Vol.3, No.3, 224-233 (2013) Open Journal of Ecology
http://dx.doi.org/10.4236/oje.2013.33026
Effectivity of arbuscular mycorrhizal fungi collected
from reclaimed mine soil and tallgrass prairie
Mark Thorne1*, Landon Rhodes2, John Cardina3
1Environmental Science Graduate Program, The Ohio State University, Columbus, USA;
*Corresponding Author:
2Department of Plant Pathology, The Ohio State University, Columbus, USA
3Department of Horticulture and Crop Science, Ohio Agricultural and Research Development Center,
The Ohio State University, Columbus, USA
Received 10 April 2013; revised 17 May 2013; accepted 23 June 2013
Copyright © 2013 Mark Thorne et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
ABSTRACT
We examined suitabilit y of arbuscular mycorrhi-
zal fungi (AMF) associated with cool-season non-
native forages on reclaimed surface-mined land
in southeast Ohio for establishment of native
warm-season grasses. The goal of establishing
these grasses is to diversify a post-reclamation
landscape that is incapable of supporting native
forest species. A 16-w eek glasshouse study com-
pared AMF from a 30-year reclaimed mine soil
(WL) with AMF from native Ohio tallgrass prairie
soil (CL). Four native grasses were examined
from seedling through 16 weeks of growth. Com-
parisons were made between CL and WL AMF
on colonized (+AMF) and non-colonized plants
(–AMF) at three levels of soil phosphorus (P).
Leaves were counted at 4 week intervals. Shoot
and root biomass and percent AMF root coloni-
zation were measured at termination. We found
no difference between WL and CL AMF. Added
soil P did not reduce AMF colonization, but did
reduce AMF efficacy. Big bluestem (Andropogon
gerardii Vitman), Indiangrass (Sorghastrum nu-
tans (L.) Nash), and tall dropseed (Sporobolus
asper (Michx.) Kunth) benefited from AMF only
at low soil P while slender wheatgrass (Elymus
trachycaulus (Link) Gould ex Shinners) exhibit-
ed no benefit. Establishment of tallgrass prairie
dominants big blue-stem and Indiangrass w ould
be supported by the mine soil AMF. It appears
that the non -nativ e f orage sp ecies h av e suppo rt-
ed AMF equally functional as AMF from a regio-
nally native tallgrass prairie. Tall dropseed and
slender wheatgrass were found to be less de-
pendent on AMF than big bluestem or Indian-
grass and thus would be useful in areas with
little or no AMF inoculum.
Keywords: Arbuscular Mycorrhizal Fungi;
Mycorrhizae; Ecosystem Restoration; Surface
Mining; Calcareous Mine Soil; Prairie Gras ses
1. INTRODUCTION
Surface coal mining negatively impacts landscapes by
altering soil structure and chemistry, and negatively af-
fects beneficial soil organisms such as AMF. Topsoil re-
moval and stockpiling prior to mining destroys active
AMF symbiosis and diminishes soil inoculum potential
and AMF species composition [1,2]. This impact may in-
hibit establishment of AMF-dependent species during re-
clamation and restoration.
Arbuscular mycorrhizal fungi benefit establishment of
many plant species on reclaimed mine soils [2-5]. The
symbiotic function of these organisms is critical for sup-
plying plants with minerals, primarily phosphorus, in ex-
change for organic energy compounds [6-10]. This rela-
tionship is critical to plant survival especially when soil
phosphorus is low [11]. In addition, AMF may affect plant
community composition and successional trajectories by
differentially benefiting some plants over others [12-20].
While AMF symbiosis is common and occurs in near-
ly every terrestrial environment [9], differences in the ef-
fectiveness of AMF occur over the landscape and in re-
sponse to management history [20,21]. Strains of AMF
from infertile soil are more effective at phosphorus trans-
fer to plants than AMF from fertile soil [22]. Greater ef-
fectivity has been found in AMF from zinc-contaminated
soil as well other stressful habitats [23,24]. These studies
suggest that in harsh, low-nutrient habitats, there is se-
lection for superior AMF strains. Furthermore, a certain

M. Thorne et al. / Open Journal of Ecology 3 (2013) 224-233 225
degree of host-plant-specificity occurs between AMF and
host plants [25-28]. Therefore, more effective strains of
AMF may benefit re-establishment of native vegetation
in disturbed habitats, as long as host-specificity is not a
barrier.
