1117

Effects of mycorrhizal inoculant, N:P supply ratio,
and water depth on the growth and biomass
allocation of three wetland plant species
Lauchlan H. Fraser and Larry M. Feinstein

Abstract: In the greenhouse, we investigated the growth and biomass allocation of three juvenile wetland species (Carex
tribuloides Wahl., Phalaris arundinacea L., and Rumex orbiculatus Gray) under three different water depths (–4, 0, and
+2 cm relative to the soil surface), three N:P supply ratios (1:30, 1:1, 30:1), and two mycorrhizal inoculant treatments (arbuscular mycorrhizal (AM) fungi present, absent). After 6 weeks, the plants were harvested, separated to above- and below-ground parts, oven-dried, and weighed. The mycorrhizal inoculant significantly increased plant growth and reduced
root:shoot ratios. At an N:P supply ratio of 30:1, plants with AM fungi had significantly greater biomass than those plants
without AM fungi. However, at 1:1 N:P supply ratio, plants without AM fungi had greater biomass. Plants without AM
fungi had higher root:shoot ratios at 0 and –4 cm water depth than plants with AM fungi. In general, C. tribuloides had
the lowest growth, and unlike P. arundinacea and R. orbiculatus, was not affected by the water depth treatment. Growth
of the wetland plants was limited more by nitrogen than by phosphorus. Our results suggest that at high N:P nutrient supply ratios and non-flooded conditions the growth of wetland seedlings can benefit by being inoculated with AM fungi.
Key words: Carex tribuloides, Phalaris arundinacea, Rumex orbiculatus, arbuscular mycorrhizae, wetland restoration, nitrogen:phosphorus ratio.
Résumé : Les auteurs ont étudié, en serre, la croissance et l’allocation de la biomasse chez trois espèces juvéniles de terres
humides (Carex tribuloides Wahl., Phalaris arundicnacea L., et Rumex orbiculatus Gray). Les traitements étaient, la profondeur d’eau (–4, 0, +2 cm) par rapport à la surface du sol, le ratio des apports N:P (1:30, 1:1, 30:1), et la présence de
deux traitements d’inoculation (champignon mycorhizien arbusculaire (AM), présent ou absent). Ils ont récolté les plants
après six semaines, séparé les parties épigées et hypogées, et les ont séchées au four avant de les peser. L’inoculant mycorhizien augmente significativement la croissance des plants et réduit les rapports racine:tige. Avec un ratio N:P de 30:1, les
plants avec les champignons AM possèdent une plus grande biomasse que les plants non inoculés. Cependant, avec un ratio
N:P de 1:1, les plants sans mycorhize ont une plus forte biomasse. Les plants sans mycorhize AM ont des ratios racine:tige,
plus élevés, à 0 et –4 cm de profondeur d’eau, que les plants inoculés. En général, le C. tribuloides montre la croissance la
plus faible, et contrairement au P. arundinaceae et au R. orbiculatus, il n’est pas affecté par la profondeur de l’eau. La
croissance de ces plantes de milieux humides est plus fortement limitée par l’azote que par le phosphore. Les résultats
montrent qu’avec des rapports en nutriments N:P élevés, et sous des conditions non immergées, la croissance de plantules
des terres humides peut bénéficier de l’inoculation mycorhizienne.
Mots clés : Carex tribuloides, Phalaris arundinacea, Rumex orbiculatus, mycorhizes arbusculaires, restauration de terres
humides, ratio azote : phosphore.
[Traduit par la Rédaction]

Introduction
Arbuscular mycorrhizal (AM) fungi are commonly found
in a wide variety of host plants (Smith and Read 1997). In
general, colonization by AM fungi is associated with an increase in plant growth, particularly when growth is limited
by phosphorus supply (Johnson 1998; McHugh and Dighton
2004). While it has been shown that flooded soils can reReceived 3 June 2005. Published on the NRC Research Press
Web site at http://canjbot.nrc.ca on 29 October 2005.
L.H. Fraser1,2 and L.M. Feinstein. Department of Biology,
University of Akron, Akron, OH 44325-3908, USA.
1
2

Corresponding author (e-mail: lfraser@tru.ca).
Present address: Department of Natural Resource Science,
Thompson Rivers University, Kamloops, BC V2C 5N3,
Canada.

