Download Different arbuscular mycorrhizal fungi alter coexistence and

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Different arbuscular mycorrhizal fungi alter coexistence
and resource distribution between co-occurring plant
Blackwell Publishing Ltd.
Marcel G. A. van der Heijden1,2, Andres Wiemken1 and Ian R. Sanders3
Botanical Institute, University of Basel, Hebelstrasse 1, CH− 4056 Basle, Switzerland; 2Department of Systems Ecology, Vrije Universiteit, De Boelelaan 1087;
1
NL−1081 HV Amsterdam, The Netherlands. Current Addresses: 3Institute of Ecology, University of Lausanne, CH−1015 Lausanne, Switzerland
Summary
Author for correspondence:
Marcel van der Heijden
Tel: +31 20 4447046
Fax: +31 20 4447123
Email: [email protected]
Received: 13 August 2002
Accepted: 6 November 2002
• It is often thought that the coexistence of plants and plant diversity is determined
by resource heterogeneity of the abiotic environment. However, the presence and
heterogeneity of biotic plant resources, such as arbuscular mycorrhizal fungi (AMF),
could also affect plant species coexistence.
• In this study, Brachypodium pinnatum and Prunella vulgaris were grown together
in pots and biotic resource heterogeneity was simulated by inoculating these pots
with one of three different AMF taxa, with a mixture of these three taxa, or pots
remained uninoculated.
• The AMF acted as biotic plant resources since the biomass of plants in pots
inoculated with AMF was on average 11.8 times higher than uninoculated pots. The
way in which the two plant species coexisted, and the distribution of phosphorus and
nitrogen between the plant species, varied strongly depending on which AMF were
present. The results showed that the composition of AMF communities determines
how plant species coexist and to which plant species nutrients are allocated.
• Biotic plant resources such as AMF should therefore be considered as one of the
factors that determine how plant species coexist and how soil resources are distributed
among co-occurring plant species.
Key words: biodiversity, mineral nutrition, mycorrhizal symbiosis, mutualism, plant
competition, plant nutrition, vesicular–arbuscular mycorrhiza (VAM).
© New Phytologist (2003) 157: 569–578
Introduction
A major goal in ecology is to search for mechanisms that
determine how plant species coexist in natural ecosystems
(Aarssen, 1983; Tilman, 1988; Zobel, 1992; Bengtsson et al.,
1994; Grime, 2001; Wardle, 2002). Coexistence is defined
here as ‘the living together of two species within the same
habitat such that neither tends to be eliminated by the other’
(Begon et al., 1996). Ecologists searching for the factors that
explain plant species coexistence have mainly focused on
habitat and plant characteristics. It has been shown that plant
species can co-occur because they vary in their resource
requirements (Aarssen, 1983; Tilman, 1988) and in other
characteristics such as rooting depth, phenology and
germination requirements (Grubb, 1977; Berendse, 1981).
Despite of this, plants basically need the same resources
(light, water and nutrients), which implies that only a limited
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number of different niches are available, causing niche overlap
among many species. As a consequence, competition among
plants occurs (Schoener, 1983; Connell, 1983), whereby the
strongest competitor can outcompete other species, thus
leading to a lower number of species that coexist.
Other factors could enable plants to coexist and, thus,
explain plant diversity. For example, it is well known that
the presence of organisms that interact with plants, such as
herbivores, pathogens and mutualists influence plant species
coexistence (Brown & Gange, 1989; Olf & Ritchie, 1998;
Clay & Holah, 1999; van der Heijden, 2002). These biotic
factors can alter plant species coexistence, together with
effects of the abiotic environment.
The influence of arbuscular mycorrhizal fungi (AMF) on
plant species coexistence has often been neglected. This is
surprising since these mutualistic soil fungi are very abundant
in soils and estimates suggest that 60% of all terrestrial plant
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species form associations with AMF (Trappe, 1987). The
AMF, fungi of the phylum Glomeromycota (Schussler et al.,
2001), can act as extensions of plant root systems and increase
nutrient uptake and plant growth (Smith & Read, 1997).
Several studies showed that AMF alter plant diversity (Grime
et al., 1987; Gange et al., 1990; van der Heijden et al., 1998a;
Hartnett & Wilson, 1999; Klironomos et al., 2000; O’Connor
et al., 2002) and change competitive relationships between
plants (Fitter, 1977; Hetrick et al., 1989; Allen & Allen,
1990; West, 1996; Marler et al., 1999).
