Does Fungal Endophyte Infection Improve Tall Fescue’s
Growth Response to Fire and Water Limitation?
Sarah L. Hall
, Rebecca L. McCulley
, Robert J. Barney
, Timothy D. Phillips
1Department of Plant and Soil Sciences, University of Kentucky, Lexington, Kentucky, United States of America, 2Community Research Service, Kentucky State University,
Frankfort, Kentucky, United States of America
Invasive species may owe some of their success in competing and co-existing with native species to microbial symbioses
they are capable of forming. Tall fescue is a cool-season, non-native, invasive grass capable of co-existing with native warm-
season grasses in North American grasslands that frequently experience fire, drought, and cold winters, conditions to which
the native species should be better-adapted than tall fescue. We hypothesized that tall fescue’s ability to form a symbiosis
with Neotyphodium coenophialum, an aboveground fungal endophyte, may enhance its environmental stress tolerance and
persistence in these environments. We used a greenhouse experiment to examine the effects of endophyte infection (E+vs.
E2), prescribed fire (1 burn vs. 2 burn vs. unburned control), and watering regime (dry vs. wet) on tall fescue growth. We
assessed treatment effects for growth rates and the following response variables: total tiller length, number of tillers
recruited during the experiment, number of reproductive tillers, tiller biomass, root biomass, and total biomass. Water
regime significantly affected all response variables, with less growth and lower growth rates observed under the dry water
regime compared to the wet. The burn treatments significantly affected total tiller length, number of reproductive tillers,
total tiller biomass, and total biomass, but treatment differences were not consistent across parameters. Overall, fire seemed
to enhance growth. Endophyte status significantly affected total tiller length and tiller biomass, but the effect was opposite
what we predicted (E2.E+). The results from our experiment indicated that tall fescue was relatively tolerant of fire, even
when combined with dry conditions, and that the fungal endophyte symbiosis was not important in governing this
ecological ability. The persistence of tall fescue in native grassland ecosystems may be linked to other endophyte-conferred
abilities not measured here (e.g., herbivory release) or may not be related to this plant-microbial symbiosis.
Citation: Hall SL, McCulley RL, Barney RJ, Phillips TD (2014) Does Fungal Endophyte Infection Improve Tall Fescue’s Growth Response to Fire and Water
Limitation? PLoS ONE 9(1): e86904. doi:10.1371/journal.pone.0086904
Editor: Martin Heil, Centro de Investigacio
´n y de Estudios Avanzados, Mexico
Received July 24, 2013; Accepted December 16, 2013; Published January 31, 2014
Copyright: ß2014 Hall et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: Funding for this project was provided by a Specific Cooperative Agreement (58-6440-7-135) between the Forage-Animal Production Research Unit of
USDA-ARS and the University of Kentucky, Kentucky Agricultural Experiment Station Award KY006045, DOE-NICCR award #08-SC-NICCR-1073, USDACSREES
Project KYX-10-05-39P, and a fellowship from the University of Kentucky Graduate School. The funders had no role in study design, data collection and analysis,
decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: firstname.lastname@example.org
¤a Current address: Department of Agriculture & Natural Resources, Berea College, CPO 2161, Berea, Kentucky, United States of America
¤b Current address: GRDI Land-Grant Institute, West Virginia State University, Institute, West Virginia, United States of America
Plant species may be considered invasive when they successfully
spread outside their native range , and may use a number of
mechanisms to gain competitive advantage over native species.
They may be released from their natural enemies and thus able to
thrive better in the new environment , they may simply be
better competitors for resources in disturbed environments ,
and/or they may use plant-soil feedbacks, including so-called
‘‘novel weapons’’ , to negatively impact co-occurring native
plants. In some cases, these effects may not be coming from the
plants alone, but may be mediated by their association with
microorganisms . Many plant functional traits have been linked
to association with bacterial and fungal microorganisms (reviewed
in ), with fungal endophytes of grasses (in the family
Clavicipitaceae) being one of the most studied associations ,
, . Association with these fungal endophytes has been linked
to success of the invasive annual Italian ryegrass , including
conferring increased herbicide resistance  (but see ). In
addition, many of the grass functional traits affected by fungal
endophytes could be considered traits that make the grasses more
competitive  and potentially more able to successfully persist
and/or invade novel habitats.
Tall fescue (Schedonorus phoenix (Scop.) Holub) is a non-native C
grass species, introduced in the late 1800’s which now covers 14
million hectares in the United States, with its adapted range being
the entire eastern U.S. and areas within the Pacific Northwest
, . In areas being managed for native warm-season
grasslands in North America, tall fescue is considered an
undesirable species, in part because it can outcompete native
grassland species , and in part due to negative effects on
wildlife . Prescribed fires are used widely in management of
many grasslands today  , either alone or in combination
with herbicide application . The persistence of tall fescue in
what are largely C
-dominated grass systems, which often undergo
frequent fire and/or water limitation (e.g. ), suggests it is
tolerant of these conditions. Other non-native cool-season
perennial grasses have been successfully eliminated with pre-
PLOS ONE | www.plosone.org 1 January 2014 | Volume 9 | Issue 1 | e86904
scribed burns , but this has not been the case for tall fescue,
which experienced no growth suppression following prescribed
burns in the field , , . One factor that may impact
growth response of tall fescue to management practices such as
prescribed fire is its frequent association with the fungal endophyte
Neotyphodium coenophialum (whose presence was unknown in the
studies reported in , , ).
