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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.

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