Arbuscular mycorrhizal fungi do not just occur in ecosystems as single species, but instead AMF communities are
found that vary in composition and diversity (Cuenca et al.,
1998; Helgason et al., 1998). Up to 37 different AMF taxa
have been found at one site (Bever et al., 2001). These AMF
taxa have a specific multidimensional niche that is determined
by the plant species that are present at a site and by edaphic
factors such as pH, moisture content, phosphorus (P), and
nitrogen (N) availability. As a result, there is large betweenand within-site variation in the composition of AMF taxa
(Burrows & Pfleger, 2002; Hart & Klironomos, 2002). Such
variations influence plants since different AMF taxa vary in
their ability to supply plants with nutrients and promote plant
growth ( Jakobsen et al., 1992; van der Heijden et al., 1998b;
Smith et al., 2000; van Aarle et al., 2002). In particular, the
plants with a high mycorrhizal dependency (those that grow
much better and acquire large amounts of nutrients when
colonized by AMF) appear to respond differently when colonized by different AMF taxa (van der Heijden, 2002). The
amount of nutrients obtained by a plant determines its competitive ability and its capability to coexist with other plants
and this suggests that the identity of the fungal associates
matters for plant species coexistence.
To date, only one study has investigated whether the
composition of AMF influences how plants co-occur (van der
Heijden et al., 1998a). In this study, we observed that the
composition of simulated grassland communities changed
when different AMF taxa were present. The growth of the
dominant plant species in this study, the grass Bromus erectus,
was hardly influenced by AMF or by different AMF taxa and
only subordinate species were affected. It appeared as if effects
of AMF on plant species coexistence were relatively small.
However, some grasslands are dominated by mycorrhizal
dependent plant species that rely on AMF for growth and
nutrient acquisition (e.g. Brachypodium pinnatum in European
calcareous grasslands exposed to atmospheric N depositions
(Bobbink, 1991)). It has been hypothesized that coexistence
between mycorrhizal dependent plant species is greatly affected
by AMF composition (van der Heijden, 2002) but this has
not yet been investigated. Effects of AMF composition on the
coexistence between such mycorrhizal dependent plants may
represent an upper limit of potential effects.
The study by van der Heijden et al. (1998a) had a high
complexity since 11 different plant species co-occurred. It was
therefore not possible to quantify how strongly different AMF
taxa affected plant species coexistence. Studies with pairs of
co-occurring species are better suited for such a quantification
and it enables ecologists to assess the importance of AMF in
relation to other factors that also affect plant species coexistence. Moreover, many AMF species are not host specific (Law,
1988) and colonize different plants at the same time, thereby
linking plant species through hyphal connections (Newman,
1988). The fungus has, thus the ability to regulate plant
species coexistence by the allocation of nutrients among cooccurring plants. The implications of this for plant species
coexistence have not yet been investigated and this is also
focus of this experiment.
Here, we present a study designed to test and quantify
(1) the extent to which different AMF taxa influence the way
nutrients are distributed between coexisting plant species
and (2) the extent that different AMF species can alter plant
species coexistence. To test this, two naturally coexisting plant
species, the grass B. pinnatum and the forb Prunella vulgaris
were grown together in pots and were inoculated either with
one of three different AMF taxa, with a mixture of these three
taxa, or remained uninoculated. Both plant species are
abundant in many European calcareous grasslands. They were
selected since they have a high mycorrhizal dependency and
rely, when grown in calcareous grassland soils, on AMF for
nutrient acquisition (Streitwolf-Engel et al., 1997, 2001; van
der Heijden, 2002). The plant species and the AMF taxa used
in this study co-occur in the same calcareous grassland,
emphasizing the ecological significance of this study. The
experiment was designed to test if different AMF species alter
the way plant species coexist. It was not our intention to investigate effects of different AMF taxa on competitive relationships between plant species. Competition studies normally
include monocultures where each of the competing species are
grown alone, in absence of competition. Such monocultures
were not included in our experimental design. Instead, we
focused on the effects of different AMF on establishment and
subsequent growth of the two coexisting plants. This mimics
conditions that are normally encountered by the plants when
they establish in species rich natural grasslands.