The tall fescue-Neotyphodium symbiosis is known to increase tall
fescue’s stress tolerance over that of endophyte-free (E2)
individuals , . Endophyte presence within tall fescue
populations can vary across the landscape: within a single field,
some areas may have no individuals infected, whereas in other
areas, all individuals present are infected. Extensive surveys of tall
fescue populations in North America show that on average .50%
of tall fescue tillers in an area test positive for endophyte presence
, , . Surveys of 17 tall fescue pastures being targeted
for restoration across the state of Kentucky found all but one had
endophyte infection frequencies (EIF) .80% . ‘Kentucky-31’,
the variety of tall fescue that is most common in pastures in this
region, has a higher occurrence of fungal endophyte symbiosis
than other varieties . The physiological benefits to tall fescue of
hosting N. coenophialum are thought to be most pronounced under
water , ,  or nutrient deficiency  (but see ), and
the fungus may actually serve as a physiological drain or sink when
the plant is not under such stress . Endophyte-infected (E+)
fescue has been shown to have larger belowground biomass
compared to E2tall fescue , , , which could serve as a
greater resource from which to recover following management
activities that negatively impact aboveground growth of the plant.
E+plants have also been shown to respond to increased nutrient
availability (which may be influenced by management) more than
E2plants , .
Prescribed fire is used as a management tool in many grasslands,
and can affect the abiotic and biotic components of the ecosystem.
In mesic grasslands effects of fire on the abiotic environment
include increased light levels and decreased soil moisture at the
surface, and increased nutrient availability . These abiotic
effects may in turn affect biotic components of the grassland
systems where they occur. Burning has been shown to reduce
cover of some non-native species , ,  and C
, ,  while simultaneously increasing native warm-
season grass tillering . The behavior of fire (which determines
impacts to the abiotic environment and effectiveness as a
management tool) can also be impacted by the vegetation present
, and areas dominated by C
’s, like tall fescue, often
experience reduced intensity of early spring burns  , as
they may have already begun to grow. In some ways, the effects of
prescribed fire on tall fescue might be similar to those of grazing
(e.g. removal of aboveground biomass), but comparative effects of
these two common grassland disturbances most likely vary
depending on the severity or intensity of the events and their
distribution in space and time . Prior studies have shown that
fungal endophyte presence within tall fescue can alter herbivory
 and improve plant persistence and performance under grazed
conditions (e.g. , , , ). However, we are aware of
no studies examining prescribed fire and its interaction with N.
coenophialum on tall fescue survival and regrowth.
Given that fire has been shown to affect some of the same
abiotic parameters also known to be important in determining
whether endophyte symbiosis increases tall fescue’s competitive
ability or reduces it (e.g., increases light availability, lowers soil
moisture, increases nutrient availability), we wanted to explore
whether endophyte infection confers greater tolerance to fire.
Given previous research indicating physiological benefits of
Neotyphodium being most pronounced under water stress, we also
incorporated two levels of water availability in the experiment.
We designed a controlled greenhouse experiment to test
differences in growth following prescribed burn and water
availability treatments for E+and E2tall fescue. This experiment
used established tall fescue plants (variety Kentucky-31, either with
(E+) or without (E2) the common toxic strain of N. coenophialum)to
which we applied a water availability treatment, providing half the
plants with adequate water supply (‘wet’), and half the plants with
half as much water (‘dry’). We included an unburned control, a
single burn treatment (1x), and a two burn treatment (2x). Based
on prior work that suggests the fungal endophyte symbiosis is
generally mutualistic, especially under stressful abiotic conditions,
we hypothesized E+plants would have higher biomass and growth
compared to E2plants, and that differences would be most
pronounced under the dry treatment. We also thought differences
between the E+and E2plant responses would be greatest for
those individuals that received the presumably more stressful 2x
Materials and Methods
This experiment consisted of a full factorial design, with E2and
E+tall fescue, three burn treatments (1x, 2x, and unburned
control), a wet and dry treatment, and six replicates per treatment
combination (2636266 = 72 plants). Replicates were arranged
into six randomized blocks.
