Positive feedback between mycorrhizal fungi and plants influences plant invasion success and resistance to invasion.

Abstract

Negative or positive feedback between arbuscular mycorrhizal fungi (AMF) and host plants can contribute to plant species interactions, but how this feedback affects plant invasion or resistance to invasion is not well known. Here we tested how alterations in AMF community induced by an invasive plant species generate feedback to the invasive plant itself and affect subsequent interactions between the invasive species and its native neighbors. We first examined the effects of the invasive forb Solidago canadensis L. on AMF communities comprising five different AMF species. We then examined the effects of the altered AMF community on mutualisms formed with the native legume forb species Kummerowia striata (Thunb.) Schindl. and on the interaction between the invasive and native plants. The host preferences of the five AMF were also assessed to test whether the AMF form preferred mutualistic relations with the invasive and/or the native species. We found that S. canadensis altered AMF spore composition by increasing one AMF species (Glomus geosporum) while reducing Glomus mosseae, which is the dominant species in the field. The host preference test showed that S. canadensis had promoted the abundance of AMF species (G. geosporum) that most promoted its own growth. As a consequence, the altered AMF community enhanced the competitiveness of invasive S. canadensis at the expense of K. striata. Our results demonstrate that the invasive S. canadensis alters soil AMF community composition because of fungal-host preference. This change in the composition of the AMF community generates positive feedback to the invasive S. canadensis itself and decreases AM associations with native K. striata, thereby making the native K. striata less dominant.

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Positive Feedback between Mycorrhizal Fungi and Plants
Influences Plant Invasion Success and Resistance to
Invasion
Qian Zhang1, Ruyi Yang1,2, Jianjun Tang1, Haishui Yang1, Shuijin Hu3, Xin Chen1*
1College of Life Sciences, Zhejiang University, Hangzhou, China, 2College of Environmental Science, Anhui Normal University, Wuhu, China, 3Department of Plant
Pathology, North Carolina State University, Raleigh, North Carolina, United States of America
Abstract
Negative or positive feedback between arbuscular mycorrhizal fungi (AMF) and host plants can contribute to plant species
interactions, but how this feedback affects plant invasion or resistance to invasion is not well known. Here we tested how
alterations in AMF community induced by an invasive plant species generate feedback to the invasive plant itself and affect
subsequent interactions between the invasive species and its native neighbors. We first examined the effects of the invasive
forb Solidago canadensis L. on AMF communities comprising five different AMF species. We then examined the effects of the
altered AMF community on mutualisms formed with the native legume forb species Kummerowia striata (Thunb.) Schindl.
and on the interaction between the invasive and native plants. The host preferences of the five AMF were also assessed to
test whether the AMF form preferred mutualistic relations with the invasive and/or the native species. We found that S.
canadensis altered AMF spore composition by increasing one AMF species (Glomus geosporum) while reducing Glomus
mosseae, which is the dominant species in the field. The host preference test showed that S. canadensis had promoted the
abundance of AMF species (G. geosporum) that most promoted its own growth. As a consequence, the altered AMF
community enhanced the competitiveness of invasive S. canadensis at the expense of K. striata. Our results demonstrate
that the invasive S. canadensis alters soil AMF community composition because of fungal-host preference. This change in
the composition of the AMF community generates positive feedback to the invasive S. canadensis itself and decreases AM
associations with native K. striata, thereby making the native K. striata less dominant.
Citation: Zhang Q, Yang R, Tang J, Yang H, Hu S, et al. (2010) Positive Feedback between Mycorrhizal Fungi and Plants Influences Plant Invasion Success and
Resistance to Invasion. PLoS ONE 5(8): e12380. doi:10.1371/journal.pone.0012380
Editor: Marcel Van der Heijden, Agroscope Reckenholz-Ta ¨nikon, Research Station ART, Switzerland
Received March 23, 2010; Accepted July 29, 2010; Published August 24, 2010
Copyright: ? 2010 Zhang et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by the Zhejiang Provincial Natural Science Foundation of China (No. Z5090089), the Research Fund for the Doctoral Program
of Higher Education of China (RFDP, No. 20070335079), and the Natural Science Foundation of China (NSFC, No. 3073020 and 30870405). The funders had no role
in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: chen-tang@zju.edu.cn
Introduction
Because of their ubiquity and presumed low level of host
specificity, arbuscular mycorrhizal fungi (AMF) have been
generally believed to play a minor role in mediating the invasion
of exotic plants [1]. However, increasing evidence indicates that
specific host-fungal pairings exist [2,3]. Some AMF species are
more beneficial to a host plant than are others [2–7], and certain
AMF are differentially promoted by different plant hosts [8,9] due
to preferential allocation of photosynthate by host plants [10].
Experiments also demonstrated that the identity of AMF species
can impact the performance of invasive plants [11,12]. This
evidence of specific host-fungal interactions suggests that AMF
could affect plant invasion.
Host-fungal specificity can lead to different AMF communities in
roots of co-occurring plant species [13,14] and has also been
presumed to enable invasive plant species to alter the density and
composition of the indigenous AMF community [15,16]. When
exposed to a mixture of indigenous AMF species, invasive plants
were colonized by one or more AMF species that differed from
those that colonized the tested native hosts [15,17]. Therefore,
invasive species could be more successful in the presence of certain
AMF species, and this could increase the abundance of those AMF
species [11] and possibly change the AMF community composition.
