RESEARCH ARTICLE

Carbon dynamics in mycorrhizal symbioses is linked to carbon costs

and phosphorus benets
Axel Olsson, Jannice Rahm & Nasser Aliasgharzad
Pal
Department of Plant Ecology and Systematics, Lund University, Lund, Sweden

Present address: Nasser Aliasgharzad,

Department of Soil Science, Faculty of
Agriculture, University of Tabriz, Tabriz, I.R. of
Iran
Received 1 April 2009; revised 17 December
2009; accepted 17 December 2009.
Final version published online 20 January 2010.

MICROBIOLOGY ECOLOGY

DOI:10.1111/j.1574-6941.2009.00833.x

Abstract
The nutrient and carbon (C) allocation dynamics in mycorrhizal hyphal networks
cause variation in costs and benefits for individual plants and fungi and influence
the productivity, diversity and C cycling in ecosystems. We manipulated light and
phosphorus (P) availability in a pot experiment with Trifolium subterraneum
colonised by the arbuscular mycorrhizal (AM) fungus Glomus intraradices. Stable
13
C-labelling was used to trace assimilated CO2 to the mycorrhizal fungus in roots
and soil using compound-specific isotope ratio mass spectrometry. We used the
neutral lipid fatty acid 16:1o5 as a signature for AM fungal storage lipids. Both P
and shading reduced the AM fungal lipid accumulation in the intraradical
mycelium, while only P reduced the amount of lipids in the extraradical mycelium.
Recently assimilated plant C was only allocated to the mycorrhizal fungus to a
small extent when plant mycorrhizal benefit was reduced by P fertilization, while
increasing the plant C cost by shading did not reduce the C flow to the fungus.
These results are of importance for our conception of mycorrhizal dynamics
during periods of shade in nature.

Editor: Philippe Lemanceau

Keywords
arbuscular mycorrhiza (AM);
carbon-13; Glomus intraradices; mycorrhizal
networks; shading; phosphorus;
stable isotopes; symbiotic costs.

Introduction
The outcome of mycorrhizal symbiosis varies in terms of the
benefits to plant hosts (Johnson et al., 1997; Klironomos,
2003). Reduced diversity of mycorrhizal fungi in agricultural
fields (Helgason et al., 1998) may result from selection for a
few, less beneficial, fungi (Johnson et al., 1992; Johnson,
1993), and fertilization may convert a mutualistic fungus
into a parasite (Johnson et al., 1997). Mycorrhizal symbiosis
has often been observed to depress plant growth, and this is
believed to be particularly important in carbon (C)-limited
systems (Hayman, 1974; Peng et al., 1993). The dynamics
of mycorrhizal symbiosis may be important for the maintenance of diversity and productivity in plant communities
(Bever, 1999; Maherali & Klironomos, 2007; Van der Heijden
et al., 2008).
Mycorrhizal fungi influence the flow and retention of C in
the rhizosphere by increasing the root allocation, speeding
FEMS Microbiol Ecol 72 (2010) 123131

up respiration and retaining C in the extraradical mycelium

(Jakobsen & Rosendahl, 1990; Staddon et al., 2003; Olsson &
Johnson, 2005). Furthermore, it has been suggested that AM
fungal networks may transport C between plants linked to a
common mycorrhizal network (Grime et al., 1987),
although this is debated, and others have reported that
fungal translocation of C to sink plants, stay in the intraradical mycelium (Robinson & Fitter, 1999; Nakano-Hylander
& Olsson, 2007; Voets et al., 2008). Still, such translocation
may be of importance for reducing the C cost of some
mycorrhizal plants in a community.
Arbuscular mycorrhizal (AM) fungi store large amounts
of C as triacylglycerides. These compounds are synthesized
only within the intraradical mycelium (mycelium inside
roots) and are then transported to the extraradical (outside
roots) mycelium (Bago et al., 2002). The production of
fungal triacylglycerides constitutes a substantial C sink for
the host plants (Bago et al., 2002). Triacylglyceride synthesis
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Axel Olsson,
Correspondence: Pal
Department of Plant Ecology and Systematics,
Lund University, Ecology Building, SE-223 62
Lund, Sweden. Tel.: 146 46 222 42 47; fax:
146 46 222 41 58; e-mail:
pal_axel.olsson@ekol.lu.se