In southeast Ohio, surface coal mine reclamation prac-
tices since 1972 have converted nearly 80,000 hectares
of native deciduous forestland to non-native forage grass-
land [29]. In 1972, reclamation laws required that over-
burden be contoured to approximate the original land-
scape form, and stockpiled topsoil be spread over the
newly-constructed landscape causing severe compaction
on the constructed landscape. Furthermore, revegetation
did not require native species if preapproved plans stated
otherwise [30]. In place of native forest species, non-
native forage species such as tall fescue (Festuca arun-
dinaceae Schreb.), Kentucky bluegrass (Poa pratensis
L.), and birdsfoot trefoil (Lotus corniculatus L.) were
planted because they established easily and tolerated soil
compaction caused by the reclamation procedures. These
cool-season forages produce a thick ground cover im-
portant for controlling erosion and have potentially main-
tained AMF across the landscape.
Replacing the non-native forage complex with region-
ally native prairie species is one alternative for increas-
ing biodiversity and ecosystem function on reclaimed
mine sites that are incapable of supporting native forest
species. Tallgrass prairies are native to parts of Ohio and
may represent a diverse set of species that could enhance
the functional quality of the mined land [31-33]. How-
ever, it is unclear if AMF associated with the cool-season
forage species currently growing on the reclaimed mined
land would be effective in supporting tallgrass prairie ve-
getation. Warm-season tallgrass species are more depen-
dent on AMF than cool-season grasses [11,34-36] and pro-
blems with host specificity or effectivity could delay or
limit their establishment [37].
This research compares the infective and effective po-
tential of AMF collected from a remnant central Ohio
tallgrass prairie with AMF from reclaimed mine soil on
growth of four native tallgrass prairie grasses. The grass
species evaluated were big bluestem, Indiangrass, tall
dropseed, and slender wheatgrass. Slender wheatgrass is
a cool-season grass while big bluestem, Indiangrass and
tall dropseed are warm-season species; all four occur
throughout the central grassland region of North America,
including tallgrass prairies [38]. The reclaimed mine soil
examined in this study has supported a low-diversity
non-native forage complex for 30 years. The tallgrass
prairie remnant contains 177 plant species including sig-
nature tallgrass prairie species big bluestem, Indiangrass,
tall dropseed, little bluestem (Schizachyrium scoparium
(Michx.) Nash), and switchgrass (Panicum virgatum L.)
[33].
The objectives of this research were 1) to determine if
AMF associated with mine soil vegetation are as effec-
tive as native tallgrass prairie AMF in supporting native
grass growth on reclaimed mine soil, and 2) to determine
how these prairie grasses respond to each source of AMF
when grown in soils with a range of soil phosphorus lev-
els. The goal of this study was to identify growth respon-
ses of prairie grasses to AMF and phosphorus that would
aid in developing strategies to increase biodiversity and
ecosystem function on compacted reclaimed mine soil
[39,40].
2. MATERIALS AND METHODS
2.1. AMF Sources and Pot Culture
Soil containing CL AMF was collected from the Clari-
don tallgrass prairie remnant near Marion, Ohio. Mine
soil containing WL AMF was collected from a reclaimed
surface mined area near Cumberland, Ohio. The CL site
is a 2.2 ha linear remnant owned by the CSX Railroad
and is overseen by the Marion County Historical Society
[33]. The WL site is located on land that had been sur-
face mined in the early 1980 s, and was once part of the
Muskingum Mine, then owned and mined by Central
Ohio Coal Company. In 1986, the land was donated to
The International Center for the Preservation of Wild
Animals, Inc. (the Wilds). The area is part of the Alle-
gheny Plateau of southeast Ohio, which extends west-
ward from the Allegheny Mountains and is a subdivision
of the Appalachian Mountain Range.
Approximately 35 liters of soil were collected from
each site during September, 2005. Soil from the surface
20 cm was collected from 15 to 20 randomly selected
locations at each site. At the CL location, samples were
collected alongside established prairie grasses big blue-
stem and Indiangrass, so that grass roots containing AMF
would be included. At the WL location, soil was col-
lected from an area supporting Kentucky bluegrass, tall
fescue, and birdsfoot trefoil. These species were domi-
nant throughout the reclaimed mined area. Pot cultures of
each AMF source were prepared by mixing soil from
each location, 1:1 by volume, with silica sand in a por-
table cement mixer, which was cleaned between mixes.