Can. J. Bot. 83: 1117–1125 (2005)

duce root and AM growth (Khan and Belik 1995; McHugh
and Dighton 2004), it has also been shown that wetland and
aquatic plants can be colonized by AM fungi (Khan and Belik 1995; Cooke and Lefor 1998). For example, of the 89
wetland species from 11 Connecticut freshwater wetlands
sampled by Cooke and Lefor (1998), only seven species
(five of which were from the family Cyperaceae) were not
colonized by AM fungi.
Plant biomass allocation patterns along an environmental
gradient can help to determine the ability of species to
grow and compete successfully, as well as predict the relative distribution and abundance of individual species (Campbell and Grime 1989; Tilman and Wedin 1991; Figiel et al.
1995; Poorter and Nagel 2000; Güsewell et al. 2003). Experiments have demonstrated that an increase in water depth
generally reduces the growth and affects morphological responses of wetland plants (Newman et al. 1996; Coops et

doi: 10.1139/b05-084

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Can. J. Bot. Vol. 83, 2005
Table 1. Modified Rorison’s nutrient solution as applied to the N:P ratio
nutrient treatments (preparation of 50 mL; Hendry and Grime 1993).
N:P ratioa
Element
Ca/N
Ca/N
Mg
K/P
K/P
Fe
Mn
B
Mo
Zn
Cu
K
Ca

mg×50 mL
4.0/2.80
0.1/0.09
1.20
7.1/2.80
0.2/0.09
0.15
0.03
0.03
0.005
0.005
0.005
3.405
27.07

–1

Stock solutions
Ca(NO3)2×4H2O
Ca(NO3)2×4H2O
MgSO4×7H2O
K2HPO4×3H2O
K2HPO4×3H2O
Fe EDTA
MnSO4×4H2O
H3BO3
(NH4)6Mo7O24×4H2O
ZnSO4×7H2O
CuSO4×5H2O
K2SO4 (0.5 mol×L–1)
CaCl2×6H2O

1:30
–
+
+
+
–
+
+
+
+
+
+
–
+

1:1
+
–
+
+
–
+
+
+
+
+
+
–
–

30:1
+
–
+
–
+
+
+
+
+
+
+
+
–

a

+, the solution was included; –, the solution was omitted from the treatment.

al. 1996; Lentz and Dunson 1998; Lenssen et al. 1999;
Miller and Zedler 2003; Fraser and Karnezis 2005). For example, a decrease in root:shoot ratios has been shown with
increasing depth in water level (Figiel et al. 1995; Coops et
al. 1996; Edwards et al. 2003). Nutrient supply also affects
growth responses, plants not only grow at a slower rate at
low nutrient supply compared with high nutrient supply, but
they also increase their biomass allocation to roots (Poorter
and Nagel 2000). However, little is known about the interacting effects of water depth, nutrient supply ratios, and, in
particular, mycorrhizal colonization on total plant biomass
and biomass allocation patterns of wetland plants (see Stevens et al. 2002).
We tested the growth and biomass allocation of three wetland perennial plant species (Carex tribuloides Wahl., Phalaris arundinacea L., and Rumex orbiculatus Gray) in a 3
(species)  3 (N:P supply ratios)  3 (water levels)  2
(mycorrhizal) experimental design located in a climate-controlled greenhouse. The three main questions we address are
the following: (1) Does the presence of AM fungi affect the
growth and biomass allocation of the three wetland species
tested? (2) Are there interacting effects between the AM
fungi treatment and different N:P supply ratios in the growth
and biomass allocation of wetland plants? (3) Are there interacting effects between AM fungi treatment and water
depth in the growth and biomass allocation of wetland plants?
In addition to these three main questions, we were also able to
ask whether water depth, different nitrogen:phosphorus ratios, and the interaction between these two factors affected
plant growth and biomass allocation. Two-week-old seedlings were used because it is already well established that
flooding is a constraint to germination (van der Valk 1981;
Keddy and Ellis 1985; Casanova and Brock 2000; Jensen
2004), but the gap in the literature regards the growth and
biomass allocation of wetland seedlings to the interacting
effects of mycorrhizal fungi infection, N:P nutrient supply
ratios, and water depth.