Field Collection of Plant Material
On 6 and 9 March 2009, tall fescue plants (cultivar Kentucky-
31) were removed from 0.08-ha plots established in 2001 at the
University of Kentucky Research Farm, Lexington, KY, USA, for
the purpose of E+and E2tall fescue seed production (n = 2 plots
of each endophyte status; common toxic strain of N. coenophialum)
(39.219167uN286.541389uW). Prior work has shown that plant
genotype can affect the nature of the grass-fungal endophyte
symbiosis , , , ; however, it was beyond the scope
of this study to evaluate plant genotype interactions. Individual
plants that had two to three overwintering tillers were selected
from E2and E+plots. When measured in 2007, these plots were
dominated by tall fescue (visual cover estimates of 92% in E+and
84% in E2) and when tested using an immunoblot assay specific
for N. coenophialum (Agrinostics Ltd., Watkinsville, GA) 96% of
tillers tested positive for endophyte infection in E+plots and 4%
for E2. PVC pipe sections (7.5cm in diameter, 22 cm in
length) were placed around each individual plant, and a hammer
was used to pound the pipe into the ground, leaving a 2-cm deep
rim above the soil surface. Each pipe section was extracted, and
contained the top 20 cm of soil and the individual tall fescue plant.
These pipe sections served as pots in the greenhouse, and will be
referred to as such. Each was marked as E2or E+, and plants
were transferred to a greenhouse at the Kentucky State University
Research Farm (38.116065uN284.890506uW).
Plants were given ambient light with a 21uC day (12 hr) and
15.6uC night (12 hr). Pots were individually numbered, and were
randomly assigned burn treatment, watering regime, and replicate
number. During this initial 2.5 week growing period (from 6 or 9
March to 25 March), all pots were watered twice a week to field
capacity. Pots were arranged in six randomized blocks across a
single greenhouse bench. On 18 March, the number of tillers for
each individual pot was counted (mean6SE = 6.9+0.3). All tillers
were clipped to 4-cm height, and clipped material was kept from
Tall Fescue Growth following Fire and Water Stress
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each pot and placed in plastic bags. The following day a wet
weight was measured for all clipped material. Eight E2and eight
E+pots were randomly selected for harvesting at this time (18
March) to estimate belowground biomass prior to the experiment,
and to obtain pre-treatment soil moisture levels. At this time, it
had been two days since the last watering event.
Burn Treatments, Watering Regime, and Fertilization
Senesced plant litter of native warm-season grasses (primarily
switchgrass (Panicum virgatum L.) and little bluestem (Schizachyrium
scoparium (Michx.) Nash)) was collected from a field at the KSU
Research Farm to serve as fuel for the prescribed burn treatments,
in an effort to mimic litter conditions in native grassland where tall
fescue is present. It was placed in the greenhouse for one week to
dry, and was then cut into approximately 3-cm pieces. In order to
evaluate heat levels of the prescribed fire treatment, we made
aluminum tags that were painted with different heat-sensitive
paints (Tempil Inc., S. Plainfield, NJ) that change appearance at
79, 163, 246, 316, 399, and 510uC. Each tag was wrapped in
aluminum foil (which melts at 644uC).
On 25 March, the first prescribed burn treatment was applied
to all pots assigned to one of the two burn treatments (1x or 2x). A
single heat-sensitive paint tag was anchored at the crown level of
each pot with a paper clip. Pots were burned in (random) groups of
12 on a concrete floor inside the headhouse. Doors were opened
on either side of the headhouse to allow for some air movement.
Pots were placed such that paint tags were all in the same direction
(with air flow). A 250-ml cup was used to scoop dried native grass
litter (approximately 5.3 g) onto each pot. Care was taken to get as
much litter as possible over each pot. A standard lighter was used
to light the native grass litter in all pots for each group. Once the
fire had burned out (#1minute), paint tags were removed and
marked with their respective pot number. Relative humidity inside
the headhouse was 100% (it was raining outside at the time of the
burns). Subsamples of native grass litter (n = 8; 250 ml each)
analyzed for fuel moisture and variance in fuel amounts indicated
that litter additions weighed 5.3460.22 g (mean6standard error
of the mean) and contained 9.8760.03% moisture.
Water regime treatments began the day of the first burn, with
only the wet pots receiving water immediately following the burn.
On 1 April, both the wet and dry treatments were watered, the wet
pots receiving 116 ml (volume of water based on long-term
average of weekly March-June precipitation for Lexington, KY,
www.weather.com calculated and applied based on the area of
each pot) and the dry pots received half this amount, 58 ml. Pots
were watered with these amounts 2–3 times per week as needed
for the rest of the experiment. On 8 May, all pots were fertilized
with 58 ml 10-20-10 NPK fertilizer (Peters 20-10-20 Greenhouse
Fertilizer Peat-lite). Wet pots were given an additional 58 ml water
without fertilizer to maintain this treatment. The same procedure
was also used on 3 June to fertilize all pots.
On 12 May, the second prescribed burn treatment was applied
to those pots assigned to that treatment. The same procedure was
followed as for the first burn, except no paint tags were used.
Relative humidity was approximately 55% at the time of the burn.
Prior to burning, all plastic markers used to identify and track
individual tillers were removed, and tillers that emerged after the
burn treatment were marked anew (as a new ‘‘cohort’’).
Subsamples of fuel (n = 8; 250 ml each) weighed 6.0860.33 and
contained 5.0560.07% moisture.