Materials and Methods
Plant and fungal material
The plants and AMF used in this study all occur in a field
study site, Nenzlinger Weide; a calcareous grassland in the
Jura Mountains, Switzerland (grid reference 255 609 of the
Landeskarte der Schweiz sheet 1067). Nenzlinger Weide is
a plant species rich calcareous grassland with a diverse
community of mycorrhizal fungi (Sanders et al., 1995). The
two plant species used in this experiment, both subordinate
plant species at Nenzlinger Weide, were B. pinnatum (Tor
Grass or Heath False-brome), a perennial grass species and
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P. vulgaris (Self-heal), a clonal forb species. The seeds of
both plant species originated from their natural populations
in Switzerland. The three AMF taxa used in this study
were isolated from Nenzlinger Weide and propagated on
Hieracium pilosella plants growing in autoclaved (121°C,
30 min) loamy sand. Two of the three AMF taxa (Glomus sp.
isolate BEG 19) and (Glomus sp. isolate BEG 21) originated
from single spores of AMF that were extracted from field soil
by a sucrose centrifugation technique (Walker et al., 1982).
The third AMF taxa (Glomus sp. isolate Basle Pi), was
obtained from roots of a P. vulgaris plant which was growing
at Nenzlinger Weide. The roots of this Prunella plant were
carefully washed to remove soil and planted into a soil mixture
of loamy sand and expanded clay. Glomus-like spores were
observed in this soil mixture. A few grams of this soil mixture
were taken and the AMF in this soil mixture were propagated
on Hieracium, as described earlier. The same AMF taxa were
used in previous experiments and further information about
these three taxa is provided there (Streitwolf-Engel et al.,
1997, 2001; Sanders et al., 1998; van der Heijden et al.,
1998a, 1998b).
AMF inoculation and plant growth conditions
Seeds of each plant species were germinated on moist
sterilized quartz sand on 20 March 1997 in the glasshouse.
On 11 April 1997, three seedlings of Brachypodium together
with three seedlings of Prunella were planted in each of 45
plastic pots (560 cm3) containing 376 g soil (dry weight) of
an autoclaved (121°C, 30 min) soil mixture of calcareous
grassland soil and quartz sand (2 : 1 v : v). The calcareous soil
originated from Dittinger Weide, a calcareous grassland near
Nenzlinger Weide. The soil mixture had a pH(H2O) of 7.15
and contained 1.02 g CaCO3 per 100 g of dried soil. The soil
was well aggregated and had an organic matter content of
7.5%. The soil contained 5.4 mg sodium acetate extractable
P per 100 g dried soil. The six seedlings were planted in a
circle and the species were alternated so that the nearest
neighbours were always different species. Pots were either
inoculated with 27 g soil inoculum of one of the three AMF
taxa, with 27 g mixed soil inoculum comprising an equal
proportion of each of the three AMF taxa, or with 27 g of an
autoclaved (121°C, 30 min) mixture of soil inoculum from
the three AMF taxa (control; nonAMF treatment). In each
pot, 15 g of soil inoculum was mixed with the soil and the
remaining 12 g of soil inoculum was distributed among six
holes that were made to plant the six seedlings. All pots
received 40 ml of inoculum washing filtrate (381 g of the
mixed inoculum was suspended in 2.12 l water and filtered
through a 10-µm nylon sieve) to correct for possible
differences in bacterial and fungal communities between
different inocula (Koide & Li, 1989). The plants were watered
three times a week with distilled water to an amount that was
equal to 28% soil mass. The plants were maintained in the
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glasshouse and additional lightning was provided with
halogen lights to a daylength of 15 h per day. No nutrients
were given during the experiment.
Measurements and harvesting
Plant variables The plants were harvested 87 d after
inoculation. The six plants (three Brachypodium plants and
three Prunella plants) in each pot were separated from each
other and the roots of each individual were washed to remove
soil and roots from the other individuals. The amount of roots
that were not connected to the plants (approximately 5.7%
and 22%, of the total plant biomass and of the total root
biomass per pot, respectively) were separated and are called
unspecified roots. The roots of each species were mixed, cut
into 1 cm pieces, and divided into two subsamples and fresh
mass was determined on both. One root subsample of
Brachypodium, one of Prunella and the shoots of each plant
species were oven dried (90°C) and weighed. The root dry
mass of the other root subsample of Brachypodium and of
Prunella was calculated by multiplying the fresh mass with
the dry mass to fresh mass ratio of the oven-dried root
subsamples. The sum of the dry mass of both root samples
and of the shoots gave the biomass for each plant species. The
sum of the biomasses of Brachypodium, Prunella and the
unspecified roots gave the biomass per pot. The coexistence
ratio was determined by dividing the biomass of Prunella by
the sum of the biomass of Prunella and Brachypodium.
The root length of both fresh subsamples and of the
unspecified roots was determined (Marsh, 1971). The root
length of each plant species was calculated based on the
known fresh mass of both root subsamples of each plant species. The root length per pot was consequently determined by
summing the root length of Brachypodium, of Prunella and of
the unspecified roots.
