cycle
A further consequence of the highly effective nutrient mo-
bilization and assimilation achieved by mycorrhizas is their
tendency to selectively remove N, P, and K from litter, leav-
ing behind the major C compounds and increasing the C/N,
C/P, and C/K ratios of the residues (Bending 2003). As a
consequence, there is intense competition, particularly for
the more labile N and P sources, between mycorrhizal and
saprotrophic fungi (Fig. 6a). This process has important im-
plications for the decomposer communities and in part may
account for the tendency of many boreal forest soils to expe
-
rience very low rates of litter decomposition and to accumu
-
late C in recalcitrant humic materials.
In particular, mycorrhizal mycelium interacts with other
large fungi such as the wood decomposers, which use unusu
-
ally large and long-lasting C resources in the form of coarse
woody debris with very low availability of N and P. These
two groups of fungi grow in decaying pieces of wood
(Tedersoo et al. 2003), forage for nutrients in the same soil
horizons, and share many similarities in growth and their nu
-
trient foraging strategies (Leake et al. 2002; Boddy 2000).
Many saprotrophic and ectomycorrhizal fungi are closely re
-
lated, as there appears to have been evolutionary instability
between them in both directions (Hibbett et al. 2000). Un
-
surprisingly, therefore, EM fungi retain many of the key
enzyme systems of saprotrophic fungi and can deploy some
of them in nutrient mobilization (Leake and Read 1997).
The increasing recognition of a broad spectrum of “sapro
-
trophic” enzyme activities in mycorrhizal fungi challenges
the conventional view that mycorrhizal fungi are fundamen
-
tally different from saprotrophs and lack the ability to
directly participate in decomposition processes. Soil micro
-
cosm studies (Lindahl et al. 1999; Leake et al. 2001, 2002)
have revealed that mycelial systems of EM and saprotrophic
wood-decomposer fungi can be antagonistic to each other,
both in growth and functioning, and this antagonism is often
greatest in the actively growing mycorrhizal mycelium fur
-
thest from root surfaces. This antagonism can lead to signifi
-
cant transfers of nutrients between the two trophic groups
when they interact (Lindahl et al. 1999).
Our studies have shown that when EM mycelium encoun
-
ters mycelium of saprotrophic wood- and litter-decomposing
basidiomycetes in natural soil there is intense territorial con
-
flict. Extension of mycelial cords of the aggressive sapro
-
troph Phanerochaete velutina was halted by dense mycelium
of Paxillus involutus symbiotic with Betula pendula (Leake
et al. 2002). The mycelial cords of the saprotroph rapidly
became truncated, their apices turned brown and senesced,
and their growth was often deflected away from the direction
of the advancing mycorrhizal mycelium, restricting their
access to ectomycorrhiza-occupied regions (Fig. 7). There
were, however, often marked reciprocal effects of the sapro
-
troph on the mycorrhizal mycelium, whose growth and C
allocation was directed away from the region of conflict in
Paxillus involutus or led to a general loss of vigour and C
allocation to ERMM in Suillus bovinus (Leake et al. 2001).
Antagonistic effects of EM on saprotrophs may serve to
further slow the process of organic matter decomposition in
forest soils and may facilitate the vitally important process
of soil C sequestration. On the other hand, decomposition
might be unaffected or accelerated (Bending 2003). Apart
from effects on nutrient supplies and territorial exclusion of
some saprotrophs, the drying of soil by water extraction by
EM has recently been implicated in reduced litter decompo-
sition in the presence of EM networks (Koide and Wu 2003),
but there are also indications that under severe surface
drought conditions, water can be supplied to mycorrhizal
hyphae from plant roots, providing the trees have access to
water via taproots in subsurface layers (Querejeta et al.
2003). How these counteracting processes actually affect nu-
trient and C cycling processes under field conditions remains
unclear, but it is likely that the hydrophobicity of many EM
fungal cords and rhizomorphs allows them to transport water
to or from the soil, depending on the moisture regime, en
-
abling efficient uptake of nutrients by the fine, absorptive
hydrophilic hyphae, even in drying soil. The supply of water
via the tree during surface drought is thought to keep the
hyphae active (Querejeta et al. 2003), making them instantly
available to absorb the flush of nutrients released into solu
-
tion as soon as the soil rewets.