All tillers were measured weekly (during the first month) and bi-
weekly thereafter. Each tiller was measured by recording the
distance from the base of the tiller to the longest green part of a
leaf blade on that tiller, and lengths for all tillers in a given pot
were combined to provide the total pot length for each
measurement. New tillers that emerged during the experiment
were measured as they appeared. Reproductive tillers were clipped
to prevent seed from developing, and date of flowering was noted
(these measurements occurred during the more frequent watering
events). This clipped material was kept to be added to oven-dry
aboveground material for biomass measurements.
On 26 Jun (100 days after experiment initiation), all control pots
were harvested. Each tiller was cut at the soil surface and placed in
a coin envelope. Tillers were stored cool, and double blotted onto
nitrocellulose paper for endophyte testing (Agrinostics Ltd.,
Watkinsville, GA). Soil from each pot was sieved, and roots were
removed by hand-picking. Individual tiller material and pot root
material were dried at 55uC for 48 hours to obtain biomass. A 5-g
subsample of soil from each pot was used to measure gravimetric
soil water content. Burned pots were harvested 10 July (2x burned)
and 13 July (1x burned) (114–117 days after experiment initiation,
and 107–110 days after the first prescribed burn), with the same
procedures followed as described for the control pots. Weights of
all crown and root material were ash-corrected by placing a 0.5-g
subsample of harvested biomass in a muffle furnace at 550uC for
We analyzed all data considering the pot as the experimental
unit (not individual tillers). Data (at the pot level) for the following
response variables were analyzed using Proc GLM to test for
effects of endophyte presence, watering regime, burn treatment,
and all interactions: final total pot tiller length, number new tillers
(difference between tiller number on 18 March, prior to treatment
implementation, and at harvest), number reproductive tillers,
oven-dry total tiller biomass, oven-dry root biomass, and total
oven-dry biomass (tillers, crowns and roots). LS Means procedure
for pairwise comparisons of means in SAS 9.2 (SAS Institute,
Cary, NC) was used to test for significant differences between
means. Means and standard errors were obtained using the Means
and Standard Deviation procedure in JMP 9.0 (SAS Institute,
In order to test whether the temperature of the first burn
treatment affected growth, Proc GLM (SAS 9.2) was used to test
for the effects of burn temperature and its interactions with water
regime and endophyte presence, on the total tiller length, number
new tillers, number reproductive tillers, and mean length per tiller
(total pot tiller length divided by number tillers present) for burned
pots as measured just prior to the second burn (12 May) for those
pots assigned to the 1x or 2x burn treatment.
In order to observe trends in growth for tall fescue tillers over
the entire experiment, total pot tiller length (61 S.E.) was
calculated at each of the nine measurement intervals (which took
place one to three days apart between treatments) and plotted on a
line graph over time. Means ANOVA procedure and Tukey-
Kramer HSD were used in JMP 9.0 to test for significant
differences between Wet/Dry, E+/E2, and 1x/2x/unburned
controls at each of these nine measurement intervals. Relative
growth rates (for total tiller length per pot) were also calculated for
the different measurement intervals by dividing the difference of
log lengths (at the beginning of the growth interval, and at the end
of the interval) by the number of days between measurements to
get the relative total tiller growth rate (cm/cm/day). For these
calculations, lengths of tillers were assumed to be 1-cm immedi-
ately following a prescribed burn. These relative growth rates were
also plotted on a line graph over time. Means ANOVA procedure
Tall Fescue Growth following Fire and Water Stress
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and Tukey-Kramer HSD were used in JMP 9.0 to test for
significant differences within measurement intervals between burn
Endophyte Infection Frequency and Initial Root Biomass
and Soil Moisture
Endophyte tests of tillers harvested at the end of the experiment
revealed twelve of 70 pots that were not 0% (E2) or 100% (E+)
endophyte-infected. Ten of the twelve were E+, two were E2.
They included six each of the wet and dry water regime. Three
were from the control treatment, two were burned once, and seven
were burned twice. Of these twelve, only two pots had endophyte
infection frequencies (EIF = total number tillers testing positive/
total number tillers) more than 50% off from what they were
supposed to be (one wet regime, 2x burn, E+pot with 40% EIF,
and one dry regime, 2x burn, E+pot with 28.6% EIF). These two
pots were removed from the dataset to ensure statistical analyses
were conducted on measurements from pots dominated by tall
fescue of the correct endophyte status.
