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Plant and Soil
An International Journal on Plant-Soil
Relationships
ISSN 0032-079X
Plant Soil
DOI 10.1007/s11104-015-2510-9
Fungal endophyte symbiosis alters nitrogen
source of tall fescue host, but not nitrogen
fixation in co-occurring red clover
Lindsey C.Slaughter, Anna E.Carlisle,
Jim A.Nelson & Rebecca L.McCulley
1 23
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REGULAR ARTICLE
Fungal endophyte symbiosis alters nitrogen source of tall
fescue host, but not nitrogen fixation in co-occurring
red clover
Lindsey C. Slaughter &Anna E. Carlisle &
Jim A. Nelson &Rebecca L. McCulley
Received: 23 December 2014 /Accepted: 6 May 2015
#Springer International Publishing Switzerland 2015
Abstract
Background and aims Infection of tall fescue with the
common toxic fungal endophyte Epichloë coenophiala
harms livestock via toxic alkaloid production; therefore,
non-toxic ‘novel’strains of the endophyte have been
developed and released. How different endophyte
strains impact biological nitrogen fixation (BNF) in
mixed species pastures is unknown. We asked whether
novel endophyte or common toxic endophyte-infected
(NE+; CTE+) tall fescue affects symbiotic and non-
symbiotic BNF, and utilization of biologically-fixed
nitrogen in tall fescue.
Methods Tall fescue was planted either endophyte-free
(E-), infected with CTE, two non-toxic strains AR542
NE, AR584 NE, or a blend of endophyte treatments. We
measured natural abundance of
15
N in plant and soil
samples, and conducted soil acetylene reduction assays.
Results Endophyte presence and strain significantly af-
fected the δ
15
N of tall fescue. Near red clover, CTE+
and AR584 NE+ tall fescue were most
15
N-depleted;
but away, E- tall fescue was most
15
N-depleted.
Endophyte strain significantly influenced N concentra-
tion in red clover, but not symbiotic or non-symbiotic
BNF.
Conclusions Endophyte strains produce different ef-
fects on tall fescue’s competitive ability and nitrogen
utilization. In mixed pastures, deployment of NE strains
for decreased alkaloid toxicity will differentially impact
use of biologically fixed nitrogen in tall fescue and
nitrogen concentration in red clover.
Keywords Acetylene reduction assay .Epichloë
coenophiala .
15
N naturalabundance .Tr ifo lium pr ate nse
L.Schedonorus arundinaceus Schreb .Tem pera te
pasture
Abbreviations
CTE+ common toxic endophyte-infected
E- endophyte-free
BNF biological nitrogen fixation
NE+ novel endophyte-infected
EMix equal mix of E-, CTE+, AR542 NE+, and
AR584 NE+ treatments within plot
PDF pasture demonstration farm
RC red clover
TF(+RC) tall fescue plant located close to red clover
TF(-RC) tall fescue plant located away from red
clover
CF-
IRMS
continuous flow isotope ratio mass
spectrometer
ARA acetylene reduction assay
Plant Soil
DOI 10.1007/s11104-015-2510-9
Responsible Editor: Kari Saikkonen.
L. C. Slaughter :A. E. Carlisle :J. A . Nelson :
R. L. McCulley
Department of Plant and Soil Sciences, University of
Kentucky, 1100 South Limestone, Lexington, KY
40546-0091, USA
L. C. Slaughter (*):A. E. Carlisle :J. A. Nelson
Department of Plant & Soil Sciences, University of Kentucky,
N-222 N Ag. Sci. North, Lexington, KY 40546-0091, USA
e-mail: lincslau@gmail.com
Author's personal copy
DWE dry weight equivalent
GC gas chromatography
FID flame ionization detector
RCBD randomized complete block design.
Introduction
Tall fescue (Schedonorus arundinaceus Schreb.) is a
widely used cool-season forage grass in the Southeast
United States. It covers over 14 million hectares of
pasture area in this region, a large proportion of which
hosts an aboveground asexual fungal endophyte
Epichloë coenophiala (Shelby and Dalrymple 1987),
previously known as Neotyphodium coenophialum
(Leuchtmann et al. 2014). The symbiotic relationship
with E. coenophiala has been shown in some cases to
increase tall fescue’s drought tolerance (Arachevaleta
et al. 1989;Boutonetal.1993;ElmiandWest1995),
insect and nematode resistance (Clay et al. 1993;
Kimmons et al. 1990), and competitive ability in mixed
species communities (Hill et al. 1991)relativetounin-
fected tall fescue, and is thus often considered a defen-
sive mutualism (Clay 1988). However, one of the de-
fensive mechanisms provided to tall fescue by common
toxic endophyte strains of E. coenophiala is production
of ergot alkaloids, and the deleterious effects of these
compounds on animal performance and health, such as
reduced heat tolerance, weight gain, and reproductive
success, have been reviewed in detail (Schmidt and
Osborn 1993; Strickland et al. 2011; Strickland et al.
1993). In hopes of retaining many beneficial character-
istics of the grass-endophyte symbiosis while reducing
toxicity to livestock, multiple strains of the endophyte
which do not produce ergot alkaloids have been isolated
from wild populations for selectionanduseintall
fescue-based pastures (Bouton et al. 2002). Whereas
common toxic endophyte effects on plant communities
have been heavily studied (e.g., Rudgers and Clay
2007), scientists have only recently begun to examine
the effects of so-called non-toxic, novel endophytes on
plant and soil communities and ecosystem dynamics
(e.g., Rudgers et al. 2010;Thometal.2014; Yurkonis
et al. 2014).