Dried plant material was ground in a ball mill, mixed thoroughly, and P and N concentrations of the shoots and roots
of each plant species and of the unspecified roots were determined. Phosphate concentrations were determined by the
molybdate blue ascorbic acid method (Watanabe & Olsen,
1965) and N concentrations were determined with a HCN
analyser (LECO instruments, model 932, St. Joseph, Michigan,
USA). The P and N contents of Brachypodium, of Prunella,
and of the unspecified roots were calculated and the sum of
these gave plant P and N contents per pot. The P and N distribution ratios were determined by dividing the total P or N
content of Prunella by the sum of the total P or N content of
Prunella and Brachypodium. The P and N distribution ratios
show the partitioning of P and N between both plant species.
For a few samples of the nonAMF treatment, especially of
Prunella, not enough plant material was available to determine root and shoot N concentrations. The means of the
N concentrations of Prunella and of Brachypodium and the
total plant N content per pot (as shown in Figs 3b and 1c,
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respectively) are therefore based on a smaller replicate number.
The data for the unspecified roots (biomass, P and N content)
were only included for analysis when total amounts (biomass,
P and N content) per pot were determined.
The leaf length of each plant was determined when the
plants were inoculated on 11 April 1997 (t = 0) and after 34,
69 and 87 d (the end of the experiment). The leaf lengths were
ln-transformed and growth rates (GRs) of each plant species
were estimated by taking the x-coefficient of the function,
which describes the increase of leaf length over time (from 0
to 87 d) (Hunt, 1982). A significant correlation occurred
between shoot biomass and leaf length in both Brachypodium
and Prunella (Pearsons correlation, n = 45, r 2 = 0.97 and 0.95,
P < 0.0001, for Brachypodium and Prunella, respectively).
Therefore, we assumed that leaf length measurements are
valid estimates of plant biomass and can be used to determine
growth rates during the experiment for both plant species.
Fungal variables After determining the root length of the
fresh subsamples of Brachypodium and Prunella, the roots
were cleared with 10% KOH and stained with Trypan blue,
in order to stain mycorrhizal structures inside the roots
(Phillips & Hayman, 1970). The percentage of root length
occupied by hyphae, arbuscules or vesicles was estimated by a
modified line intersection method (McGonigle et al., 1990),
where a minimum of 50 line intersections per root sample
were scored for the presence of AMF. The length of hyphae
occurring in the soil was estimated by an aqueous extraction
and membrane filter technique ( Jakobsen et al., 1992). The
hyphal length was determined in three subsamples of each
pot, the average was taken, and hyphal length per pot and the
hyphal length per gram dried soil was estimated.
Experimental design and statistical analysis
The experiment was set up as a complete randomized design
with five mycorrhizal treatments and nine replicates per
treatment. The position of the pots was randomized weekly.
We performed analysis of variance (ANOVA) or, in case of
unequal variation among treatments, nonparametric analysis
of variance (NPAR-ANOVA) to test whether treatments varied
from each other. A one-way ANOVA was executed on the
biomass per pot, on the plant P content per pot, on the plant
N content per pot, on the root length per pot, on the log(x)
transformed hyphal length per pot and on the P distribution.
A one-way NPAR-ANOVA was performed on the coexistence
ratio and on the N distribution ratio.
A two-way ANOVA was performed on the P concentration,
N concentration, percentage of root length occupied by
hyphae and arbuscules and on log(x + 1)-transformed P
content per species. A two-way NPAR-ANOVA was performed
on biomass, percentage root length occupied by vesicles, N
content per species and on growth rates (GR). The two-way
NPAR-ANOVA was performed by ranking the data, followed
by a normal ANOVA, and the accompanying H-ratio was determined (Zar, 1984). The two-way ANOVA and the two-way
NPAR-ANOVA were executed with two factors: PLANT, containing both plant species, and AMF, containing the four
treatments which were inoculated with AMF. The non-AMF
treatment was not included in these analyses because we
wanted to test if treatments inoculated with AMF varied from
each other. Inclusion of the nonAMF treatment, in which
plant growth was extremely low, may affect the outcome of
the analysis of variance strongly (van der Heijden et al.,
1998b). Inclusion of this treatment increased the statistical
significance of AMF effects in van der Heijden et al. (1998b)
and in this experiment (data not shown) without having any
ecological relevance. Arbuscular mycorrhizal fungi are present
in almost all terrestrial ecosystems (including the grassland
under investigation) and comparisons with a nonmycorrhizal
situation only points to the potential importance of AMF and
not to current effects of AMF. Current effects of AMF on
plants are most likely determined by changes in AMF composition or abundance. A probability of P < 0.05 was considered
to represent a significant difference among treatments in this
study.