For the 16 randomly selected pots that were harvested prior to
the implementation of water regime and burn treatments (18
March), soil moisture was significantly higher (P= 0.0385) for the
E+pots (mean6standard error of the mean; 17.4461.57%)
compared to E2pots (13.6360.55%). E2plants were extracted
from the field three days earlier than E+plants, but all had been
watered to field capacity two days prior to harvesting in the
greenhouse, so it seems unlikely that differences in soil moisture
were due to extraction date differences. Biomass of belowground
material for these 16 pots harvested prior to implementation of the
treatments revealed significantly (P= 0.0499) higher root biomass
for E+(0.9860.15 g) compared to E2(0.6360.06 g). The wet
weight of the plant material clipped and removed at this time (any
material .4-cm tall) for these same pots was not significantly
different between E+and E2plants (P= 0.4617). Therefore,
endophyte-related differences prior to the initiation of the
treatments were only apparent belowground (root biomass and
Effects of Prescribed Burn Treatments on Growth
The heat-sensitive paint tags used during the first prescribed
burn revealed that fire created temperatures ranging from ,79uC
(no paints melted) to .316uC but ,399uC (the fourth paint
melted). Of the 48 pots that were burned, five were ,79uC,
twenty-three were .79uC but ,163uC, one was .163uC but
,246uC, fifteen were .246uC but ,316uC, and four were
.316uC but ,399uC. Because the number of replicates was low
in several burn temperature categories, pots were categorized into
those that had experienced fire temperature of ,162uC and those
that had experienced fire temperatures of 246–398uC in order to
allow for LS Means comparisons (the one pot that was .163uC
but ,246uC was removed from the dataset). Burn temperature did
not have a significant effect for any of the measured variables using
this binned Proc GLM approach. Given the variability of
temperatures within each of these ranges, we also ran linear
regressions to see if any of the growth variables might be
significantly correlated to fire temperature. Again, no significant
relationships were identified. This lack of burn temperature effect
suggests that the variability observed in fire temperature at the
crown level did not result in differences in tall fescue growth as
measured in a greenhouse for 48 days after the burn treatment;
however, additional replicates would help further assess this claim.
Surprisingly, burn treatment (1x, 2x, or control/no burn) did not
significantly affect soil moisture averaged across wet/dry treat-
ments, which was similar (10.460.5% for 1x, 10.860.5% for 2x,
and 11.460.4% for control) in soils at the end of the experiment
across burn treatments.
Burn treatment had significant main effects on total pot tiller
length, number of reproductive tillers, tiller biomass, and total
biomass as measured at the end of the entire experiment period
(Table 1). The once and twice burned pots had greater total tiller
length than the unburned control at the final harvest, while the
opposite was true for number of reproductive tillers. The control
had more reproductive tillers than either of the burn treatments
(Table 2). Tiller biomass was greatest for the 1x burn treatment,
intermediate for the control, and lowest for the 2x burn treatment
(Table 2), but there was no effect of burning on root biomass
(Table 1). Total biomass was greater for the control and the 1x
burn treatment compared to the 2x burn treatment (Table 2).
When trends in total pot tiller length were compared over time,
they varied by burn treatment for the first five measurement
intervals following the 25 March prescribed burn (Figure 1). At
each of the first four measurement intervals during this period, the
control pots had greater tiller length compared to the burned pots,
but for the last measurement during this period (12 May, just prior
to the second burn), the 1x burn pots remained lower than the
controls, but the 2x burn pots had become similar to the control
(despite the fact that both 1x and 2x burn treatments had both
received the same treatment of one burn at this point in time). By
26 May, two weeks after the second burn was performed on the 2x
pots, there was no significant difference in total tiller length
between any of the burn treatments. Burn treatment did not have
a significant effect on total pot tiller length throughout the rest of
When relative total tiller growth rates were calculated over the
experiment by burn treatment, a number of trends emerged.
Immediately following the first prescribed burn, growth rates for
burned pots were significantly greater compared to the controls
(for the two weeks following the burn), and they remained
significantly greater at the next measurement interval (third week
after the burn) (Figure 2). At the fourth week after the burn these
differences had disappeared, and all growth rates were similar.
Similar growth rates persisted until the second burn was applied to
the 2x pots. Burning a second time stimulated higher tiller growth
rates in 2x pots than 1x pots for the month following the second
burn (Figure 2). For the two last measurement intervals (mid to late
June for all treatments, and late Jun to mid July for the 1x, 2x
burned pots) there were no significant differences in growth rates.
Two pots had no aboveground live material at the final harvest-
both were E2pots under the dry water regime that were burned
once or twice. One of these had no aboveground material at the
first measurement following the first prescribed burn, and the
other had very low growth following the first burn that declined
over time (no material present when second burn was applied).
Given this low number of pots that experienced mortality, and the
fact that one of them did have growth following the first prescribed
burn, no conclusions can be made as to why these plants
experienced mortality. The higher total tiller length at the final
harvest for burned pots compared to the controls (Table 2) clearly
suggests that burning did not negatively impact tall fescue growth,
even when applied twice in a single season. Fire stimulated growth
rate (but decreased biomass) initially following the first burn, with
even higher growth rate following the second burn (Figure 2), and
no depression in biomass accumulation (Figure 1).
