Benefits of a fungal endophyte in Leymus chinensis depend more on water than on nutrient availability

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Benefits of a fungal endophyte in Leymus chinensis depend more on water than on nutrient availability Anzhi Ren ∗ , Maoying Wei, Lijia Yin, Lianjie Wu, Yong Zhou, Xia Li, Yubao Gao ∗ College of Life Sciences, Nankai University, Tianjin 300071, PR China

a r t i c l e Keywords: Biomass Drought Fertilizer N allocation Photosynthetic rate

i n f o

a b s t r a c t Symbiotic relationships with microbes may influence how plants respond to environmental change. In this study, we tested the hypothesis that symbiosis with the endophyte promoted drought tolerance of the native grass and hypothesized the drought tolerance was affected by nutrient availability. In the greenhouse experiment we compared the performance of endophyte-infected (EI) and endophyte-free (EF) Leymus chinensis, a dominant species native to the Inner Mongolia steppe, under altered water and nutrient availability. The results showed that the benefits of the endophyte to the host depended on water availability. Under well-watered conditions, total biomass was not affected by endophyte infection. Under drought stress conditions, however, EI had significantly more total biomass than EF plants. In contrast to expectations, the beneficial effect of endophyte infection was less dependent on fertilizer supply. In the well-watered treatment, there were no significant differences in total biomass between EI and EF plants regardless of fertilizer levels, and their differences occurred only in biomass allocation. Under drought conditions, EI produced significantly more biomass than EF plants regardless of fertilizer levels. Endophyte infection tended to reduce leaf nitrogen (N) concentration of host leaves but made the host allocate significantly higher fractions of N to photosynthetic machinery, and thus EI plants had higher photosynthetic N use efficiency and shoot biomass than did EF plants when fertilizer was limited. Our results support the idea that the endophyte–grass interactions are dependent on available resources – but in this case they depended more on water than on nutrient availability. © 2013 Elsevier B.V. All rights reserved.

1. Introduction Drought is a naturally occurring climate phenomenon affecting human and environmental activity globally. It is among the costliest and most widespread of natural disasters (Sheffield et al., 2009). Climate change is expected to exacerbate current stresses on water resources, and many semi-arid areas will suffer a decrease in water resources. Drought-affected areas are projected to increase in extent, duration and intensity (IPCC, 2007). Symbiotic relationships with microbes may influence how plant species respond to environmental change (Kannadan and Rudgers, 2008). The fossil record indicates that plants have been associated with endophytic fungi for >400 Myr (Krings et al., 2007) and were likely associated when plants colonized land, thus playing a long and important role in mediating the responses of host plants to variable environments. Recently, Rodriguez et al. (2009) differentiated endophytes into four functional classes based on several traits including fitness benefits conferred to hosts. To date, most researches have focused on

∗ Corresponding authors. Tel.: +86 22 23508249; fax: +86 22 23508249. E-mail addresses: [email protected] (A. Ren), [email protected] (Y. Gao).

clavicipitaceous endophytes (Class 1), with a much more intimate relationship to their host plant species than other three classes. In Class 1, the best know endophytes are the genera Neotyphodium and its sexual stage Epichloë, which are systemic symbionts of at least 80 genera and 300 species of cool-season and warm-season grasses (Saikkonen et al., 2006). Up to now, however, the majority of studies have focused on N. lolii and N. coenophialum, which colonizes two introduced and economically important grass species, perennial ryegrass (Lolium perenne L.) and tall fescue (L. arundinaceum Darbyshire ex. Schreb.), respectively (Saikkonen et al., 2006). Here endophyte infection frequently increase plant biomass and confer drought tolerance of endophyte-infected (EI) relative to endophyte-free (EF) grasses (Malinowski and Belesky, 2000; Clay and Schardl, 2002). Systemic fungal endophytes are estimated to occur in 20–30% of all grass species (Leuchtmann, 1992; Faeth, 2002). However, the question of whether endophytes improve drought tolerance in natural grass populations has been examined only in several native grass species (Ahlholm et al., 2002; Morse et al., 2002; Kannadan and Rudgers, 2008; Rudgers and Swafford, 2009). The limited research suggests that the benefits conferred by these fungi are less frequent and more variable in wild grasses (Saikkonen et al., 1999; Faeth and Sullivan, 2003). Furthermore, there is

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Please cite this article in press as: Ren, A., et al., Benefits of a fungal endophyte in Leymus chinensis depend more on water than on nutrient availability. Environ. Exp. Bot. (2013), http://dx.doi.org/10.1016/j.envexpbot.2013.11.019