Results
Variation among plants inoculated with different AMF
The biomass produced in the pots inoculated with one of
the three AMF taxa or with the combination of these three
taxa was much higher than in the uninoculated non-AMF
treatment (Fig. 1a). The mean biomass across all four
treatments with AMF was 11.8 times higher than the mean
biomass of the non-AMF treatment. The mean plant P and
mean plant N content of the treatments with AMF were,
respectively, 25 and 10 times higher than the contents of
the plants in the non-AMF treatment (Fig. 1b,c), indicating
that the presence of AMF enhanced resource acquisition. The
root length and the length of external hyphae were greater in
pots with AMF than in the non-AMF treatment (Fig. 1d,e).
The hyphal length per pot was, on average, 49 times greater
than the root length per pot (Fig. 1d,e), indicating that
mycorrhizal hyphae exploited the soil more intensively than
plant roots. The hyphal length in the non-AMF treatment
represents a background count of dead fungal hyphae already
present in the soil or of nonAM fungi introduced through the
sieved washing of AMF soil inoculum. The external AMF
hyphae present in the non-AMF treatment were apparently all
dead since we did not observe that any of the plant roots were
colonized by AMF.
The plant biomass, plant P content, plant N content, root
length and hyphal length per pot also differed significantly
among the different AMF taxa treatments and not just compared with the non-AMF treatment (Fig. 1). Pots inoculated
with Glomus sp. isolate BEG 19 had a lower biomass, plant P
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content, plant N content, root length and hyphal length compared with the other AMF treatments (Fig. 1). Furthermore,
the plant P content of pots inoculated with Glomus sp. isolate
BEG 21 was significantly lower than that of pots inoculated
with Glomus sp. isolate Basle Pi (Fig. 1b).
Effects of different AMF taxa on plant species
coexistence and resource distribution
Fig. 1 Plant biomass (a), plant phosphorus content (b), plant
nitrogen content (c), root length (d) and hyphal length (e) in pot
mixtures with both Brachypodium pinnatum and Prunella vulgaris.
Pots were inoculated with one of three arbuscular mycorrhizal fungi
(AMF) taxa, a mixture of all three taxa or were not inoculated with
AMF. Glomus sp. isolate Basle Pi (light tinted columns), Glomus sp.
isolate BEG 21 (closed columns), Glomus sp. isolate BEG 19 (hatched
columns), all three taxa (patterned columns) or non-AMF (open
columns). Bars represent ± 1 SE. The F-ratios of ANOVA for the
dependent variables presented in a–e were F3,32 = 38.5 (P < 0.0001),
F3,32 = 14.5 (P < 0.0001), F3,32 = 32.6 (P < 0.0001), F3,32 = 23.9
(P < 0.0001) and F3,32 = 22.7 (P < 0.0001), respectively. Different
letters above bars indicate a significant difference among the means
of the treatments (P < 0.05) according to Tukey’s test, with the
non-AMF treatment excluded from the analysis.
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The way in which the two plant species coexisted and in
which the nutrients were distributed, differed significantly
among the AMF treatments (Figs 2 and 3). Prunella was
dominated by Brachypodium, and contributed to only 14% of
the biomass, when pots were inoculated with Glomus sp. BEG
21 (Figs 2a and 3a). Prunella was also dominated by
Brachypodium in pots that were inoculated with all three AMF
taxa, although here Prunella contributed to 23% of the
biomass (Figs 2a and 3a). However, the proportional biomass
of Prunella was significantly higher in pots inoculated with
Glomus sp. isolate Basle Pi and Glomus sp. isolate BEG 19
(Figs 2a and 3a), where it contributed to 39% and 53% of the
biomass, respectively (Figs 2a and 3a). These effects of AMF
on plant species coexistence were also indicated by the
significant PLANT × AMF interaction term for biomass
(Table 1). Interestingly, the influence of the different AMF
taxa on the coexistence of the two plant species was
independent of the AMF effects on the total biomass per pot.
For example, plants inoculated with Glomus sp. isolate Basle
Pi and Glomus sp. isolate BEG 21 had an equal total biomass
per pot (Fig. 1a), but the coexistence ratio was significantly
different (Fig. 2a). The coexistence ratio is, for comparative
reasons, also shown for the non-mycorrhizal treatment.
