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ORIGINAL PAPER
Effects of arbuscular mycorrhizal fungi on seedling growth
and development of two wetland plants, Bidens frondosa L.,
and Eclipta prostrata (L.) L., grown under three levels
of water availability
Kevin J. Stevens &Christopher B. Wall &Joel A. Janssen
Received: 24 June 2010 / Accepted: 4 July 2010
#Springer-Verlag 2010
Abstract To identify the importance of arbuscular mycor-
rhizal fungi (AMF) colonizing wetland seedlings following
flooding, we assessed the effects of AMF on seedling
establishment of two pioneer species, Bidens frondosa and
Eclipta prostrata grown under three levels of water
availability and ask: (1) Do inoculated seedlings differ in
growth and development from non-inoculated plants? (2)
Are the effects of inoculation and degree of colonization
dependent on water availability? (3) Do plant responses to
inoculation differ between two closely related species?
Inoculation had no detectable effects on shoot height, or
plant biomass but did affect biomass partitioning and root
morphology in a species-specific manner. Shoot/root ratios
were significantly lower in non-inoculated E. prostrata
plants compared with inoculated plants (0.381 ±0.066 vs.
0.683±0.132). Root length and surface area were greater in
non-inoculated E. prostrata (259.55±33.78 cm vs. 194.64±
27.45 cm and 54.91± 7.628 cm
2
vs. 46.26 ±6.8 cm
2
,respec-
tively). Inoculation had no detectable effect on B. frondosa
root length, volume, or surface area. AMF associations
formed at all levels of water availability. Hyphal, arbuscular,
and vesicular colonization levels were greater in dry
compared with intermediate and flooded treatments. Meas-
ures of mycorrhizal responsiveness were significantly de-
pressed in E. prostrata compared with B. frondosa for total
fresh weight (−0.3± 0.18 g vs. 0.06± 0.06 g), root length
(−0.78±0.28 cm vs.−0.11±0.07 cm), root volume (−0.49±
0.22 cm
3
vs. 0.06± 0.07 cm
3
), and surface area (−0.59±
0.23 cm
2
vs.−0.03±0.08 cm
2
). Given the disparity in species
response to AMF inoculation, events that alter AMF
prevalence in wetlands could significantly alter plant
community structure by directly affecting seedling growth
and development.
Keywords Arbuscular mycorrhizal fungi .Wetlands .
Mycorrhizal responsiveness .Flooding .Bidens frondosa .
Eclipta prostrata
Introduction
While the effects of AMF on plant physiology (Auge 2001;
Evelin et al. 2009; Smith et al. 2010), soil stability and
nutrient cycling (Bethlenfalvay and Linderman 1992;
Bethlenfalvay and Schüepp 1994; Jastrow and Miller
1991; Rillig and Mummey 2006), and plant community
structure (Daleo et al. 2008; Escudero and Mendoza 2005;
Jackson and Mason 1984; van der Heijden 1998)in
terrestrial environments are well known, the importance of
AMF in aquatic and wetland habitats has received little
attention (Cornwell et al. 2001; Muthukumar et al. 2004;
Stevens et al. 2002; Stevens and Peterson 2007; Turner and
Friese 1998). Historically, AMF were thought to be absent
or rare in wetland plants (Crawford 1992; Khan and Belik
1995; Khan 2004; Peat and Fitter 1993). In part, this was
attributable to a perceived inability of AMF to survive
anaerobic conditions found in reduced wetland soils and/or
a decreased need for nutrient augmentation by AMF since
plants could potentially acquire nutrients from water and
the substrate across the leaf and root surfaces (Cooke et al.
1993; Peat and Fitter 1993). An increasing number of
studies have, however, revealed that many wetland plant
species harbor AMF and AMF have been found in wetland
K. J. Stevens (*):C. B. Wall :J. A. Janssen
Department of Biological Sciences, Institute of Applied Sciences,
University of North Texas,
Denton, TX 76203-0559, USA
e-mail: kjstevens@unt.edu
Mycorrhiza
DOI 10.1007/s00572-010-0334-2
habitats (Cooke and Lefor 1998) ranging from bottom-
land hardwood forest (Stevens et al. 2010), degraded
Cypress swamps (Kandalepas et al. 2010), marshy
environments (Bohrer et al. 2004; Radhika and Rodrigues
2007), groundwater-fed wetlands (Turner et al. 2000),
freshwater fens (Bohrer et al. 2004;Cornwelletal.2001;
Šraj-Kržičet al. 2006), calcareous fens (Wolfe et al.