This assumption, however, has rarely been experimentally tested.
A shift in the AMF community driven by invasive plants may
impact invasive and native plants differently because, as noted
earlier, AMF do vary in host preference [2,3]. Thus, predicting
how the shifted AMF communities will affect the outcome of
competition between invasive and native plants may depend on
understanding the positive feedback between specific AMF and
specific invasive hosts [18,19]. When an invading species
encounters and develops strong mutualisms with specific AMF,
benefits to the AMF may generate positive feedback that enhances
the persistence and abundance of the invasive host [20], helping
the invasive host to compete with native plants. On the other
hand, if the invaders are less responsive to the AMF species or are
not mycorrhizal hosts at all, populations of AMF fungi could
decline as plant invasion proceeds [16]. This decline could then
reduce the formation of native plant mutualisms and thereby
reduce the growth and competitive ability of native hosts, again
resulting in positive feedback to invasive hosts.
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Although there is a great deal of evidence demonstrating
changes in AMF communities during exotic plant invasion [15,21–
23], the mechanisms underlying these changes and the conse-
quences of these changes have not been well elucidated. Here we
wanted to test how invasive plants alter indigenous arbuscular
mycorrhizal fungal (AMF) communities and how the changed
AMF communities affect invasive plants in their competition with
native plants. We hypothesize that invasive plants may alter
indigenous AMF communities by establishing preferred mutual-
isms that favor the invasive plant itself (positive feedback). These
new mutualisms with invasive plants may lead to the decline in
mutualisms with native plants and may favor invasive plants in
their competition with native plants. We tested these hypotheses in
greenhouse experiments by using the invasive forb Solidago
canadensis L. and the native legume forb Kummerowia striata (Thunb.)
Schindl.
Solidago canadensis L. (goldenrod) is a successful worldwide
invader of North American origin [24] that has established in
Europe, large parts of Asia, Australia, and New Zealand [25,26].
This invasive weed spreads rapidly in southeastern China,
invading abandoned fields and disturbed habitats [26]. In a
previous three-year field study, we compared the AMF associated
with several native species in the presence or absence of the
invasive S. canadensis by analyzing soil and root samples from field
sites. We found that S. candensis reduced AMF colonization of some
native plants (K. striata, Lolium perenne, Echinochloa crusgalli, and
Ageratum conyzoides) and altered AMF spore composition [27,28].
Richness and abundance of AMF species that colonized the roots
of the native K. striata differed in fields dominated by S. canadensis
than in fields without S. canadensis [28].
In the present study, we first created an AMF community with
five species that commonly exist in the field and examined the
divergence of the constructed AMF community when pots were
planted with the invasive S. canadensis or with the native K. striata
(experiment 1). Changes in AMF community composition were
assessed using spore counts (experiment 1) and with molecular
tools (experiment 2). To measure feedback, we then examined the
effects of the altered AMF community on the mutualisms formed
with native K. striata and on the interaction between invasive and
native plants (experiment 3). The host preference of the five AMF
was also assessed to test whether the AMF form preferred
mutualistic relations with the invasive species (experiment 4). All
experiments were performed in the greenhouse with plants and
AMF that coexist in the field.
Results
Effects of a native and an exotic plant on AMF
community composition (experiment 1)
The spore composition of the AMF community differed under
the two hosts (Fig. 1): after the two growing seasons in
experiment 1, Glomus geosporum spores were dominant under the
invasive host (F1,14=37.64, P=0.000) while Glomus mosseae
spores were dominant under the native host (F1,14=89.71,
P=0.000) (Fig. 1A). There was no significant change in spore
numbers of Glomus versiforme (F1,14=1.61, P=0.225), but
significantly different spore numbers of Glomus diaphanum
(F1,14=5.28, P=0.038) and Glomus etunicatum (F1,
P=0.009) were found under the two host plants after the two
growing season (Fig. 1A). The total numbers of AMF spores
(F1,14=4.04, P=0.064, Fig. 1A) were not different, but AMF
communities diverged (in terms of Bray-Curtis similarity
decreased, F1,6=77.15, P=0.000, Fig. 1B) between the two
host plants after the two growing seasons.
14=9.29,
Changes in the abundance of G. mosseae in native roots
based on DNA (experiment 2)
According to the nested PCR-DGGE-sequencing method in
experiment 2, the relative abundances of DNA G. mosseae and G.
geosporum in roots of native K. striata were changed when K. striata
was grown in the soil conditioned by S. canadensis under both
monoculture (F1,6=56.50, P=0.000 and F1,6=590.79, P=0.000
for G. mosseae and G. geosporum respectively) and mixture
(F1,6=110.52, P=0.000 and F1,6=84.98, P=0.001 for G. mosseae
and G. geosporum respectively). The S. canadensis-altered AM fungal
community (SC-A-AMF) treatment reduced the relative abun-
dance of DNA of G. mosseae but increased that of G. geosporum in
roots of K. striata compared to the initial AMF community (I-AMF)
treatment with both monoculture and mixed plantings (Fig. 2).