Methods
A common wet meadow sedge, C. tribuloides, a wetland

forb, R. orbiculatus, and an invasive wet meadow grass,
P. arundinacea, were grown in a factorial experiment with
four treatments: 3 plant species  3 water depths (+2, 0,
and –4 cm relative to soil level)  3 N:P ratios (30:1, 1:1,
and 1:30)  2 mycorrhizal treatments (with or without an
AM fungal inoculant), replicated three times for a total of
162 individual potted plants. A randomized block design
was used.
We selected C. tribuloides, P. arundinacea, and R. orbiculatus because they occur together in wet meadow communities in the American Midwest and are all rhizomatous
perennials. Carex tribuloides, bristleback sedge, has culms
ranging from 3–9 mm wide and 60–90 cm tall. Short lateral
rhizomes, and rapid growth of the culms often cause the formation of a robust tussock (Crow and Hellquist 2000).
Cooke and Lefor (1998) measured a 50% mycorrhizal colonization for C. tribuloides. Phalaris arundinacea, reed canary grass, has culms ranging from 60–150 cm tall
(Hitchcock 1950). This fast-growing grass is considered an
invasive plant species. Mycorrhizal colonization for P. arundinacea has been shown to vary from 34% to 80% (Wetzel
and van der Valk 1998; Cooke and Lefor 1998). Rumex orbiculatus, great water dock, is a forb ranging in height from
80 to 200 cm, with flat, elongate leaves that can reach
60 cm in length (Crow and Hellquist 2000). We could find
no accounts of mycorrhizal colonization for R. orbiculatus,
but R. altissimus had colonization between 10% and 20%
(Cooke and Lefor 1998).
Seeds were germinated on filter paper in Petri dishes and
seedlings were transplanted to 473-mL pots (diameter of
8.9 cm) filled with autoclaved sand (11.4 cm depth). We
used sand, as opposed to a potting soil, to better control nutrient supply ratios (Wetzel and van der Valk 1998; Güsewell et al. 2003; McHugh and Dighton 2004). We sterilized
the sand in a steam autoclave (9 kg bag for 3 h) to kill any
live fungal spores. For the pots that received the AM fungal
inoculum, we removed the top 3 cm of sand for each cup,
mixed in 2.5 cm3 of a commercially supplied AM fungal inoculant (Root Boost Mycorrhizal Inoculant1, Gardens
Alive, Lawrenceburg, Indiana; includes mycorrhizal spores
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1119
Table 2. Percent mycorrhizal colonization by species, water level, and N:P ratio for plants
grown in the mycorrhizal inoculant treatment.
Water level
(cm)
–4
–4
–4
0
0
0
+2
+2
+2

N:P ratio
30:1
1:1
1:30
30:1
1:1
1:30
30:1
1:1
1:30

Carex tribuloides
(% colonization)
8.33
8.08
7.69
12.90
4.50
10.45
1.00
4.40
4.10
6.83 (1.22)

Phalaris arundinacea
(% colonization)
13.70
4.41
11.28
7.80
6.67
26.15
11.10
49.02
25.55
17.30 (4.73)

Rumex orbiculatus
(% colonization)
22.69
27.66
16.13
9.30
8.50
7.69
NA
NA
NA
15.33 (3.41)