2006), and salt marshes (Brown and Bledsoe 1996;
Carvalho et al. 2003). The prevalence of AMF in
wetlands is now recognized; the dependency of wetland
plants on their AMF partners and the factors that affect
levels of AMF colonization in wetland habitats are poorly
understood (Cornwell et al. 2001; Muthukumar et al.
2004; Stevens et al. 2002; Stevens and Peterson 2007;
Turner and Friese 1998).
Wetlands are characterized by the presence of flooded
or saturated soils for at least part of the growing season
(Cowardin et al. 1979). Interspecific differences in the
capacity to tolerate or avoid conditions associated with
flooded or saturated soils (i.e., reduced soil oxygen
availability, altered nutrient availability, and the build up
of toxic ions) are a major determinant of wetland plant
community structure (Keddy 2002; Mitsch and Gosselink
2000). While seasonal or episodic flooding maintain
wetland plant community structure through elimination
of less flood tolerant upland species (Middleton 1999),
prolonged flooding can result in widespread vegetation
loss. Following severe flooding and vegetation loss,
wetland plant communities may reestablish through con-
tributions from the soil seed bank (see Keddy 2002;van
der Valk 1981). Several studies of terrestrial habitats
suggest that plants established following disturbance tend
to be non-mycorrhizal (see Reeves et al. 1979; Smith et al.
2010; Smith and Read 2002), however, Stevens et al.
(2010) found AMF colonization in 31 plant species
established following a prolonged flood in a remnant
bottomland forest in north central Texas. While this
implies a role of AMF in the reestablishment of wetland
plant species following disturbance, experimental support
is lacking.
The seedling stage is the most vulnerable stage in a
plant’s life cycle (Grubb 1977), and responses to flooding at
the seedling stage are considered one of the most important
determinants of species composition in bottomland swamps
(Bedinger 1978), and possibly other types of wetlands
(Middleton 1999). While understanding the factors affect-
ing seedling establishment and survival are crucial for
wetland restoration and management (Keddy 2002; Mid-
dleton 1999), as well as community/population diversity
and dynamics (Grime and Hiller 1992), little is known of
the responses of seedlings to various hydrologic treatments
(Fraser and Feinstein 2005), and we are unaware of any
studies that have evaluated the effects of AMF on wetland
seedlings. Previous studies that have sought to quantify
effects of AMF on wetland plant growth and identify
interaction effects of AMF and water availability have
examined effects on established plants (see Carvalho et al.
2003; Garcia et al. 2008; Ipsilantis and Sylvia 2007; Miller
2000; Miller and Sharitz 2000; Osundina 1998;Šraj-Kržič
et al. 2006; Stevens and Peterson 1996; Stevens and
Peterson 2007; Wolfe et al. 2006). Consequently, several
questions remain regarding the role of AMF in early
seedling establishment in wetlands and the conditions
required for AM colonization of wetland seedlings. While
there is a general trend towards a reduction in AM
colonization with increasing water availability (Escudero
and Mendoza 2005; Miller 2000; Osundina 1998; Stevens
and Peterson 1996), the high incidence of AM colonization
in seedlings established immediately after a 100-year flood
in a remnant bottomland hardwood forest suggests that
colonization can occur rapidly and possibly under wet
conditions (Stevens et al. 2010).
To identify the importance of AMF colonizing wetland
plants following flooding, we assess the effects of AMF on
seedling establishment of two pioneer species, Bidens
frondosa L. and Eclipta prostrata (L.) L. grown under
three levels of water availability. We ask the following:
1. Do inoculated seedlings differ in their growth and
development from non-inoculated plants?
2. Are the effects of inoculation and degree of coloniza-
tion dependent on water availability?
3. Do plant responses to inoculation differ between two
closely related species?