Common toxic endophyte-symbiotic (CTE+) tall fes-
cue has often demonstrated enhanced competitive abil-
ity relative to other plant species over time (e.g., Clay
et al. 2010), reducing plant diversity in mixed species
stands (Iqbal et al. 2013; Rudgers et al. 2010)compared
to uninfected (E-) tall fescue. This could be a particular
challenge for utilization of legumes, which are added to
pastures to provide increased forage quality and added
N fertility via biological N fixation (BNF) in root nod-
ules with diazotrophic bacterial symbionts such as
Rhizobium spp. (e.g., Sleugh et al. 2000, see Nelson
and Moser 1994). Few studies have examined the spe-
cific effect of CTE+ tall fescue on clover when grown
together, though a recent greenhouse study found no
effect (Dirihan et al. 2014). In contrast, three genotypic
strains of Epichloë lolii (previously Neotyphodium lolii
Leuchtmann et al. 2014), another asexual fungal endo-
phyte species, infecting perennial ryegrass decreased
growth of white clover when grown in mixture, but
differences between endophyte strains were not attrib-
uted to strain-specific alkaloid profiles (Sutherland et al.
1999). Root and leaf extracts of red fescue infected with
Epichloë festucae reduced seed germination of both red
and white clover (Vázquez-de-Aldana et al. 2011), and
E. festucae-infected red fescue has also been shown to
inhibit red clover biomass production and reduce
growth of other legumes when grown in mixture
(Vázquez-de-Aldana et al. 2013). Furthermore, in tall
fescue, Peters and Mohammed Zam (1981) found re-
duced germination and root growth of red clover and
birdsfoot trefoil when subjected to tall fescue extracts of
unknown endophyte status, and Springer (1996) later
found that extracts from both E- and CTE+ tall fescue
reduced red clover germination and root growth.
Inhibition of forage legumes when grown in mixture
with CTE+ fescue may be due to allelopathic effects
(Springer 1996; Sutherland et al. 1999; Vázquez-de-
Aldana et al. 2013; Vázquez-de-Aldana et al. 2011), or
to other competitive effects such as increased soil mois-
ture stress or decreased light interception (Staley and
Belesky 2004). Yet, because formation of bacterial sym-
biosis for BNF and fixation activity is linked to legume
growth and development (e.g., Delves et al. 1986;
Robson et al. 1981), we must consider whether
endophyte-infected tall fescue influences these charac-
teristics that may contribute to inhibition of legumes.
Alterations in nutrient dynamics both in neighboring
tall fescue plants and the surrounding soil may influence
legume growth and N fixation activity. CTE+ tall fescue
has been shown to accumulate more nutrients such as P,
Ca, Zn, and Cu in root tissue than uninfected plants
(Malinowski et al. 2000), though specific nutrient up-
take dynamics vary widely according to both host and
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endophyte genotype, especially in response to nutrient
limitation (Malinowski and Belesky 1999). Increased N
use efficiency and activity of N assimilation enzymes in
CTE+ tall fescue (Arachevaleta et al. 1989; Lyons et al.
1990) may alsoalter long-term N pools in mixed species
stands.
Fungal endophyte symbiosis with tall fescue can also
impact soil microorganisms and alter C and N cycles
(pools and trace gas flux) (Buyer et al. 2011;
Franzluebbers et al. 1999; Iqbal et al. 2013;Rojas
2014). Stands with higher endophyte-infection frequen-
cies contain more soil C and N than E- stands or stands
with low frequencies of infected tall fescue, presumably
due to decreased microbial activity or altered plant
inputs (Franzluebbers et al. 1999; Guo et al. 2015;
Iqbal et al. 2012). Therefore, because factors such as
nutrient availability have been shown to influence non-
symbiotic N fixation in grassland soils (Zechmeister-
Boltenstern and Kinzel 1990), non-symbiotic N fixing
soil microorganisms may also be affected by CTE+ tall
fescue, which has further implications for altered N-
pools and dynamics in pastures.
Characteristics of N cycling in terrestrial systems can
be assessed bymeasuring the ratio of naturally occurring
15
and
14
N stable isotopes in plant or soil material and
expressing the results as δ
15
N, or deviation in the ratio
of
15
N:
14
N natural abundance measured in each sample
from the standard ratio of 0.0036765 measured in atmo-
spheric N
2
and calculated in parts per thousand, also
called per mil (‰) (Junk and Svec 1958; Mariotti 1983).
One key assumption with this approach is that rapid
biological transformations of N discriminate against
the heavy
15
N form, resulting in products that are
15
N-
depleted relative to the lighter
14
N isotope, and these
products may be leached, volatilized, or taken up by
plants (Pörtl et al. 2007; Templer et al. 2007).
Substances enriched in
15
N thus generally accumulate
in soil over time, and include highly stable soil organic
matter (Shearer et al. 1974). The δ
15
N of plant or soil
material may be interpreted as reflecting the integrated
δ
15
N of its N source, in addition to isotopic fraction-
ation, gains, losses, or mixing of N pools within the
plant (Evans 2001; Robinson 2001). For example, some
studies in grasslands have utilized the
15
N natural abun-
dance method, where depleted foliar
15
N in plant spe-
cies growing in mixed stands with clover demonstrate
transfer of
15
N-depleted clover-fixed N to non-legumes
(e.g., Gubsch et al. 2011; Temperton et al. 2007), and the
same method has been utilized to examine transfer
between N-fixing and non-N-fixing trees (Hoogmoed
et al. 2014). Legumes rely heavily on atmospheric N
2
,
which is fixed through bacterial symbiosis and un-
dergoes further slight fractionation toward the lighter
14
Nform(DelwicheandSteyn1970); thus, legumes
naturally exhibit more depleted δ
15
Nthannon-fixing
plants in most ecosystems (Virginia and Delwiche
1982).