Note: Mean percent colonization by species are presented at the bottom of each species column with ±1
SE in parentheses. NA, not available.

of Glomus spp. and Gigaspora spp.) and reapplied the sand
– AM-inoculant mixture as a top layer in the pots.
The commercial inoculant and the sterilized sand were analyzed for nitrate nitrogen, total nitrogen, and available
phosphorus. Nitrate N was determined on an autoanalyzer
using the copper–cadmium method (Technicon Industrial
Systems 1978). Total N was determined using the Dumas
automated combustion technique (McGill and Fiqueiredo
1993) using a CNS analyzer (Carla Erba, Milan, Italy).
Available phosphorus (ortho-P) was extracted using a 1:25
ratio of 1 g of sample and 25 mL of Kelowna extract (Van
Lierop 1988) after shaking at low speed for 1 h. Available P
was analyzed on the autoanalyzer using the ascorbic acid
method (Technicon Industrial Systems 1974).
The 473 mL sand-filled pots had holes in the bottom for
water uptake and a filter paper under the sand to prevent
loss of sand. Each of the sand-filled pots was placed within
an outer 946 mL pot that had holes drilled at the appropriate
level relative to the soil surface (either –4, 0, or +2 cm; see
Fraser and Karnezis 2005).
We waited for the development of primary leaves, which
was 14 d, before initiating the water depth and nutrient
treatments. The three different N:P ratio nutrient solutions
were made by modifying Rorison nutrient solution (Hendry
and Grime 1993; Table 1). Every fourth day, 50 mL of the
appropriate nutrient solution was applied to each pot: 30:1,
2.80 mg N and 0.09 mg P; 1:1, 2.80 mg N and 2.80 mg P;
1:30, 0.09 mg N and 2.80 mg P (relative amount of nutrients
per 50 mL solution). The 30:1 and 1:1 N:P nutrient supply
ratios are commonly used experimental combinations (Güsewell et al. 2003; Güsewell 2005). The 1:30 N:P ratio is less
commonly used in experimental designs and is also uncommon in natural conditions; however, we chose to compare
these extreme nitrogen and phosphorus limitations as treatment variables as a first step to understanding the interactions among mycorrhizal inoculation, N:P supply ratio, and
water depth on biomass accumulation and root:shoot ratio.
To better isolate the effects of the water depth treatment
on the three species, other abiotic factors were standardized.
A daily, 16-h photoperiod was provided by four, 1000 W,
High Pressure Sodium bulbs providing an average photosynthetically active radiation (measured with a LiCor LI-250
light meter) of 150.5 mmol×s–1×m–2 (± 8.2 SD) on the exper-