The soil/sand mix was poured into 3.8-L plastic nursery
containers and sown with white clover (Trifolium repens
L.) as a host plant [41]. By using a legume inoculated
with nitrogen fixing bacteria, instead of another grass,
the pot cultures could be grown without having to add
supplemental N. But, more importantly, this would re-
duce the chance of propagating pathogens specific to gra-
minoids along with the AMF. The containers were placed
on benches in a 20˚C to 27˚C glasshouse with artificial
lighting 12 hr·day−1. The pot cultures were watered daily
without fertilizer for 10 months. Inoculum was prepared

M. Thorne et al. / Open Journal of Ecology 3 (2013) 224-233
226
by chopping up soil and roots from each pot, discarding
course roots and tops, then mixing all soil and fine roots
together for each AMF source.
Sterile growing medium soil was prepared by mixing
topsoil collected from the surface 20 cm at the WL site
with silica sand, 1:1 by volume, in a portable cement
mixer. The soil/sand mix was then steamed for 5 hr at
100˚C. The sterile soil was stored in plastic bins for 21
days at 20˚C prior to use in the experiment. Soil from
each pot culture, the sterilized growing medium soil, and
original WL topsoil were analyzed for pH, P, K, Ca, and
Mg content by the Service Testing and Research Labora-
tory (STAR lab), The Ohio State University/Ohio Agri-
cultural Research and Development Center, Wooster, OH
(Table 1). Identification of AMF species was not attemp-
ted in this study; however, several Glomus species were
noted in early examination of the source soils (personal
observation). It is likely that WL AMF species were part
of deciduous forest ecosystem present before mining; al-
though much of the forested biome was deforested from
agriculture in the late 1800s. The CL AMF was associ-
ated with a historical tallgrass prairie in Northwest Ohio.
2.2. Experiment Establishment and Design
Experimental units consisted of individual grass seed-
lings growing in 660-cm3 pots (D40 Deepot®, Stuewe
and Sons, Inc., Corvallis, OR) containing 500 cm3 sterile
growing medium soil plus one of four AMF inoculum
treatments, and one of three P levels. Inoculum treat-
ments included 100 cm3 of CL or WL pot-culture soil, or
100 cm3 of sterilized CL (CLS) or sterilized WL (WLS)
pot culture soil. Sterilized inoculum soil was added to the
AMF pots to control for possible fertilizer effects from
the pot culture soil. The sterilized soil was prepared by
autoclaving 8 L of each pot culture soil for 70 min at
130˚C, and then resting the soil in plastic bags at 4˚C for
96 h.
Three levels of P (P1, P2, P3) were established by
mixing 0.0, 0.1, and 0.3 g triple super phosphate (0 - 45 -
0) (Bonide Products Inc., Oriskany, NY) per 500 cm3
sterile soil plus the 100 cm3 inoculum soil to reach target
P levels of 5, 13, and 27 mg·kg-1, respectively. Calcula-
tions were based on the recommendation that 10 mg·kg-1
P is required to increase available soil P 1 mg·kg-1 (Dr.
Donald Eckert, The Ohio State University, personal com-
munication). Each pot was standardized for bacteria by
adding 100 ml of sievate corresponding to each particu-
lar AMF inoculum. The sievate for each inoculum was
prepared by mixing 1000 cm3 pot culture soil and 16 L
water, allowing the slurry to briefly settle, and pouring
the liquid and suspended matter through a 53-µm sieve.
The experiment was designed as a randomized com-
plete block with a factorial arrangement of four levels of
grass species, four levels of AMF source (CL, CLS, WL,
and WLS), and three levels of P (P1, P2, and P3). Each
treatment was replicated six times. The four grass species
(SPP) were “Bison” big bluestem (Andropogon gerardii
Vitman), “Tomahawk” Indiangrass (Sorghastrum nutans
(L.) Nash), “Revenue” slender wheatgrass (Elymus trach-
ycaulus (Link) Gould ex Shinners), and tall dropseed
(Sporobolus asper (Michx.) Kunth). Big bluestem, India-
ngrass, and slender wheatgrass were purchased from
Western Native Seeds, Coaldale, CO USA, and tall drop-
seed was purchased from Oak Prairie Farm, Pardeeville,
WI USA. Seeds of each species were sown 10 - 20 per
pot, and thinned to leave a single seedling in each pot.