Materials and methods
Experimental design
The experiment was a 2×2 ×3 randomized complete block
design with two wetland plant species (B. frondosa L. and
E. prostrata (L.) L.), two AMF treatments (inoculated and
non-inoculated), and three levels of water availability
(water levels maintained at the soil surface, 3.5 cm below
the soil surface, and no-standing water but watered twice
daily). Individually potted seedlings were placed in 29.3 L
(67.8×40.1 ×17.5 cm) plastic trays and grown on shelves in
a growth room at the University of North Texas. A total of
12 plants were grown in each tray (two species ×two levels
of AMF×three subsamples (plants)/tray); all plants were
randomized within trays and all trays were randomly
assigned a position and treatment within shelves. Each of
the five shelves used constituted one block and contained
one tray for each of the three water level treatments.
Shelves were lit by a bank of eight high intensity
Mycorrhiza
fluorescent lights (Sun System Tek Light T-5 high output
fluorescent fixture with three VitaLume Plus Bloom and
three VitaLUME Plus Grow bulbs) providing an average of
459 μmols/m
2
PAR on 16/8-light/dark cycle. Temperature
was maintained at 23°C.
Establishment of AMF cultures
Mycorrhizal cultures were established using riparian
soils obtained from the Elm Fork of the Trinity River,
Denton, Texas. Five 5-gallon buckets of soil were
obtained and spores extracted following the methods
described by Brundrett et al. (1996). Five trays (60 ×
30×15 cm) were filled with locally obtained masonry
sand to which the isolated, washed spores were added.
Three native, locally abundant wetland species (B.
frondosa,E. prostrata and Sesbania herbacea (Mill.)
McVaugh) were germinated in Petri dishes on the
surface of moist filter paper then transplanted to trays.
Cultures were maintained under growth room condi-
tions. To prepare the inoculum, seedlings were uprooted
from the culture trays, the roots excised, washed, and
blended to obtain a slurry.
Seedling establishment
Seeds of B. frondosa and E. prostrata were collected in the
fall of 2007 from the floodplain of the Elm Fork of the
Trinity River and stored at room temperature. Seeds were
germinated on the surface of moist filter paper in sealed
15 cm Petri dishes under growth room conditions.
Germination began within 2 days, and after 5 days the
germinated seeds were transplanted into an 8 ×8× 9 cm
plastic pots. Pots were filled with masonry sand and for the
inoculated treatments, 15 ml of inoculum was added to a
small well made in the sand at the center of the pot. Each
pot was internally lined with a piece of Whatman # 41 filter
paper to retain the sand and prevent cross-contamination.
For the two wettest treatments, a 6 mm stand-pipe was used
in each tray to maintain water levels at the soil surface and
3.5 cm below the soil surface. A Manostat Carter Multi-
Channel Precision 12/6 cassette pump (Cole-Parmer Instru-
ment Co., Vernon Hills, IL) maintained a continuous flow
of 1/64 Long Ashton’s nutrient solution (Hewitt 1966)—
delivering approximately 6.4 mg/l of phosphorous—at an
average flow of 0.085 L/h for the intermediate and wet
microcosms. Nutrient solution was made up in a 70 L
reservoir and refilled every 48 h. For the driest treatment,
plants were watered twice daily with 25 ml of 1/64 strength
Long Ashton nutrient solution. Prior studies indicate that
this level of nutrient availability is sufficient to maintain
plant growth without inhibiting AM colonization (Stevens
et al. 2002; White and Charvat 1999).
Harvesting and assessment
Harvesting began 50 days after seedlings were trans-
planted and continued for a 48-h period, with each block
being harvested within a 2-h period. Stems were removed
at the soil surface and main stem height and fresh weight
was recorded. Stems were bagged and dried at 40°C for
dry mass determination. Roots were freed of the soil
substrate by gentle agitation of the root system under
water. To prevent root loss water was filtered through
250 μm sieve and any severed roots collected. Root fresh
weight was determined and the root system was digitized
using an Epson Expression 10000 XL color photo
scanner at 400 dpi. After scanning, roots were stored in
50% ethanol. Root length, volume and surface area were
determined using WinRHIZO PRO (ver 2007c Regent
Instruments, Quebec, Canada). A subsample of non-
woody lateral roots was obtained for determination of
AMF colonization levels. Roots were cleared by auto-
claving in 10% potassium hydroxide for 20 min and then
stained with 0.1% Chlorazol Black E for 40 min in an
autoclave at 121°C (Brundrett et al. 1996). Roots were
destained and stored in 50% glycerol prior to mounting on
slides in 50% glycerol (Phillips and Hayman 1970). Slides
were viewed with 200× magnification using a Zeiss Axio
image microscope and images obtained with a Zeiss
Axiocam MRC-5 camera. Colonization levels were
assessed using a modified grid line intersect procedure
(McGonigle et al. 1990). A total of 100 fields of view
were assessed for each sample.
