Post on 18-Aug-2020
transcript
.sciencedirect.com
f u n g a l e c o l o g y 1 3 ( 2 0 1 5 ) 4 4e5 2
available at www
ScienceDirect
journal homepage: www.elsevier .com/locate/ funeco
An asexual Epichlo€e endophyte enhanceswaterlogging tolerance of Hordeum brevisubulatum
Meiling SONGa, Xiuzhang LIa, Kari SAIKKONENb, Chunjie LIa,*,Zhibiao NANa
aState Key Laboratory of Grassland Agro-ecosystems, College of Pastoral Agriculture Science and Technology,
Lanzhou University, Lanzhou 730000, ChinabMTT Agrifood Research Finland, Plant Production Research, FI-31600, Jokioinen, Finland
a r t i c l e i n f o
Article history:
Received 28 April 2014
Revision received 12 July 2014
Accepted 16 July 2014
Available online
Corresponding editor:
James White Jnr
Keywords:
An asexual Epichlo€e endophyte
Hordeum brevisubulatum
Performance
Physiology
Tolerance
Waterlogging
* Corresponding author. Tel.: þ86 931 891423E-mail address: chunjie@lzu.edu.cn (C. Li
http://dx.doi.org/10.1016/j.funeco.2014.07.0041754-5048/ª 2014 Elsevier Ltd and The Britis
a b s t r a c t
Using field collected asexual Epichlo€e endophyte infected (Eþ) and endophyte free (E�)
Hordeum brevisubulatum plants in a greenhouse experiment, we demonstrate that endo-
phyte infection increases host plant resistance to waterlogging. All plants assigned to
waterlogging treatment started to wither and lose their root vitality, and consequently lost
considerable photosynthesizing and root tissues. However, Eþ plants showed significantly
less symptoms of damage, and they produced significantly greater content of chlorophyll,
more tillers, higher shoots and higher under-ground biomass compared to E� plants.
Waterlogging induced osmoprotective proline production particularly in Eþ plants and had
lower malondialdehyde content and electrolyte leakage, suggesting that endophyte
infection positively affects osmotic potential and oxidative balance of the host plant. We
propose that higher resistance of Eþ plants of H. brevisubulatum to waterlogging should be
acknowledged in breeding programmes and the scenarios of changes in grassland eco-
systems as a result of climate change.
ª 2014 Elsevier Ltd and The British Mycological Society. All rights reserved.
Introduction responses to waterlogging, in which roots and some portion of
Temporary or continuous flooding is one of the major abiotic
stresses determining the adaptive radiation of plants and agri-
cultural productivity worldwide (Kozlowski, 1984; Armstrong
et al., 1985; Jackson et al., 2009; Ahmed et al., 2013). Water-
logging, referring to saturation of soil with water, rapidly
depletesoxygencausingsoilhypoxia (deficiencyofO2)oranoxia
(absence of O2) (Ricard et al., 1994; Blokhina et al., 2003; Zabalza
et al., 2009), and alters soil physiochemical properties such as
soil pH and redox potential directly or indirectly bymodulating
the microbial community of the soil (Ashraf, 2012). Plant
3; fax: þ86 931 8910979.).
h Mycological Society. Al
the shoot are submerged, varywith plant species and genotype
(e.g. agricultural variety) as well as with water level, duration
and timing of waterlogging (Kozlowski, 1984; Pucciariello and
Perata, 2013). Only 1e2 % of angiosperms are aquatics (Cook,
1999). A number of studies on terrestrial plants show water-
logging alters homeostasis and metabolism, thereby inducing
leaf senescence, reducing chlorophyll content and leaf area,
inhibiting photosynthesis and plant growth (Kozlowski, 1984;
Armstrong et al., 1985; Colmer et al., 2001; Jackson et al., 2009;
Gibbs et al., 2011). At present waterlogging is regarded as
one of the most hazardous natural occurrences in low-lying
l rights reserved.
Epichlo€e endophyte enhances waterlogging tolerance 45
countries (Ahmed et al., 2013), and accumulating evidence
suggests thatclimatechangewill increasetheriskofgeographic
coverageoffloods in the future (Milly etal., 2002;Woodruff etal.,
2013).
In this study, we examined whether systemic fungal
endophytes increase waterlogging tolerance of their host
grasses. Epichlo€e endophytes [(ex.Acremonium) Clavicipitaceae,
Hypocreales, Ascomycota] live asymptomatically and inter-
cellularlywithin aerial parts of the host grass tissues including
the developing inflorescence and seeds (Saikkonen et al., 2004;
Schardl et al., 2004). Consequently the symbiotic fungus is
vertically transmitted in the mother plant lineage (Saikkonen
et al., 2002). Empirical evidence has demonstrated that
endophyte-host plant interactions are widespread and com-
mon in many cool season grasses (Leuchtmann, 1992), but
variable and labile ranging from antagonistic to mutualistic in
both ecological and evolutionary time scales (Clay, 1988;
Saikkonen et al., 1998, 2006, 2010a; Schardl et al., 2004). The
mutual benefits to the partners appear to be dependent on
genetic variation in the host and endophyte, and on environ-
mental conditions (Saikkonen et al., 2006; Davitt et al., 2010;
Gundel et al., 2010; Saikkonen et al., 2010a, 2010b). Many
studies have demonstrated that endophytes can significantly
increase plant tolerance to environmental stresses such as
drought, salt, cold, heat, heavy metal, insects and diseases
(Redman et al., 2002; Schardl et al., 2004; Kuldau and Bacon,
2008; Wang et al., 2009; Zhang et al., 2010; Peng, 2012). For
water stress, many studies have found that endophytes play a
key role in the host plant’s tolerance to drought (Malinowski
and Belesky, 2000; Zhang and Nan, 2007; Hahn et al., 2008;
Nagabhyru et al., 2013). Furthermore, field surveys also have
indicated that the asexual Epichlo€e endophyte-infection fre-
quencies are high in Lolium arundinacea and L. multiflorum
populations growing in the flooding Pampa (De Battista, 2005).