Little work has yet investigated the effects of novel
endophyte strains on the plant and soil biological pro-
cesses described above, but some studies suggest that
both cultivar and endophyte type influence community-
scale effects of the symbioses. Whereas stands of novel
endophyte-symbiotic (NE+) or E- tall fescue are more
beneficial for animal performance, having reduced tox-
icity (Bouton et al. 2002), they are not necessarily as
persistent as CTE+ tall fescue (Hopkins and Alison
2006). NE+ tall fescue may impact plant species abun-
dance and invertebrate community structure differently
than CTE+, but specific effects also differ between tall
fescue cultivars (Rudgers et al. 2010; Yurkonis et al.
2014). In addition, because some consequences of en-
dophyte infection, such as increased drought resistance
(Elmi and West 1995), the inhibition of legume seed
germination (Peters and Mohammed Zam 1981;
Springer 1996), or effects on soil microbial community
composition (Rojas 2014), are not specifically linked to
alkaloid production, which is a primary difference be-
tween the novel and common toxic strains, the question
remains whether novel endophytes elicit similar effects
on both symbiotic and non-symbiotic BNF.
To examine the effects of CTE+ and NE+ tall
fescue on symbiotic and non-symbiotic BNF and
concomitant N-usage in tall fescue, we measured
the natural abundance of
15
N stable isotope ratios in
plant and soil samples in addition to estimating po-
tential N
2
-fixation activity in free-living, non-
symbiotic soil bacteria using the acetylene reduction
assay. We hypothesized that in mixed species plots:
1) tall fescue infected with CTE and NE strains will
competitively utilize more N and differentially inter-
act with red clover and soil microbial communities
compared to endophyte-free tall fescue, reducing
both symbiotic and non-symbiotic BNF through de-
creased abundance and growth of neighboring red
clover and altered soil microbial communities, and
2) differential effects on BNF between endophyte
strains will elicit long-term changes in size and iso-
topic signature of soil N pools.
Plant Soil
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Materials and methods
Site description and experimental design
This study was conducted at the University of Kentucky
Spindletop Research Farm in Lexington, Kentucky
(38°6’29^N, 84°29’31^W). The location receives an
average annual precipitation of 1163 mm, and has an
average annual summer temperature of 23.8 °C and a
mean annual winter temperature of 1.6 °C (Ferreira et al.
2010). The soil type was a well-drained Bluegrass-
Maury silt loam, which is a fine, mixed, semi-active,
mesic Typic Paleudalf that weathered from a silty loess
mantle over clayey phosphatic limestone residuum (Soil
Survey Staff). Prior to site preparation, this location was
an established hayfield containing predominantly tall
fescue (‘Select’variety, endophyte-free), and <5 % each
of Kentucky bluegrass (Poa pratensis L.), nimblewill
(Muhlenbergia schreberi J.F. Gmel.), and alfalfa
(Medicago sativa L.) (Flynn et al. 2008). After site
clearing and before plot establishment, seven T
0
soil
samples from 5.0 cm diameter soil cores collected
across the study area to a depth of 10 cm were charac-
terized as having 5.81 pH , 2.25 %C, 0.25 total %N, and
184 mg P kg
−1
soil (Iqbal et al. 2013).
A randomized complete block design (RCBD) con-
taining a total of 30, 2×2 m square plots divided among
six blocks with five plots each was established on April
10, 2008. Each of the five plots within the six blocks
were broadcast with 11.2 kg/ha tall fescue (Schedonorus
arundinaceus Schreb) seeds in monoculture containing
one of the following five fungal endophyte treatments:
endophyte-free (E-), infected with the common toxic
endophyte E. coenophiala (CTE+), infected with one
of two novel non-toxic endophyte strains (AR542 NE+
or AR584 NE+; AR=AgResearch, Hamilton, New
Zealand), or a seed mixture containing 25 % each of
the four previous treatments (EMix). Tall fescue seeds
planted in this experiment were from a pasture demon-
stration farm (PDF) variety provided by the Samuel
Roberts Noble Foundation, which recently registered
the PDF-AR584 endophyte combination as ‘Te xoma’
MaxQ II tall fescue (Hopkins et al. 2011). Individual
plots in this study were spatially separated by 1 m
alleyways sown with Kentucky bluegrass (Poa
pratensis L.). All aboveground vegetation in the plots
was mowed to a height of 10 cm once per year during
the winter (December—February) after plot establish-
ment in 2008. Collection of aboveground plant biomass
to a height of 10 cm within a randomly placed 50×
20 cm quadrat in each plot occurred in September 2011.
Endophyte treatments were checked in May 2010, with
20 individual tillers harvested per plot and assayed for
endophyte presence using an immunoblot assay and for
alkaloid potential using genetic screening (Takach and
Young 2014a, b). At that time, endophyte infection
frequencies of the plots were as follows: E- 0.83 %
infected, CTE+ 84.2 % infected, AR 542 NE+ 83.7 %
infected, AR584 NE+ 96.9 % infected, and EMix
75.9 % infected overall, with 49 % NE+ and 27.5 %
CTE+.
Sample collection and handling
Plant composition and forage types
Whereas only tall fescue was planted at establishment in
2008 and remained the dominant species in each plot,
plant community composition across plots had di-
verged, especially in E- plots, to include an abundance
of other graminoid and forb species by 2010 (Iqbal et al.
2013) and included up to 20 species by the time of plant
sampling for this study in 2011 (McCulley et al., un-
published data). Plant species commonly found in the
plots included Kentucky bluegrass (found in 100 % of
plots with an average of 4.8 % relative abundance),
crabgrass (Digitaria spp.; 97, 8.5 %), marestail
(Conyza canadensis L.; 93, 8.5 %), and nimblewill
(87, 3.7 %). These species are presumed to have either
germinated from the seedbank or arrived through vari-
ous natural mechanisms of plant succession such as
wind-blown seeds or other vectors. One species present
at the time of this study in each of the plots was red
clover (Trifol iu m prate ns e L.), a cool-season perennial
legume which has agronomic value for use in mixed
species pastures for forage and animal production (e.g.,
Tay lor 2008). Despite the presence of other legumes in
this location in previous years, such as alsike clover
(Trifolium hybridum L.) in treatment plots in 2010
(Iqbal et al. 2013), and alfalfa (Medicago sativa L.)
presence prior to study establishment (Flynn et al.