124

Materials and methods

P-fertilization (experiment 1)
Clover (T. subterraneum L.) seeds were surface sterilized
in 5% calcium hypochlorite for 15 min and pregerminated
in aerated sterile water for three days. Seedlings were
then planted in 48 pots (two seedlings per pot) with 230 g
of a 1 : 1 w/w mixture of washed quartz sand (fraction
o 0.55 mm). The field soil had been g-irradiated (10 kGy)
in order to eliminate indigenous AM fungi and had properties as described by Gavito & Olsson (2003). Mild radiation
has been shown to have little influence on soil P availability
(Jakobsen & Andersen, 1982). The field soil had a pH of 6.6,
contained 5.8% organic matter and 16 mg kg 1 available P
(Olsen-P). Half of the pots were inoculated with a 3 cm2
phytagel disc of G. intraradices propagated in vitro (Schenck &
Smith, BEG 87) on Ri T-DNA-transformed carrot root (AM),
and the rest were left uninoculated (NM). All pots were
inoculated with a bacterial inoculum as described below.
The plants were allowed to grow in a greenhouse at
temperatures of approximately 22 1C during the daytime
and approximately 18 1C at night-time. The photosynthetic
photon flux density was at least 270 mmol m 2 s 1 and the
photoperiod 18 h. The light intensity was kept constant
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Table 1. The designs of the two experiments with nonmycorrhizal and

AM-inoculated Trifolium subterraneum
Time

Experiment 1

Day 1

Planting of seedlings half of pots inoculated

with Glomus intraradices
All pots fertilized three times with 5 mg N
Shading treatment
to half of the pots
P amendment to half of the pots
Harvest 1
Harvest 2
Harvest
Harvest 2

Days 2036
Day 37
Days 4344
Days 4413
Days 4417
Days 44114

Experiment 2

Both experiments had a full factorial design: Experiment 1 with AM

inoculation, P fertilization and Harvest time as factors (n = 4), and
Experiment 2 with AM inoculation, Shading and P fertilization as factors
(n = 5).

through automatic sunshine protection during sunny days.

The plants were watered daily with distilled water to 60%
water-holding capacity. An overview of the experimental
set-ups is given in Table 1. On days 24, 31 and 36, 5 mg of
nitrogen (N) was added to each pot using a NH4NO3
solution with a concentration of 28.6 g L 1. At 44 days after
sowing, 22.5 mg P was added to 24 pots (P), 12 AM and 12
NM using a KH2PO4 solution with a concentration of
44 g L 1. This corresponds to 90 mg P kg 1 soil. The pots were
arranged in a factorial randomized block design consisting of
two factors (AM inoculation and P fertilization) with 12
replicates for each treatment.
The contents of the pots were harvested 3, 7 and 14 days
after P fertilization (four replicates in each harvest). The
shoots were dried at 40 1C for 4 days and their dry weight
noted; they were then subjected to Kjeldahl digestion with
4 mL concentrated H2SO4 in the presence of a catalytic
mixture (K2SO4 and CuSO4  5H2O), which was boiled at
350 1C for 2 h. Ammonium from the digested samples were
then determined using flow injection analysis according to
Lima et al. (1999) and phosphate with a flow injection
application of the stannous chloride-molybdate method.
The soil was removed from the roots and a sample was taken
and frozen at 20 1C for later fatty acid analysis. The roots
were washed and weighed and were then divided into two
fractions, one for fatty acid analysis, which was frozen at
20 1C, and one for mycorrhizal colonization measurements, which was fixed in 50% ethanol for later staining.