Tall Fescue Growth following Fire and Water Stress
PLOS ONE | www.plosone.org 4 January 2014 | Volume 9 | Issue 1 | e86904
Effects of Water Regime on Growth
Water regime had the most pronounced and widespread effects
on measured growth variables, being significant for all parameters
(Table 1). In all cases, the dry watering regime had significantly
lower measured growth responses at the final harvest than the wet
treatment (Table 3). This was also true for the total tiller length at
all measurement intervals, the dry pots had less tiller length than
the wet (data not shown). Water regime was the only treatment
that significantly affected root biomass and new tiller number, with
the dry regime reducing both variables. A total of 29 reproductive
tillers appeared in 24 pots over the course of the experiment, and
all emerged in May. The ‘wet’ treatment had two times the
number of reproductive tillers than ‘dry’ (Table 3). Water regime
significantly affected date of flower during May (P= 0.0156), with
plants under the wet treatment flowering earlier (on average, ‘wet’
plants flowered on May 661 days) than those under the dry
treatment (average ‘dry’ date of flowering May 1263 days).
Clearly, tall fescue in those pots under the dry water regime was
limited in growth compared to those under the wet water regime,
as intended. Water regime had a significant effect on soil moisture
of the pots at the end of the experiment, with the dry pots having
significantly lower soil moisture than the wet pots (P= 0.0003;
9.9960.39% (‘dry’) vs. 11.8360.29% (‘wet’)).
Effects of Endophyte Presence on Growth
Endophyte status significantly affected total tiller length
(P= 0.0256) and final tiller biomass (P= 0.0129) (Table 1). In
both cases, the E2tall fescue plants had greater growth than E+.
Total tiller length was 193.9612.6 cm for E+, and
225.3614.8 cm for E2. Oven-dry tiller biomass was
2.1260.13 g for E+and 2.3760.13 g for E2. Surprisingly, we
did not find any significant interactions between the watering
regime and endophyte presence or burn treatment (Table 1). The
difference in soil moisture between E+and E2pots observed at
the initial harvest prior to implementation of the experimental
treatments was no longer present at the end of the experiment
(P= 0.3865), indicating that effects of the water regime treatment
on soil moisture had over-ridden any differences present at the
beginning of the experiment related to endophyte presence. The
root biomass differences were also no longer significant at the end
of the experiment (P= 0.8475).
Of the different treatments imposed during this experiment
(endophyte status, water regime, burn), water regime had the most
pronounced and consistent effect on tall fescue growth, with those
plants under the dry water regime having less growth than those
under the wet regime throughout the entire course of the
experiment. This result was not surprising given that tall fescue
is a C
species that cannot perform well during warm temperatures
unless adequate water is supplied . However, contrary to our
hypothesis and expectations, the effects of water stress imposed by
the dry regime were equally detrimental for both E+and E2
plants and across burn treatments. This was surprising, given that
others have observed endophyte-related differences in growth
responses, especially under dry conditions , , , ,
, although in some cases these effects have been varied by host
plant genotype , . It is possible that if we had controlled for
plant genotype (e.g. using genetic clone pairs of E+and E2
individuals) we would have found a different result. It is also
possible that our ‘dry’ treatment was not dry enough to stimulate
such endophyte effects, although it should be noted it was dry
enough to depress tall fescue growth (total biomass) by approx-
imately 32% at the end of the experiment. The relatively cool
temperatures of the greenhouse and the frequency of watering (dry
Table 1. F-value and degree of significance for effects of burn treatment (1x, 2x, unburned control), water regime (dry, wet), and
endophyte infection status (E+,E2) and their interactions on biomass measurements and tiller number at the final harvest.
(1) Trtmt*Endo (2)
FP.FF P.FF P.FF P.FF P.FF P.FF P.F
Total Length 5.48 ** 64.66 *** 5.25 * 0.99 ns 0.24 ns 1.43 ns 0.65 ns
Number New Tillers 0.19 ns 31.02 *** 1.67 ns 0.25 ns 0.04 ns 1.13 ns 0.82 ns
Number Reproductive Tillers 7.26 ** 5.41 * 3.59 ns 2.37 ns 0.48 ns 1.3 ns 1.87 ns
Tiller Biomass 14.83 *** 101.41 *** 6.58 * 0.02 ns 0.07 ns 0.85 ns 0.18 ns
Root Biomass 0.41 ns 17.37 *** 0.51 ns 0.58 ns 2.94 ns 0.69 ns 0.29 ns
Total Biomass 9.78 *** 73.14 *** 1.83 ns 0.14 ns 0.36 ns 1.84 ns 0.28 ns
Degrees of freedom are indicated in parentheses.
ns, not significant.
Table 2. Mean measured growth response variables (61 S.E.)
for tall fescue plants exposed to 1 prescribed burn (1x), 2
prescribed burns (2x), or no prescribed burn (control),
averaged across watering regimes and endophyte status.
1x 2x Control
Total Tiller Length (cm) 230.9617.9 a 221.3619.5 a 178.8612.4 b
Number Reproductive Tillers 0.2560.1 b 0.2360.1 b 0.7560.2 a
Tiller Biomass (g) 2.6060.16 a 1.8960.15 c 2.2360.13 b
Total Biomass (g) 5.6060.31 a 4.4360.23 b 5.2860.30 a
Parameters shown are those for which burn treatment had a significant main
effect (see Table 1 above). Letters represent LS Means differences (a= 0.05) for
the main burn treatment effect.