Culture types (monoculture and mixture) did not affect the
relative DNA abundance of G. mosseae in roots of native K. striata
grown in treatments of I-AMF (F1,6=0.58, P=0.391) and SC-A-
AMF (F1,6=2.12, P=0.196) (Fig. 2A). For G. geosporum, culture
types did not change the relative abundance of DNA in roots of K.
striata under treatment of I-AMF (_F1,6=1.92, P=0.238), but
mixture reduced the relative abundance of DNA in roots of K.
striata under the treatment of SC-A-AMF compared to monocul-
ture (F1,6=250.89, P=0.000) (Fig. 2B).
Interaction between invasive and native plants under the
changed AMF community (experiment 2)
AMFtreatmentssignificantly
(F5,18=49.23, P=0.000 and F5,
canadensis in monoculture and mixture, respectively; F5,
49.45, P=0.000 and F5,18=28.25, P=0.000 for K. striata in
monoculture and mixture, respectively). AMF treatments also
significantly affected shoot
F5,23=6.62, P=0.001 for S. canadensis in monoculture and
mixture, respectively; F5,18=31.81, P=0.001 and F5,18=27.19,
P=0.006 for K. striata in monoculture and mixture, respectively).
No significant differences in both shoot biomass and shoot15N
were found among the three no-AMF controls for both hosts
under monoculture or mixture (Fig. 3, P.0.05). Compared to the
I-AMF treatment, the SC-A-AMF treatment enhanced (P,0.05)
but the K. striata-altered AM fungal community (KS-A-AMF)
treatment did not change (P.0.05) the shoot biomass (Fig. 3A)
and shoot15N (Fig. 3B) of S. canadensis. For K. striata, however,
shoot biomass (Fig. 3A) and shoot
(P,0.05) under the SC-A-AMF treatment but increased (P,0.05)
under the KS-A-AMF treatment relative to the I-AMF treatment.
AMF communities significantly affected the ratio of K. striata to
S. canadensis biomass in mixture (F5,18=7.97, P=0.000). There
was no significant difference in biomass ratio among the three
non-AMF controls (Fig. 4A, P,0.05). The biomass ratio of K.
striata to S. canadensis was reduced by SC-A-AMF treatment
(P,0.05), but was enhanced by the KS-A-AMF treatment
(P,0.05) (Fig. 4A).
AMF communities also significantly affected the aggressivity
index in the K. striata–S. canadensis competition (F5,18=14.67,
P=0.000). No significant difference in aggressivity index was
found among the three non-AMF controls (Fig. 4B, P,0.05).
Aggressivity index was enhanced by the KS-A-AMF treatment
(P.0.05), but was reduced by the SC-A-AMF treatment (P,0.05)
(Fig. 4B) relative to the I-AMF treatment.
affected
18=33.86, P=0.000 for S.
shoot biomass
18=
15N (F5, 18=162.76, P=0.000 and
15N (Fig. 3B) decreased
Host preference of AMF (experiment 3)
No AMF spores or other indications of colonization were found
in non-AMF controls in both host plants. Differences in host
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preference in the AMF population growth rates were detected in
experiment 3 (F4,15=36.46, P=0.000 and F4,
P=0.000 for spore density of S. canadensis and K. striata,
respectively; F4, 15=3.09, P=0.049 and F4, 15=3.87, P=0.023
for colonization rates of S. canadensis and K. striata, respectively). For
G. geosporum, spore numbers (Fig. 5A, P,0.05) and colonization
rates (Fig. 5B, P,0.05) were higher with S. canadensis than with K.
striata. For G. mosseae, however, spore number (Fig. 5A, P,0.05)
and colonization rates (Fig. 5B, P,0.05) were higher with K. striata
than with S. canadensis.
Plant growth responses (in terms of the dependency index and
shoot
among the five AMF species (F4,
F4, 15=4.65, P=0.012 for dependency indices of S. canadensis
and K. striata, respectively; F5, 18=3.12, P=0.034 and F5, 18=
3.95, P=0.014 for shoot
respectively). For S. canadensis, the dependency index and shoot
15N were highest with G. geosporum (Fig. 6A and 6B, P,0.05 in
both cases). For K. striata, the dependency index and shoot15N
were highest with G. mosseae (Fig. 6A and B, P,0.05 in both cases).
15=23.91,
15N) of the two host plants to AMF inoculation differed
15=14.07, P=0.000 and
15N of S. canadensis and K. striata,
Discussion
Here we demonstrate that positive feedback between specific
mycorrhizal fungi and the invasive plant (Solidago canadensis)
promoted the invasion success of the invader. The invader altered
the spore composition of the AMF communities in that AMF
species that were most beneficial for its own growth were
promoted at the expense of AMF species that were most beneficial
to the native plant species (Kummerowia striata).
In our experiment 3, all five AMF species were capable of
infecting both S. canadensis and K. striata. But AMF spore density in
soil and hyphal colonization of roots (Fig. 5), both of which reflect
AMF population growth [2], indicated that the five AMF species
responded differently to the two host plants. S. canadensis promoted
G. geosporum while K. striata promoted G. mosseae. The dependency
index [29] also indicated a high dependency of S. canadensis on G.
geosporum and a high dependency of K. striata on G. mosseae (Fig. 6A).