Data analysis
Plant growth responses were analyzed using a three-way
analysis of variance (ANOVA) in SAS 9.1 (SAS Institute
Cary, NC), with species, water availability and AMF
colonization as main effects and species x water availabil-
ity, species x AMF colonization, water availability x AMF
colonization and species x water availability x AMF
colonization as interaction effects. Blocks and subsamples
within blocks were treated as random effects. To meet
requirements of normality and equal variance shoot height,
shoot fresh and dry weight, root length, surface area and
volume were log transformed, root fresh weight was square
root transformed and shoot/root (S/R) fresh weight ratios
were analyzed using ranked data. AMF colonization levels
were analyzed using a two-way ANOVA in SAS with
species and water availability as main effects and species x
water availability as the interaction effect. To meet
assumptions of normality and equal variance analyses were
conducted on ranked data. When significant main effects or
interaction effects were detected, multiple comparisons
were conducted using the Tukey–Kramer option in SAS.
Mycorrhiza
Mycorrhizal responsiveness (MR) was assessed as the
difference in morphometric characteristics of inoculated
and non-inoculated plants relativized through division by
the response of inoculated plants (Janos 2007). Since there
were three plants for each treatment combination in each
block, MR was calculated for each species at each level of
water availability in each block. MR was assessed for shoot
height, shoot fresh and dry weight, root and total fresh
weight, root length, volume and surface area. MR was
analyzed using a two-way ANOVA with species and water
availability as main effects and species x water availability
as interaction effect. When significant main effects or
interaction effects were detected, multiple comparisons
were conducted using the Tukey–Kramer option in SAS.
For all figures untransformed means are presented ±1
standard error.
Results
Shoot height was affected by water availability, species and
the water availability x species interaction (Table 1). Shoot
height was consistently higher for B. frondosa compared
with E. prostrata at all levels of water availability (Fig. 1)
and for both species was significantly higher in the dry
treatment compared with the intermediate and flooded
treatment. Shoot fresh and dry weight, root fresh weight
and total fresh weight were affected by water availability,
species and the water availability x species interaction
(Table 1). While there were no significant differences in
shoot fresh weight, root fresh weight or total fresh weight
between species in the dry treatment (Fig. 2a–c), all were
significantly lower in E. prostrata compared with B.
frondosa in the intermediate and wet treatments. For both
species, shoot fresh weight and root fresh weight were
significantly higher in the dry compared with the interme-
diate and wet treatments. Shoot dry weight was significant-
ly lower in the intermediate and wet treatments compared
with the dry treatments for both E. prostrata and B.
frondosa, and was significantly lower in E. prostrata
compared with B. frondosa at all levels of water availability
(Fig. 2d).
Shoot/root fresh weight ratio (S/R) differed among
species, inoculation (AMF), and was affected by the
interaction of water availability x species and species x
AMF (Table 1). S/R was lower in E. prostrata compared
with B. frondosa in the dry treatment but did not differ
among species in the intermediate and wet treatments
(Fig. 3). Within species, S/R was significantly lower in
the dry treatment compared with the wet treatment for B.