However, it is unlikely that both plant species interacted in
the non-mycorrhizal treatments since they hardly grew
without AMF. The P distribution ratio varied significantly
among the AMF treatments (Fig. 2b) and this was also
reflected by the significant PLANT × AMF interaction term
on plant P concentration and content (Table 1). Both
Brachypodium and Prunella contributed approximately equal
parts to the plant P content when pots were inoculated with
Glomus sp. isolate Basle Pi or Glomus sp. isolate BEG 19
(Fig. 2b). Prunella, however, contributed to only 21% and 31%
of the total plant P content when pots were inoculated, respectively, with Glomus sp. isolate BEG 21 or all three isolates
together. The P content of Prunella was 3.8 or 2.1 times lower
than that of Brachypodium in pots inoculated with Glomus sp.
isolate BEG 21 or all three isolates together, respectively.
The N distribution ratio also varied significantly among
the AMF treatments (Fig. 2c) and a significant PLANT
× AMF interaction term for the plant N concentration and
content confirmed that the two plant species did not respond
in the same way to different AMF treatments (Table 1). The
pattern resembled that for the P distribution among the
plant species and AMF treatments (Fig. 2c).
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Fig. 2 Coexistence ratio (a), phosphorus distribution ratio (b)
and nitrogen distribution ratio (c), between Prunella vulgaris and
Brachypodium pinnatum in pots that were not inoculated with
arbuscular mycorrhizal fungi (non-AMF), inoculated with one of three
AMF taxa or with a mixture of these three AMF taxa: Glomus sp. isolate
Basle Pi (light tinted columns), Glomus sp. isolate BEG 21 (closed
columns), Glomus sp. isolate BEG 19 (hatched columns), all three taxa
(patterned columns) or non-AMF (open columns). Bars represent
± 1 SE. The coexistence ratio shows the percentage biomass of
P. vulgaris compared with summed biomass of P. vulgaris and
B. pinnatum. The phosphorus and nitrogen distribution ratios
show the percentage of phosphorus or nitrogen in P. vulgaris relative
to the total amount of phosphorus or nitrogen in P. vulgaris and
B. pinnatum. The χ2 and F-ratio for data presented in a–c were
χ2 = 26.7 (P < 0.0001), F3,31 = 18.8 (P < 0.0001) and χ2 = 29.4
(P < 0.0001), respectively. The non-AMF treatment is not included
in the statistical analysis (see methods section). Different letters
above bars indicate a significant difference among the means
of the treatments (P < 0.05) according to Wilcoxon’s test (a and c)
or Tukey’s test (b).
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Table 1 Results of ANOVA and nonparametric
ANOVA (ψ) with corresponding F-ratios and
H-ratios for six variables
Response variable
Source of variation
PLANT (df = 1)
Plant biomassψ
24.1***
Growth rateψ
19.99***
Plant P content
37.6***
Plant P concentration
95.5***
Plant N contentψ
20.4***
Plant N concentration
233.5***
Root length occupied by:
Hyphae
147.5***
Arbuscules
0.257ns
Vesiclesψ
11.76**
AMF (df = 3)
PLANT × AMF (df = 3)
12.7**
16.91**
10.2***
30.7***
10.4*
73.1***
20.7***
20.41***
22.0***
3.5*
25.3***
10.8***
44.7***
10.42***
41.83***
3.5*
0.54ns
5.0ns
*,**,***P < 0.05, P < 0.01 and P < 0.0001, respectively; ns, not significant. Note: error
df = 66. The factor PLANT comprises two plant species and the factor AMF comprises four
treatments with arbuscular mycorrhizal fungi (AMF; nonAMF treatment excluded).
Growth rates
The growth rate (GR) was significantly different between
plants that were inoculated with different AMF, independent
of plant species (Fig. 3b; Table 1). This indicates that different
AMF taxa or a combination of these AMF taxa induced a
variation in the growth rate of the two plant species. In these
analyses, the non-AMF treatment was excluded because we
were testing if the GR varied among treatments with different
AMF. A significant PLANT × AMF interaction term also
occurred for the GR (Table 1) showing that the growth rate
also depended on the specific combination of plant species
and AMF taxa, or combination of AMF taxa.
Differences between the two plant species
The P and N concentration and the percentage of root
colonization by AMF varied significantly between the two
plant species (the PLANT factor in Table 1) and this also
depended on the AMF taxa that colonized the plant roots (the
AMF factor in Table 1). Prunella had, on average, higher P
and N concentrations than Brachypodium (Fig. 3c,d; Table 1).