Another study by Arachevaleta et al. (1989) demonstrated that
endophytes could decrease leaf width and increase leaf
thickness of tall fescue under flooding. However, exper-
imental approaches to reveal whether fungal endophytes
mediate host grass tolerance to waterlogging are lacking.
Materials and methods
Plant materials and experimental design
We selected wild barley (Hordeum brevisubulatum) as a model
species for our study because it is: (1) commonly infected by
the asexual Epichlo€e endophytes (Moon et al., 2004;
Leuchtmann et al., 2014); (2) known for its high tolerance to
several abiotic stresses including drought, salinity and alka-
linity (Guo et al., 1998), and can be found commonly in wet-
lands in Linze county, Gansu province of China. In these H.
brevisubulatum populations, infection frequencies are com-
monly high ranging from 80 % to 90 % (Song et al., 2010).
We collected naturally asexual Epichlo€e endophyte infected
(Eþ) and uninfected (E�) wildH. brevisubulatum plants growing
in Linze county, Gansu province of China in 2007. Plants were
transplanted to a common garden at the Yuzhong Exper-
imental Station (E103�360, N36�280), Lanzhou University. The
infection status of the plants was determined by the
microscopic examination of stained seeds/leaf sheaths (Li
et al., 2004) in the State Key Laboratory of Grassland Agro-
ecosystems. In Nov. 2012, seeds were harvested from Eþ and
E� plants and sown in 100 pots (50 pots for Eþ plants and the
others for E� plants; six plants per pot; and each pot with a
diameter of 21 cm and a height of 16 cm) filled with compound
soil (with a sand: sierozem: peat ratio of 2:3:5) in a greenhouse
of College of Pastoral Agriculture Science and Technology,
Lanzhou University. The temperature and light cycle in the
greenhouse was adjusted to 22:18 �C and a 14:10 hr light:dark
cycle, respectively.
Six weeks after sowing, half of the Eþ and E�seedlings
were subjected to a waterlogging treatment (W) by submerg-
ing half of the pots with Eþ and E�plants in water in plastic
containers (diameter 34 cm � height 28 cm) filled with tap
water 2 cm above the soil level of the pots for 16 d. The water
level was checked and water added if needed every other day.
Untreated control pots (C) were placed in empty containers.
To be able to follow plant performance five times during the
experiment (in the beginning of the experiment (day 0), 4, 8, 12
and 16 d after starting the waterlogging treatment), we had
five separate pots for each combination of endophyte infec-
tion status andwaterlogging treatment (E�/C, Eþ/C, E�/W and
Eþ/W) which were replicated in five blocks according to a
randomized block design. Thus, each treatment had five pots
on every sampling day.
We acknowledge that naturally infected Eþ and uninfected
E�plants can be genetically distinct from each other because
genetic compatibility can determine endophyte-grass combi-
nations (Saikkonen et al., 2010b). Thus, detected differences
between Eþ and E�plants can be due to the effects of the
endophyte infection and plant genotype. Whether this caveat
biases our interpretation of the results remains to be tested in
future studies using both naturally Eþ and manipulatively
E�plants.
Plant performance
To test the effect of endophyte on plant performance, dry
weight of shoots and roots, leaf wilt rate and root vitality were
determined. To determine dry weight of shoots and roots,
samples were gently washed and weighed separately after
oven-drying at 80 �C until a constant weight was reached. Leaf
wilt rates were measured by visual observation. Leaf wilt rate
(%) ¼ (wilt leaves per plant)/(all leaves per plant) � 100. Root
vitality was determined by triphenyltetrazolium chloride
(TTC) method (Clemensson-Lindell, 1994), in which 200 mg of
the fresh root tissue was cut into 1e2 mm long pieces and
incubated with 5 ml of 0.4 % (weight/volume) TTC and 5 ml
0.06 M Na2HPO4eKH2PO4 at 37 �C for 3 hr. The samples were
then extracted in 95 % ethanol at 80 �C for 15 min. Absorption
at 485 nm was measured using a SP-723-type visible spec-
trophotometer (Shanghai, China). At the final harvest (day 16),
we recorded plant height, root length, number of tillers, leaf
length and leaf width of plants.
Physiological parameters
In addition to plant performance, we measured chlorophyll,
proline and malondialdehyde (MDA) contents and electrolyte
Fig 1 e Variation in (A) leaf wilt rates and (B) root vitality of
H. brevisubulatum during the entire experiment. Asterisks
indicate significant differences (P < 0.05) between ED and
EL on the same day within the waterlogging treatment
(W). C is control treatment.
46 M. Song et al.
leakage (EL) of the plants to estimate possible endophyte-
mediated physiological responses to waterlogging stress.