Not all interactions between mycorrhizal fungi and
saprotrophs are likely to be antagonistic. Indeed, where the
main N sources are highly recalcitrant tanned-protein com
-
plexes, which most EM fungi tested to date have very lim
-
ited abilities to degrade, the action of saprotrophs appears
to facilitate N recovery by the mycorrhizas (Wu et al.
2003).
Since AM are most common in environments in which
organic matter decomposition is relatively quick and N is
readily mineralized, interactions between their mycelia and
those of saprotrophs is likely to be less important with re
-
spect to competition for N, but they may interact with other
organisms, particularly in competition for P. In vitro studies
of bacteria and fungi in the presence and absence of AM
mycelial networks have revealed modest positive and nega
-
tive effects (Fortin et al. 2002), but most studies in soil
© 2004 NRC Canada
1034 Can. J. Bot. Vol. 82, 2004
(sandy soil) have often shown little or no effect (e.g., Olsson
et al. 1996; Olsson and Wilhelmsson 2000).
Selective ion uptake and exclusion of toxic elements
from plants by mycorrhiza and their external mycelium
Both AM (Joner et al. 2000b) and EM (Meharg and
Cairney 2000) mycelial systems can immobilize and reduce
plant uptake of nonessential and toxic metals such as Cd and
Pb and thereby decrease the passage of these elements
through the food chain. These effects are strain specific, and
those isolates that are most effective at tolerating metals and
excluding them from their tissues are typically found on
contaminated sites (see, e.g., Malcova et al. 2003). The abil
-
ity of mycorrhizal mycelium to selectively take up and pass
on nutrients to their hosts whilst excluding toxic metals en
-
ables mycorrhizas to play an important role in revegetation
of metal-contaminated sites. It allows some EM fungi to mo
-
bilize P from aluminosilicates and aluminium phosphate, in
particular, whereby they exclude potentially phytotoxic alu
-
minum, whilst supplying their host plants with P (Cumming
and Weinstein 1990).
Protective effect of mycorrhizal mycelium against
attack by root pathogens
There is evidence that both AM and EM fungi can reduce
root-pathogen attack, and some of this effect may be due to
interactions between external mycelium and mycelium of
pathogens. Root-organ culture studies have demonstrated ef
-
fects of AM mycelia on spore germination and growth of
root pathogens, but the effects may be positive or negative
and are different for different pathogens (see Fortin et al.
2002). In greenhouse studies beneficial effects of AM
mycorrhizas have often been shown (e.g., Abdel-Fattah and
Shabana 2002), but the importance of ERMM versus intra
-
radical infection in AM is unclear.
In the case of EM, a few studies have demonstrated a pro
-
tective antibiotic role of external mycelium against patho
-
gens, in addition to the benefits of the mycorrhizal sheath.
For example, Paxillus involutus can be antagonistic to root
pathogens in the soil and suppress their growth before they
have the opportunity to encounter roots (Duchesne et al.
1989). If ERMM is generally inhibitory to many root patho
-
gens this will have important consequences for management
practices in agriculture that disrupt and damage these net
-
works (see next section).
Effects of mycorrhizal networks on plant
community composition and ecosystem
functioning
Plant community diversity and biomass production
correlate with length of ERMM
In experimental macrocosms, AM mycorrhizal fungal di
-
versity was found to have a strong positive effect on plant
community diversity and plant productivity (van der Heijden
© 2004 NRC Canada
Leake et al. 1035
Fig. 7. The effect on carbon allocation in the extraradical mycelium of the mycorrhizal fungus Paxillus involutus when in contact and
interaction with the wood-decay fungus Phanerochaete velutina, growing from a piece of wood (PV). The mycorrhizal fungus is grow
-
ing in association with Betula pendula, which was pulse-labelled with
14
C, and this was quantified in a 20 cm × 24 cm belowground
area by digital autoradiography. There were two patches of litter (L) in the microcosm to provide resources for the fungi. Note the
truncation and browning of the Phanerochaete hyphal cords in contact with Paxillus (small arrowheads) and the deflection of the
growth of the saprotroph on the right of the wood block (large arrowhead). The mycorrhizal fungus allocates the carbon it receives
from the host plant away from the area of territorial combat, and its growth is locally stopped by contact with Phanerochaete. Mod
-
ified from Leake et al. (2002).