2008), no legumes other than red clover were detected
in our study plots in fall 2011.
In September 2011, one sample each of the following
three forage types was collected from within the 30
study plots, yielding a total of 90 forage samples: a red
clover plant (RC), a tall fescue plant growing in close
association with the collected red clover (maximum
Plant Soil
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8 cm distance between plants; TF(+RC)), and a tall
fescue plant spatially isolated from red clover within
the plot (minimum 45 cm distance; TF(-RC)). All forage
samples were oven-dried at 55 °C for 48 h and ball-
ground for storage until analysis. At the time of
plant harvest in September 2011, the relative per-
centage abundance of every plant species present in
each study plot was visually estimated (to 0.1 %
cover) using the vegetative canopy coverage scale
of Daubenmire (1959).
Soil samples over time
Two or three composited 1.5 cm diameter soil cores
taken to a depth of 10 cm from each of the 30 plots
had been sampled periodically after site establishment
T
0
sampling in 2008. Available soil samples collected
for previous research which were used to assess long-
term soil N pools from each treatment in this study were:
seven ball-ground, dried T
0
soils from pre-establishment
in April 2008, March 2010 soils from each plot (n=30) ,
and May 2011 soils (n=30) which were sieved to 2 mm
and stored fresh at −80 °C. Soils were also collected
from each plot (n=30) during October 2012 and 2013
by compositing three 1.5 cm soil cores per plot taken to
a depth of 10 cm, and sieved to 2 mm and stored fresh at
−80 °C (2012) or −20 °C (2013). We therefore utilized a
total of 127 soil samples throughout this study.
Stable isotope analysis in forage and soil samples
Before measuring the natural abundance of
15
N, dried
and ball-ground forage material was stored in glass
vials. Field-fresh soil subsamples from each study year,
which were previously sieved and frozen for storage,
were dried at 105 °C for 48 h, ball-ground, and then
further dried at 55 °C overnight immediately before
15
N
analysis. Based on preliminary tests for appropriate
sample weights to avoid measurement errors and max-
imize precision, 5 mg of forage or 30 mg of soil material
was weighed into pre-cleaned tin cups and combusted
on a Costech Elemental Analyzer (ECS 4010) attached
to a Finnigan Delta
Plus
XP continuous flow isotope ratio
mass spectrometer (CF-IRMS). CF-IRMS analysis pro-
vided measurements of total N concentration (%) and
15
N:
14
N isotopic ratio for each sample. Then, for each
sample, δ
15
N was calculated as: δ
15
N(‰)=((R
sample
/
R
standard
)−1)× 1000), where R
sample
and R
standard
are the
15
N:
14
Nratiosmeasuredineachsampleandin
atmospheric N
2
, respectively. Repeated measurements
of in-house and international standards were included
throughout each run sequence (n=4) in order to calibrate
sample values against known ‰values of δ
15
N. Isotope
measurements were generally reproducible within ±
0.2‰(standard error) for δ
15
Nvalues.
Acetylene reduction assays (ARA) in soil samples
To evaluate the potential activity of free-living N fixing
microorganisms in soil samples, laboratory incubation
assays of acetylene reduction to ethylene, where acety-
lene is provided as an alternative substrate for the nitro-
genase enzyme responsible for biological N
2
-fixation
activity, were performed using a method adapted from
Hardy et al. (1968) and Döbereiner et al. (1972).
Because most soil samples available for this study were
previously sieved and fresh-frozen (e.g., May 2011
soils), this study utilized sieved bulk soil samples rather
than soil cores assayed in situ, as are often done in field
studies of nitrogen dynamics (e.g., Keuter et al. 2014;
Strauss et al. 2012). In addition, because free-living
biological N fixation by soil microorganisms varies
seasonally (Belnap 2002; Watanabe et al. 1978), only
soils from October 2012 and 2013 were compared for
changes in activity over time, whereas soils from
May 2011 were used only to detect differences resulting
from endophyte treatments. Six grams dry weight equiv-
alent (DWE) each of thawed, field-moist soil samples
from May 2011, October 2012, and October 2013 were
weighed into 50 ml plastic centrifuge tubes with O-rings
and septum installed in the caps, adjusted to 30 %
gravimetric soil moisture content, and allowed to pre-
incubate at 20 °C, uncapped and covered with Parafilm,
for 2 days to equilibrate from storage conditions. To
avoid any physiological effects of long-term acetylene
exposure on microorganisms (David and Fay 1977)or
possible long-term selection for acetylene use with-
in the soil microbial community, which might in-
terfere with treatment effects, we chose an assay
incubation time of 6 h.
Acetylene (C
2
H
2
) gas was generated by adding dis-
tilled H
2
O to evacuated calcium carbide granules
(Fisher Scientific, #C57-500) in a glass serum bottle.