Pots were placed in trays and arranged so that +AMF
treatments were adjacent to −AMF control pots to allow
for paired-pot comparisons of AMF sources. Trays were
placed on a glasshouse bench in a randomized-block
design such that blocks controlled for distance from the
cooling/heating source on one end and exhaust fan at the
other. Artificial lighting was set to maintain a minimum
of 300 W·m−2 16 h·day−1, and temperature ranged be-
tween 19˚C - 27˚C.
2.3. Grass Leaf and Biomass Measurements
At 4, 8, 12, and 16 weeks following germination, liv-
ing and dead leaves were counted on each plant. To re-
duce confusion in successive censuses, dead leaves were
removed and stored for later biomass measurement. At
the end of the 16-week experiment, plants were destruc-
tively harvested to assess aboveground and belowground
biomass. Culms and leaves were clipped at the soil sur-
face and put in paper bags along with dead leaves from
earlier censuses. Roots were washed to remove the soil
and then bagged separately from shoots. Biomass sam-
ples were dried at 55˚C for a minimum of 96 h, and then
weighed. Three small sub-samples were cut fresh from
each root system to assess AMF colonization. The root
sub-samples were approximately 10 × 25 mm each and
cut from the top, middle, and bottom third of the root
length. Root sub-samples were stored in a 48% ethanol
solution until being processed for AMF evaluation.
2.4. AMF Colonization Assessment
Root samples were cleared and stained according to a
modified Phillips and Hayman [42] procedure. During
processing, root samples from each plant were contained
in 28 × 5-mm tissue processing cassettes (Canemco Inc.,
Quebec, Canada). Roots were cleared in 10% KOH solu-
tion and autoclaved at 130˚C for 10 min, and then acidi-
fied in a 1% HCL solution for 20 min at room tempera-
ture. Roots were stained in 0.05% Trypan blue staining
solution containing 1:2:1 distilled water, lactic acid, and
glycerin, and autoclaved for 7 min at 130˚C. Following
staining, roots were rinsed in tap water and stored in pla-

M. Thorne et al. / Open Journal of Ecology 3 (2013) 224-233 227
stic Petri dishes covered with a 1:1 solution of distilled
water and glycerin and kept in a 4˚C cooler. Colonization
was assessed using a gridline intersect method [43,44] in
a plastic Petri dish with gridlines scored 13 mm apart on
the bottom. For each sample, the first 50 roots bisecting
gridlines were examined. Roots were designated coloniz-
ed if the root segment contained hyphae, arbuscules, or
vesicles; otherwise, they were designated non-colonized.
Percent colonization was calculated by dividing the num-
ber of colonized roots by 50, then multiplying by 100.
The root sample was then dried at 55˚C and weighed,
and the dry weight was added back to the total root bio-
mass.
2.5. Statistical Analysis
Data were analyzed using PROC GLM in SAS/STAT®
software [45] and significance was accepted at α = 0.05.
Main effects were SPP, AMF, and P. Post-hoc compari-
sons were made using protected Fisher’s LSD test where
differences were accepted only if the P-value calculated
by PROC GLM was equal to or less than 0.05 [46]. De-
pendent variables were leaf number, shoot, root, and total
biomass, root-to-shoot ratio (RSR), difference between
+AMF and −AMF for shoot biomass difference (SDIFF),
root biomass difference (RDIFF), total biomass differ-
ence (TDIFF), and AMF root colonization percent. Dif-
ference in biomass was calculated as a separate continu-
ous random variable for each paired-pot comparison [47].
The null hypothesis for a paired-pot analysis is that the
difference (D) between the two pairs is zero. Accepting
the alternative hypothesis is based on the deviance from
zero. Benefit from AMF inoculation was indicated by a
positive outcome after subtracting the −AMF value from
the +AMF value. Analysis of colonization percent only
included the inoculated treatments in order to accurately
reflect the level of infectivity of each inoculum.