et al. 1998). With increasing AM fungal diversity, hyphal
length in soil rose from 2.8–6.5 m·g
–1
, reaching a plateau
with 8–14 species. The responses to AM diversity seen in
both the plant productivity and community diversity (Simp
-
sons’s diversity index) correlated almost exactly with the
pattern of hyphal length increase with increasing AM diver
-
sity, and this in turn was inversely related to the quantities
of P remaining in the soil. This experiment supports the hy
-
pothesis that under P-limiting conditions maximum produc
-
tivity and plant biodiversity is dependent upon the amounts
of external mycorrhizal mycelium produced, and diverse as
-
semblages of AM fungi provide more external mycelium
and, consequently, more P uptake than species-poor AM
communities. Further work is require to test these hypothe
-
ses but there is increasing evidence that the uptake of P by
AM mycorrhiza is often closely related to total AM hyphal
lengths (see later section).
The role of interplant C and nutrient transport
through mycorrhizal mycelial networks
A feature of mycorrhizal networks that has recently at
-
tracted enormous interest and generated the most contro
-
versy has been their transporting of nutrients and C from one
plant to another through interconnecting mycelium (Simard
et al. 1997, 2002). These interspecies resource transfers have
far-reaching implications for evolutionary theories applied to
plant communities and suggest that a radical reappraisal of
conventional concepts of competition is required (Perry
1998; Wilkinson 1998). The controversy has centred on the
evidence for net transport of C between plants linked by a
common mycelial network, and Robinson and Fitter (1999)
have gone as far as to state “it is not possible to conclude
that plant–plant C transfer via a common mycorrhizal net-
work has any significance for the composition and function-
ing of plant communities”. However, it has been known for
over 150 years that some achlorophyllous plants, called
myco-heterotrophs gain all their C from fungi (Leake 1994).
Over 400 species of such plants exist, and they are derived
from multiple independent lineages of green plants. In recent
years, using DNA-based identification, fungal partners of
many of these plants have been identified for the first time,
and most have proved to be EM (Taylor et al. 2002) or AM
fungi (Bidartondo et al. 2002) that are coinfecting the roots
of adjacent green plants. Pulse-labelling studies, and bio
-
mass measurements in experimental microcosms with and
without mycelial interconnections between autotrophs and
myco-heterotophs, have confirmed that C is transported via
the shared EM mycelium (McKendrick et al. 2000b; Bidar
-
tondo et al. 2003).
The fungal partnerships of myco-heterotophs are charac
-
terized by extreme specificity (Taylor et al. 2002; Bidar
-
tondo et al. 2002; Young et al. 2002) and unusual host-plant
combinations. Many orchids switch from associations with
soil saprotrophs and pathogens, such as fungi in the form ge
-
nus Rhizoctonia, to associate with EM fungi (Taylor et al.
2002; McKendrick et al. 2002) that are better able to supply
large amounts of C over long periods of time. The exchang
-
ing of “normal” partnerships to gain direct access to plant-
derived C has also been demonstrated for the achlorophyllous
liverwort Cryptothallus mirabilis, whose fungal partner is
a Tulasnella, a genus of fungi that often form orchid
mycorrhizas and are normally considered saprotrophic, but in
this case the fungus forms ectomycorrhizal associations with
birch (Bidartondo et al. 2003).