For each soil sample, C
2
H
2
was injected into assay tubes
to 0.1 atm. Blank tubes, containing no soil but receiving
C
2
H
2
, were included during each assay to correct for
ethylene (C
2
H
4
) impurities in laboratory-generated
C
2
H
2
gas. Assay tubes were incubated at 20 °C in the
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dark for 6 h after injection. Gas sub-samples were with-
drawn from each tube at 6 h and placed in pre-evacuated
13 mm crimp-top glass vials, then stored under water to
prevent leakage until gas chromatography (GC) analysis
within 24 h. The C
2
H
4
concentration in 100 μLinjec-
tions of each stored sample was measured on a
Shimadzu GC-14A (Shimadzu Scientific, Columbia,
Maryland, USA) equipped with a Poropak R column
(80—100 mesh, 2×2 mm). Samples were passed
through a flame ionization detector (FID) using an in-
jection temperature of 70 °C, an initial column temper-
ature of 50 °C, and a final detector temperature of
155 °C, and using N
2
as a carrier gas at 200 kPa. After
calculating injected sample concentrations using pure
C
2
H
4
gas (100 ppm C
2
H
4
in He, Matheson Tri-Gas
Inc., #GMT10325TK, Twinsburg, OH) as a standard
and subsequently adjusting for C
2
H
4
impurity from
blank assay tubes, the amount of C
2
H
4
evolved from
C
2
H
2
during the 6 h incubation assay per gram DWE
soil was calculated for each sample as nmol C
2
H
4
g
−1
dry soil.
Statistical analysis
We tested for statistically significant effects (α=0.05) of
endophyte treatment, forage type, and year of soil sam-
pling, where applicable, on measured plant and soil
parameters using the PROC MIXED procedure in SAS
(9.3 SAS Institute Inc., Cary, NC, USA). To examine
differences in δ
15
N between forage types from each plot,
the data were analyzed as a split-plot design within the
experimental randomized complete block design
(RCBD), with endophyte treatment and forage type as
fixed effects, and both block and the interactive effects
of treatment and block specified as random effects.
Significant endophyte treatment effects and changes
over time were analyzed for δ
15
N in soil samples using
the previously described mixed modeling procedure in
SAS, though with no split-plot designation. Endophyte
treatment and year of sampling were modeled as fixed
effects, with block specified as a random effect, and a
repeated measures statement for each block×treatment
by year was added to detect significant changes over
time. Results from soil ARAs were statistically analyzed
two ways where: 1) the fixed effects of endophyte
treatment and sampling year (October 2012 and 2013)
were examined using repeated measures as described
above for analysis of soil δ
15
N, and 2) 2011 soils were
individually analyzed for only the fixed effects of
endophyte treatment without the repeated measures
statement. Individual relative abundance estimates of
tall fescue and red clover in 2011 were also analyzed
for fixed effects of endophyte treatment and random
effects ofblock using PROC MIXED. Where significant
main or interactive effects were found, significant dif-
ferences between individual treatments, years, or forage
types for all analyses were determined by comparing the
LSMEANS using the PDIFF option in SAS.
Results
Plant composition
Endophyte infection treatments resulted in significant
differences in the relative abundance of tall fescue
(Fig. 1a;p=0.0021; F
4, 20
=6.17), where CTE+ plots
contained approximately 42 % more tall fescue cover
than E- plots and approximately 32 % more than in
AR542 NE+ plots. The relative abundance of red clover
cover was not significantly affected by endophyte treat-
ments (Fig. 1b;p=0.1241; F
4, 20
=2.06) and averaged
15 % (±1.86 S.E.) across plots, although red clover was
more abundant in plots with significantly reduced abun-
dance of tall fescue, such as E- and AR542 NE+ plots.
Endophyte infection significantly reduced the abun-
dance of graminoid species other than tall fescue
(Fig. 1c;p=0.0379; F
4, 20
=3.12), with CTE+ and both
NE+ treatments containing approximately 12 % less
other graminoid cover, on average, than E- plots. The
relative cover of forb species, excluding red clover, was
also significantly affected by endophyte infection
(Fig. 1d;p=0.0021; F
4, 20
=6.18), in that CTE+ plots
contained approximately 20 % less forb cover than E-
plots, and 7.5 % less forb cover than EMix plots.
Stable isotope analysis in plant and soil samples
Forage types
While endophyte infection status did not significantly
alter the natural abundance of
15
N(δ
15
N) within asso-
ciated red clover (RC) samples (p>0.05), δ
15
Nintall
fescue samples differed significantly within both TF(+
RC) and TF(-RC) forage type and endophyte treatment
(Fig. 2a,Endophyte×Foragep=0.016; F
8, 45
=2.71).As
expected, δ
15
N of RC samples were significantly more
depleted than all tall fescue samples (Fig. 2a;all
Plant Soil
Author's personal copy
p<0.05), indicating RC utilization of primarily
15
N-
depleted N products via symbiotic N
2
-fixation. This
forage type effect was consistent acrossendophyte treat-
ments. For tall fescue growing near red clover, TF(+
RC), samples from plots infected with either the com-
mon toxic endophyte (CTE+) or the novel endophyte
AR584 (AR584 NE+) were significantly more depleted
in δ
15
N compared to AR542 NE+ plots (Fig. 2a), but
were not different than E- tall fescue. However, when
located away from red clover, TF(-RC), samples from
only endophyte free (E-) plots were significantly deplet-
ed compared to all other endophyte treatments
(p<0.05).
The N concentration (%) of RC samples differed
significantly as a result of endophyte treatment, with
RC from E-, AR542 NE+, and EMix plots containing
Fig. 1 Estimates of relative cover (%) in each treatment plot for a
tall fescue, bred clover, cother graminoid species, excluding tall
fescue, and dforb species, excluding red clover in September
2011. Within each panel, a, b, c denote significant differences
between endophyte treatments (α= 0.05), while bars indicate±
1S.E.ofeachaverage
Fig. 2 a
15
N natural abundance (δ
15
N) and btotal aboveground
plant tissue nitrogen concentration (%) measured in red clover
(RC), tall fescue associated with red clover (TF + RC), and tall
fescue not associated with red clover (TF-RC) harvested from each
endophyte treatment plot in September 2011. Within each forage
type, a, b, c indicate significant differences between endophyte
treatments (α<0.05; NS not significant). A, B, C, indicate signif-
icant differences between forage type across endophyte treatments
in the x-axis labels
Plant Soil
Author's personal copy
significantly higher N than from CTE+ plots (Fig. 2b,
Endophyte × Forage p= 0.0446; F8, 45= 2.21). Within
tall fescue samples, no significant differences in N were
measured between endophyte treatments, and this effect
was consistent regardless of whether tall fescue was
located near red clover or not.