3. RESULTS
3.1. AMF Colonization as Affected by Soil P,
Inoculum, and Grass Species
The reclaimed mine soil used in this experiment ini-
tially averaged 12 mg·kg−1 P, 3768 mg·kg−1 calcium (Ca),
and pH of 7.3 (Table 1). These values indicate a cal-
careous soil with limited available P. Mixing the soil
with silica sand reduced the available P to 5 mg·kg−1 cre-
ating critically low soil P for plant growth. Low P is con-
ducive for testing and comparing the efficacy of the
AMF strains. Furthermore, the addition of P acts as a
control for the activity of AMF because it tests the effi-
cacy of the AMF. Other nutrients were likely limiting, i.e.
N, but P is the primary nutrient associated with AMF
Ta ble 1. Soil properties of reclaimed mine and AMF inoculum
soil used to compare growth of prairie grasses with different
concentrations of phosphorus (P) and different sources of arbu-
scular mycorrhizal fungi (AMF).
AMF sourcec
Soil ParameteraMineb
topsoil
(0 - 20 cm)
Sterile mine
soil/sand mix
(1:1) CL WL
pH 7.3 7.3 7.7 7.9
P (mg·kg-1)12 5 <1 7
K (mg·kg-1)161 80 77 41
Ca (mg·kg-1)3768 1722 1345 1262
Mg (mg·kg-1)321 198 235 198
aSoil analyzed by STAR lab, Wooster, OH. P analyzed with Bray P1 method;
K, Ca, and Mg analyzed with ammonium acetate extract method. bSoil col-
lected from the Wilds 30-yr reclaimed surface-mined land near Cumberland,
OH. cCL collected from Claridon tallgrass prairie remnant near Marion, OH;
WL collected from the Wilds mine soil supporting non-native forage grasses.
AMF inoculum soil prepared as pot-cultures containing a 1:1 mix of soil and
silica sand.
function. Adding N to the pots would have increased
growth, but then the activity of the AMF may have been
confounded by amount of N taken up by each species,
and in turn, how each species converted the added N into
photosynthates to fuel the AMF. In our study, we found
that neither AMF source nor P concentration had any
effect on colonization percent when averaged over all
other factors (Table 2). Grasses inoculated with either
CL or WL averaged slightly greater than 50% AMF co-
lonization, indicating that both AMF cultures were equal-
ly accepted by the host grasses. The AMF colonization
trended lower from 56% to 49% as P increased, but those
differences were not significant (Table 2). Colonization
differed among species, as tall dropseed and slender wheat-
grass had the highest percentages with 70% and 55%, re-
spectively (Tab le 2). Big bluestem and Indiangrass had
lowest colonization with 51% and 36%, respectively.
3.2. AMF and Soil P Effect on Plant Growth
Response to soil P concentration was predictable, as an
increase in P resulted in an increase in biomass produc-
tion, and was most consistent for shoot biomass (Table
3). Total biomass also followed the same pattern, increas-
ing with each increased level of P. However, root bio-
mass did not increase from P2 to P3, thus P2 had the
highest RSR, as the increased shoot growth at the higher
P was not matched by a corresponding increase in root
growth. This is likely a result of space limitation in the
pots and not a lack of response to increased P, as pots
with the highest P level were densely packed with roots
when harvested.
Slender wheatgrass produced the greatest total bi-
omass, which was similarly split between root and shoot

M. Thorne et al. / Open Journal of Ecology 3 (2013) 224-233
228
Table 2. Percent colonization by arbuscular mycorrhizal fungi
(AMF) as affected by AMF sources, soil phosphorus level (P),
and grass species (SPP) in a 16-week glasshouse experiment.
Parameter Colonizationa
AMF (%)
CL 52.3 a
WL 53.1 a
(P = 0.7917)
Pb
P1 55.6 a
P2 53.7 a
P3 48.9 a
(P = 0.2271)
SPP
Big bluestem 51.0 bc
Indiangrass 35.5 c
Tall dropseed 69.9 a
Slender wheatgrass 54.7 ab
(P = 0.0226)
aColonization percents reflect only plants inoculated with AMF. Non-ino-
culated plants had 0% AMF colonization. Differences within each variable
are determined using protected Fisher’s LSD (α = 0.05). Means followed by
the same lower-case letter (a, b, c) are not different. bP target levels were P1 =
5 mg·kg−1 P; P2 = 13 mg·kg−1 P; P3 = 27 mg·kg−1 P.
Table 3. Biomass production as affected by grass species (SPP)
and three levels of soil phosphorus (P) in a 16-week glasshouse
experiment.