Chlorophyll content was determined using a colorimetric
method modified by Li (2000). 0.2 g of plant leaf samples were
extracted in 80 % acetone mixed with about 0.2 g calcium
carbonate powder and centrifuged at 12 000� g for 25min. The
chlorophyll contents were computed by absorbance of the
sample which were measured at 645 and 663 nm. To measure
proline content, fresh plant samples were homogenized with
3 % sulfosalicylic acid and then centrifuged at 3 000� g for
20 min; the supernatant was treated with acetic acid and acid
ninhydrin, boiled for 1 hr and then absorbance at 520 nm was
determined (Bates et al., 1973).
To measure MDA concentrations, 0.5 g plant samples were
mixedwith 5ml TCA (5 %), centrifuged at 12 000� g for 25min,
then 2 ml of supernatant was mixed with 2 ml of a 0.67 %
thiobarbituric acid (TBA) solution, incubated in boiling water
for 30 min, and the reaction stopped by placing the reaction
tubes in an ice bath. Then the samples were centrifuged at
12 000� g for 5 min and the absorbance of supernatant was
used for the determination of the MDA content by using a SP-
723-type visible spectrophotometer (Shanghai, China) (for
more detailed description, see Li (2000)).
To determine electrolyte leakage, 100 mg fresh leaf sam-
ples were cut into 5 mm lengths and placed in test tubes
containing 10 ml distilled deionized water. After 2 hr in the
32 �C water bath, the initial electrical conductivity of the
medium (EC1) was measured using an electrical conductivity
meter (DDSJ-308A, Shanghai, China). Then, the samples were
soaked in boiling water for 30 min to completely kill the tis-
sues and release all electrolytes. The final electrical con-
ductivity (EC2) was measured after samples were cooled to
25 �C. The electrolyte leakage was computed following the
formula EL ¼ EC1/EC2 � 100 (Dionisio-Sese and Tobita, 1998).
Statistical analysis
Data analyses were performed with SPSS 17.0 for windows
(SPSS, Inc., Chicago, IL). The independent t-test was employed
to analysis the effect of endophyte infection on all parameters
measured. The interactions between waterlogging and
endophyte-infection on tillers, plant height, root length, leaf
width and length, as well as chlorophyll, MDA and EL were
determined by two-way ANOVA. In addition, a repeated
measures ANOVA with Fisher’s LSD test was employed to
estimate the effect of waterlogging, endophyte-infection and
time on shoot/root biomass, leaf wilt rates, root vitality and
proline contents. Statistical significancewasdefinedat the95%
confidence level.Meansarereportedwith their standarderrors.
Results
Plant performance
Waterlogging negatively affected plant performance. Plants
started to gradually wither and their roots lost vitality soon
after the start of the waterlogging treatment (Fig 1A and B,
Table 1). Consequently, waterlogging decreased shoot and
root biomass (Fig 2A and B), the number of tillers (Fig 2C),
shoot height (Fig 3A), and root length (Fig 3B) (Table 2). The
leaves of waterlogged plants tended to be wider than untrea-
ted control plants, while leaf length was indistinctive (Fig 3C
and D, Table 2). In contrast, untreated control plants showed
no symptoms of withering, loss of root vitality, plant height,
root length, leaf length or width, and they gained shoot and
root biomass during the study (Figs 1e3).
Eþ plants generally performed better than, or equal to,
E�plants in the waterlogging treatment (Figs 1e3, Tables 1
and 2). Leaves and roots of Eþ plants withered and lost their
vitality significantly less than E�plants in the waterlogging
treatment, and leaf wilt rate had significant waterlogging-
endophyte interactions (Fig 1, Table 1). Similarly, untreated
Eþ and E�plants grew equally whereas Eþ plants tended to
produce more tillers, taller shoots, longer roots and broader
leaves in the waterlogging treatment (Fig 2C, Fig 3AeC), also,
under-ground dry mass of Eþ plants was greater than that of
E� plants in the later period of waterlogging (Fig 2B, Table 1).
Table 1 e Repeated measures ANOVA of root vitality, leaf wilt rate, shoot and root biomass during the whole study period
Treatments Df Root vitality Leaf wilt rate Shoot biomass Root biomass
F P F P F P F P
Wa 1 92.3 <0.001 539.1 <0.001 159.9 <0.001 139.0 <0.001
Eb 1 8.0 0.022 21.3 <0.001 1.8 0.212 40.2 <0.001
Tc 4 20.9 <0.001 83.3 <0.001 15.7 0.002 332.8 <0.001
W � E 1 4.9 0.058 21.3 <0.001 1.8 0.212 1.0 0.346
W � T 4 12.2 <0.001 83.3 <0.001 202.7 <0.001 930.7 <0.001
E � T 4 1.4 0.258 3.4 0.047 2.9 0.117 9.2 0.013
W � E � T 4 0.4 0.802 3.4 0.047 2.5 0.140 4.5 0.060
a Waterlogging.
b Endophyte.
c Time (days after treatment).
Epichlo€e endophyte enhances waterlogging tolerance 47
Physiological parameters
Untreated Eþ plants did not differ from E� plants in chlor-
ophyll, proline and malondialdehyde (MDA) contents or
Fig 2 e Biomass of H. brevisubulatumwith (ED) and without
endophyte (EL) during the entire experiment. (A) shoot
biomass, (B) root biomass, and (C) tiller numbers after 16 d.