Table 1 Summary table of three-way ANOVA assessing the effects of water availability (Water) and AMF inoculation (AMF) on the growth of
Eclipta prostrata and Bidens frondosa
Response
variable
Water Sp AMF Water×Sp Water×AMF Sp×AMF Water×Sp×
AMF
Fp
Value
Fp
Value
Fp
Value
Fp
Value
Fp
Value
Fp
Value
Fp
Value
Shoot
height
55.97 <.0001 437.77 <.0001 0.97 0.3252 12.76 <.0001 0.24 0.7864 0.52 0.4732 0.66 0.5158
Shoot
fresh
weight
115.81 <.0001 115.71 <.0001 1.33 0.2498 12.91 <.0001 0.28 0.7571 0.41 0.5205 0.63 0.5316
Shoot dry
weight
132.58 <.0001 162.27 <.0001 1.92 0.1681 11.08 <.0001 0.53 0.5870 2.27 0.1341 1.30 0.2745
Root fresh
weight
114.24 <.0001 46.37 <.0001 0.60 0.4397 8.92 0.0002 0.63 0.5351 1.10 0.2949 0.14 0.8720
Shoot/root
fresh
weight
2.61 0.0764 5.39 0.0215 9.71 0.0022 7.11 0.0011 1.68 0.1901 9.65 0.0022 1.52 0.2224
Total fresh
weight
120.23 <.0001 57.62 <.0001 0.00 0.9619 6.92 0.0013 0.24 0.7880 1.12 0.2919 0.11 0.8993
Root
length
55.70 <.0001 30.34 <.0001 9.24 0.0028 13.37 <.0001 0.45 0.6391 4.33 0.0391 0.31 0.7324
Root
volume
98.50 <.0001 59.21 <.0001 1.71 0.1927 11.31 <.0001 1.28 0.2799 5.12 0.0250 0.18 0.8362
Root
surface
area
76.77 <.0001 40.45 <.0001 4.51 0.0352 13.28 <.0001 1.18 0.3115 4.43 0.0369 0.16 0.8537
Significant effects (p=< 0.05) are in bold
Sp species
Mycorrhiza
frondosa. Although there were no significant differences in
S/R among species in inoculated plants, in non-inoculated
plants S/R was significantly lower in E. prostrata compared
with B. frondosa (Fig. 3). For E. prostrata S/R was
significantly lower in non-inoculated compared with inoc-
ulated plants.
Root length, volume and surface area differed among
species, AMF, and were affected by the interactions of
water availability x species and species x AMF (Table 1).
While there were no significant differences in root length,
volume and surface area between species in the dry
treatment, these were significantly greater in B. frondosa
in the intermediate and wet treatments compared with E.
prostrata (Fig. 4a–c). For both species root length, volume
and surface area was significantly greater in the dry
treatment compared with the intermediate and wet treatment
(Fig. 4a–c). There were no detectable effects of inoculation
on root length, volume and surface area of B. frondosa
(Fig. 4a–c), however, root length and surface area were
greater in non-inoculated compared with inoculated treat-
ments for E. prostrata. With the exception of root length in
the non-inoculated plants, root length, surface area and
volume were significantly greater in B. frondosa compared
with E. prostrata.
Hyphal, vesicular and arbuscular colonization levels
were affected by water availability, however, there were
no significant differences attributable to interspecific
responses or the interaction of species x water availability
for any measure of colonization (Table 2). All three
measures of colonization were significantly greater in the
dry treatment compared with the intermediate and dry
treatments with no significant differences between interme-
diate and wet treatments (Fig. 5). There were no significant
effects of water availability, species or the interaction of
species x water availability on mycorrhizal responsiveness
(MR) for shoot height, shoot fresh and dry weight and root
fresh weight (Table 3; Fig. 6). The MR of each species
differed for total fresh weight, root length, volume and
surface area, however MR was not significantly affected by
E. prostrata
B. frondosa
Shoot height (cm)
Dry Int Wet
A
B
b
B
b
**
*
a
Fig. 1 Effect of water availability on shoot height of Eclipta prostrata
and Bidens frondosa grown under three levels of water availability
(Dry, intermediate (Int), and Wet). Different uppercase letters indicate
significant differences (p<0.05) among E. prostrata plants grown
across levels of water availability. Different lowercase letters indicate
significant differences among B. frondosa plants grown across levels
of water availability. Asterisk indicate significant differences between
species within levels of water availability. Raw means are presented
with bars indicating standard error
(b)
(c)
(a)
(d)
Dry Int Wet
A
a
B
b
B
b
**
E. prostrata
B. frondosa
Shoot fresh weight (g)
a
Dry Int Wet
B
A
B
bb
**
Root fresh weight (g) Total fresh weight (g)
B
Dry Int Wet
Dr
y
Int Wet
Aa
B
bb
**
Shoot dry weight (g)
A
a
B
b
B
b
**
*
Fig. 2 Effect of water availability on shoot fresh weight (a), root
fresh weight (b), total fresh weight (c), and shoot dry weight (d)of
Eclipta prostrata and Bidens frondosa grown under three levels of
water availability (Dry, intermediate (Int), and Wet). Different
uppercase letters indicate significant differences (p<0.05) among E.