Whilst myco-heterotrophy is recognized as an extreme
“cheating” type of mycorrhiza (Bidartondo et al. 2002), it is
surprisingly common and not confined to the approximately
400 species that are entirely achlorophyllous. It characterizes
the early developmental stages of germination of most or
-
chids, which number over 17 500 species, and the game
-
tophyte stages of many club mosses and ferns. In these cases
there is net C flow to the plant during the early stages of
growth from seeds or spores, but the plants eventually go on
to photosynthesize (Leake 1994). These “initially myco-
heterotrophic” plants receive a modest C investment from
their fungal partners to establish themselves as autotrophs,
but providing the same fungi are retained in the adult plants,
the fungi in the long term may stand to gain much more C
back from these partnerships once the plants mature into
autotrophs. There is increasing evidence that obligate myco-
heterotrophy represents one extreme end of a continuum of
C transfer from fungi to plants, and the multiple evolution
-
ary origins of this form of nutrition is consistent with the
view that a large number of plants can gain C, at least tran
-
siently, by this pathway. Myco-heterotrophs exemplify the
ability of mycorrhizal networks to transport substantial
quantities of C and nutrients and the potential importance of
these networks for the control of species composition of nat-
ural communities. In the absence of their critical fungal part-
ners, myco-heterotrophic plants typically fail to germinate
and certainly fail to establish (McKendrick et al. 2000a;
2002). In these cases, mycorrhizal mycelial networks pro-
vide the ultimate control on composition of myco-
heterotroph plant communities.
What remains unclear is the extent to which these kinds of
interplant transfers occur routinely between interlinked
green plants in the field and over what scale. Experimental
evidence is fragmentary, and as Robinson and Fitter (1999)
note, crucial experiments have not been conducted to ex
-
clude the possible fixation in the shoots of “receiver” plants
of labelled C tracers respired from the roots and mycorrhizas
of the “donor” plants supplying most of the C to the plant-
interlinking mycorrhizal network in the soil. Thus, even in
the most recent studies of interplant C transfers (Wu et al.
2001; Lerat et al. 2002) there remains uncertainty about the
pathway of labelled C uptake by shoots, whether from re
-
fixation of C respired from below ground or through direct
transfer through mycorrhizas interlinking the roots. How
-
ever, there is already clear evidence that at the seedling-
establishment phase many plants receive, at the very least,
an indirect C subsidy from the established plants. Newly
emerging seedling roots link to an established nutrient-
absorbing mycelial network that has already been “paid for”
by C supplied by the established plants, and the fungal colo
-
nization of their roots may also be C subsidized (Simard et
al. 2002). In addition, ERMM networks provide pathways
for interplant transfers of nutrients, and in some cases, de
-
pending upon the species and circumstances, substantial
amounts of N, and to a lesser extent P, have been demon
-
strated to move through mycorrhizal mycelium interlinking
plants (Simard et al. 2002). Recent studies of the
15
N and
13
C enrichment of fully myco-heterotrophic plants (Trudell
© 2004 NRC Canada
1036 Can. J. Bot. Vol. 82, 2004
et al. 2003) and of both green and fully myco-heterotrophic
orchids (Gebauer and Meyer 2003) have shown that the
myco-heterotrophs have distinctive enrichment in both
13
C
and
15
N relative to co-occurring autotrophic plants. More
importantly, the characteristic heavy-isotope enrichment of
myco-heterotrophs has revealed that within green orchids
there is a wide range of dependency upon fungal-C. Some
green-leaved species previously assumed to be autotrophs
are clearly gaining most of their C by myco-heterotrophy
(Gebauer and Meyer 2003), confirming the view that there
is a continuum between full autotrophy and full myco-
heterotrophy.
Effects of mycorrhizal mycelial networks on plant
community composition: costs and benefits are not
shared equally
It is increasingly apparent that the C costs and functional
“benefits” to plants of linking to ERMM networks are fungal
specific and, because of variations in physiology and host
specificity, are not shared equally amongst plants (Hartnett
and Wilson 2002). Grasses, for example, with their extensive
fibrous root systems with long root hairs, are much less my
-
corrhiza dependant than coarser rooted forbs, but may con
-
tribute substantially to C supply to the ERMM (Grime et al.