Asterisks indicate significant differences (P < 0.05)
between ED and EL on the same day within the
waterlogging treatment (W). C is control treatment.
electrolyte leakage (EL) (Figs 4 and 5), but endophyte infection
interacted with the waterlogging treatment affecting all
physiological parameters (Table 3, W � E effect). First,
although waterlogging significantly decreased chlorophyll
content of both E� and Eþ plants, the chlorophyll content of
Eþ plants was significantly greater than that of E� plants
(Fig 5A, Table 3). Second, waterlogging rapidly increased pro-
line contents of plants, but after a few days the proline con-
tents normalized back again to the original level (Fig 4). The
proline burst of Eþ plants was two-fold compared to E� plants
(Fig 4). Third, the increased malondialdehyde (MDA) content
and electrolyte leakage (EL) of E� plants was significantly
greater than that of Eþ plants in the waterlogging treatment
(Fig 5B and C, Table 3).
Discussion
Our results show that endophyte infection increased plant
resistance to waterlogging. Both Eþ and E� H. brevisubulatum
plants assigned to 16 d waterlogging treatment lost consid-
erable leaf activity and root tissues. However, Eþ plants con-
sistently outperformed E� plants in waterlogging treatment;
the leaves and roots of Eþ plants withered and lost their
vitality significantly less than E� plants, Eþ plants had higher
chlorophyll content than E� plants resulting in potentially
higher photosynthetic capacity of Eþ plants. In contrast to the
general presumption that endophyte infection confers bene-
fits to the host grass in terms of higher growth performance
over uninfected plants (Clay, 1990; Clay and Holah, 1999;
Zaurov et al., 2001; Rudgers et al., 2005), untreated (control)
Eþ plants were equivalent to untreated E� plants in per-
formance in our experiment (see also Saikkonen et al., 2004;
Saikkonen et al., 2006; Cheplick and Faeth, 2009). Neither Eþnor E� control plants showed symptoms of withering or loss
of root vitality, and both plant types equally gained shoot and
root biomass during the study. Endophyte promoted plant
resistance to abiotic environmental stress, including drought,
has been demonstrated inmany studies (see e.g. Zhou and Lin,
1995; Monnet et al., 2001; Newman et al., 2003; Hunt et al.,
2005; Gou, 2007; Li et al., 2008; Soleimani et al., 2010; Zhang
et al., 2010). However, to our knowledge this is the first study
demonstrating that asexual Epichlo€e endophyte infection can
increase host grass resistance to waterlogging stress.
Fig 3 e Differences in plant height (A), root length (B), leaf width (C) and leaf length (D) under different treatments after 16 d.
Asterisks indicate significant differences (P< 0.05) between ED and EL on the same day within the waterlogging treatment
(W). C is control treatment.
48 M. Song et al.
Hypoxia and anoxia caused by waterlogging affect plant
metabolism and induce adaptive responses in plants (Ricard
et al., 1994; Blokhina et al., 2003; Zabalza et al., 2009). Aerobic
respiration is affected through the slowdown of glycolysis in
waterlogged plants leading to decreased adenosine triphos-
phate (ATP) production and switch to less energy yielding
anaerobic fermentation (Bramley et al., 2007; Zabalza et al.,
2009). Consequently, oxygen deficiency due to waterlogging
is predicted to lead to reduced plant growth, development
and survival (Grimoldi et al., 1999; Ashraf et al., 2011;
Krishnamurthy et al., 2011). In addition to reduced growth,
plants that are subject to waterlogging often develop symp-
tomsof leafyellowing,wilting, root rotting, androotblackening
(Jackson, 2002; Dodd et al., 2013; Shaw et al., 2013). Our results
support these findings. H. brevisubulatum plants subjected to
Table 2 e Two-way ANOVA of tillers, plant height, root length
Treatments Df Tillers Plant height
F P F P
Wa 1 15.0 0.005 85.9 <0.001
Eb 1 4.0 0.082 8.9 0.017
W � E 1 1.3 0.282 1.1 0.328
a Waterlogging.
b Endophyte.
waterlogging treatment had lower chlorophyll content, and
they started to gradually wither and their roots lost vitality
leading to decreased biomass of the plants at the end of the
experiment. In addition, waterlogging treatment decreased
plant growth in terms of shoot and root length and tiller
number (Fig 2C, Fig 3A and B, Table 2).We also found that plant
leaves were broader in the waterlogging treatment, which is
presumably an evolutionary adaption of H. brevisubulatum to
frequentflooding (seealsoMommeretal., 2005; Luoetal., 2007).
Defensive mutualism has been a predominant framework
for endophyte-grass studies since alkaloids produced by sys-
temic fungal endophytes associated with agricultural grasses
were found to cause livestock disorders in themid 1970s (Clay,
1988; Saikkonen et al., 2006; Cheplick and Faeth, 2009;
Saikkonen et al., 2010a, 2013). Generally, fungal-origin acting
, leaf width and leaf length in day 16
Root length Leaf width Leaf length
F P F P F P
36.6 <0.001 31.7 0.001 4.2 0.075
1.0 0.340 2.1 0.183 0.4 0.564
1.9 0.210 6.3 0.037 0.1 0.960
Fig 4 e Variation of proline contents of H. brevisubulatum
during the entire experiment. Asterisk means significant
difference (P < 0.05) between ED and EL on the same day
within the waterlogging treatment (W). C is control
treatment.
Fig 5 e Contents of chlorophyll (A), malondialdehyde
(MDA, B) and electrolyte leakage (EL, C) under different
treatments after 16 d. Asterisks indicate significant
differences (P< 0.05) between ED and EL on the same day
within the waterlogging treatment (W). C is control
treatment.