imental plants. Windows in the room were covered to prevent incident solar radiation. Temperature ranged between
20 and 24 8C and was maintained by air-conditioner units.
Humidity ranged between 40% and 50% and was self-maintained because of the evaporation of the water from all the
microcosms. After the initial 2 weeks of germination, plants
were watered every other day. A watering wand was used to
apply distilled water to the outer sleeve of each individual
microcosm. Algae grew in some of the flooded microcosms
but were physically removed immediately so that algae were
never present longer than 24 h.
Plants were harvested after 8 weeks of growth (6 weeks in
experimental treatment conditions). Each plant’s biomass
was divided into above-ground and below-ground biomass,
placed in a drying oven for 48 h at 80 8C, and weighed to
the nearest ten-thousandth gram. Plants that died during the
experiment were removed at time of death and weighed.
We randomly selected one of the three replicates that had
been treated with the mycorrhizal inoculant (for a total of 27
plants: 3 species  3 water depths  3 N:P supply ratios) to
measure mycorrhizal colonization. We also randomly selected one of each of the three wetland species that were
not inoculated with AM fungi for a measurement of mycorrhizal colonization to ensure the sterility of the autoclaved
sand. Ten 1-cm pieces of fine roots were cut from each of
the 30 plants and stained in 0.05% trypan blue in lactophenol following the methodology of Norris et al. (1994).
Stained roots were mounted on slides in lactophenol, and
colonization was estimated at  100 magnification using
the cross-hair method (McGonigle et al. 1990), with a minimum of 100 observations per slide, categorizing colonization by vesicle, arbuscule, both vesicle and arbuscule,
hyphae, or absence of fungal tissue.
Three one-way ANOVAs were done to test the effect of
species, N:P ratio, and water depth on percent mycorrhizal
colonization. A general linear model was applied to measure
for block effects by treatment variables on total biomass and
root:shoot ratios. No block effects were detected, so we proceeded by conducting four-way fixed-effects ANOVAs for
the combined AM fungi by species by water depth by N:P
ratio nutrient addition effect on total biomass and root:shoot
ratio. Tukey’s HSD test was run to determine statistical significance between means. Logarithmic transformations were
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Can. J. Bot. Vol. 83, 2005
Table 3. Results from two 4-way ANOVAs for dry biomass (mg) (log) and root:shoot
ratio (log), with species, N:P ratio, water level, and mycorrhizas as the independent
factors.
Log biomass
Source
Species
NP
Water
Myc
Species  NP
Species  water
Species  myc
NP  water
NP  myc
Water  myc
Species  NP  water
Species  NP  myc
Species  water  myc
NP  water  myc
Species  NP  water  myc
Error

df
2
2
2
1
4
4
2
4
2
2
8
4
4
4
8
108

F ratio
21.757
45.981
69.074
4.359
5.972
39.517
1.063
27.977
32.647
0.362
4.216
2.198
0.376
13.291
1.989

Log root:shoot
p
<0.001
<0.001
<0.001
0.039
<0.001
<0.001
0.349
<0.001
<0.001
0.697
<0.001
0.074
0.825
<0.001
0.055

F ratio
4.441
10.041
5.332
24.156
3.046
1.050
0.301
6.148
1.733
3.641
1.434
2.362
0.492
1.337
1.613

p
0.014
<0.001
0.006
<0.001
0.020
0.385
0.741
<0.001
0.182
0.029
0.190
0.058
0.741
0.261
0.129

Note: Error mean squares were 0.507 for log biomass and 0.021 for log root:shoot.

done on the biomass and root:shoot ratio data to improve
homoscedasticity and normality of residuals. The general
linear model, all ANOVAs and the post-hoc analyses were
conducted using Systat 8 (SPSS 1998).

Results
Mycorrhizal colonization was observed in all the species
and in all of the experimental treatments, with the exception
of R. orbiculatus in flooded conditions (+2 cm) because of
one hundred percent mortality resulting in insufficient roots
for staining (Table 2). None of the ANOVAs to test mean
differences in percent mycorrhizal colonization were significant (p < 0.05); however, the effect of species on percent
colonization was significant at a p < 0.10 (F = 2.752; p =
0.087; Table 2). Carex tribuloides had the lowest percent
colonization. Mycorrhizal colonization was not found in the
three plants randomly sampled from those plants grown
without mycorrhizal inoculant.
The soil chemical analysis showed that the inoculant had
6 ppm NO3, 0.03% total N, and 5 ppm available P. Therefore, by volume, the inoculant contained 0.015 mg NO3 and
0.013 mg available P, which were very low levels and unlikely to cause indirect nutrient addition effects especially
considering the rate of nutrient solution applied as treatment.
The autoclaved sand had 4 ppm NO3, 0.01% total N, and
1 ppm available P.
The two four-way fixed-effect ANOVAs showed that dry
biomass and root:shoot ratio were significantly affected
(p < 0.05) by AM fungi, species, N:P ratio, and water
depth (Table 3). The biomass of plants grown in the presence of AM fungi was significantly greater than those
plants grown without AM fungi (Fig. 1a). Root:shoot ratio
in the presence of mycorrhizal fungi was significantly
lower than the mycorrhizal absent treatment (Fig. 1a).
Carex tribuloides had a significantly lower dry biomass