1987). Whilst the former often show limited growth benefit
from mycorrhiza, the latter can be highly responsive, and
their response may vary greatly depending upon which fun-
gal partners are present, so that mycorrhizal fungal species
composition can have a major effect on individual plant per-
formance and overall plant community structure (see, e.g.,
van der Heijden et al. 1998). The evidence of extreme speci-
ficity and fungal dependence in myco-heterotophs reinforces
the need to investigate the functional importance and com-
munity consequences of host–fungus specificity that has
been revealed in a number of recent studies (Vanden-
koornhuyse et al. 2002; Bever 2002). In autotrophs such
specificity may help to ensure that interplant C and nutrient
transfers and the costs and benefits of linking to a common
mycelial network are not shared too widely and preferen
-
tially benefit co-linked plants of the same species. An alter
-
native perspective is that specialization is primarily of
advantage for the specialist, not its target, since “cheats” and
parasites are typically characterized by very high host speci
-
ficities (Bruns et al. 2002).
Mycorrhizal mycelial networks in
sustainable agriculture
Pollution and disturbance affect the extent and
functioning of mycorrhizal mycelium
The environmental impact of intensive agriculture on bio
-
diversity, soil and water pollution, and soil erosion and sus
-
tainability has become a major concern (Robinson and
Sutherland 2002). As awareness of these issues has in
-
creased in recent years together with consumer concerns
about food safety, such as possible health risks of pesticide
and fertilizer residues, there has been significant expansion
of alternative forms of agriculture, such as reduced tillage
and organic and biodynamic farming. Under increasing in
-
tensification of agriculture there may be little scope for AM
involvement in agricultural production (Ryan and Graham
2002), as phosphate fertilizer applications and soil distur
-
bance have very adverse effects on AM mycelial networks
(McGonigle and Miller 2000), and most AM fungal species
are eliminated (Daniell et al. 2001). The loss of diversity is
characterized by the virtual elimination of certain key genera
such as Acaulospora and Scutellospora, both of which tend
to be abundant in undisturbed communities (Daniell et al.
2001), and the dominance of communities by a single spe
-
cies, Glomus mosseae. It is suggested that the abundance of
the latter may relate to its ability to form anastomoses and
repair severed hyphae (see Daniell et al. 2001) and the readi
-
ness of Glomus sp. to form infections from hyphae and root
pieces (Klironomos and Hart 2002). This contrasts with
Scutellospora, which does not readily form anastomoses
and, in the absence of intact hyphal networks, appears to
depend upon spore inoculum (Klironomos and Hart 2002).
There are indications that the loss of AM genera in intensive
agriculture is likely to have much greater functional signifi
-
cance than loss of species within the same genus, since there
appear to be larger functional differences at the genus level
(see Hart and Klironomos 2002). This hypothesis requires
more rigorous testing, as the taxonomy of Glomeromycota is
currently undergoing rapid revisions with the increasing de
-
velopment of molecular taxonomy.
Studies of the impacts of anthropogenic pollutant N depo-
sition on mycorrhizas have mainly focused on EM in forest
ecosystems that are normally N limited and very sensitive to
N enrichment. EM diversity, biomass, and fruit-body pro-
duction are adversely affected by N enrichment (Erland and
Taylor 2002). In short-term N addition experiments in the
laboratory, EM fungi showed large increases in respiration in
response to mineral N (Ek 1997; Bidartondo et al. 2001), and
Suilloid species, which are particularly sensitive to forest N
enrichment, gave up to five-fold greater increase in respiration
compared with Paxillus involutus, which is considered rela-
tively N tolerant. In the field, long-term (>10 years) N fertil
-
ization of forest plots reduced EM mycelial growth into sand
ingrowth bags by approximately 50% (Nilsson and Wallander
2003). In highly N enriched soils the large C drain imposed
on the hyphae by sustained uptake of high concentrations of
mineral N is highly detrimental to the fungi, and the supply
of C from the host plants to the fungi may be down-
regulated when the plants are no longer N-limited (Hobbie
and Colpaert 2003). However, as N supply increases, P limi
-
tation typically becomes more important. In N-fertilized for
-
ests plots, Nilsson and Wallander (2003) found that, in mesh
bags of sand mixed with apatite, the growth of EM myce
-
lium was not inhibited in the N-fertilized forest plots, where
demand for P may have increased.