Tall Fescue Growth following Fire and Water Stress
PLOS ONE | www.plosone.org 5 January 2014 | Volume 9 | Issue 1 | e86904
treatment received 50% less water than the wet treatment but was
applied at the same frequency) may have played a role in not
seeing the expected endophyte x water interaction.
Endophtye effects on biomass were opposite those expected
(E2.E+), and as stated previously, there were no significant
interactions with water regime or burn treatment. The only time
E+plants had higher biomass than E2was at the beginning of the
experiment for initial root weight. E+fescue has been shown in a
number of cases to have greater shoot , , , , ,
 and root , , ,  mass compared to E2.
However, the magnitude of these differences observed in the
previously mentioned studies varied widely (e.g., E+plants 4.4%
 to 70%  more biomass than E2), and there are a few
studies in which no endophyte effect was observed. It is possible
that enhanced root biomass reservoir might increase the ability of
E+tall fescue to regrow following aboveground biomass removal,
through either fire or grazing. However, in our study, greater root
biomass in E+individuals at the start of the study appeared to have
no effect on growth responses following disturbance. Similarly,
endophyte presence did not affect leaf elongation, tiller density or
dry weight per tiller in studies conducted by Elbersen and West
 and Newman et al. . It did result in earlier flowering in
the Newman et al. study , but in our experiment, date of
flower was not significantly affected by endophyte presence either.
Some might speculate that endophyte effects are better seen in
field studies than in greenhouse studies, but in a climate change
Figure 1. Average total pot tiller length (61 S.E.) across the duration of the experiment, as measured at each of nine measurement
intervals (measurements for all treatments were made within a two-day window for each interval). Asterisks indicate dates for which
there was a significant difference between treatment means. Flame symbols indicate when the two prescribed burn treatments were applied to
either both the 1x and 2x treatments for the first burn (25 March), or the 2x treatment only for the second burn (12 May).
Figure 2. Mean relative growth rate of tillers (cm/cm/day) (61 S.E.) across the duration of the experiment, as measured at each of
nine measurement intervals (measurements for all treatments were made within a two-day window for each interval). Asterisks
indicate dates for which there was a significant difference between treatment means. Flame symbols indicate when the two prescribed burn
treatments were applied to either both the 1x and 2x treatments for the first burn (25 March), or the 2x treatment only for the second burn (12 May).
Tall Fescue Growth following Fire and Water Stress
PLOS ONE | www.plosone.org 6 January 2014 | Volume 9 | Issue 1 | e86904
experiment in the field at the same research farm where the tall
fescue used here originated from (and using tall fescue propagated
from seed collected in the plots from which our material came),
Brosi also observed relatively few endophyte effects on tall fescue
tiller growth . Host plant genotype , , , , ,
 and fungal genotype , , , ,  have both
been shown to influence the dynamics of symbiosis within the tall
fescue-N. coenophialum system. It may be that the combination used
in our study simply does not exhibit the differences in growth seen
in other cases, although it should be noted that our combination
(variety ‘Kentucky-31’ and common toxic endophyte) was the
same as in some of this previous work and is the most common
pairing of tall fescue cultivar-endophyte on the landscape.
Physiological benefits of symbiosis with N. coenophialum to host
plants can vary depending on soil fertility , , , but the
results are not consistent. Cheplick et al. found higher biomass of
E+seedlings compared to E2at high nutrient levels and lower
biomass for E+at low nutrient levels , but Arechavaleta et al.
 and Malinowski et al.  saw higher biomass for E+at
lower nutrient levels and no difference  or reduced biomass
 for E+at high nutrient levels. The plants used in the current
study were grown in the relatively fertile (especially for phospho-
rus; see ) soil from which they originated. Malinowski et al.
 and Rahman and Saiga  looked at tall fescue growth in
response to different P levels, and our results are consistent with
what both studies found in high P soils, E+biomass was lower than
E2. It may be that if we had performed this experiment in less
fertile soil we would have seen a different outcome with regard to
the potential endophyte effects on growth. Given the variability in
growth responses in previous studies and this one, it seems there is
still much to be learned about the conditions under which fungal
endophyte symbiosis is strongly mutualistic for this species.
The response of tall fescue to fire might be dependent on its life
history (specifically life form and bud characteristics), which Pyke
et al. used to characterize plant species’ fire tolerance . With
tall fescue being a cryptophyte (sensu ), Pyke et al. predicted
the growth response following fire to be neutral or positive if buds
are insulated by soil, but negative if buds are closer to the surface
and fire temperatures are hot enough . In a review of fire
effects on invasive weeds, DiTomaso et al. list cool-season
perennial grasses as a category that can be controlled with
burning, and while they do not specifically address tall fescue; they
do cite successful reductions in Kentucky bluegrass with mid-late
spring burns . However, in our study, tiller length was greater
for the burned pots (1x or 2x) compared to the control, but
biomass (tiller and pot total) was suppressed in 2x compared to 1x
or unburned control, so there was a slight reduction in material in
the pots burned twice at the end of the experiment (leaf sheaths
were the same lengths but apparently not as thick). The rapid
growth rate following the second burn was surprising, and likely
indicates that given more time prior to harvest (2x burned plants
were harvested only 59 days after the second burn, but 1x burned
plants were harvested 117 days after the first burn) the 2x burn
pots may have regrown all, if not more than, the material lost to
fire. Our study did not aim to detect whether fire could actually kill
the endophyte, but when we tested for endophyte presence at the
end of our experiment, we found more pots in the 2x burn
treatment that differed from either 0 or 100% infection (1 such pot
in control, 2 pots in 1x, and 7 pots in 2x burn). In fact, the two pots
that were excluded from the study were 2x burn that had less than
100% infection. Neotyphodium coenophialum is known to be sensitive
to heat, as heat treatments are regularly employed to remove the
fungus from infected seed lots . It is possible that prescribed
fires may negatively affect the fungus, but more work exploring
this topic is required.