and root:shoot ratio than P. arundinacea and R. orbiculatus
(Fig. 1b). The N:P ratio with the lowest biomass was 1:30,
and the 1:1 N:P ratio had the greatest biomass (Fig. 1c).
The N:P nutrient supply ratio of 1:1 had the lowest root:shoot
ratios compared with 1:30 and 30:1 (Fig. 1c). The lowest
biomass was found in the flooded (+2 cm) water level
treatment, which was significantly lower than the biomass
at 0 and –4 cm water depths (Fig. 1d). The lowest root:shoot ratios were found in the elevated (–4 cm) water level
treatment (Fig. 1d).
The significant two-way interaction effects on dry biomass shown by the four-way fixed-effect ANOVA were
N:P ratio  AM fungi; species  N:P; species  water
depth; and, N:P  water depth (Table 3). Dry biomass was
significantly greater when the mycorrhizal innoculant was
present at the 30:1 N:P ratio compared with the mycorrhizal
absent treatment (Fig. 2a). However, biomass was greater in
the AM fungi absent treatment at the 1:1 N:P ratio compared with the AM fungi present treatment. There was no
significant interaction effect on biomass between water
depth and AM fungi (Fig. 2b). R. orbiculatus and P. arundinacea biomass responded similarly to N:P nutrient supply
ratio, with maximum biomass at the 1:1 ratio and minimum
biomass at the 1:30; whereas C. tribuloides biomass showed
no response to N:P ratios (Fig. 2c). Carex tribuloides also
showed no growth response to water depth (Fig. 2d), but
P. arundinacea and R. orbiculatus had greater biomass at 0
and –4 cm water depth. Figure 2e, the N:P  water depth
interaction, demonstrated that the dry biomass at 1:30 N:P
ratio was not affected by the depth of the water level,
whereas biomass at +2 cm water depth was significantly
less at N:P ratios of 1:1 and 30:1 compared with water
depths at 0 and –4 cm.
The significant two-way interaction effects on root:shoot
ratios shown by the four-way fixed-effect ANOVA were the
following: water depth  AM fungi, species  N:P ratio,
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1121

Fig. 1. Mean dry biomass and root:shoot ratio of plants separated by treatment: AM fungi (a), species (b), N:P ratio (c), and water depth
(d). Error bars represent ±1 SE. The species are Carex tribuloides, Phalaris arundinacea, and Rumex orbiculatus. N:P ratios are 1:30, 1:1,
and 30:1. Water depths are +2, 0, and –4 cm relative to soil level. AM fungi are either absent or present. Bars in figures sharing the same
letter are not significantly different using Tukey’s HSD.

and N:P ratio  water depth (Table 3). The water depth 
mycorrhizal fungi interaction showed that when the mycorrhizal inoculant was absent, the root:shoot ratio was significantly lower at 0 and –4 cm water level compared with the
AM fungi absent treatment, but there was no such difference
between water depth treatments in the presence of AM fungi
(Fig. 2b). For C. tribuloides, the root:shoot ratio was signifi-

cantly lower in the N:P supply ratio treatment of 1:30 compared with treatment of 1:1 and 30:1 (Fig. 2c). The lowest
root:shoot ratio for P. arundinacea was at 1:1 N:P ratio, and
R. orbiculatus showed no significant difference in root:shoot
ratio across the N:P supply ratios (Fig. 2c). The N:P 
water depth interaction showed that the root:shoot ratio
under the 1:30 and 1:1 N:P ratio was lowest at +2 cm
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Can. J. Bot. Vol. 83, 2005

Fig. 2. Mean dry biomass and root:shoot ratio of plants separated by two-way treatment interactions of N:P ratio  AM fungi (a), water
depth  AM fungi (b), species  N:P ratio (c), species  water depth (d) , and N:P ratio  water level (e). The species are Carex tribuloides, Phalaris arundinacea, and Rumex orbiculatus. N:P ratios are 1:30, 1:1, and 30:1. Water depths are +2, 0, and –4 cm relative to soil
level. AM fungi are either absent or present.

water depth, while there was no significant effect on root:shoot
ratios at 30:1 N:P supply ratio (Fig. 2e). There were no
significant two-way interactions on root:shoot ratios between N:P ratio  AM fungi (Fig. 2a) and species 
water depth (Fig. 2d).