Whilst grasslands are typically P rather than N limited
and might be considered less sensitive to pollutant or fertil
-
izer N inputs, recent studies of effects of long-term N en
-
richment of species-rich grasslands have shown major
effects on AM functioning (Ames 2002). Under these condi
-
tions, P becomes the key nutrient that limits plant growth,
and in grasslands plant P limitation can be exacerbated by
the impairment of mycorrhizal functioning through exces
-
sive N enrichment (Ames 2002). It is likely that N fertilizer
is one of a number of factors that contributes to the low di
-
versity of AM fungi in intensive agriculture, but this requires
further confirmation.
© 2004 NRC Canada
Leake et al. 1037
The importance of mycorrhizal mycelium in sustainable
agriculture
AM hyphal lengths in soil show strong positive correla
-
tions with soil-aggregate stability (Rillig et al. 2002; Kabir
and Koide 2002), P uptake efficiency (Schweiger and Jakob
-
sen 2000), and crop-yield improvements (Kabir and Koide
2002). Interest in the development of less intensive manage
-
ment systems is presenting new opportunities for adapting
agricultural production systems to enhance these benefits
that can be gained from AM networks. Substantial improve
-
ments in “soil health” and AM functioning in field crops are
gained by the doubling of lengths of AM hyphae in soil
when tillage is reduced (Kabir et al. 1998a, 1998b). Similar
gains are achieved by growth of AM-compatible cover crops
in place of winter fallow (Kabir and Koide 2002). Frost-
sensitive cover crops can be equally effective as wintergreen
crops for maintaining AM inoculum potential (Kabir and
Koide 2002) and are ideally suited to organic and bio
-
dynamic management systems, where herbicide use is not
permitted. Improved productivity in mycorrhiza-demanding
crops, for example, maize, can be achieved by substitut
-
ing mycorrhizal-compatible crops (e.g., sunflower) for
mycorrhiza-incompatible crops, (e.g., members of the Bras
-
sicaceae) in the preceding year of the rotation (Karasawa et
al. 2002).
Reducing management intensity of farmland can increase
the importance of mycorrhizal functioning in P acquisition.
Using mesh-compartmented pots containing turf from winter
wheat fields and the uncultivated field margins, Muckle
(2003) has shown that the uptake of P from root-free mycor-
rhizal hyphal compartments is increased with decreasing
management intensity. These effects can extend beyond the
ploughed fields to include the field margins, too (Fig. 8).
Mycorrhizal networks can contribute to sustainability by
increasing nutrient-use efficiency, reducing infections by
root-pathogens, and increasing soil-aggregate stability and
soil physical properties. These effects provide impetus for
the development of new forms of sustainable agricultural
management that seek to optimize benefits gained from
these symbioses.
Future directions
The progress made in our understanding of ERMM has
brought the importance of these networks to the fore, but at
the same time has highlighted the enormous gaps in our
knowledge and uncertainties as to the networks’ nature, ex
-
tent, biomass, and functioning in the field. Whilst reports of
AM hyphal lengths in agricultural soils are becoming in
-
creasingly common, our knowledge of them in natural eco
-
systems remains scant. It is particularly important that
hyphal lengths and activities are now recorded in field-
collected soils, particularly from under woodland, forests,
and permanent grasslands, where we currently have few or
no data. We also require observations from crops under
different kinds of agriculture, especially in less intensive
management. There is clearly a need to devise less labour-
intensive methods for the counting and identification of ex
-
tracted AM hyphae, and the use of image analysis systems
to do this should be developed. It is also important to de
-
velop and apply methods for the identification and quantifi
-
cation of the biomass of different mycorrhizal species in soil
and to assess their functioning in the field, for example, us-
ing isotope tracers and root-free hyphal compartments. Pro-
tocols for molecular identification of species from hyphae in
soil have been successfully developed (Landeweert et al.