Light-manipulation (experiment 2)
Seedlings of T. subterraneum were pregerminated in Petri
dishes overnight and planted in 40 pots containing 230 g
of a 1 : 1 w/w mixture of washed quartz sand (fraction
o 0.55 mm) and g-irradiated field soil with the properties
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can be estimated using the major fatty acid in these lipids,

16:1o5 (Olsson et al., 2005). 13C-labelling followed by
compound-specific isotope ratio mass spectrometry (IRMS)
can be used to trace the allocation of C specifically to the
AM fungus in complex matrices such as soil and roots
(Olsson et al., 2005).
We conducted two experiments to investigate how C
allocation to intra- and extraradical mycelium is influenced
by C and phosphorus (P) availability to the plant host. By
increasing the soil P availability the plant benefit of mycorrhizal P uptake is expected to be reduced (Pacovsky et al.,
1986) and by reducing the light intensity the C cost of the
symbiosis is expected to increase since the mycorrhizal
activities will consume a higher proportion of the C-flow
into shaded plants (Peng et al., 1993; Johnson et al., 1997).
We manipulated light and soil-P in Trifolium subterraneum
(clover) colonised by the relatively P-tolerant mycorrhizal
fungus Glomus intraradices (Douds & Schenck, 1990). The
experiments were performed under conditions that have
previously been shown to provide little benefit, and sometimes mycorrhizal-induced growth reduction in host plants
(Soderberg et al., 2002). We tested the hypothesis that when
plants in established mycorrhizal symbiosis experience
decreased mycorrhizal benefit (through P addition) or
increased mycorrhizal cost (through shading) the fungal
symbiont will experience decreased availability of plant
assimilated C.

P.A. Olsson et al.

125

Carbon dynamics in mycorrhizal symbioses

FEMS Microbiol Ecol 72 (2010) 123131

analysis, which was frozen, and one for mycorrhizal colonization measurements, which was processed immediately.

Preparation of bacterial inoculum

Field soil (200 g) was mixed with 1000 mL sterile distilled
water and stirred for 1 h. The soil suspension was then
passed through filter paper (Munktell no. 3). All pots
received 5 mL of the soil filtrate (Van Aarle & Olsson, 2003).

AM fungal root colonization assessment

Roots were washed twice with distilled water, cleared in 10%
KOH at 90 1C for 30 min. After rinsing in water, roots were
stained at room temperature in 0.1% Trypan blue in lactic
acid, glycerol and water (1 : 1 : 1, v/v/v), rinsed in water and
destained in 50% glycerol. The magnified intersections
method, as described by McGonigle et al. (1990), was used
to determine the total proportion of the root length with
AM fungal root colonization and the frequency of AM
fungal arbuscules and vesicles.

Lipid analysis
Neutral lipids are storage compounds that may comprise up
to 20% of the biomass of hyphae, vesicles and spores of AM
fungi (Olsson & Johansen, 2000). The neutral lipid fatty acid
(NLFA) 16:1o5 is an effective signature for AM fungal lipids
(Van Aarle & Olsson, 2003) and the C-flow to this NLFA can
be used to track the C-flow to the AM fungi in soil and roots
(Olsson et al., 2005).
Roots were freeze-dried and then ball-milled in stainless
steel beakers. The lipids from roots (30 mg dry mass) and
soil (3 g wet weight) were extracted and quantified as
described by Olsson et al. (1997). Briefly, samples were
vortexed (1 min) in a one-phase mixture of citrate buffer,
methanol and chloroform (0.8 : 2 : 1, v/v/v, pH 4.0). The
lipids were fractioned into neutral lipids, glycolipids and
phospholipids on prepacked silica columns (100 mg sorbent
mass, Varian Medical Systems, Palo Alto, CA) by eluting
with chloroform, acetone and methanol, respectively. The
fatty acid residues of the lipids were then subjected to mild
alkaline methanolysis, to transform the fatty acids in the
neutral lipids and phospholipids into free fatty acid methyl
esters. Identification and quantification were performed
using gas chromatography (Hewlett Packard 5890, 50 m
HP5 capillary column, Palo Alto, CA, and H2 as carrier gas)
and measuring the retention times in comparison with that
of an internal standard (fatty acid methyl ester 19:0).
13

CO2 enrichment in crude tissue samples and

fatty acids
Approximately 200 mg ball-milled freeze-dried shoot material
was enclosed in tin capsules and analysed by continuous-flow
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described above. Twenty of the pots were inoculated with