We found that the mutualism formed between the AMF species
G. mosseae and the native plant K. striata decreased when this native
plant grew in soil in which the AMF spore community had been
Figure 1. Numbers of AMF spores (total and by AMF species) in soil grown with invasive S. canadensis or native K. striata in two
growing years in experiment 1. Bars represent total spore numbers of the five AMF. Inset pie charts represent spore composition of the AMF
species. Values are means 6 standard error. Means with different letters are significantly different at the 5% level.
doi:10.1371/journal.pone.0012380.g001
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changed by invasive S. canadensis (Fig. 2). Prior experiments have
shown that invasive plants degraded AMF mutualisms of native
hosts because most invasive species are not mycorrhizal hosts or
are less dependent than the native hosts on the mutualism and
therefore invest less carbon in maintaining the AMF community
[12,16,23,30]. Also, some invasive species can inhibit native
mutualisms through allelopathy [31]. In our study, however, the
invasive S. canadensis is a highly mycorrhizal host [27], and it did
Figure 2. The relative abundance of DNA of G. mosseae (A) or G. geosporum (B) in roots of K. striata grown in soil containing the initial
AMF community (I-AMF) or the AMF community altered by the invasive S. canadensis (SC-A-AMF) under monoculture and mixed
planting with S. canadensis in experiment 2. The relative abundance of DNA of G. mosseae, or G. geosporum (%), =Ig/It,6100, where Igis the
intensity of the G. mosseae band or G. geosporum band, and Itis the total intensity of all the AMF species bands in one profile. Values are means 6
standard error. Within monoculture or within mixed plantings, means with different lower case letters are significantly different at the 5% level.
Within each AMF treatment (I-AMF or SC-A-AMF), means with different capital letters are significantly different at the 5% level.
doi:10.1371/journal.pone.0012380.g002
Figure 3. Shoot biomass (A) and shoot15N (B) of invasive and native plants under various AMF communities in experiment 2. I-N-
AMF: the initial non-AMF control; SC-N-AMF: the S. canadensis-altered non-AMF control; KS-N-AF: the K. striata-altered non-AMF control; I-AMF: the
initial AMF community; SC-A-AMF: the AMF community altered by the invasive S. canadensis; KS-A-AMF: the AMF community altered by the native K.
striata. Values are means 6 standard error. Within each set of six AMF treatments (within monoculture or mixture for each host plant), means with
different letters are significantly different at the 5% level.
doi:10.1371/journal.pone.0012380.g003
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not decrease the total abundance of AMF spores in soil after two
growing seasons (Fig. 1A). Studies also showed that plant
neighbors were important in structuring AMF communities [32]
in roots and that the presence of invasive plants changed AMF
assemblages in roots of their native neighbors [15]. However,
when coexisting with native plants in the same AMF communities,
this invasive S. canadensis did not change the abundance of G.
mosseae in native roots (Fig. 2). Based on these results, we suggest
that the decrease in mutualism between G. mosseae and native
plants was due to the decrease of spore density of G. mosseae in the
AMF community that had been changed by S. canadensis.
The degradation of the mutualism between AMF G. mosseae and
native plants resulted in reductions in nutrient uptake and growth
of native plants in our study. The presence of shoot15N indicates
that AMF can absorb and deliver N to plants through hyphae
because the15N was added to the microcosm compartment that
excluded roots but did not exclude AMF hyphae [33]. Our data
on shoot15N demonstrated that N uptake by AMF-colonized K.
Figure 4. Biomass ratio of K. striata to S. canadensis (A) and aggressivity index of competition between K. striata and S. canadensis (B)
in response to various AMF communities in experiment 2. The ratio of biomasses and aggressivity index were calculated based on shoot
biomass. I-N-AMF: the initial non-AMF control; SC-N-AMF: the S. canadensis-altered non-AMF control; KS-N-AF: the K. striata-altered non-AMF soil
control; I-AMF: the initial AMF community; SC-A-AMF: the AMF community altered by the invasive S. canadensis; KS-A-AMF: the AMF community
altered by the native K. striata. Values are means 6 standard error. Means with different letters are significantly different at the 5% level. Inset pie
charts represent biomass composition of the two plant species.
doi:10.1371/journal.pone.0012380.g004
Figure 5. Spore production (A) and root colonization (B) by the five AMF species with K. striata or S. canadensis as hosts in
experiment 3. Values are means 6 standard error. For each host plant, means with different letters are significantly different at the 5% level. In the
non-AMF treatment, spore numbers and colonization rate were zero (data not shown).
doi:10.1371/journal.pone.0012380.g005
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striata was reduced when the AMF community had been altered by
S. canadensis (Fig. 3B). Moreover, host-fungal preference detected in
experiment 3 further demonstrated that this reduction was due to
a decrease in the abundance of G. mosseae, which was the most
effective AMF species for K. striata (Fig. 6). It is well documented
that invasion by non-mycorrhizal or less mycorrhizal species can
reduce AMF abundance and disrupt mutualism of native plants
[16,23,30]. By using15N in the current study, we have expanded
this understanding by demonstrating that an invasive plant can
reduce the nutrient-acquiring functions of mutualisms by degrad-
ing the preferred mutualisms of native plants.
AMF can mediate competition between some invasive and native
plants by differently affecting growth of hosts or by transferring
carbon between hosts via a shared mycorrhizal network [34–37].