Soil samples
Averaged across endophyte treatments, soil δ
15
Nsteadi-
ly and significantly declined during each year of analy-
sis (Fig. 3a, Year p<0.0001; F
3, 92
=41.62), whereas no
significant differences were measured between endo-
phyte treatments either individually (Endophyte p=
0.8785; F
4, 92
=0.30) or over time (Endophyte × Year
p=0.1303; F
12, 92
=1.52).
The N concentration measured in soil samples was
significantly affected by the interaction between endo-
phyte treatment and year of analysis (Fig. 3b, Endophyte
× Year p=0.0425; F
12, 92
=1.91), but increases over time
were small (on average, +0.0263 % N between 2010 and
2013). The Endophyte × Year interaction also appeared
to be driven by slightly higher N in AR584 NE+ and
AR542 NE+ plots in most years, with the least N t
contained in EMix plots (Fig. 3b).
Acetylene reduction in soil samples
No significant endophyte effects on potential free-living
N fixing activity were detected in either May 2011
(Fig. 4a, Endophyte p=0.1928; F
4, 10
=1.87), or
October 2012 and 2013 soils (Fig. 4b, Endophyte p=
0.9176; F
4, 24
=0.23). In May 2011 soils, free-living N-
fixing organisms showed slightly higher activity in
CTE+ and AR584 plots, but differences were not sig-
nificant. Overall potential activity significantly in-
creased between October 2012 and 2013 when analyzed
together in a repeated measures model (Fig. 4b, Year p=
0.0001; F
1, 24
=21.35), though no significant endophyte
effects or interactive effects of endophyte treatment and
year (Endophyte × Year p=0.0936; F
4, 24
=2.25) were
found.
Discussion
In this study, the infecting strain of E. coenophiala and
the proximity of red clover influenced the proportion of
biologically-fixed N
2
utilized by tall fescue, as indicated
by δ
15
N in tall fescue tissue. However, there were no
significant effects of either CTE+ or NE+ tall fescue on
δ
15
N within red clover, δ
15
N in soil samples, or the
potential activity of non-symbiotic N
2
-fixing soil micro-
organisms. The δ
15
N in soil samples from each treat-
ment at this site steadily declined over time, while non-
symbiotic N
2
fixation activity increased significantly
between the last two study years. These results suggest
that endophyte infection in tall fescue may not signifi-
cantly influence symbiotic or non-symbiotic N
2
-fixation
capacity in mixed species pastures, but different
Fig. 3 a
15
N natural abundance (δ
15
N) and btotal N concentra-
tion measured in bulk soil samples collected from each endophyte
treatment plot over time. a,b,cindicate significant differences
between average soil δ
15
N across treatments for the main effect of
year (α=0.05) in panel A, although data are presented by endo-
phyte treatment to aid interpretation. Data in panel B are arranged
to illustrate the significant interactive effect of endophyte treatment
and year (α=0.05). Points and bars represent treatment average±
1 S.E. In both panels, the dashed line represents the average site
δ
15
N or N measured in T
0
bulk soil samples collected immediately
prior to plotestablishment in 2008, which isprovided for reference
and thus not included in statistical analyses, and the grey shaded
area represents±1 S.E
Plant Soil
Author's personal copy
endophyte strains can affect the ability of tall fescue to
utilize fixed-N
2
produced by neighboring red clover or
free-living soil microorganisms.
Our first set of hypotheses, in which we expected that
CTE and NE infection would increase uptake of biolog-
ically fixed N in tall fescue and alter biological N
cycling both in neighboring red clover and in free-
living soil microorganisms, were unsupported by the
results of our study. Uptake of
15
N-depleted N products
in tall fescue grown near red clover was not altered
solely by endophyte infection, although endophyte
strain did appear to influence uptake of
15
N-depleted
products in tall fescue (Fig. 2a). When grown near red
clover, tall fescue infected with the novel AR542 endo-
phyte accessed significantly fewer
15
N-depleted prod-
ucts than either CTE+ or AR584 NE+ tall fescue. This
suggests that although neither endophyte infection nor
toxicity of endophyte strain in tall fescue alters access to
immediately proximate products of biological N
2
fixa-
tion compared to E- plots, plants with different endo-
phyte strains differ in their ability to gain fixed N from
neighboring red clover. However, this dynamic was not
observed in tall fescue samples collected within the
same endophyte treatment plots yet were spatially iso-
lated from red clover. In TF(-RC) samples, only E- tall
fescue exhibited significant δ
15
N depletion compared to
either CTE+ or NE+ tall fescue. The N isotope signature
of tall fescue may be altered if mycorrhizal networks
were impacted by endophyte strain or proximity to red
clover, since transfer of N through mycorrhizal net-
works is known to fractionate against
15
N (Hobbie and
Ouimette 2009). For example, greater transfer of
biologically-fixed N from red clover to tall fescue may
have occurred through increased mycorrhizal networks
(Haystead et al. 1988;Mårtenssonetal.1998) in TF(+
RC) samples compared to TF(-RC) samples or in E-
plots compared to endophyte-infected plots (Chu-Chou
et al. 1992; Guo et al. 1992).