20 g G. intraradices (BEG 87) inoculum and 20 g of the
sand:soil mixture was added to the other 20 pots. All pots
were provided with a bacterial inoculum as described below.
These plants were also grown in a greenhouse with a
daytime temperature of approximately 22 1C and a night-time
temperature of approximately 18 1C. The photosynthetic
photon flux density and photoperiod were as described above.
The plants were watered with distilled water to 60% of waterholding capacity. On days 20, 27 and 30, 5 mg of N was added
to each pot using a NH4NO3 solution with a concentration of
28.6 g L 1. Thirty-seven days after sowing, 20 of the 40 pots
were shaded with a net to 25% of the photosynthetic photon
flux density of the control pots (Table 1).
P was added to 20 pots on day 43 (22.5 mg, corresponding to 90 mg P kg 1 soil) as described above. Ten pots were
kept under normal light conditions without extra P (controls); 10 were given extra P; 10 were shaded, and the
remaining 10 pots were given extra P and shaded. In half
the number of pots of each treatment, the soil had been
inoculated with the AM fungus G. intraradices. There were
thus five replicates of each treatment. The light saturation
point of T. subterraneum is 650 mmol m 2 s 1 (Tester et al.,
1986) and the light reduction was expected to cause growth
reduction in mycorrhizal plants (Soderberg et al., 2002) and
reduced root allocation (Tester et al., 1986).
After 43 days (see Table 1), the plants were transferred to a
greenhouse seedling propagation box and were labelled with
13
CO2 under normal light conditions (Olsson et al., 2005).
The pots were divided into two groups of 20 pots each, both
containing at least two replicates of each treatment. The box
was then sealed with paraffin to make it airtight. The lid
of the box was equipped with a fan, a thermometer and a
gas-sampling tube connected to an infrared gas analyzer.
The CO2 concentration inside the box was recorded, and
125 mL 13CO2 was injected through a septum in the lid
using a gas-tight syringe. The CO2 concentration in the box
increased from 100190 to 430500 mL L 1 after injection,
and the labelling period was 2 h (during the middle of the
day, between 10:00 and 14:00 hours), by which time the CO2
concentration was the same as, or below, the initial level.
After labelling, the pots were returned to the same conditions as before for 7 days, which was the chase period for the
13
C-labelling. This time was chosen due to earlier experiences with the same plantfungus combination when 13C
enrichment peaked at 7 days (Nakano-Hylander & Olsson,
2007, fig. 5a).
On day 50, the contents of the pots were harvested. The
shoots were cut, their fresh weights were noted and they
were then frozen until further analysis. The soil was removed
from the roots and a soil sample was taken and frozen for
later fatty acid analysis. The roots were washed and weighed
and were then divided into two fractions, one for fatty acid

126

P.A. Olsson et al.

IRMS using an ANCA-NT-20-20 Stable Isotope Analyser

interfaced to a solid/liquid preparation module (PDZ Europa
Scientific Instruments, Crewe, UK). The 13C/12C ratios of the
CO2 in the combusted samples (total C) were determined
with 0.01% precision. The 13C abundance in the NLFA 16:1o5
was determined using the isotope analyser interfaced to a gas
chromatograph (6890, Hewlett-Packard), which was equipped
with a 50-m column (HP-5, Agilent, Palo Alto, CA) using He
as carrier gas. 13C enrichment (atom % 13C excess) was
calculated as described by Olsson et al. (2005).