Epichlo€e endophyte enhances waterlogging tolerance 49
secondary metabolites (primarily alkaloids) are regarded to
play a significant role in endophyte improved plant resistance
to environmental stresses (Kuldau and Bacon, 2008;
Saikkonen et al., 2013). For example, the loline alkaloid lev-
els increased in response to water deficit stress in endophyte-
infected tall fescue (Nagabhyru et al., 2013). Here, we assume
that our results of endophyte improved host plant tolerance to
waterlogging might be related to the secondary metabolites
induced by the endophyte. However, chemical ecology medi-
ated by endophytes in grasses has been revealed to be far
more complex (Saikkonen et al., 2013). Our findings indicate
that pronounced waterlogging induces a proline burst partic-
ularly in Eþ plants, and stronger increase in malondialdehyde
(MDA) content and electrolyte leakage (EL) of E� plants (Fig 4,
Fig 5B and C, Table 3), and suggest that endophyte infection
affects osmotic potential and oxidative balance of the host
plant. Consistent with our results, Bush et al. (1997) found that
loline alkaloids produced by endophytes could affect osmotic
potential and thereby increase plant resistance to drought. In
addition, oxidative balance might play a crucial role in the
resistance of endophyte-plant symbiotum to a wide range of
environmental stresses (Tanaka et al., 2006; Hamilton et al.,
2012).
Proline is well known to accumulate in plants in response
to a variety of environmental stresses, such as drought, sal-
inity, high temperature, nutrient deficiency, and exposure to
heavy metals and high acidity (Hare et al., 1999; Gou, 2007; Li,
2007; Szabados and Savoure, 2010), and as an important
osmolyte thought to be involved in stress resistance mecha-
nisms (Pyngrope et al., 2013). Similar to our study with H.
brevisubulatum, waterlogging increased proline contents in
Bambara groundnut (Vurayai et al., 2011), jute (Parvin and
Karmoker, 2013) and Solanum lycopersicum (Horchani et al.,
2010). Similarly, Gou (2007) and Li (2007) reported that salt
and drought stress induced more proline accumulation in Eþ
than in E� A. inebrians. Although proline contents normalized
in less than 10 d, our results support the idea that endophyte
infection can enhance plant tolerance to waterlogging by
increased osmoprotective proline production.
Table 3 e Statistical results of chlorophyll, proline, malondialdehyde (MDA) content and electrolyte leakage (EL) during thewhole study period
Treatments Df Chlorophyll Proline MDA EL
F P F P F P F P
Wa 1 31.2 <0.001 39.7 <0.001 1061.5 <0.001 24.0 0.001
Eb 1 5.9 0.042 20.3 0.002 12.9 0.007 11.8 0.009
Tc 4 29.4 <0.001
W � E 1 17.2 0.003 17.3 0.003 22.1 0.002 7.4 0.027
W � T 4 29.2 <0.001
E � T 4 7.6 0.004
W � E � T 4 6.7 0.007
a Waterlogging.
b Endophyte.
c Time (days after treatment).
50 M. Song et al.
Similarly, stronger increase in malondialdehyde (MDA)
content and electrolyte leakage (EL) of E� plants suggest
positive endophyte-mediated physiological host plant
responses to waterlogging stress. MDA and EL have been used
to estimate the peroxidation of lipids inmembrane and loss of
membrane integrity (Delong and Steffen, 1997; Dionisio-Sese
and Tobita, 1998). Lipid peroxidation of membranes causes
impaired membrane function, decreased fluidity, and inacti-
vation of membrane-bound receptors and enzymes (Zhang
et al., 2013). In general, increased MDA and EL indicate lipid
peroxidation in response to abiotic stress (see e.g. Wang et al.,
2009; Zhang et al., 2010). Thus, lower MDA and EL of Eþ plants
in our study suggest that endophytes protect host cell mem-
branes against oxidative degradation of lipids.
Our results have both theoretical and applied importance.
First, endophyte-improved tolerance to waterlogging stress
may partly explain high endophyte infection frequencies
(80e90 %) in H. brevisubulatum populations, adaptive radiation
of the symbiotum and effects of endophytes on grassland
communities. Second, we propose that endophyte-mediated
grass resistance to flooding should be taken into account
when forecasting changes in grassland ecosystems as a result
of climate change. Third, systemic grass-endophytes should
be taken into account in grass breeding (Gundel et al., 2013)
aiming to combat the increasing risk of floods in grass pro-
duction due to climate change.
Acknowledgments
This study is supported by National Basic Research Programof
China (2014CB138702), Natural Science Foundation of China
(31372366) and Program for Changjiang Scholars and Innova-
tive Research Team in University, China (IRT13019). The
authors would like to thank Qing Chai, Chunxia Hu and Xiang
Yao for their help of samples collecting and analyzing.
r e f e r e n c e s
Ahmed, F., Rafii, M.Y., Ismail, M.R., Juraimi, A.S., Rahim, H.A.,Asfaliza, R., Latif, M.A., 2013. Waterlogging tolerance of crops:
breeding, mechanism of tolerance, molecular approaches, andfuture prospects. BioMed Research International 2013, 963525.
Arachevaleta, M., Bacon, C., Hoveland, C., Radcliffe, D., 1989.Effect of the tall fescue endophyte on plant response toenvironmental stress. Agronomy Journal 81, 83e90.
Armstrong, W., Wright, E., Lythe, S., Gaynard, T., 1985. Plantzonation and the effects of the spring-neap tidal cycle on soilaeration in a Humber salt marsh. Journal of Ecology 73, 323e339.
Ashraf, M.A., 2012. Waterlogging stress in plants: a review. AfricanJournal of Agricultural Research 7, 1976e1981.