Fig. 3 Plant biomass (a), growth rate (b), phosphorus concentration
(c), nitrogen concentration (d) and percentage of root length
occupied by hyphae (e), vesicles (f) and arbuscules (g) of
Brachypodium pinnatum and Prunella vulgaris. Both plant species
grew together in pots and were inoculated with one of three
arbuscular mycorrhizal fungi (AMF) taxa, with a mixture of these
three AMF taxa or in pots without AMF: Glomus sp. isolate Basle Pi
(light tinted columns), Glomus sp. isolate BEG 21 (closed columns),
Glomus sp. isolate BEG 19 (hatched columns), all three taxa
(patterned columns) or nonAMF (open columns); bars represent
± 1 SE. Corresponding F-ratios of ANOVA are shown in Table 1. The
nonAMF treatment is only given for comparative reasons and is not
included in the statistical analysis (see Materials and Methods
section). Different letters above bars indicate a significant difference
among the means of the treatments (P < 0.05) according to
Wilcoxon’s test (a, b and g) or Tukey’s test (c, d, e and f).
© New Phytologist (2003) 157: 569 – 578 www.newphytologist.com
Of the plants inoculated with AMF, the P and N concentration of each of the two plant species was lowest in
pots that were inoculated with Glomus sp. isolate BEG 21
(Fig. 3c,d). The percentage root length that was occupied by
hyphae and vesicles was higher in Prunella (Fig. 3e,f ). The
lowest amount of root colonization by hyphae and vesicles
was found in pots that were inoculated with Glomus sp. BEG
19 (Fig. 3e,f ). The percentage of root length that was
occupied by arbuscules did not vary between both plant
species (Fig. 3g). These differences between the two plant
species and among the AMF species could explain why
different AMF species affect plant species coexistence and
resource distribution. Life history traits of both plant species
also varied. Brachypodium formed tillers (between six and
32 tillers per pot, depending on AMF treatment) while
stolons were formed by Prunella (between 0 and 17 stolons
per pot, depending on AMF treatment). The amount of
root colonization in Prunella and Brachypodium roots were
comparable with that observed in the field. Average observed
levels of infection in plant roots at the field site were 72.0%
(SD = 5.4% n = 7) for Prunella and 56.3% (SD 8.9%; n = 7)
for Brachypodium.
Discussion
Plant species coexistence and the importance of AMF
It has been known for some time that the presence of AMF
enhances growth and resource acquisition of many plant
species (Grime et al., 1987; Smith & Read, 1997; van der
Heijden et al., 1998a) and our results confirm this for
Brachypodium and Prunella. Arbuscular mycorrhizal fungi can
therefore be considered as biotic plant resources since they
enhance biomass as well as the mineral nutrition of plants.
However, it is not only the presence of AMF that is important
for plants. Previously, it has been shown that the presence of
different AMF taxa alters the performance of individual plant
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576 Research
species (Roldan-Fajardo, 1994; Streitwolf-Engel et al., 1997;
van der Heijden et al., 1998b; Klironomos, 2000; Helgason
et al., 2002) and that it changes plant community structure
and plant diversity (van der Heijden et al., 1998a). From the
results of this study, we are now also able to conclude that
different AMF taxa have a potential impact on the way plant
species coexist. We observed that Prunella was dominated by
Brachypodium, contributing to only 14% of the biomass,
when pots were inoculated with one AMF taxon (Glomus sp.
isolate BEG 21) while it contributed to 39% and 53% of the
biomass when, respectively, the AMF taxa Glomus sp. isolate
Basle Pi and Glomus sp. isolate BEG 19 were present (Fig. 2a).
The roots of both plant species investigated were extensively
colonized by the three AMF isolates in this study suggesting
that no selectivity for infection existed as found elsewhere
(Helgason et al., 2002). Despite this, we were unable to
determine whether the AMF isolates actually colonized both
plant species in the field (with the exception of Glomus sp.
isolate Basle Pi that was obtained from the roots of a P. vulgaris
plant that was growing at the field site). Molecular identification tools are important in this respect since they
enable us to determine which plants in the field are colonized
by which fungi (Helgason et al., 2002; Kowalchuk et al.,
2002). Future studies therefore need to combine such
descriptive molecular field data with laboratory experiments
in order to test the functionality of various AMF.
A comparison of these results with other biotic factors that
affect plant species coexistence, such as insect herbivores,
pathogens and fungal endophytes (see Bentley & Whittaker,
1979; Windle & Franz, 1979; Cottam, 1986; Paul, 1989;
Clay et al., 1993), indicates that the impact of different AMF
taxa on plant species coexistence is comparable to those biotic
factors.
species. Prunella contained 56% of total plant P when pots
were inoculated with one of the AMF taxa but it contained
only 21% with another AMF taxa. These observations suggest
that different AMF taxa, have the ability to alter the distribution of nutrients between coexisting plants species. Such an
unbalanced resource distribution is an intriguing aspect of
our study, which provides a mechanistic explanation as to
how different AMF taxa altered the way these plant species
coexisted. Competition and coexistence between plants
that receive different amount of resources from AMF can be
modelled and in this way it is possible to make predictions
about the coexistence of plants in multispecies communities
(van der Heijden, 2002).