Results

Table 2. Shoot biomass, P and N concentrations of Trifolium subterraneum growing with (AM) or without (NM) mycorrhizal inoculation

NM
C

AM
C

Time

Dry shoot
mass (mg)

P concentration
(mg g 1)

N concentration
(mg g 1)

1
2
3
1
2
3

0.53  0.031
0.63  0.050
0.79  0.057
0.52  0.012
0.65  0.037
0.86  0.021

0.12  0.011
0.11  0.008
0.12  0.009
0.26  0.010
0.33  0.015
0.35  0.029

2.5  0.12
2.2  0.13
1.9  0.08
3.1  0.07
2.7  0.13
2.2  0.10

1
2
3
1
2
3

0.54  0.021
0.67  0.016
0.82  0.016
0.58  0.011
0.66  0.021
0.80  0.022

0.24  0.013
0.24  0.015
0.24  0.020
0.27  0.013
0.31  0.015
0.35  0.020

2.9  0.15
2.2  0.12
2.1  0.05
2.7  0.08
2.3  0.09
2.1  0.05

NS
NS
P o 0.001
NS
NS

P o 0.001
P o 0.001
P o 0.001
P o 0.001
P o 0.001

NS
P o 0.01
P o 0.001
P o 0.001
NS

ANOVA

AM
P
T
AM  P
PT

Half of the pots had been P fertilized, while the other half was not
fertilized (C) and they were harvested at three different times (harvest
times 13; see Table 1). Values are means (  SE) and the level of
significance was calculated with a three-factor ANOVA.

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Neither P amendment nor AM fungal inoculation influenced shoot biomass significantly in the first experiment
(Table 2). Shoot biomass increased with time, but there was
only a weak tendency for a positive effect of P fertilization in
the nonmycorrhizal plants. Shoot P concentration was
increased by mycorrhizal inoculation of plants to which
no P had been added, but not in plants fertilized with P
(Table 2). N concentrations in plants were not influenced by
AM colonization, but increased with P fertilization. The
positive effect of P fertilization on N concentration was only
found for nonmycorrhizal plants, as seen from a significant

AM  P interaction (Table 2). We found that P fertilization

resulted in lower amount of NLFA 16:1o5 in roots within
only 3 days, and in the soil mycelium within 7 days,
compared with the control (Fig. 1). After 14 days the effect
of P fertilization was strongest on the NLFA 16:1o5 in soil,
reflecting the extraradical mycelium. P addition stopped the
increase in vesicle colonization rate between 7 and 14 days
after fertilization (Fig. 1), but it did not influence total AM
fungal colonization (Table 3) or arbuscule formation.
The increase in vesicle colonization occurred later than the
increase in NLFA 16:1o5 in the roots.
In the second experiment, mycorrhizal colonization significantly reduced the growth of clover, (Fig. 2) and shading
significantly reduced clover shoot weight, regardless of P
treatment. The addition of P did not influence the growth
rate significantly. A significant AM  P interaction was
found; P addition stimulated nonmycorrhizal plants, but
not mycorrhizal plants.
Mycorrhizal colonization of roots ranged from 77% in the
control plants (normal light conditions) to 42% in the shaded
plants (Table 3). P reduced the colonization rate, but only in
plants under normal light (a significant shade  P interaction). Mycorrhizal colonization of the clover plants increased
the AM fungal NLFA 16:1o5 from 0.009 nmol mg 1 to
between 1 and 9 nmol mg 1, compared with nonmycorrhizal
controls. Mycorrhizal colonization, as indicated by the NLFA
16:1o5, was reduced by both shading and P fertilization (Fig.
3a). The lipid accumulation in the soil mycelium was strongly
reduced by P fertilization, while shading did not influence the
accumulation of fungal lipids (Fig. 3b).
There was no effect of AM inoculation on 13C enrichment
in shoots or roots of T. subterraneum. The 13C enrichment in
shoots was increased by shading, and particularly so in P
fertilized plants (Table 4). Shading also increased the 13C
enrichment in roots, while P alone had no effect on 13C
enrichment in shoots or roots. There was also a significant
P  Shading interaction for 13C enrichment in shoots resulting from the fact that P increased 13C enrichment in shaded
plants, but not in plants under normal light conditions.
Shading resulted in a much higher rate of 13C enrichment
by the fungal lipids inside the roots than under normal light
conditions (Fig. 3c). The 13C enrichment in the soil mycelium after 13C-labelling was not significantly influenced by
either P or shading (Fig. 3d).
We found no significant effect of P or shading on total
retention of labelled C in the plant shoots although a
decreasing effect was indicated (Fig. 4a). P fertilization
under shaded conditions, on the other hand, increased the
uptake in shoots (Fig. 4a). The transfer of C by plants to AM
fungi was considerably reduced by P fertilization, particularly under normal light conditions (Fig. 4b). Shading, on
the other hand, increased the C allocation to the fungus
under fertilized conditions and it seemed as if P fertilization