We found no evidence of endophyte-associated sig-
nificant differences in N dynamics from analysis of
plant or soil δ
15
N or in assays of free-living bacteria
activity. Endophyte presence and strain had no signifi-
cant effect on δ
15
N in red clover grown adjacent to tall
fescue in this study (Fig. 2a), despite significant changes
in tissue N (Fig. 2b). Though our findings support
earlier reports suggesting that neither endophyte nor
alkaloid presence in tall fescue is the mechanism respon-
sible for reduced legume seedling germination and
growth (Dirihan et al. 2014;Springer1996;Staleyand
Belesky 2004), we had originally hypothesized that
differences in plant competition between legumes and
tall fescue resulting from endophyte presence or strain
would subsequently affect N
2
-fixation capacity.
However, although the effects of endophyte presence
and strain on utilization of
15
N-depleted products, such
as biologically-fixed N
2
, in red clover were not signifi-
cant in this study, the significant differences measured in
N concentration of red clover (Fig. 2b) reflect the trends
observed in δ
15
N(Fig.2a). N concentration of red
clover tissue was significantly lower in plots containing
CTE+ or AR584 NE+ tall fescue compared to AR542
NE+ plots, and somewhat lower than E- or EMix plots.
Fig. 4 Potential non-symbiotic N
2
-fixation results determined via
assays of C
2
H
2
reduction to C
2
H
4
in bulk soil samples from a
May2011andbOctober 2012 and 2013. No significant effects of
endophyte treatment were detected in May 2011 soils, which were
not compared to October 2012 and 2013 soils because of con-
founding differences in seasonal variation of microbial activity. A,
B, C denote significant main effects of year between October 2012
and 2013 (α=0.05). Bars in each panel indicate averages± 1 S.E
Plant Soil
Author's personal copy
In a reversal of this trend, δ
15
N of red clover was
most heavily depleted in CTE+ or AR584 NE+
plots, though not significantly. This suggests that
although red clover in CTE+ or AR584 NE+ plots
relied most heavily on biological N fixation, less N
was incorporated into aboveground tissue.
Schipanski and Drinkwater (2012) estimated that in
red clover-orchardgrass mixtures, N fixation activity
increasedby15%duetotransferoffixedNbe-
tween species. García Parisi et al. (2014) also found
that asexual Epichloë spp. infection of annual rye-
grass almost doubled N fixation activity and bio-
mass in neighboring white clover despite a reduction
in nodulation. These could explain our results,
where products of higher N
2
-fixation in red clover
may have been increasingly transferred to other
plant species such as tall fescue in the TF(+RC)
CTE+ and AR584 NE+ treatments, especially com-
pared to AR542 NE+, while competitive ability of
E- tall fescue for N seemed little impacted by prox-
imity to red clover. Although this endophyte-specific
mediated increase in tall fescue’s competitive ability
is supported by significantly increased CTE+ tall
fescue cover compared to AR542 NE+, coupled
with trends for decreased red clover cover in CTE+
plots compared to AR542 NE+ (Fig. 1), no such
biomass trends were observed for AR584 NE+ plots,
suggesting other competitive mechanisms were in-
fluenced by this strain of the endophyte. We suggest
that differential mechanisms and effects of endo-
phyte strain will impact nutrient transfer dynamics
and legume N
2
-fixation, as well as legume and for-
age nutritive value.
Few studies have investigated the effects of
endophyte-infected tall fescue on soil microbial
communities, and, to our knowledge, no studies
have examined these effects on biological N
2
-fixing
activity by free-living soil diazotrophs. Iqbal et al.
(2012) found higher total microbial biomass in
CTE+ plots compared to E-, whereas Franzluebbers
et al. (1999) measured lower microbial biomass and
respiration in soils associated with tall fescue with a
high endophyte infection frequency compared to
low endophyte infection frequency. We were there-
fore surprised to find no significant effects of endo-
phyte infection on assays of acetylene reduction in
soils from each treatment over multiple years.
Though unmeasured in this study, we expected po-
tential endophyte-associated differences in soil
microbial biomass, as measured in other studies, to
elicit differences in activity of non-symbiotic N
2
-
fixing soil microorganisms. However, grasslands
are known for having lower global rates of non-
symbiotic N fixation relative to other ecosystems
such as tropical rainforests (Cleveland et al. 1999),
and thus the proportion of N
2
-fixing microbes within
the microbial biomass may have been too small at
our site to be affected by potential changes in total
microbial biomass. The low overall activity at this
site may have also resulted from incubating samples
in the dark, which excluded autotrophic diazotrophs
such as cyanobacteria, and from lack of glucose
amendment prior to incubation to decrease carbon
limitation and increase activities. In May 2011 soils
(Fig. 4a), non-significant trends in our results
showed increased non-symbiotic N
2
-fixation activity
in CTE+ soils compared to E- soils. This result
complements a study by Franzluebbers and Hill
(2005), who found increased microbial biomass N,
but reduced microbial biomass C, in soils exposed to
E+ tall fescue litter relative to E- tissue, although we
caution that we did not consistently observe this
effect across years (Fig. 4b). It is of interest to note
that trends observed in non-symbiotic N
2
-fixation
from May 2011 soils closely followed those ob-
served in δ
15
N of red clover tissue, which indicated
a greater degree of reliance of N
2
fixation due to
competitive demand by CTE+ tall fescue. Non-
symbiotic N
2
-fixation activity in May 2011 was
higher in CTE+ and AR584 NE+ soils than in other
treatments, potentially providing further support, al-
though not significant, for our above discussion of
stimulated biological N
2
fixation resulting from in-
creased competition for N and N transfer between
grasses and legumes in mixed stands. However, we
again caution that these trends were not observed in
soils from October of 2012 or 2013, so it is also
possible that some
15
N-depleted N utilized by red
clover was a product of non-symbiotic soil microor-
ganisms rather than symbiotic BNF.