Ashraf, M.A., Ahmad, M.S.A., Ashraf, M., Al-Qurainy, F.,Ashraf, M.Y., 2011. Alleviation of waterlogging stress in uplandcotton (Gossypium hirsutum L.) by exogenous application ofpotassium in soil and as a foliar spray. Crop and Pasture Science62, 25e38.
Bates, L., Waldren, R., Teare, I., 1973. Rapid determination of freeproline for water-stress studies. Plant and Soil 39, 205e207.
Blokhina, O., Virolainen, E., Fagerstedt, K.V., 2003. Antioxidants,oxidative damage and oxygen deprivation stress: a review.Annals of Botany 91, 179e194.
Bramley, H., Turner, D.W., Tyerman, S.D., Turner, N., 2007. Waterflow in the roots of crop species: the influence of rootstructure, aquaporin activity, and waterlogging. Advances inAgronomy 96, 133e196.
Bush, L.P., Wilkinson, H.H., Schardl, C.L., 1997. Bioprotectivealkaloids of grass-fungal endophyte symbioses. PlantPhysiology 114, 1e7.
Cheplick, G.P., Faeth, S.H., 2009. Ecology and Evolution of theGrass-Endophyte Symbiosis. Oxford University Press, NewYork, USA.
Clay, K., 1988. Fungal endophytes of grasses: a defensivemutualism between plants and fungi. Ecology 69, 10e16.
Clay, K., 1990. Fungal endophytes of grasses. Annual Review ofEcology and Systematics 21, 275e297.
Clay, K., Holah, J., 1999. Fungal endophyte symbiosis and plantdiversity in successional fields. Science 285, 1742e1744.
Clemensson-Lindell, A., 1994. Triphenyltetrazolium chloride asan indicator of fine-root vitality and environmental stress inconiferous forest stands: applications and limitations. Plantand Soil 159, 297e300.
Colmer, T.D., Lambers, H., Schortemeyer, M., 2001. Changes inphysiological and morphological traits of roots and shoots ofwheat in response to different depths of waterlogging.Functional Plant Biology 28, 1121e1131.
Cook, C.D., 1999. The number and kinds of embryo-bearing plantswhich have become aquatic: a survey. Perspectives in PlantEcology, Evolution and Systematics 2, 79e102.
Davitt, A.J., Stansberry, M., Rudgers, J.A., 2010. Do the costs andbenefits of fungal endophyte symbiosis vary with lightavailability? New Phytologist 188, 824e834.
Epichlo€e endophyte enhances waterlogging tolerance 51
De Battista, J., 2005. Neotyphodium Research and Application inSouth America. Neotyphodium in Cool-Season Grasses,pp. 65e71.
Delong, J., Steffen, K., 1997. Photosynthetic function, lipidperoxidation, and a-tocopherol content in spinach leavesduring exposure to UV-B radiation. The Canadian Journal of PlantScience 77, 453e459.
Dionisio-Sese, M.L., Tobita, S., 1998. Antioxidant responses of riceseedlings to salinity stress. Plant Science 135, 1e9.
Dodd, K., Guppy, C., Lockwood, P., Rochester, I., 2013. Impact ofwaterlogging on the nutrition of cotton (Gossypium hirsutum L.)produced in sodic soils. Crop and Pasture Science 64, 816e824.
Gibbs, D.J., Lee, S.C., Isa, N.M., Gramuglia, S., Fukao, T.,Bassel, G.W., Correia, C.S., Corbineau, F., Theodoulou, F.L.,Bailey-Serres, J., Holdsworth, M.J., 2011. Homeostatic responseto hypoxia is regulated by the N-end rule pathway in plants.Nature 479, 415e418.
Gou, X.Y., 2007. Effect of Neotyphodium Endophyte on SaltTolerance to Drunken Horse Grass (Achnatherum inebrians)(MSc dissertation). Lanzhou University, China (In Chinese,with English abstract).
Grimoldi, A., Insausti, P., Roitman, G., Soriano, A., 1999.Responses to flooding intensity in Leontodon taraxacoides. NewPhytologist 141, 119e128.
Gundel, P.E., Omacini, M., Sadras, V.O., Ghersa, C.M., 2010. Theinterplay between the effectiveness of the grass-endophytemutualism and the genetic variability of the host plant.Evolutionary Applications 3, 538e546.
Gundel, P.E., Perez, L.I., Helander, M., Saikkonen, K., 2013.Symbiotically modified organisms: nontoxic fungalendophytes in grasses. Trends in Plant Science 18, 420e427.
Guo, J.X., Jiang, S.C., Sun, G., 1998. Comparative study on remedyways of saline alkali grassland in Songnen Plain. ChineseJournal of Applied Ecology 4, 425e428 (In Chinese, with Englishabstract).
Hahn, H., McManus, M.T., Warnstorff, K., Monahan, B.J.,Young, C.A., Davies, E., Tapper, B.A., Scott, B., 2008.Neotyphodium fungal endophytes confer physiologicalprotection to perennial ryegrass (Lolium perenne L.) subjected toa water deficit. Environmental and Experimental Botany 63,183e199.
Hamilton, C.E., Gundel, P.E., Helander, M., Saikkonen, K., 2012.Endophytic mediation of reactive oxygen species andantioxidant activity in plants: a review. Fungal Diversity 54,1e10.
Hare, P., Cress, W., Van Staden, J., 1999. Proline synthesis anddegradation: a model system for elucidating stress-relatedsignal transduction. Journal of Experimental Botany 50, 413e434.