127

Carbon dynamics in mycorrhizal symbioses

NLFA 16:15
P roots
+P roots

Vesicles

P soil

P roots
+P roots

14
40
12
30

10
8

20
6

10
2
0

5
10
Time (days after P addition)

Table 3. Development of AM fungal root colonisation following P

fertilization of Trifolium subterraneum colonised by the mycorrhizal
fungus Glomus intraradices

Treatment
C

ANOVA

Experiment 2

Time
(days)

AM
colonization (%)

3
7
14
3
7
14
P
T
PS

44  3.4
54  2.7
59  6.7
52  6.3
59  3.6
51  5.8
NS
NS
NS

Treatment

AM
colonization (%)

C
P
S
PS

77  3.1
52  6.2
42  4.7
44  5.4

P
S
PS

P o 0.05
P o 0.001
P o 0.05

600

400

200

0
In Experiment 1 (n = 4) we tested the effect of P fertilization (P) with time
(T) after amendment and in Experiment 2 (n = 5) the effects of P
fertilization and shading (S). Means (  SE) are given as well as the P
values following two-way ANOVA.

had no negative effect on C retention in the AM fungal

mycelium under shaded conditions.

Discussion
The allocation of C to the mycorrhizal fungus was strongly
reduced by P fertilization under good light conditions.
When shading induced C limitation there was no reduction
FEMS Microbiol Ecol 72 (2010) 123131

800
Shoot dry weight (mg per pot)

Experiment 1

15

PS

Mycorrhizal

PS

Nonmycorrhizal
Treatment

Fig. 2. Effects of mycorrhizal inoculation, P fertilization (P) and shading

(S) on the dry weight of Trifolium subterraneum shoots. Controls (C)
were neither shaded nor fertilized. Three-way ANOVA revealed significant
effects of the AM fungus (P o 0.001) and shading (P o 0.001), and a
significant AM  P effect (P o 0.05). P fertilization alone had no significant effect, and no other significant interactions were observed.

in the amount of C retained in the mycorrhizal mycelium. It

has earlier been shown that T. subterraneum allocate much
less resources to roots under shaded conditions (Tester et al.,
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Vesicle incidence (% of root length)

Fig. 1. Development of vesicles and the

AM fungal signature, NLFA 16:1o5, following
P fertilization of 44-day-old Trifolium
subterraneum plants inoculated with Glomus
intraradices. The background of NLFA 16:1o5 in
nonmycorrhizal roots was 0.020 nmol mg 1
and in soil with nonmycorrhizal plants
0.044 nmol g 1. P had a significant effect on NLFA
16:1o5 in roots and soil (P o 0.001, two-way
ANOVA), while harvest time had no effect. Shading
and P together had no effect on the 16:1o5
concentration. Neither P nor time alone had any
effect on vesicle incidence, but a significant
P  time interaction was seen (P o 0.01).

NLFA 16:1w5 (nmol g1 soil or mg1 root dw)

+P soil

128

P.A. Olsson et al.

Roots

NLFA 16:1w5 (nmol mg1)

(a)

NLFA 16:1w5 (nmol g1)

10
8
6
4
2
0

(c)

P
S
Treatment

(d)
13C enrichment in NLFA
16:1w5 (%)

2
1.5
1
0.5
0

P
S
Treatment

PS

4
3
2
1
0

PS

Roots

Soil

0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0

13

C enrichment (%)

Treatment

Shoot

Root

0.62  0.15
0.38  0.088
0.43  0.18
1.1  0.51

0.35  0.048
0.38  0.025
0.59  0.14
0.65  0.086

0.44  0.16
0.45  0.14
1.0  0.19
1.5  0.46

0.43  0.045
0.42  0.036
0.53  0.048
0.58  0.082

NS
NS
P o 0.01
P o 0.05

NS
NS
P o 0.001
NS

ANOVA

AM
P
S
PS

Controls (C) were neither shaded nor fertilized. The 13C enrichment was
calculated as the % 13C above the background level after a 7-day chase
period. Means are given (  SE) and P values from three-way ANOVA.