The identity of AMF species influencing the performance of
invasive plants [11] suggests that the species composition of AMF
communities is also important in this mediation. We found that
under the S. canadensis-changed community in which G. geosporum
became dominant, the competitive ability of K. striata (as indicated
by the biomass ratio of K. striata to S. canadensis and by the
aggressivity index [38] was reduced but that of S. canadensis was
enhanced (Fig. 4). These results suggest that, by shifting the AMF
community, the invasive S. canadensis generates two kinds of positive
feedback that increased its own competiveness: it increased those
AMF species that favored its own growth while it decreased the
AMF species that favored the growth of its native competitor. Bever
[39] found that negative feedback through changes in the
composition of the AM fungal community inhibited the dominant
plant species leading to the coexistence of the competing plant
species. Here, we indicated that the positive feedback through
changes in the composition of the AM fungal community promoted
the invasive S. canadensis, leading to the dominance of the invasive
plant and the decline of the native K. striata.
One may argue that it is difficult to measure how changes in
AMF affect or produce feedback on the interaction between
invasive and native plants because of the confounding effects of
abiotic factors (i.e., soil nutrients; [40]) and biotic factors (i.e.,
allelopathy and soil pathogens [17,41,42]). The invasive plant used
in our study, S. canadensis, does exude allelochemicals that interfere
with neighboring plants [43] and soil pathogens [44]. We designed
non-AMF control treatments (I-N-AMF, SC-N-AMF and KS-N-
AMF) corresponding to AMF treatments (I-AMF, SC-A-AMF and
KS-A-AMF) during the whole study. We thus can separate
allelopathy and other effects from mycorrhizal effect by comparing
the biomass ratio and aggressivity index of K. striata to S. canadensis
in each AMF treatment to its corresponding non-AMF control.
Our work increases the understanding of the ecological
mechanisms underlying how AMF interactions can participate in
feedback affecting plant invasion. Invasive plants may encounter
certain novel AMF that facilitate the establishment of the invasive
plants [45] or the invasive plants may disrupt AMF mutualisms so
as to inhibit native species [16,23]. Our results indicate that, under
greenhouse conditions, an invasive species (S. canadensis in our
study) can change the dominant species in the AMF community as
a consequence of host-AMF preference. This shift in the AMF
community generates positive feedback to the invasive plant while
reducing preferred mutualisms and competitiveness of native
plants (K. striata in our study), thus modifying the outcome of the
competition in favor of the invasive plant.
Materials and Methods
Plants, soil, and AMF species
We used the invasive forb S. canadensis L. and the native legume
forb Kummerowia striata (Thunb.) Schindl. as model plants, and we
used soil conditions that matched those of an abandoned
agriculture field (about 15 ha) in Zhejiang, China (29u89N,
121u59E), where S. canadensis has been invasive for 3 years
[28,44]. Kummerowia striata is a common weed in crop fields,
orchards, and abandoned land [46]. Both S. canadensis and K. striata
Figure 6. Mycorrhizal dependency index (A) for K. striata or S. canadensis, and mycorrhizally enhanced15N in shoot biomass (B) as
affected by the five AMF species in experiment 3. Note that the15N was added to the compartment without roots and presumably entered the
plant via AMF hyphae. Mycorrhizal dependency index=(BAMF2Bnon-AMF)/BAMF, where BAMFis biomass of the plants in mycorrhizal inoculation
treatment and Bnon-AMFis biomass of the control plants. Values are means 6 standard error. For each host plant in (B), means with different letters are
significantly different at the 5% level.
doi:10.1371/journal.pone.0012380.g006
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are highly dependent on AMF in low nutrient soil [27,47]. The
propagules (ungerminated buds from rhizomes) of S. canadensis and
seeds of K. striata were collected from the abandoned agricultural
field. Before the S. canadensis invasion, native K. striata was the
dominant species in this abandoned field.
Five common AMF species (Glomus mosseae, Glomus versiforme,
Glomus diaphanum, Glomus geosporum, and Glomus etunicatum) were
selected. These species naturally exist in the abandoned field [28]
where the propagules of S. canadensis and seeds of K. striata were
collected. A culture of each of the five AMF was established from a
single spore. Cultures were propagated on a common host (Zea
mays L.) that was grown in sterilized sand (0.45 to 1 mm dia.) in a
growth chamber for 4.5 months until sporulation. Each of these
AMF has been deposited in Glomales Germplasm Bank in China
(Institute of Plant Nutrient & Resources, Beijing Academy of
Agriculture & Forestry Sciences). Equal numbers of spores
incorporated in soil from five pure cultures were then mixed to
create the initial AMF communities (I-AMF) used for the
experiments. The soil with spores was used as inoculum.