prostrata plants grown across levels of water availability. Different
lowercase letters indicate significant differences among B. frondosa
plants grown across levels of water availability. Asterisk indicate
significant differences between species within levels of water
availability. Raw means are presented with bars indicating standard
error
Mycorrhiza
water availability or the interaction of species x water
availability (Table 3). Total fresh weight, root length,
volume and surface area were significantly depressed in
E. prostrata compared with B. frondosa (Fig. 6).
Discussion
Reductions in AMF colonization levels with increasing
levels of water availability are consistent with previous
field and greenhouse/growth-room studies (Stevens and
Peterson 1996; Rickerl et al. 1994). Water availability is not
the sole factor affecting AMF colonization levels in wetland
plants, however. Stevens and Peterson (2007) found that
phosphorus availability and not water availability led to
reduced levels of AMF colonization in the amphibious
species Lythrum salicaria, while Carvalho et al. (2003)
found that salinity had a greater effect on colonization of
Aster tripolium L. than flooding. Furthermore, seasonal
variability in colonization levels attributable to species-
specific differences in phenology have been found in salt
marsh (Carvalho et al. 2001), fen and fresh water marsh
plants (Bohrer et al. 2004). While AMF colonization levels
may be reduced in flooded soils compared with non-
flooded soils, colonization levels of E. prostrata and B.
frondosa in our flooded treatments were relatively high
(<20%) compared with colonization in comparable treat-
ments of other wetland plant species (i.e., <6% in Typha
latifolia L., Ipsilantis and Sylvia 2007) but comparable to
levels found in continuously flooded A. tripolium,a
member of the same family as E. prostrata and B. frondosa.
The survival of non-inoculated E. prostrata and B. frondosa
plants indicates that these are facultative mycorrhizal
species.
AMF colonization has been documented in flooded roots
of several emergent wetland plant species (i.e., Bagyaraj et
al. 1979; Stevens and Peterson 1996; Weishampel 2005;
Šraj-Kržičet al. 2006), yet what is not clear from these
studies is when initial colonization occurred. In field studies
it is often not possible to discern between colonization
occurring when soils were flooded or during drawdown
(Stevens and Peterson 1996; Ray and Inouye 2006). In
greenhouse/growth room studies inoculation is often fol-
lowed by a pretreatment period to facilitate AMF coloni-
ab A
b
B
A
aA
*
E. prostrata
B. frondosa
Shoot / root fresh weight
Dry Int Wet -AMF +AMF
A
a
a
*
Fig. 3 Effect of water availability and AMF inoculation on shoot/root
fresh weight ratio of Eclipta prostrata and Bidens frondosa grown
under three levels of water availability (Dry, intermediate (Int), and
Wet). Different uppercase letters indicate significant differences (p<
0.05) among E. prostrata plants grown across levels of water
availability. Different lowercase letters indicate significant differences
among B. frondosa plants grown across levels of water availability.
Bold and italics letters indicate differences in AMF inoculation.
Asterisk indicate significant differences between species within levels
of water availability and inoculation. Raw means are presented with
bars indicating standard error
Root length (cm)
a
Dry Int Wet -AMF +AMF
a
E. prostrata
B. frondosa
*
A
B
b
B
b *
Aa
B
*
a
a
Dry Int Wet
A
B
b
B
b
**
-AMF +AMF
A
a
A
*
*
Root volume (cm3)
A
a
Dry Int Wet -AMF +AMF
B
b
B
b
**
Aa
B
a
*
*
Root surface area (cm2)
(a)
(b)
(c)
Fig. 4 Effect of water availability and AMF inoculation on root
length (a), root volume (b), and root surface area (c)ofEclipta
prostrata and Bidens frondosa grown under three levels of water
availability (Dry, intermediate (Int), and Wet). Different uppercase
letters indicate significant differences (p<0.05) among E. prostrata
plants grown across levels of water availability. Different lowercase
letters indicate significant differences among B. frondosa plants grown
across levels of water availability. Bold and italics letters indicate
differences in AMF inoculation. Asterisk indicate significant differ-
ences between species within levels of water availability or AMF
inoculation. Raw means are presented with bars indicating standard
error
Mycorrhiza
zation prior to water level manipulation (i.e., Garcia et al.