Tall fescue experiences two periods of growth during a single
season with a period in the mid-summer of slow growth , ,
and it is possible that the timing of fire might interact with the
seasonal growth cycle of tall fescue to alter the plant’s response.
Based on growth rates prior to burns, this experiment imposed the
first burn during the period of early summer growth, and tall
fescue took longer to recover compared to when the second burn
applied, which occurred as the plants were entering their slower
growth mid-summer period. Prescribed fires are most often
conducted in February or March in the eastern U.S., which
coincided with the timing of our first prescribed burn (during the
initial spring growth period). Based on our data, a burn applied at
this time appears to allow plenty of time for plants to recover
aboveground material, and they can do so in a relatively short
period of time (,3 weeks in this greenhouse experiment). A burn
during the mid-summer period (which is when the second burn in
this experiment occurred) resulted in rapid recovery in length (2x
burn plants had the same tiller length as 1x and unburned control
within 2 weeks following the second fire), although it should be
noted that the greenhouse was maintained at a daytime
temperature lower than ambient summer temperatures which
normally produce a ‘‘summer slump’’ or drop in production for
tall fescue and other cool-season grasses , . A summer
prescribed burn applied to a field dominated by another C
Texas wintergrass, resulted in 2x higher yield of that species
compared to a winter (Feb/Mar) burn or no burn . A burn
during the autumn growing period would allow less time for
recovery before the winter dormant period, and might be
predicted to reduce tall fescue dominance better over the long-
term than summer or spring burns, but Madison et al. found that
fall burning did not reduce tall fescue cover . Our results
indicate that tall fescue is able to readily recover following fire,
even if applied twice in a single growing season, under wet or dry
conditions, and irrespective of endophyte status.
Our data suggest that regardless of endophyte status, tall fescue
growth was stimulated after being burned. Water stress negatively
affected tall fescue growth, and did so equally for E+and E2
plants in this experiment. When we did observe significant effects
of endophyte on growth of fescue plants, it was opposite that
expected, with E2plants having greater tiller length and biomass
Table 3. Mean measured growth response variables (61 S.E.)
at the final harvest for tall fescue plants exposed to Wet and
Dry water regimes and averaged across burn treatments and
Total Tiller Length (cm) 264.9610.8a 155.2610.2b
Number New Tillers 11.060.9a 4.060.8b
Number Reproductive Tillers 0.660.1a 0.360.1b
Tiller Biomass (g) 2.7860.08a 1.7260.10b
Root Biomass (g) 1.8460.08a 1.3660.08b
Total Biomass (g) 6.1060.17a 4.1660.18b
Parameters shown are those for which watering regime had a significant main
effect (see Table 1 above). Letters represent LS Means differences (a= 0.05) for
the main water regime effect.
Tall Fescue Growth following Fire and Water Stress
PLOS ONE | www.plosone.org 7 January 2014 | Volume 9 | Issue 1 | e86904
compared to E+(one notable exception being belowground
biomass prior to the treatments being applied). These results add
to the growing body of literature that shows differences in E+and
E2tall fescue plant response to stress may depend on a number of
factors (i.e., soil fertility, tall fescue and fungal endophyte genotype
interactions, climatic factors, etc.) and are not universal across its
range in the Eastern U.S. Our study indicated no apparent role of
symbiosis with Neotyphodium in the ability of tall fescue to regrow
following fire even under dry conditions such as are commonly
experienced in North American grasslands. This result suggests
that the persistence of tall fescue in native grassland ecosystems
may be linked to other endophyte-conferred abilities not measured
here (e.g., herbivory release) or not related to this plant-microbial
We greatly appreciate the efforts of A. Breton, A. Cooke, E. Carlisle, J.
Earnest, P. May, and J. Nelson in the field and processing samples for this
study. M. Arthur and G. Sovkoplas provided guidance and supplies for the
heat-sensitive paint tags. T. Tillery of University of Kentucky Regulatory
Services provided endophyte testing.
Conceived and designed the experiments: SLH TDP RLM. Performed the
experiments: SLH RJB RLM. Analyzed the data: SLH RLM. Contributed
reagents/materials/analysis tools: TDP RJB RLM. Wrote the paper: SLH
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