The surface soil (0–15 cm depth) used in the experiments was
obtained from the same abandoned field where the propagules of
S. canadensis and seeds of K. striata were collected. The soil is a
sandy loam with a pH of 6.62 (2.5:1, KCl aqueous solution: soil),
42.14 g kg21
organic matter,
98.23 mg kg21soil of extractable N (NH4-N and NO3-N, [48]),
extractable P [49] and extractable K (extracted by 2 M HNO3
and determined by flame atomic absorption spectrophotometry,
flame-AAS), respectively. The soil was air dried, passed through a
5-mm-mesh sieve, and uniformly moistened to constant water
content. Then, the prepared soil was mixed with sand (1:1 by
weight) and sterilized by gamma (c)-radiation.
and38.07, 22.99,and
Experiment 1
In this experiment, S. canadensis and K. striata were grown
separately in soil containing the mixed initial AMF community (I-
AMF). After two growing seasons, the effect of host plant on the
AMF community composition was evaluated. The experiment had
two plant species (S. canadensis and K. striata), two AMF treatments
(non-AMF [N-AMF] and I-AMF), and four replicates. The N-
AMF treatment was not analyzed in experiment 1 but the soil was
used as a non-mycorrhizal control in experiment 2.
Each rectangular mesocosm (45 cm long630 cm wide620 cm
high) with a volume of 27 L was filled with 16 kg of the sterilized
loam-sand mixture described above. The soil in half of the
mesocosms was inoculated with 500 g of soil containing I-AMF
inoculum. The remaining mesocosms received 500 ml of filtrate
from 500 g of inoculum (with no mycorrhizal spores) and 500 g
sterilized inoculum to correct for possible differences between the
microbial communities in mycorrhizal and non-mycorrhizal
treatments. Eight propagules of S. canadensis or eight seeds of K.
striata were planted in each mesocosm. Mesocosms were arranged
in a greenhouse in a completely randomized block design. Plants
were maintained with ambient light and temperature and with air
temperature ranging from 18 to 30uC. Plants were watered daily
to keep soil moisture at 70–90% of water-holding capacity. No
additional nutrients were added.
All the plants were harvested when they naturally senesced after
an 8-month growing season (from March to November). Five soil
samples (each 100 g) were collected from each mesocosm for
monitoring AMF spore density and composition of AMF
communities. All of the mesocosms with the remaining soils were
stored at 4uC until the following March, when the mesocosms
were returned to the greenhouse and planted with the same host as
in the first growing season. At the end of the second growing
season, soil was again sampled for monitoring density and
composition of AMF communities.
Spores were separated from the soil by the wet-sieving method
[50]. Spores were counted and identified to species according to
the taxonomic information provided by the Glomales Germplasm
Bank in China and the VAM website (http://invam.caf.wvu.edu).
To estimate the similarity of AMF communities between the
two host plants at the end of each growing season, we subjected
the data to analysis by the program PAST (Version, 1.94) [51] and
calculated Bray-Curtis similarity.
The data for total spore density of AMF community and spore
density of each AMF species under each host plant were first
subjected to a homogeneity test and then to a multivariate analysis
of variance (MANOVA) (plant hosts as factor and growing seasons
as block) using SPSS V.17.0. Treatments were compared by the
LSD at the 5% significance level. Bray-Curtis similarity was
analyzed with a one-way ANOVA using SPSS V.17.0.
Experiment 2
Experiment 2 examined how the host-induced alteration in
mycorrhizal communities affected the host plants when grown
separately or together. Soil samples containing AMF communities
under S. canadensis (SC-A-AMF) and K. striata (KS-A-AMF) from
the end of experiment 1 were used as inocula. One kg of soil from
each mesocosm at the end of the second growing season in
experiment 1 was collected and passed through a sterilized 2-mm
sieve to mix the inoculum. The inoculum from each original
replication in experiment 1 was used for one replication of
experiment 2.
To separate the effects of allelopathy, nutrients, and other
rhizosphere factors induced by host plants from the effects of AMF
communities, the soils from N-AMF controls under S. canadensis
(SC-N-AMF) and K. striata (KS-N-AMF) in experiment 1 were
used as no-AMF inoculum controls corresponding to SC-A-AMF
and K.S-A-AMF in experiment 2. Overall, experiment 2 had three
kinds of AMF communities and their corresponding non-AMF
controls, three kinds of host plants (S. canadensis, K. striata, and their
mixture), and four replicates. The AMF communities and their
corresponding non-AMF controls were: the initial non-AMF
control (I-N-AMF) and the initial AMF community (I-AMF); the S.
canadensis-altered non-AMF control soil (SC-N-AMF) and the S.
canadensis-altered AM fungal community (SC-A-AMF); and the K.
striata-altered non-AMF soil control (KS-N-AM) and the K. striata-
altered AM fungal community (KS-A-AMF).
A microcosm containing two compartments (Fig. 7) was
designed to assess mycorrhizal contribution to nutrient uptake.
Each compartment was 20 cm long615 cm wide620 cm high,
and the two compartments were separated by two pieces of
replaceable nylon mesh (20-mm openings, Tetko/Sefar mesh,
Sefar America, New York). To prevent the diffusion of mobile
nutrients between the compartments, a stainless wire net (1.5 mm
thick and with 6-mm openings) was inserted between the two
pieces of replaceable mesh to create an air gap (Fig. 7, modified
from Tanaka & Yano [33]). As described in the next paragraph,
the compartment containing a plant and AMF was called the
HOST compartment, and the other was called the SOIL
compartment. The mesh permitted AMF hyphae but not roots
to penetrate from the HOST to the SOIL compartment to obtain
nutrients.