Horchani, F., Hajri, R., Khayati, H., Aschi-Smiti, S., 2010.Physiological responses of tomato plants to the combinedeffect of root hypoxia and NaCl-salinity. Journal of Phytology 2,36e46.
Hunt, M.G., Rasmussen, S., Newton, P.C., Parsons, A.J.,Newman, J.A., 2005. Near-term impacts of elevated CO2,nitrogen and fungal endophyte-infection on Lolium perenne L.growth, chemical composition and alkaloid production. PlantCell and Environment 28, 1345e1354.
Jackson, M.B., 2002. Long-distance signalling from roots to shootsassessed: the flooding story. Journal of Experimental Botany 53,175e181.
Jackson, M.B., Ishizawa, K., Ito, O., 2009. Evolution andmechanisms of plant tolerance to flooding stress. Annals ofBotany 103, 137e142.
Kozlowski, T., 1984. Plant responses to flooding of soil. Bioscience34, 162e167.
Krishnamurthy, L., Upadhyaya, H.D., Saxena, K.B., Vadez, V.,2011. Variation for temporary waterlogging response within
the mini core pigeonpea germplasm. The Journal of AgriculturalScience 150, 357e364.
Kuldau, G., Bacon, C., 2008. Clavicipitaceous endophytes: theirability to enhance resistance of grasses to multiple stresses.Biological Control 46, 57e71.
Leuchtmann, A., 1992. Systematics, distribution, and hostspecificity of grass endophytes. Natural Toxins 1, 150e162.
Leuchtmann, A., Bacon, C.W., Schardl, C.L., White, J.F.,Tadych, M., 2014. Nomenclatural realignment of Neotyphodiumspecies with genus Epichlo€e. Mycologia 106, 202e215.
Li, C.J., Nan, Z.B., Gao, J.H., Tian, P., 2004. Detection anddistribution of Neotyphodium-Achnatherum inebriansassociation in China. In: Proceedings of 5th InternationalNeotyphodium/Grass Interactions Symposium. Arkansas,p. 210.
Li, C.J., Nan, Z.B., Li, F., 2008. Biological and physiologicalcharacteristics of Neotyphodium gansuense symbiotic withAchnatherum inebrians. Microbiology Research 163, 431e440.
Li, F., 2007. Effects of Endophyte Infection on Drought Resistanceto Drunken Horse Grass (Achnatherum inebrians)(MSc dissertation). Lanzhou University, China(In Chinese, with English abstract).
Li, H.S., 2000. Principle and Techniques of Botanic, Chemical andPhysiological Experiments. Senior Education Press, Beijing,China (In Chinese).
Luo, W.B., Xie, Y.H., Song, F.B., 2007. Survival strategies ofwetland plants in flooding environments. Chinese Journal ofEcology 9, 1478e1485 (In Chinese, with English abstract).
Malinowski, D.P., Belesky, D.P., 2000. Adaptations of endophyte-infected cool-season grasses to environmental stresses:mechanisms of drought and mineral stress tolerance. CropScience 40, 923e940.
Milly, P., Wetherald, R., Dunne, K., Delworth, T., 2002. Increasingrisk of great floods in a changing climate. Nature 415, 514e517.
Mommer, L., De Kroon, H., Pierik, R., B€ogemann, G.M., Visser, E.J.,2005. A functional comparison of acclimation to shade andsubmergence in two terrestrial plant species. New Phytologist167, 197e206.
Monnet, F., Vaillant, N., Hitmi, A., Coudret, A., Sallanon, H., 2001.Endophytic Neotyphodium lolii induced tolerance to Zn stress inLolium perenne. Plant Physiology 113, 557e563.
Moon, C.D., Craven, K.D., Leuchtmann, A., Clement, S.L.,Schardl, C.L., 2004. Prevalence of interspecific hybridsamongst asexual fungal endophytes of grasses. MolecularEcology 13, 1455e1467.
Nagabhyru, P., Dinkins, R.D., Wood, C.L., Bacon, C.W.,Schardl, C.L., 2013. Tall fescue endophyte effects on toleranceto water-deficit stress. BMC Plant Biology 13, 127.
Newman, J., Abner, M., Dado, R., Gibson, D., Brookings, A.,Parsons, A., 2003. Effects of elevated CO2, nitrogen and fungalendophyte-infection on tall fescue: growth, photosynthesis,chemical composition and digestibility. Global Change Biology 9,425e437.
Parvin, D., Karmoker, J., 2013. Effects of waterlogging on ionaccumulation and sugar, protein and proline contents inCorchorus capsularis L. Bangladesh Journal of Botany 42, 55e64.
Peng, Q.Q., 2012. Effect of Neotyphodium Endophyte on ChillingTolerance to Festuca sinensis (MSc dissertation). LanzhouUniversity, China (In Chinese, with English abstract).
Pucciariello, C., Perata, P., 2013. Quiescence in rice submergencetolerance: an evolutionary hypothesis. Trends in Plant Science18, 377e381.
Pyngrope, S., Bhoomika, K., Dubey, R., 2013. Reactive oxygenspecies, ascorbate-glutathione pool, and enzymes of theirmetabolism in drought-sensitive and tolerant indica rice(Oryza sativa L.) seedlings subjected to progressing levels ofwater deficit. Protoplasma 250, 585e600.
52 M. Song et al.
Redman, R.S., Sheehan, K.B., Stout, R.G., Rodriguez, R.J.,Henson, J.M., 2002. Thermotolerance generated by plant/fungal symbiosis. Science 298, 1581e1581.