1986) and we can show here that this does not reduce the C
allocated to mycorrhizal structures, although the mycorrhizal colonization may decrease, as observed by Tester et al.
(1986) and this study. Mycorrhizal networks are extensive
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PS

Soil

Table 4. Influence of P fertilization (P) and shading (S) on the 13C

enrichment in shoots and roots of Trifolium subterraneum colonised
(AM) or not (NM) by the mycorrhizal fungus Glomus intraradices

AM
C
P
S
PS
NM
C
P
S
PS

P
S
Treatment

P
S
Treatment

PS

Fig. 3. Influence of P fertilization (P) and shading

(S) on the amount of NLFA 16:1o5 in roots (a) and
soil (b) (values from nonmycorrhizal treatments
were subtracted), and the 13C enrichment in
16 : 1o5 in roots (c) and soil (d). Controls (C)
were neither shaded nor fertilized. The 13C
enrichment was calculated as the % 13C above the
background level after a 6-day chase period.
(a) P o 0.001 for P fertilization, o 0.001 for shading and o 0.01 for the P  shading interaction;
(b) P o 0.05 for P fertilization; neither shading nor
interactions had any significant effect; (c)
P o 0.001 for shading, neither P nor interactions
had any significant effect; (d) no significant effects.

(Whitfield, 2007), and our results can help in understanding

the sourcesink dynamics in these networks. Several lines of
evidence suggest that the plants in this study were C-limited:
mycorrhizal colonization reduced plant growth, phosphorus
fertilization did not increase plant growth, and shading
reduced plant growth. This C limitation did not reduce
colonization by the mycorrhizal fungus G. intraradices, a
fungus known to be tolerant to high P levels (Douds &
Schenck, 1990), which may indicate that it is able to act as a
parasite under certain conditions. Johnson et al. (1997)
suggested that either fertilization (reduced benefits) or
shading (increased costs) could generate mycorrhizal parasitism. The results of our study indicate that plants can
control C allocation to their associated fungus when the soil
is enriched with P, but not when light is limited. However, P
fertilization did not reduce C allocation to the AM fungus in
shaded plants as shown by a significant P  shade interaction. This could be an effect of higher total C uptake in those
plants (Table 1), but it also emphasizes the ability of AM
fungi to obtain C from shaded plants. When we transplanted
T. subterraneum to the shaded conditions we also moved it
to conditions far below its saturation point, which has been
shown to be 650 mmol m 2 s 1 (Tester et al., 1986). One
reason for the lack of a negative P effect in shaded plants
could be that they did not have the energy requirement
binding further C.
The reduced AM colonization in shaded and P fertilized
plants was as could be expected from earlier studies (Tester
FEMS Microbiol Ecol 72 (2010) 123131

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13C enrichment in NLFA

16:1w5 (%)

(b)