Dependent variablesa
Main effects Shoot Root Total RSRb
SPP (g dry weight) (g/g)
Big bluestem 0.7 c 1.5 a 2.2 b 2.4 a
Indiangrass 0.7 c 1.1 b 1.8 c 1.8 b
Tall dropseed 1.1 b 0.7 c 1.8 c 0.7 c
Slender
wheatgrass 1.5 a 1.3 ab 2.8 a 0.9 c
(P < 0.0001) (P < 0.0001) (P < 0.0001) (P < 0.0001)
Pc
P1 0.6 c 0.7 b 1.4 c 1.5 ab
P2 1.0 b 1.4 a 2.4 b 1.6 a
P3 1.3 a 1.4 a 2.7 a 1.3 b
(P < 0.0001) (P < 0.0001) (P < 0.0001) (P = 0.0231)
aP-values represent the probability of differences within each dependent va-
riable for each main-effect. Differences within each variable are determined
using protected Fisher’s LSD (α = 0.05). Means followed by the same
lower-case letter (a,b,c) are not different. bRoot to shoot ratio (RSR) calcu-
lated by dividing root weight by shoot weight. cSee Table 2 for target P levels.
biomass (Table 3). Big bluestem had similar root bio-
mass compared with slender wheatgrass, but only pro-
duced half the shoot biomass. The difference in alloca-
tion of resources between these species was reflected in
the RSR, as slender wheatgrass averaged 0.9 while big
bluestem averaged 2.4 (Table 3). Indiangrass and tall
dropseed produced the least total biomass; however, In-
diangrass allocated more resources to root biomass,
while tall dropseed allocated more resources to shoot
production. This indicates that big bluestem and Indian-
grass appear to direct more resources, proportionately, to
root growth during seedling establishment, compared with
slender wheatgrass and tall dropseed.
3.3. AMF Effectivity in Paired-Pot
Comparison
To compare the effectiveness of the AMF cultures, a
paired-pot arrangement was used to examine the differ-
ence in biomass accumulation between colonized and
non-colonized plants. By subtracting the biomass of a
–AMF plant from an adjacent +AMF plant for each com-
ponent (shoot/root/total), new variables were created that,
if positive, indicated AMF benefit, and if negative, indi-
cated AMF detriment. The GLM analysis indicated that
SPP and P had the greatest influence on shoot difference
(SDIFF), root difference (RDIFF), and total difference
(TDIFF), with P-values < 0.0001 (Ta b l e 4 ). In contrast,
AMF had a significant impact only on SDIFF (Table 4).
There was a significant interaction between AMF and
P for RDIFF and TDIFF, influenced mainly by RDIFF
(Table 4). For SDIFF, AMF benefit was positive for both
CL and WL at P1, but decreased for CL with each increase
in P, whereas there was no decreased benefit for WL at
P3 (Figure 1(a)). There was no difference in AMF bene-
fit for RDIFF between P1 and P2 for CL AMF, which
was negative at all three P levels (Figure 1(b)); however,
WL AMF was positive at P1 but negative at both P2 and
P3, with the least benefit occurring at P2 (Figure 1(b)).
For TDIFF, the benefit of both AMF sources was positive
at P1, but negative at P2 and P3 (Figure 1(c)). The in-
teractions between AMF and P, for all three difference
variables, occurred as the response to increasing P dif-
fered between AMF sources.
Significant interactions were also found between SPP
and P for all three difference variables. Overall, the only
AMF benefit occurred with big bluestem, Indiangrass
only at the lowest P level (Figure 2). For SDIFF, benefit
decreased as P increased for big bluestem, Indiangrass,
and slender wheatgrass, but tall dropseed was not affect-
ed (Figure 2(a)). In addition, slender wheatgrass experi-
enced negative benefit at all three P levels. For RDIFF,
both tall dropseed and slender wheatgrass had negative
benefit at all three P levels and was not affected by an
increase in P (Figure 2(b)). Big bluestem RDIFF declin-
ed with each increase in P while Indiangrass declined
only from P1 to P2. The interactions with TDIFF were
very similar to RDIFF (Figure 2(c)).