2008; Ipsilantis and Sylvia 2007). Consequently, while the
effects of water availability on AMF levels have been
documented after initial colonization has occurred, there are
few studies that determine if colonization can occur under
conditions of excess water availability. Miller and Sharitz
(2000), utilizing vegetative propagules of Panicum hemi-
tomon Schult. and Leersia hexandra Sw., exposed plants to
soil inundation immediately following AMF. They found
soil inundation inhibited AMF establishment and exposure
to constant soil inundation resulted in total colonization
levels near zero. Carvalho et al. (2003) transplanted 4-
week-old A. tripolium plants to field collected soils and
imposed three levels of water availability. Total coloniza-
tion levels in their continuously flooded treatments con-
ditions were significantly lower than in pulsed or drier
treatments, however, they were quite high (>20%) com-
pared with the results of Miller and Sharitz (2000) and
comparable to the results obtained in the current study with
E. prostrata and B. frondosa. Although methodologies
differ among studies by Miller and Sharitz (2000), Carvalho
et al. (2003) and the current study, together they indicate
that while soil inundation may inhibit AMF formation in
some emergent wetland species under certain conditions,
this is not always the case and AMF associations can
establish in inundated soils.
Regardless of water availability we were not able to
detect a significant effect of AMF inoculation on shoot
height or biomass of B. frondosa or E. prostrata. The
documented effects of AMF inoculation on wetland plant
performance are, however, inconsistent. Whereas an in-
crease in aboveground measures of plant performance were
found in inoculated Carex tribuloides Wahlenb., Phalaris
arundinacea L., and Rumex orbiculatus A. Gray (Fraser
and Feinstein 2005), Casuarina equisetifolia L. (Osundina
1998), P. hemitomon, and T. latifolia (Dunham et al. 2003),
a reduction in aboveground measures of plant performance
was found in inoculated L. salicaria plants (Stevens et al.
2002; Stevens and Peterson 2007). Shoot height and
biomass are often used as surrogates of fitness, though an
absence of a significant effect on shoot height and biomass
does not imply an absence of a fitness contribution. Field
grown plants interact with their biotic and abiotic environ-
ments in complex ways, therefore any potential contribu-
tion to fitness may only manifest when assessed under a
broader range of conditions (Smith et al. 2010).
Since AMF are generally able to forage for resources
more economically than host plant roots, colonized plants
tend to invest fewer resources in root system development
compared with non-colonized plants, and a trend towards
an increase in S/R ratios and reduced root biomass has been
found in inoculated terrestrial plants (Smith and Read
2002). Although similar responses have been shown to
occur in wetland plant species (Fraser and Feinstein 2005;
Cerligione et al. 1988; Neto et al. 2006), White and Charvat
(1999) found a reduced S/R ratio in inoculated L. salicaria
plants. In the current study, AMF inoculation was associ-
ated with an increase in S/R fresh weight in E. prostrata but
not B. frondosa, yet there were no significant effects on root
biomass. Root morphology also differed between species in
response to AMF inoculation; whereas inoculation resulted
in reduced root length and surface area for E. prostrata,
there were no significant differences in these parameters in
inoculated and non-inoculated B. frondosa plants. In contrast,
Percent colonization
Hyp Ves Arb
A
BB
B
B
Int
Dry
Wet
B
B
AA
Fig. 5 Effect of water availability on arbuscular mycorrhizal
colonization levels on plants grown under three levels of water
availability (Dry, intermediate (Int), and Wet). Different uppercase
letters indicate significant differences (p<0.05) within each classifi-
cation of AMF colonization (Hyp hyphal, Ves vesicular, Arb
arbuscular colonization). Pooled raw means of E. prostrata and B.
frondosa plants are presented± standard error
Table 2 Summary table of two-way ANOVA assessing the effects of water availability (Water) on levels of AMF colonization of Eclipta
prostrata and Bidens frondosa
Response variable Water Sp Water×Sp
FpValue F pValue F pValue
Hyphal colonization 16.01 <.0001 0.23 0.6326 2.15 0.1227
Vesicular colonization 19.59 <.0001 1.04 0.3117 2.80 0.0666
Arbuscular colonization 10.26 0.0001 2.02 0.1592 2.22 0.1153
Significant effects (p=< 0.05) are in bold
Sp species
Mycorrhiza
a trend towards increased root length in inoculated L.
hexandra and P. hemitomon plants was found by Miller
and Sharitz (2000), while no effect of inoculation on root
length or root surface area of L. salicaria was found by
Stevens et al. (2002). It must be emphasized that the
preceding studies differed in many respects (i.e., plant
species, flooding duration, frequency and depth of flooding,
nutrient availability, study duration), however, the results
suggest site and species-specific differences in root system
morphology and biomass portioning in response to AMF
inoculation.