129

Carbon dynamics in mycorrhizal symbioses

Clover shoots

1.4

5
4
3
2
1
0

P
S
Treatment

1.2
1
0.8
0.6
0.4
0.2
0

PS

P
S
Treatment

PS

ing P addition. Instead, we found that P fertilization reduced

the flow of C to the fungus indicating that a mechanism in
the carbohydrate transfer system was involved.
In some systems, indications of plant-to-plant transfer of
C via the mycorrhizal network have been observed, perhaps
toward shaded plants (Francis & Read, 1984; Grime et al.,
1987; Simard et al., 1997). Our results, showing that extraradical AM fungal mycelium connected to a shaded plant
has a high C sink capacity, support the suggestion that the
transfer of C is unlikely in many species (Robinson & Fitter,
1999; Zabinski et al., 2002; Voets et al., 2008), but still
certain species may have special adaptations to achieve
plant-assimilated C (Cameron et al., 2008). The significant
allocation of C to extraradical mycelium in the shaded
plants may be a strategy that has evolved to find new hosts
when the C allocation capacity of the current host is
reduced. Such a strategy may be important when vernal
flora is shaded later in the season by plants of other
functional types. This kind of C flow may also explain the
finding that seedlings connected to mature plants with a
common mycorrhizal network benefit when mature plants
are defoliated (Pietikainen & Kytoviita, 2007).
The fact that C was reallocated to extraradical mycelium
in shaded hosts may lead to more aggressive colonization of
other plants. The more rapid turnover in soil mycelium
could also lead to a reduction in C flow to other plants, but
the possibility of fungusplant C transfer is still under
debate. We propose that the high C sink capacity of certain
fungi may draw C from shaded plants, thus regulating the
community structure in shaded environments.
Although vesicles are lipid storage organs, their formation
is not correlated with lipid accumulation over time. Earlier
results indicate that vesicles are formed first, and then later
filled with lipids (Van Aarle & Olsson, 2003). The vesicles
also showed a rather late response to the high P fertilization
in the current study, while the lipid indicators reacted much
faster. Overall, our results showed that using 13C-labelling of
indicator lipids is an efficient way of tracing C flows under
symbiotic conditions.
2010 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved

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et al., 1986; Bruce et al., 1994; Olsson et al., 1997). Here we

show that these effects can be detected as rapidly as within
12 weeks after environmental change. Usually structures do
not disappear, but root growth without new colonization
will decrease the colonization rate in roots. The low colonization in the shaded plants did not prevent a high mycorrhizal C allocation. Therefore the reduced mycorrhizal C
allocation in P fertilized plants could be due not to reduced
colonization rate, but instead to specific regulation of Cflow over the mycorrhizal interface.
We have shown that parasitic behavior can be independent
of P fertilization, and that perhaps shaded plants are more
likely to be subject to the parasitic action of AM fungi. Fitter
(2006) suggested that plants allocate C to places in the root
where there is an in-flow of P, a mechanism that would
prevent the plants from being parasitized by cheating fungi.
The reduced mycorrhizal C allocation in P-fertilized plants
under normal light conditions is in line with this hypothesis.
Still, the mycorrhizal plants had a higher P content than
nonmycorrhizal ones. Indeed, it is well-known that AM fungi
may contribute significantly to plant P uptake without
increasing the growth (Smith et al., 2003). Therefore, it could
be that both AM inoculation and P fertilization may in the
long run increase fitness even when no growth response was
found during the time-span of this experiment and in
particular during the short time-span following P fertilization.
P-mediated benefits in the long run could be due to increased
seed quality or pathogen resistance (Newsham et al., 1995).
Such benefits from a better P status could be one reason for
the relatively high C allocation to the fungus in spite of a lack
of an obvious P limitation in the plants in this study.
The reduction in colonization often observed following P
fertilization (see e.g. Bruce et al., 1994) has been explained
by a reduction in germ tube formation by AM fungal spores,
reduced exudation of branching factors by roots, and by
faster root growth (Smith & Read, 2008). None of these
factors were, however, possible in our study since the AM
colonization was already established when P fertilization was
applied, and because we saw no growth stimulation followFEMS Microbiol Ecol 72 (2010) 123131

Glomus intraradices

(b)

6
Total NLFA 16:15 13C
excess (g)

Total shoot 13C excess (mg)

(a)
Fig. 4. Effects of mycorrhizal colonization (AM)
P fertilization (P) and shading (S) on C retention in
(a) shoots of Trifolium subterraneum and (b) the
AM fungus Glomus intraradices. Controls (C)
were neither shaded nor fertilized. The total C
uptake in plant shoots and in the AM fungal
signature NLFA 16:1o5 was calculated by
multiplying the enrichment by the amount.
Neither P nor shading had any effect on C
retention in T. subterraneum, but P (P o 0.01)
and the P  shade interaction (P o 0.05)
had significant effects on C retention in G.
intraradices.

130

Acknowledgements
Financial support from FORMAS and The Swedish Research
Council is gratefully acknowledged. We thank Nancy Johnson and Ylva Lekberg for valuable comments on the manuscript.

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