2003; Dickie et al. 2002) and clearly have enormous poten
-
tial to reveal detailed spatial structure in communities of EM
extraradical mycelia in soil, but the methods are not quanti
-
tative, and effective procedures for routine identification of
AM hyphae in soil still need to be developed. The linking of
mycelial location, identification, and function in soil remains
a major challenge but is starting to be achieved (see, e.g.,
Wallander et al. 2003).
Studies of the functioning of ERMM in the field are in
their infancy, but the availability of methods that enable root
and hyphal activities to be distinguished should now be
more widely applied to establish the quantities of nutrients
and C passing through mycorrhizal mycelia in a wider range
of plant communities and farmland under different types of
management. It is clearly of vital importance that the quanti
-
ties of C passing through ERMM are determined for a wider
range of plant communities and soils to fill this major gap in
our knowledge of the terrestrial C cycle. It has been sug
-
gested that mycorrhizal mycelia may comprise a third of the
soil microbial biomass in forests (Högberg and Högberg
2002), and these may receive 10%–20% of net photosynthe
-
sis (Birdartondo et al. 2001). Accurate estimates of typical C
allocation to mycorrhizal mycelia in the field must now be
an absolute priority. Their major contribution to soil C pools,
which is unknown, but thought to be substantial (Högberg
and Högberg 2002), must also be quantified. The recent evi
-
© 2004 NRC Canada
1038 Can. J. Bot. Vol. 82, 2004
Fig. 8. The effect of management intensity on the mean shoot
33
P concentration in turfs 30 d after being provided with
radiolabelled orthophosphate on anion-absorption membranes
buried 1 cm inside a root-free hyphal compartment. The turfs
were sown with a wildflower seed mix onto monoliths of soil re
-
moved from conventional (CONV.) or organic (ORG) managed
winter wheat fields and their uncultivated margins (Margin). The
monoliths came from five replicate fields from each management
type and originated from North Yorkshire, two locations in
Leicestershire, and one in Norfolk, all in the UK. Vertical bars
indicate the SE of the means. From Muckle (2003).
dence of the accumulation of large amounts of glomalin-C
under AM plants, and its fate following land use changes
(Rillig et al. 2003), highlights the need to understand more
fully this important potential C store in soil and the effects
of different management practices on its production and ac
-
cumulation.
Some of the most exciting future prospects for studies of
the functioning of mycorrhizal networks, their environmental
sensing, and their physiological responses include applica
-
tions of genomic and proteomic approaches. Unearthing the
spatial and temporal regulation of gene expression involved
in nutrient sensing, uptake and transport within mycorrhizal
mycelial networks, and their localized interactions with
other organisms presents a fascinating prospect. The recent
analysis of expressed sequence tags in two ectomycorrhizal
fungi (Peter et al. 2003) opens the door on these entirely
new avenues of study. We already know that some EM fungi
can sense nutrients and direct growth and C allocation into
resource patches of litter (Bending and Read 1995; Leake et
al. 2001; Hodge et al. 2001), mineral N (Ek 1997), or apatite
(Nilsson and Wallander 2003). However, the signalling and
control of resource allocation, the control of gene expression
for the production of specific enzymes, secretions, and
hyphal growth forms within the networks, and nutrient parti
-
tioning within the networks, including to the host plants, is
currently not adequately understood. There are intriguing
indications that EM growth may be tightly regulated by the
specific nutrient limitations affecting the host plants:
mycelial production was stimulated in mesh bags containing
apatite mixed with sand but only in a forest where the trees
were highly P limited (Hagerberg et al. 2003). The recent
confirmation of a motile tubular vacuole system in AM
(Uetake et al. 2002) as well as in EM mycelial networks
(Allaway and Ashford 2001) demonstrates highly sophisti-
cated internal structures for bidirectional transport of C and
nutrients, but much has yet to be learned about the nature of
the substances transported and the manner in which this is
controlled and regulated.