Discussion
Our primary objective was to test the effects of the mycorrhizal inoculant on 8-week-old wetland plant growth and
biomass allocation by plant species, water level, and N:P
supply ratios. Our examination of percent mycorrhizal colonization was to ensure that the inoculant was effective, and
that the non-mycorrhizal treatment plants were indeed free
from colonization by AM fungi, which seemed to be satisfied on both counts.
Our first question was whether the AM fungi affected
growth and biomass allocation of the wetland plants. We
found that the total dry biomass of the three wetland plants
combined (C. tribuloides, P. arundincacea, and R. orbiculatus) was greater in the presence of AM fungi, and that
root:shoot ratios were reduced nearly by half in the presence of AM fungi. These results are concordant with other
studies investigating the mycorrhizal effects on plant growth
(Oliver et al. 1983; McHugh and Dighton 2004; see also
Smith and Read 1997 for review). The low root:shoot ratio
we found in the presence of AM fungi is a typical response of terrestrial plants (Smith and Read 1997), and
has also been documented in two salt marsh grasses
(McHugh and Dighton 2004). However, in some cases the
growth of juvenile plants has been shown to be negatively
associated with mycorrhizal inoculation owing to the carbon demand of the fungi (Smith et al. 1979). Indeed, the
dependency of a mycorrhizal association may vary not
only by plant species but with the stage of growth of the
plant, and can therefore potentially influence plant establishment and the responsiveness of the plant to mycorrhizal
colonization (Allsopp and Stock 1992).
Our second question addressed whether there was an interacting effect of AM fungi and N:P supply ratios on
growth and biomass allocation. The interaction between the
mycorrhizal treatment and N:P supply ratio was significant
for total dry biomass, but not the root:shoot ratio. Plants
grown at 30:1 N:P ratio had significantly greater biomass
when grown in the treatment containing the mycorrhizal inoculant. Plants exposed to AM fungi in our study seemed to
be more efficient at using very low levels of phosphorus
than plants without AM fungi, which was also demonstrated
by Oliver et al. (1983), Dhillion (1992), and McHugh and
Dighton (2004). White and Charvat (1999), though, found
that mycorrhizal colonization of Lythrum salicaria (a fastgrowing wetland plant) had no effect on total biomass under
low phosphorus supply. Stevens et al. (2002) found increased AM colonization of L. salicaria under low phosphorus supply (<5 mg/L), but the presence of AM fungi did not