The interaction between SPP and P was also evident in
the number of leaves produced during the 16-week expe-

M. Thorne et al. / Open Journal of Ecology 3 (2013) 224-233 229
Ta b le 4. Analysis of variance table (PROC GLM) for the full model with a factorial arrangement testing the difference in biomass
production for grass species (SPP) colonized with arbuscular mycorrhizal fungi (+AMF) and non-AMF (–AMF) plants. Dependent
variables shoot difference (SDIFF), root difference (RDIFF), and total difference (TDIFF) were produced by subtracting biomass of
–AMF plants from +AMF plants in a paired-pot glasshouse experiment examining the effects of AMF source and P on growth of prairie
SPP grown in sterilized mine soil.
SDIFF RDIFF TDIFF
Model DF F value P > F F value P > F F value P > F
BLOCK 5 0.4 0.8753 2.2 0.0601 1.2 0.3225
SPP 3 15.5 <0.0001 19.2 <0.0001 25.5 <0.0001
AMF 1 6.0 0.0162 0.6 0.4294 0.2 0.6244
P 2 38.9 <0.0001 29.4 <0.0001 51.2 <0.0001
SPPxAMF 3 0.7 0.5354 2.8 0.0461 2.8 0.0428
SPPxP 6 3.9 0.0013 3.3 0.0053 5.2 <0.0001
AMFxP 2 2.8 0.0629 15.6 <0.0001 13.1 <0.0001
SPPxAMFxP 6 0.5 0.8391 0.7 0.6449 0.9 0.5276
TOTAL 141
riment (Figure 3). At P1, leaf production at each census
was greater for mycorrhizal big bluestem, Indiangrass,
and tall dropseed compared with non-mycorrhizal plants.
Indiangrass appeared to have the greatest AMF benefit,
whereas +AMF slender wheatgrass produced slightly
more leaves only at 4 weeks (Figure 3).
At P2 and P3, AMF effect was less evident for all
grass species except slender wheatgrass where –AMF
plants produced the greatest number of leaves (Figure 3).
The interaction between SPP and P at these levels of P
would indicate that the cool-season slender wheatgrass is
negatively affected by AMF when P is abundantly avail-
able, whereas the warm-season grasses (big bluestem, In-
diangrass, and tall dropseed) are less affected (Figure 3).
Slender wheatgrass appears to gain little, if any, benefit
from AMF, which would suggest this species would be
useful in restoration plantings where AMF is not initially
present.
4. DISCUSSION
A number of studies have shown that AMF coloniza-
tion is reduced by higher soil P [48-51], but that was not
evident in this study. If plants can obtain P on their own
then symbiosis would be less beneficial. However, there
is often no clear relationship between colonization per-
cent and P uptake or plant growth response [52-54],
meaning that efficacy is not necessarily related to the
magnitude of colonization. It is known that warm-season
grasses tend to be more dependent on AMF than cool-
season grasses, especially when P is limited [35]. Cool-
season grasses tend to have finer root systems that are
better suited for P uptake, while warm-season grasses
tend to have more coarse root systems. The high abun-
dance of AMF in slender wheatgrass roots was unex-
pected. Big bluestem is known to be very dependent on
AMF [55] and is a dominant species in tallgrass prairies
across North America. The mycorrhizal status of tall drop-
seed has not been reported, but a related species, Sporo-
bolus heterolepis, is mycorrhizal [56].
Big bluestem and Indiangrass responded to AMF and
P as expected according to previous research [55]. Both
species benefited from AMF when soil P was low, and
showed less benefit as P increased. Both of these grasses
allocated more resources to roots than aboveground tis-
sue, which is important for access to nutrients and water
during periods of stress. Harris [57] determined that com-
petitive success of non-native downy brome (Bromus tec-
torum L.) was due to its ability to establish a deep root
system during autumn and winter when native bluebunch
wheatgrass (Pseudoroegneria spicata (Pursh) A. Löve)
was dormant. During spring, a downy brome infestation
depleted soil moisture before bluebunch wheatgrass was
able to complete its reproductive cycle. The dominance of
big bluestem and Indiangrass in tallgrass prairies is likely
due to their ability to establish deep root systems over time,
as well as their association with AMF when soil P is limited.
Tall dropseed and slender wheatgrass both appear fa-
cultative in their response to AMF. Tall dropseed is a
warmseason prairie grass, but seems to respond to AMF
and P similarly to facultative cool-season grasses. Great-
er production of aboveground biomass compared with
root biomass and low dependence on AMF would sug-
gest that tall dropseed can quickly establish following
disturbance in habitats where P and AMF may be limit-
ing. Slender wheatgrass forms association with AMF, but
is clearly not dependent on AMF. It is able to access P
when soil levels are low, and can be very productive
when soil P is higher.