Mycorrhizal responsiveness, as defined by Janos (2007)
is “the difference in growth between mycorrhizal and non-
mycorrhizal plants at a designated level of phosphorus
availability”and can be relativized by expression in terms
of growth of either inoculated or non-inoculated plants. In
this study, total productivity, measured as total fresh weight,
and root morphology were more responsive to AMF
colonization in E. prostrata compared with B. frondosa.
Whereas productivity and root morphology of B. frondosa
showed little response to inoculation, a negative response
was displayed in E. prostrata. These results should not,
however, be construed as indicating differences in host
species intrinsic capabilities to respond to various mycor-
rhizal fungal species (Janos 2007), nor should they be
interpreted as indicating that overall fitness of colonized E.
prostrata plants was at a disadvantage (Smith et al. 2010);
such conclusions require assessments conducted over a
much broader range of conditions. Our results reveal
differences in mycorrhizal responsiveness among two
sympatric wetland Asteraceae. Although once thought to
be absent in wetlands, AMF have now been found in many
diverse wetland types (Stevens et al. 2010; Kandalepas et
al. 2010; Radhika and Rodrigues 2007). Furthermore, while
their role in secondary succession has been thought to be
minimal, Stevens et al. (2010) documented widespread
colonization in 31 out of 37 wetland plant species
establishing in a bottomland hardwood forest following
prolonged flooding. This study has shown that AMF can
colonize plants at a very early stage in their development
across a wide range of water availabilities—including
inundated soils—and have the capacity to affect patterns
of resource allocation and root morphology that is species
and environment specific. The study also shows that two
closely related wetland species both in the Asteraceae differ
in mycorrhizal responsiveness. If a wide disparity in
mycorrhizal responsiveness among seedlings of wetland
plant species exists, as has been found in some upland
species (i.e., Janos 1980; Saif 1987), then it could be
expected that events that alter AMF prevalence in wetlands
could significantly alter plant community structure by
directly affecting seedling growth and development and
the interactions of seedlings with other organisms. Since
wetland plant seedlings are generally more responsive to
hydrology than adult plants (Bedinger 1978), understanding
interactions among AMF, seedling growth and develop-
ment, and hydrology in wetlands may provide greater
insight into the factors shaping wetland plant community
structure.
Mycorrhizal responsiveness
shhgt rtln rtvol
shdw rtfw rtsa
totfw
shfw
E. prostrata
B. frondosa
*
*
*
*
Fig. 6 Comparisons of mycorrhizal responsiveness of E. prostrata
and B. frondosa grown at three levels of water availability. Shhgt
shoot height, shfw shoot fresh weight, shdw shoot dry weight, rtfw
root fresh weight, totfw total fresh weight, rtln root length, rtvol root
volume and rtsa root surface area. Asterisk indicate significant
differences (p<0.05) between species. Pooled means for all levels of
water availability for each species are presented± standard error
Response variable Water Sp Water x Sp
FpValue F pValue F pValue
Shoot height 0.290 0.7536 0.04 0.840 1.10 0.3509
Shoot fresh weight 0.670 0.5213 0.01 0.905 0.62 0.5471
Shoot dry weight 0.100 0.9085 0.02 0.898 1.37 0.2776
Root fresh weight 0.770 0.4763 2.03 0.170 0.79 0.4676
Total fresh weight 0.040 0.9564 4.54 0.046 0.31 0.7335
Root length 0.003 0.0968 5.58 0.029 0.29 0.7517
Root volume 0.720 0.4993 5.98 0.024 0.53 0.5992
Root surface area 0.420 0.6610 5.31 0.032 0.60 0.5565
Table 3 Summary table of two-
way ANOVA assessing the
effects of water availability
(Water) on mycorrhizal respon-
siveness (MR) of Eclipta pros-
trata and Bidens frondosa
Significant effects (p=< 0.05)
are in bold
Sp species
Mycorrhiza
Acknowledgements We thank Sajag Adhikari, Johanna Blaszczak,
Cheryl Harrell, Seon-Young Kim, Tiffany Limmanjing, Amanda
Turley, and Misty Wellner.
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