Our second set of hypotheses, which predicted
that differences in biological N fixation and N up-
take as a result of endophyte presence or strain
infecting tall fescue would produce long-term effects
on soil N pools, were also unsupported by our
results. No consistent differences between endo-
phyte treatments were observed either in soil δ
15
N
(Fig. 3a) or potential non-symbiotic N
2
fixation
Plant Soil
Author's personal copy
(Fig. 4b) over time. Lack of endophyte effects on
long-term soil N pools was likely due to the fact
that, while we did observe differences in pool access
in tall fescue (Fig. 2a), no changes were observed in
tall fescue tissue N (Fig. 2b), which was the domi-
nant plant species in each treatment (Fig. 1).
Although one may expect to see long-term changes
in δ
15
N resulting from differences observed both in
δ
15
N and N concentration in red clover, in 2011,
these changes may have been too small for detection
because of relatively low abundance of red clover in
subsequent years (data not shown), and the absence
of endophyte treatment effects on the relative abun-
dance (and N fixation) of red clover in 2011 (Fig. 1).
Differences in soil δ
15
N between endophyte treat-
ment plots from 2010—2013 were inconsistent and
not statistically significant, although interannual dy-
namics appeared to pair E- with CTE+ plots, and
AR542 NE+ with EMix plots (Fig. 3a). Statistically
significant differences in soil N concentration did
occur between endophyte treatments over time
(Fig. 3b), but these changes in N were very small
and did not reflect the treatment patterns observed in
δ
15
N. The relative subtlety of endophyte effects on
soil δ
15
N in this study could potentially be due to
relationships between BNF and soil phosphorus.
Although much of the relationship between legume
N and P requirements across ecosystem characteris-
tics and plant species remains unclear, studies have
often shown that adequate P levels are an important
control of biological N fixation (Vitousek et al.
2002). Because we assumed BNF to be the primary
source of
15
N-depleted N, and low N:P ratios
increase N
2
-fixation (Eisele et al. 1989; Vitousek
and Field 1999), it is possible that differences in
N
2
-fixation between treatments in this study were
minimized by naturally high levels of P in our
soils from phosphatic limestone parent material
(Karathanasis 1991).
Although no significant endophyte effects on BNF
were measured in this study, we found significant
changes over time for both δ
15
N and the activity of
free-living N fixing soil microorganisms. This site
exhibited significant declines in soil δ
15
N between
2010 and 2013, which may suggest that either
15
Nis
being lost or
14
N is accumulating. Many soils exhibit
15
N enrichment over time, because
15
N-depleted
forms of N produced through biologically mediated
transformations are discriminated against and
accumulate in soils as stable organic N while
14
N-
enriched inorganic N is lost (e.g., Brenner et al. 2001;
Menge et al. 2011). However, Brenner et al. (2001)
also attributed increased δ
15
N of older soils to even-
tual P-limitation. Our site’s naturally high soil P
levels discussed above may have also resulted in
relatively less N loss over time compared to other
studies. Temperton et al. (2007) further observed that
increasing species richness in pasture soils decreased
soil δ
15
N independently of legume effects, and we
have also observed increased plant diversity across
treatment plots since planting only tall fescue in 2008
(Iqbal et al. 2013). In addition, decline of δ
15
Nacross
pasture soil chronosequences was observed by
Piccolo et al. (1996), who attributed decreased δ
15
N
to increased inputs of BNF over time. We glimpsed a
similar effect in our study through the significant
increase in non-symbiotic BNF between 2012 and
2013 (Fig. 4b). Thus, adequate soil P and potentially
increased BNF inputs over time may have contribut-
ed to steadily decreasing soil δ
15
N at our temperate
grassland site regardless of endophyte treatments.
Conclusions
The results of this study suggest that regardless of
alkaloid profile or toxicity, specific endophyte strain-
tall fescue combinations differentially impact the
amount of biologically-fixed N
2
utilized by tall fescue,
though not resultant tissue N, when grown in close
association with red clover in mixed species pastures.
However, when spatially distant from red clover, only
E- tall fescue maintained the ability to utilize more
products of biological N fixation. Assays of non-
symbiotic soil microbial N
2
-fixation in bulk soils did
not reveal any endophyte treatment effects, and there
were no differences between treatments in soil δ
15
N
over time. A steady decline in average soil δ
15
Nover
time at this site might be attributable either to successive
closure to N-loss over time, increased biological N
fixation inputs, or to minimized P limitations due to
phosphatic parent material and adequate rainfall.
Different effects of endophyte strain on tall fescue com-
petitive ability and utilization of N produced by N-
fixing symbioses are likely to impact nutrient cycling
of pastures and therefore should be considered in the
development and adoption of new grass-endophyte
combinations.
Plant Soil
Author's personal copy
Acknowledgments This research was supported by a grant to
L.C. Slaughter from the Karri Casner Environmental Sciences
Fellowship, which is sponsored by the University Of Kentucky
College Of Agriculture’s Environmental and Natural Resources
Initiative. L.C. Slaughter was supported by an assistantship from
the Department of Plant and Soil Sciences at UK. The authors
would like to thank Dr. Suvankar Chakraborty and the Stable
Isotope Laboratory at UK, Slone Research Building, for his patient
guidance in conducting analyses for this project. We also thank Dr.
Elisa D’Angelo, who provided laboratory equipment and assis-
tance with acetylene reduction assays, and Kristen McQuerry of
the Applied Statistics Laboratory at UK, for assistance on statisti-
cal analyses for this research. We thank the Noble Foundation for
providing seed and endophyte treatment assessments. This field
project was supported with funds from the Kentucky Agricultural
Experiment Station and a cooperative agreement between UK’s
College of Agriculture, Food, and the Environment and the
USDA-ARS-Forage Animal Production Research Unit.
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