Conclusions: looking beyond the
rhizosphere into the mycorrhizosphere
The extraradical mycelium of mycorrhizal fungi is the
“Cinderella” of soil microbial communities whose time has
now come. The past decade of research has clearly estab
-
lished that, although often unacknowledged, ERMM is the
hidden power behind plant community composition and eco
-
system functioning through the major processes it carries
out, such as nutrient uptake, weathering of minerals, soil-
aggregate stability, and the way in which it alters competi
-
tion between plants (van der Heijden et al. 1998). Whilst the
key roles of ERMM are appreciated by increasing numbers
of ecologists, soil biologists, agronomists, and foresters, its
multifunctional importance needs to gain much wider recog
-
nition. This is particularly true in sustainable agriculture,
where the benefits of appropriate management systems to
ensure effective functioning of mycorrhizal networks are
now being demonstrated in yield increases (Kabir and Koide
2002). To many, however, ERMM remain the “hidden half”
of the symbiosis. The well-established concept of the rhizo
-
sphere was developed from observations on bacterial enrich
-
ment on exudates around the surfaces of roots grown with
-
out their normal mycorrhizal associations in the laboratory
(e.g., Whipps and Lynch 1983). We must now recognize that
mycorrhizal hyphae carry carbohydrates from plants into
soil regions far beyond the conventional rhizosphere, and
they release exudates, enzymes, hydrophobic glycoproteins,
chelators, and dead cells and interact with other soil micro
-
organisms (e.g., Leake et al. 2001) to create their own
mycorrhizosphere with distinct microbial populations
(Timonen et al. 1998; Bomberg et al. 2003).
As mycorrhizal mycelium is now known to constitute as
much as 20%–30% of microbial biomass, it is necessary to
critically reevaluate the value of standard measures of soil
microbial biomass such as substrate-induced respiration,
which selectively discriminate against detecting its major
contribution to soil C pools and fluxes. The vital roles of the
ERMM in soil science and plant nutrition need to be prop
-
erly recognized. With few exceptions, undergraduate texts
on plant physiology, plant nutrition, and agronomy pay scant
attention to external mycorrhizal mycelium. Those that dia
-
grammatically illustrate mycorrhizal mycelium typically
misrepresent it as comprising highly simplistic, unbranched
structures modelled on thinner versions of root-hairs, beyond
which they are shown to extend, at the most, by a few milli
-
metres. Diagrams of EM typically focus on the mantle and
root-tip structure, whilst the mycorrhizal mycelium, which
may account for 75% of the absorptive area (Rousseau et al.
1994), is often depicted by a few wisps of simple hyphae.
The reality that external mycorrhizal mycelium form exten-
sive and complex networks, interlink plant roots, extend far
beyond the conventional “rhizosphere”, and provide ex-
tremely important pathways for nutrient and C movements
needs to eclipse such gross oversimplifications. Many of the
multiple functions of ERMM result directly from the com-
plex structural properties of mycelial networks, so that sim-
plistic representations of them implicitly deny these
functions. The accumulating evidence of the importance of
ERMM in biogeochemical cycles, soil microbial ecology,
plant communities, and agroecosystem functioning indicates
that in future an even higher priority must be placed on stud
-
ies of this part of the symbiosis. Conventional views on
plant community functioning have been challenged by the
evidence of transport of C and nutrients between some
plants interlinked by a common mycelial network and by the
demonstration of EM mycelia short-circuiting of the miner
-
alization path of the N cycle. These are indeed networks of
power and influence, and the time has come for us to pay
due attention to them.
Acknowledgements
I wish to thank the organizers of the 4th International
Conference on Mycorrhizae, especially Dr. André Fortin for
inviting me to present this paper. Funding for our own re
-
search work, included in this review, was provided by the
Natural Environment Research Council (NERC), UK, grants
GR3/11059, NER/T/S/2001/00177, and NER/A/S/2000/00411.
Dr. Gemma Muckle was funded by an NERC–CASE (Coop
-
erative Awards in Sciences of the Environment) studentship
(GT 04/99/TS/246) in association with Dr. A.R. Leake at Fo
-
© 2004 NRC Canada
Leake et al. 1039
cus on Farming Practice and sponsored by CWS Agriculture
UK, Profarma, and Hydro.
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