Each compartment was filled with 3 kg of sterilized 1:1 soil and
sand as described for experiment 1. For treatment SC-A-AMF and
its corresponding control (SC-N-AMF), and for treatment KS-A-
AMF and its corresponding control (KS-N-AMF), 100 g of soil
containing AMF inocula from the end of experiment 1 was
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Page 8

incorporated into the soil of each HOST compartment. For the I-
AMF treatment, the inocula as used in experiment 1 were
incorporated into the soil of HOST compartment. For its
corresponding no-AMF control (I-N-AMF), microcosms received
100 ml of washing filtrate from 100 g of AMF inoculum (with no
mycorrhizal spores) and equal amounts of inoculum sterilized with
c-radiation to correct for possible differences between the
microbial communities in I-AMF and I-N-AMF treatments.
Four healthy germinated seeds of K. striata, four propagules of S.
canadensis, or their combination (two plants of each species) were
planted in each HOST compartment. The microcosms were
placed in a growth chamber in the greenhouse. All growth
conditions were the same as in experiment 1.
The
mediated plant N uptake’’ 3 weeks before the experiment was ended.
The15N tracer was injected uniformly as15N-enriched mineral N
((NH4)2SO4, 99.7% atom
3.0 mg N kg21soil into each SOILcompartment. When plantswere
harvested (see next paragraph), the N isotope fraction (14N or15N) in
shoots was determined using a ThermoFinnigan DELTAPlus
continuous flow isotope ratio mass spectrometer (CF-IRMS, Thermo
Finnigan DELTA Plus, Waltham, MA, USA). Sample15N (%) was
converted to excess N isotope (mg) based on the atom ratio of
atmospheric N. Sample
fractional abundance (15N/(14N+15N)) and total N content [52].
The plants were harvested 6 months after planting when both
invasive and native plants were flowering. Root systems were
separated from shoots, and the fresh roots were weighed
immediately. Half of each root sample was frozen at 280uC for
molecular analysis. The remaining half of each sample was used
for measurement of dry root biomass. The shoots and roots were
dried at 65uC for 48 h and weighed to determine dry shoot and
root biomass. Shoot biomass of S. canadensis and K. striata in
mixture were used to calculate the ratio of K. striata to S. canadensis.
Several methods can be used to quantify the abundance of
specific AMF in roots, and these methods include real-time
polymerase chain reaction (PCR) [53–56]. Here we used a nested
15N tracer was introduced to quantify ‘‘mycorrhizally
15N) in deionized water at a rate of
15N content was then calculated from
PCR-denaturing gradient gel electrophoresis (DGGE)-sequencing
method [54–56] to measure the relative abundance of DNA of the
AMF species G. mosseae and G. geosporum in the native roots. Two
kinds of primers were used. One primer, AM1/NS31 [57], is
specific for all AMF species, and the other primer, NS31-GC/Glol
[58], is specific for the AMF species in the genus Glomus.
Briefly, total DNA of root samples was extracted using a DNA
Extraction Kit and following the manufacturer’s protocol (Axygen
Biosciences). Isolated DNA was subjected to nested PCR with
primers AM1/NS31 and NS31-GC/Glol. Thermocycling pro-
gram and conditions for the first PCR with primers AM1/NS31
were 95uC 5 min; followed by 35 cycles of 95uC 30 sec, 64uC
1 min, and 72uC 2 min; and a final extension at 72uC for 10 min.
The 50-ml reaction volume contained 1 ml of dNTP, 1 ml of each
primer (10 pmol), 5 ml of 106 buffer, 1 ml of template, 0.5 ml of
Taq polymerase, and ddH2O. The 550-bp PCR product [57] and
primer specificity were analyzed by agarose gel electrophoresis
(1.0% (w/v) agarose, 100V, 60min) and ethidium bromide staining
in the presence of the PUC19 DNA marker. The thermocycling
program and conditions for the second PCR with primers NS31-
GC/Glol were 94uC for 5min; followed by 35 cycles of 94uC
45 sec, 55uC 1min, and 72uC 45 sec; and a final extension at 72uC
for 10 min. The PCR reaction was carried out with a Tgradient
DNA thermal cycler (Whatman Biometra, Germany). The nested
PCR amplicons were first checked by agarose gel electrophoresis
(1.7% (w/v) agarose, 100V, 60 min) and ethidium bromide
staining to determine size (approximately 270 bp) and yield in
the presence of the pBR322 DNA/Alul Marker. Then the nested
PCR products were used for DGGE analysis following the
procedure described by Muyzer et al. [59] and Liang et al. [55]
and by using a D-Gene system (Bio-Rad Laboratories, Hercules,
CA, USA) at a constant temperature of 60uC. Electrophoresis was
for 10 min at 200 V, after which the voltage was lowered to 150 V
for an additional 7 h. Gels were stained in 16TAE containing
4 ml Sybr Green per 20 ml TAE, and gel images were digitally
captured using the ChemiDoc EQ system. The DGGE band
pattern and intensity were analyzed by Quantity One Software
Figure 7. Diagram of a microcosm used in experiments 2 and 3. Each microcosm had two equal-sized compartments, termed the HOST
compartment and the SOIL compartment. The compartments were separated by two pieces of nylon mesh with 20-mm openings. A stainless wire net
(1.5 mm thick and with 6-mm openings) was inserted between the two pieces of mesh to create an air gap that prevented the diffusion of mobile
nutrients between the compartments.
doi:10.1371/journal.pone.0012380.g007
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(Bio-rad, Hercules, CA, USA). To obtain sequences from DGGE
bands, each band in DGGE was excised. Then the DNA in the
band was eluted and reamplified with primer Glo1/NS31 (no GC-
clamp added) following the PCR procedure described above. The
reamplified PCR products were sequenced by the Shanghai
Sangon Biological Engineering Technology & Services Co., Ltd.