affect plant performance. The apparent cost to mycorrhizal
infection was demonstrated in our experiment within the 1:1
N:P ratio treatment, with greater biomass found in plants
without AM fungi.
It is well documented that wetland plant seedlings have
reduced growth and survivorship under flooded conditions
(Casanova and Brock 2000; Edwards et al. 2003; Fraser and
Karnezis 2005). Our third question was whether there was a
significant interaction between the mycorrhizal inoculant
and water depth on biomass and root:shoot ratios. Our results showed no such affect on biomass. Root:shoot ratios
were affected such that the allocation of biomass to roots
was greater in the absence of AM fungi at 0 and –4 cm
water depths compared with +2 cm. We suggest that such a
change in biomass allocation may affect long-term biomass
accumulation, survivorship, and competitive ability. For example, plants with AM fungi that allocate more resources to
above-ground growth may exhibit a greater ability to maintain photosynthesis and a greater competitive advantage for
light.
There was no difference in biomass or root:shoot ratios
between the presence and absence of AM fungi under
flooded conditions. Therefore, mycorrhizal inoculation of
the three seedling species under flooded conditions did not
affect the growth parameters tested. However, our experiment was not designed to tell us whether seedlings inoculated with mycorrhiza under elevated conditions (–4 cm)
are then more or less resistant to a flooding treatment. Hydrologic regimes in natural wetlands are highly variable;
therefore, it would be interesting to test whether plants
colonized by mycorrhizal fungi under non-flooded conditions are then more resistant to flooding. Mycorrhizal colonization has been shown to alter water relations of
terrestrial plants (Fitter 1988; Koide 1993). Perhaps wetland
plant water relations are also affected by mycorrhizas resulting in an increased tolerance of flooded conditions; that is,
through (1) increases in transpiration rates and stomatal conductance (Fitter 1988; Koide 1993), and (2) increased root
aeration due to increased photosynthesis from a greater allocation to above-ground growth.
There was no difference in total biomass or root:shoot ratios between plant species with regard to mycorrhizal treatment, which suggests that C. tribuloides, P. arundinacea,
and R. orbiculatus respond similarly in terms of growth and
biomass allocation to mycorrhizal inoculation. Before 1987,
it was generally thought that the sedge family was non-mycorrhizal, but studies since have proven otherwise (see Muthukumar et al. 2004 for review). The sedge in our study
(C. tribuloides) was mycorrhizal and performed similarly,
with respect to the mycorrhizal inoculant treatment, to the
other species. There is no evidence from our study to suggest that P. arundinacea, the fast-growing invasive, had a
comparatively greater growth benefit accruing from the mycorrhizal inoculation than C. tribuloides and R. orbiculatus.
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Pairwise competition experiments under similar experimental conditions involving mycorrhizal inoculation would be
needed to better characterize species-level effects due to
AM fungi colonization.
We found that the effects of water level and N:P supply
ratio on the growth and biomass allocation of the three wetland plants tested generally confers with other studies.
Flooding has been shown to cause a reduction in growth and
a reduction in the allocation to root tissue resulting in reduced root:shoot ratios for these same wetland species
(Fraser and Karnezis 2005). The exception in our study was
C. tribuloides, which showed no response in growth or root:shoot ratio to water level. However, this may be explained
by the slower growth rate of C. tribuloides in combination
with the short time-length of the experiment (only eight
weeks). Fraser and Karnezis (2005) grew C. tribuloides,
P. arundinacea, and R. orbiculatus over a six-month period.
Nitrogen seemed to limit growth to a greater degree than
phosphorus. Despite the 30-fold greater phosphorus supply,
plants in the 1:1 treatments produced only a twofold biomass compared with the 30:1 treatment. This suggests that
plants were able to utilize low levels of phosphorus, or that
the phosphorus levels at the N:P 1:1 treatment was excessive. Other studies that manipulated N:P supply ratios also
found that biomass increased with an increase in the N:P
supply ratio up to 45:1 (Güsewell et al. 2003). Root:shoot
ratios were also significantly greater when N:P supply ratios
were at 1:30, demonstrating an increase in root allocation
under limited nutrient conditions (Poorter and Nagel 2000).
Only limited statistical analyses could be conducted on
the percent mycorrhizal colonization data, but the percent
mycorrhizal colonization of C. tribuloides was generally
less than the other two plants. Although this difference was
significant at p < 0.10 it did not seem to have an effect on
plant growth and biomass allocation as evidenced by the
data discussed above. However, more research is needed to
determine whether there is a correlation between percent
colonization and plant growth performance.

Acknowledgements
We thank Traci Branch who autoclaved the sand. Two
Akron City Public school teachers, Morgan Greene and
Stacy Beck-Ross, helped prepare and run the experiment.
Comments from two anonymous reviewers improved the
manuscript. Funding was provided by the National Science
Foundation (GK-12 grant No. 0086378).

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