Ricard, B., Cou�ee, I., Raymond, P., Saglio, P.H., Saint-Ges, V.,Pradet, A., 1994. Plant metabolism under hypoxia and anoxia.Plant Physiology and Biochemistry 32, 1e10.
Rudgers, J.A., Mattingly, W.B., Koslow, J.M., 2005. Mutualisticfungus promotes plant invasion into diverse communities.Oecologia 144, 463e471.
Saikkonen, K., Faeth, S., Helander, M., Sullivan, T., 1998. Fungalendophytes: a continuum of interactions with host plants.Annual Review of Ecology and Systematics 29, 319e343.
Saikkonen, K., Gundel, P.E., Helander, M., 2013. Chemical ecologymediated by fungal endophytes in grasses. Journal of ChemicalEcology 39, 962e968.
Saikkonen, K., Gyllenberg, M., Ion, D., 2002. The persistence offungal endophytes in structured grass metapopulations. In:Proceedings of the Royal Society of London. B, pp. 1397e1403.
Saikkonen, K., Lehtonen, P., Helander, M., Koricheva, J.,Faeth, S.H., 2006. Model systems in ecology: dissecting theendophyte-grass literature. Trends in Plant Science 11, 428e433.
Saikkonen, K., Saari, S., Helander, M., 2010a. Defensivemutualism between plants and endophytic fungi? FungalDiversity 41, 101e113.
Saikkonen, K., W€ali, P.R., Helander, M., 2010b. Geneticcompatibility determines endophyte-grass combinations. PLoSOne 5, e11395.
Saikkonen, K.,Wali, P., Helander, M., Faeth, S.H., 2004. Evolution ofendophyte-plant symbioses. Trends in Plant Science 9, 275e280.
Schardl, C.L., Leuchtmann, A., Spiering, M.J., 2004. Symbioses ofgrasses with seedborne fungal endophytes. Annual Review ofPlant Biology 55, 315e340.
Shaw, R.E., Meyer, W.S., McNeill, A., Tyerman, S.D., 2013.Waterlogging in Australian agricultural landscapes: a reviewof plant responses and crop models. Crop and Pasture Science64, 549e562.
Soleimani, M., Afyuni, M., Hajabbasi, M.A., Nourbakhsh, F.,Sabzalian, M.R., Christensen, J.H., 2010. Phytoremediation ofan aged petroleum contaminated soil using endophyteinfected and non-infected grasses. Chemosphere 81, 1084e1090.
Song, M.L., Li, C.J., Peng, Q.Q., Liang, Y., Nan, Z.B., 2010. Effects ofNeotyphodium endophyte on germination of Hordeumbrevisubulatum under temperature and water stress
conditions. Acta Agrestia Sinica 6, 833e837 (In Chinese, withEnglish abstract).
Szabados, L., Savoure, A., 2010. Proline: a multifunctional aminoacid. Trends in Plant Science 15, 89e97.
Tanaka, A., Christensen, M.J., Takemoto, D., Park, P., Scott, B.,2006. Reactive oxygen species play a role in regulating afungus-perennial ryegrass mutualistic interaction. Plant Cell18, 1052e1066.
Vurayai, R., Emongor, V., Moseki, B., 2011. Physiological responsesof bambara groundnut (Vigna subterranea L. Verdc) to shortperiods of water stress during different developmental stages.Asian Journal of Agricultural Sciences 3, 37e43.
Wang, Z.F., Li, C.J., Jin, W.J., Nan, Z.B., 2009. Effect of Neotyphodiumendophyte infection on salt tolerance of Hordeumbrevisubulatum (Trin.) Link. Acta Agrestia Sinica 17, 88e92(In Chinese, with English abstract).
Woodruff, J.D., Irish, J.L., Camargo, S.J., 2013. Coastal flooding bytropical cyclones and sea-level rise. Nature 504, 44e52.
Zabalza, A., Van Dongen, J.T., Froehlich, A., Oliver, S.N., Faix, B.,Gupta, K.J., Schm€alzlin, E., Igal, M., Orcaray, L., Royuela, M.,2009. Regulation of respiration and fermentation to controlthe plant internal oxygen concentration. Plant Physiology 149,1087e1098.
Zaurov, D., Bonos, S., Murphy, J., Richardson, M., Belanger, F.,2001. Endophyte infection can contribute to aluminumtolerance in fine fescues. Crop Science 41, 1981e1984.
Zhang, N., Zhao, B., Zhang, H.J., Weeda, S., Yang, C., Yang, Z.C.,Ren, S., Guo, Y.D., 2013. Melatonin promotes water-stresstolerance, lateral root formation, and seed germination incucumber (Cucumis sativus L.). Journal of Pineal Research 54,15e23.
Zhang, X.X., Li, C.J., Nan, Z.B., 2010. Effects of cadmium stress ongrowth and anti-oxidative systems in Achnatherum inebrianssymbiotic with Neotyphodium gansuense. Journal of HazardousMaterials 175, 703e709.
Zhang, Y.P., Nan, Z.B., 2007. Growth and anti-oxidative systemschanges in Elymus dahuricus is affected by Neotyphodiumendophyte under contrasting water availability. Journal of AgroCrop Science 193, 377e386.
Zhou, W.J., Lin, X.Q., 1995. Effects of waterlogging at differentgrowth stages on physiological characteristics and seed yieldof winter rape (Brassica napus L.). Field Crops Research 44,103e110.