Similarity comparison of each DNA sequence recovered from the
DGGE gel was performed using an online program (BLAST,
http://www.ncbi.nlm.gov/BLAST). The specific bands for G.
mosseae and G. geosporum in the DGGE were identified through this
sequence similarity comparison. The sequences of G. mosseae and
G. geosporum were submitted to GenBank database for verification.
The accession numbers are GU978970 for G. mosseaes and
HM853685 for G. geosporum.
The total intensity of all bands and the bands representing G.
mosseae and G. geosporum in the same profile were used to calculate
the relative abundances of DNA G. mosseae and G. geosporum. The
relative abundance DNA of G. mosseae or G. geosporum (%)=Ig/
It6100, where Igis the intensity of the G. mosseae band or G.
geosporum band, and Itis the total intensity of all the AMF species
bands in one profile.
The aggressivity indices of plants [38] were calculated using shoot
biomass of K. striata and S. canadensis in monoculture and mixture.
Aggressivity index=(Yij/Yii)2(Yji/Yjj), where Yijand Yiiare the
shoot biomass of K. striata in monoculture and mixture, and Yjiand
Yjjarethe shoot biomassof S. canadensis inmonoculture and mixture.
Differences in shoot biomass and shoot
treatments for each host plant in monoculture and mixture were
separately analyzed (one analysis for monoculture and one for
mixture) with a one-way ANOVA using the general linear model
procedure in SPSS (V.17.0). Differences in shoot biomass ratio (K.
striata: S. canadensis) and aggressivity indices in the competition
between K. striata and S. canadensis, as affected by AMF community,
were also analyzed with a one-way ANOVA. The relative
abundance of DNA of G. mosseae or G. geosporum in roots of K.
striata, as affected by AMF community or plant culture types, was
analyzed separately with a one-way ANOVA. The relative
abundance of DNA of AMF species, biomass ratios, and
aggressivity indices were arcsine transformed to satisfy variance
assumptions before ANOVAs were performed. When ANOVAs
were significant, means were compared by least significant
difference (LSD) at the 5% significance level.
15N between AMF
Experiment 3
Experiment 3 determined whether any of the five AMF species
preferred the invasive host to the native host or vice versa. There
were five AMF species and a non-AMF control, two plant species,
and four replications. AMF treatments received 100 g of soil
containing AMF inocula. The non-AMF control received equal
amounts of inoculum sterilized with c-radiation plus non-AMF
filtrate from the inoculum, thereby controlling for potential
mineral and non-mycorrhizal microbial components of the
inoculum. S. canadensis or K. striata were grown in the HOST
compartment of the microcosms described for experiment 2.
Three kg of sterile soil and sand mix (1:1 w/w) plus the AMF
inoculum was added to each compartment. The15N tracer was
introduced to the SOIL compartment of the microcosms as
described for experiment 2.
The plants were grown under the same conditions as described
for experiments 1 and 2. Six months after planting, the plants were
harvested. Root systems were separated from shoots, and the fresh
roots were weighed immediately. Half of each root sample was
used for quantification of AMF colonization (see next paragraph).
The remaining half of each sample was oven-dried (65uC for 48 h)
and used for measurement of dry root biomass. Measurements for
plant biomass and15N in shoots were the same as described for
experiment 2.
AMF colonization of roots was quantified using a microscope
(620 magnification) and the gridline intersection method devel-
oped by Giovannetti & Mosse [60]; 200 transects were examined
per replicate. Measurement of spore density was the same as
described for experiment 1. The mycorrhizal dependency index
(DI) of host plants for each AMF species [29] was calculated using
biomass of S. canadensis and K. striata in the AMF inoculation
treatments and the non-AMF control. DI=(BAMF2Bnon-AMF)/
BAMF, where BAMFis biomass of the plants in the mycorrhizal
inoculation treatment and Bnon-AMF is biomass of the control
plants.
For each host plant species, one-way ANOVAs (with AMF
species as the factor) were performed on the dependent variables of
shoot N15and DI. In the non-AMF treatments, spore numbers
and colonization rate were always zero, and this treatment was not
included when one-way ANOVAs were used to compare spore
numbers and colonization rates between AMF treatments.
Treatments were compared using LSD at the 5% significance
level. Data for AMF colonization rate and DI were arcsine
transformed before ANOVAs were performed.
Acknowledgments
We thank Leyi Li and Fan Zhang at Institute of Botany, the Chinese
Academy of Science for technical assistance in measuring15N, Dr. Huixia
Shou and Ms. Lu Wang at Zhejiang University for molecular test, and Dr.
Bruce Jaffee in the USA for helpful comments on the text and English
revision.
Author Contributions
Conceived and designed the experiments: JT SH XC. Performed the
experiments: QZ RY. Analyzed the data: QZ HY XC. Wrote the paper:
QZ JT SH XC.
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