The difference among AMF taxa is also outlined by our
result that plants inoculated with one of the AMF taxa
(Glomus sp. isolate BEG 21) had a lower P content than plants
inoculated with another AMF taxon (Glomus sp. isolate Basle
Pi). This reduced ability of Glomus sp. isolate BEG 21 to provide plants with P was independent of AMF effects on biomass and root length because both total pot biomass and total
pot root length did not vary between pots inoculated with
these AMF taxa. Glomus sp. isolate BEG 21 might therefore
be ineffective in terms of P acquisition for the plants and this
confirms an earlier study (van der Heijden et al., 1998b).
Several other studies showed that AMF taxa differ in their
capacity to supply plants with P (Jakobsen et al., 1992). The
effectiveness of soil nutrient resource acquisition therefore
varies among AMF taxa. It is important to note that Brachypodium performed best when this ineffective P provider was
present (Figs 2a and 3a). Field studies suggest that Brachypodium can cope with low levels of available P and this ability
likely contributed to its dominance in calcareous grasslands
exposed to N deposition (Bobbink, 1991).
Resource uptake, resource distribution and the
importance of AMF
Significance of mycorrhizal diversity
We show here that the ability of a plant species to obtain
soil nutrients depends on the identity of the AMF taxa that
are present. The results also indicate that the outcome of
competition between plants for soil resources would not
necessarily be dependent on the competitive abilities of each
plant species for soil resources, but also on the species of
mycorrhizal symbionts that occupy the plant roots. Different
AMF taxa could, thus, in themselves be considered as biotic
resources that differently affect plant species coexistence by
altering acquisition of abiotic resources and by allocating
these resources to different plant species.
The roots of both plant species were colonized by considerable amounts of fungal hyphae (between 30% and 90%
of the root length of both plants) and the coexisting plants
shared the same fungal mycelium in treatments inoculated
with one AMF taxon. Despite of this, different AMF taxa
altered the distribution of nutrients between the two plant
The relationship between biodiversity and ecosystem
functioning has received considerable attention in recent years
(Loreau et al., 2002). An increasing number of studies suggest
that mycorrhizal fungi are an integral part of this relationship
(van der Heijden & Sanders, 2002). However, increased
mycorrhizal diversity (three AMF taxa compared with one
AMF taxa) did not result in a greater biomass or in increased
nutrient acquisition in this experiment. This observation
followed that in Experiment 1 of van der Heijden et al.
(1998a) where plant productivity did not vary among
experimental grasslands inoculated with one or four different
AMF taxa. The three AMF taxa used in this study belonged
to the same genus (Glomus). Variations in growth effects by
different AMF taxa appear to be greatest at the genus level and
not at the species or isolate level (Hart & Klironomos, 2002;
Hart & Reader, 2002). This indicates that it is more likely to
find complementary effects of AMF diversity when different
AMF genera (with different strategies) are present (Smith
www.newphytologist.com © New Phytologist (2003) 157: 569 – 578
Research
et al., 2000). Also, because of complementarity between
specific plant-fungal pairs (Ravnskov & Jakobsen, 1995; van
der Heijden et al., 1998b) it is likely that positive effects of
AMF diversity increase when the number of plant and fungal
species increases. It is also possible that complementarity of
different AMF occurs after a longer growth period (and not
during 12 wk, as in this study) when nutrient availabilities
have dropped to low levels.
Conclusions
This study provides evidence that the composition of AMF
communities can influence plant species coexistence.
Coexistence of plants in a homogenous abiotic environment
could be explained by resource partitioning (Aarssen, 1983;
Tilman, 1988), but it could, according to this study, also be
explained by heterogeneity in the biotic environment.
Moreover, we showed that the ability of a plant to obtain soil
nutrient resources, such as N and P, depends on the AMF taxa
that are present. The competitive ability of a plant for soil
resources is therefore not only determined by its own ability
to capture nutrients, but it is interlinked with the mycorrhizal
symbionts colonizing the roots.
Acknowledgements
We thank W. Weisser and W. Ernst and the anonymous
reviewers for comments on this manuscript. This research was
supported by grant from the Priority Program Environment
from the Swiss National Science Foundation.
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