5. CONCLUSIONS
Results of this research indicate that AMF associated
with reclaimed mine soil are not likely a barrier for es-

M. Thorne et al. / Open Journal of Ecology 3 (2013) 224-233
230
Figure 1. Interaction of arbuscular mycorrhizal fungi (AMF)
from the Claridon tallgrass prairie remnant (CL) and the Wilds
reclaimed mine soil (WL) in Ohio and soil phosphorus level
(P1, P2, P3). Values represent the difference in biomass be-
tween AMF-colonized and non-colonized grasses ain a 16-week
glasshouse experiment measuring shoot biomass difference
(SDIFF), root biomass difference (RDIFF), and total biomass
difference (TDIFF). Solid lines (—) represent CL-AMF com-
parisons; dotted lines (····) represent WL-AMF comparisons.
Differences at each phosphorus level within each graph (α =
0.05) are shown to the right.
tablishing tallgrass prairie species. Colonization levels
were similar between the two AMF inoculums. This would
also suggest that host specificity is not a deterrent for na-
tive grass establishment even though the mine soil AMF
have been associated with non-native cool-season forage
species for 30 years. It appears that poor soil conditions
of the mine soil, i.e. compacted calcareous soil with low
available phosphorus, may have selected an effective AMF
community, which could benefit native tallgrass prairie
grasses.
Tall dropseed and slender wheatgrass both appear to
establish well when P is low, with or without AMF, and
would be useful in early establishment of a prairie com-
munity on reclaimed mine soil. Big bluestem and Indian-
grass are more dependent on AMF, but did benefit from
Figure 2. Interaction of grass species (SPP) and soil phospho-
rous concentration (P) on the difference in biomass between
grasses colonized with arbuscular mycorrhizal fungi (AMF) and
non-colonized grasses in a 16-week glasshouse experiment mea-
suring shoot (SDIFF), root (RDIFF), and total biomass meas-
urements (TDIFF). Solid lines (−−) represent big bluestem; dot-
ted lines (····) represent Indiangrass; dashed lines (− −) repre-
sent tall dropseed; dashed and dotted lines (− ·· −) represent
slender wheatgrass. Differences at each phosphorus level with-
in each graph are shown to the right and determined using a
protected Fisher’s LSD (α = 0.05).
the mine soil AMF in this study. This study suggests that
years of growth by non-native cool-season forage species
on reclaimed compacted mine soil in southeast Ohio have
propagated AMF that would aid in the establishment of
native AMF-dependent warm season prairie grasses. The
ecological significance of these findings is that in highly
disturbed landscapes there are many potential ways for
ecosystems to self organize. The non-native species plant-
ed during reclamation were a valuable nurse crop for the
indigenous AMF, but did not yield a diverse landscape.
By adding more species that can utilize the AMF, the low
diversity issue may be addressed and a new era of self
organization could lead to more function, structure, and

M. Thorne et al. / Open Journal of Ecology 3 (2013) 224-233 231
Figure 3. Arbuscular mycorrhizal fungi (AMF) effect on num-
ber of leaves produced by prairie grasses at three concentra-
tions of soil phosphorus (P) level during a 16-week glasshouse
experiment. Solid lines (—) represent AMF colonized; dotted
lines (····) represent non-AMF colonized plants. P-values are
signified as follows:  = 0.05 < P < 0.001,  = 0.001 < P <
0.0001,  = P < 0.0001, and ns = P > 0.05 between AMF and
non-AMF plants at each sampling.
productivity from the landscape.
6. ACKNOWLEDGEMENTS
We thank Dr. Nicole Cavender for access to soil at the Wilds; Dr.
Robert Klips for access to soil at the Claridon Prairie; Dr. Marc Evans
for statistical consultation; and Drs. Craig Davis and James Metzger for
comments on the manuscript. We also thank David Snodgrass and Jim
Vent of the Howlett Greenhouse facility at The Ohio State University
for assistance with this project. Salaries and research support were
provided by state and federal funds appropriated to the Ohio Agricul-
ture Research and Development Center, The Ohio State University.
Manuscript No. HCS13-04.
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