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increasing evidence that the response of EI grasses to drought depends largely on the availability of other resources, in particular nutrients (McCormick et al., 2001). Saona et al. (2010) found EI Festuca rubra outperformed EF grasses only in dry, nutrient-rich conditions. Saari and Faeth (2012) reported that EI Arizona fescue had greater biomass than EF plants only in dry, nutrient-poor conditions. In contrast, Ahlholm et al. (2002) found no significant effect of endophyte infection on total biomass of F. pratensis in high nutrient and low water soils. Unlike agricultural settings, where resources, particularly nutrients and soil water content, are usually high; resources in natural conditions are typically variable. Resource availability may affect the ratio of costs and benefits of endophyte infection to the host, and thus may modify the nature of the host–endophyte relationship from mutualistic to neutral or even antagonistic (Saikkonen et al., 1998, 1999; Cheplick, 2007). Among nutrients related to grass–endophyte associations, nitrogen (N) has mostly been investigated, since it is not only a constituent of alkaloids in infected plants but also one of the most important limiting resources for plant growth in nature (Porter, 1994). As stated above, many studies that have found improved growth in EI grasses were done in moderate to high N soils. However, there are beneficial effects of endophyte infection in low N soils. For example, Louis and Faeth (1997) found EI Arizona fescue had a higher herbage dry matter at low soil N level, but no responses were observed at high soil N level compared with EF plants. Ravel et al. (1997) also reported that the advantage of EI over EF plants was greater at low N levels. These results suggested a more efficient use of N by EI plants (Malinowski and Belesky, 2000). In fact, the photosynthetic apparatus is the largest sink for N in plants (Poorter and Evans, 1998). Photosynthetic N use efficiency (PNUE) correlates strongly with N allocation to the photosynthetic machinery (Niinemets et al., 2003). Small changes in N allocation can greatly influence PNUE, and therefore plant performance (Onoda et al., 2004; Feng et al., 2008, 2009). To expand understanding of the ecological consequences of grass–endophyte symbiosis and examine potential functional traits involved in these interactions, we investigated the symbiosis between a dominant native grass, Leymus chinensis, and a systemic endophytic fungus, Epichloë bromicola, under different levels of water and nutrient availability in a greenhouse. We tested the hypothesis that symbiosis with the endophyte promoted drought tolerance of the host and investigated whether the tolerance was affected by nutrient availability. Specifically, we addressed the following questions: (1) does the endophyte ameliorate drought resistance of the native grass host? If yes, (2) does nutrient availability affect the symbiosis-dependent drought tolerance? (3) Could endophyte infection change N allocation strategy of the host grass?

steppe, China. We found that endophyte infection rates in most sites were low, with range of 0–9.1%. Only in the Abaga Banner population, the west distribution of L. chinensis in the Inner Mongolia steppe, was the endophyte infection rate relatively high (63.3%). In this area, the annual mean precipitation is about 250 mm, which is the lowest among 12 populations. L. chinensis and Stipa krylovii are dominant species. This area used to be grazing land, but has been fully enclosed for recovery since 2001. Within this population, plant individuals were sampled with an interval of 5–10 m between them. Three tillers were cut off from each plant, and the outermost non-senescent leaf sheath of each tiller was used for the detection of endophytes, using the aniline-blue staining method (Latch et al., 1984). To investigate endophyte diversity and taxonomy, 96 fungal isolates were obtained from tillers of EI plants. The isolates were classified into three morphotypes based on morphological characters and phylogenetic analyses of sequences of genes for b-tubulin (tubB), translation elongation factor 1-a (tefA) and actin (actG). The dominant morphotype was identified as E. bromicola (Zhu et al., 2013). For this experiment, thirty EI (infected by E. bromicola) and 30 EF plants were chosen and maintained in a greenhouse at Nankai University in August 2009. The endophyte was first reported in L. chinensis in Wei et al. (2006), and was identified as E. bromicola (Zhu et al., 2013). L. chinensis in this area seldom produce seeds. No stromata have ever been observed in natural populations, thus the endophyte is highly likely transmitted via vegetative propagation.

2. Materials and methods

2.3. Drought and nutrient treatments

2.1. Study system

Every morning, a soil moisture probe (ECH2 O Check, Decagon Devices, Pullman, WA, USA) was inserted to a depth of 5 cm into each pot to record the volumetric water content percentage, i.e. the amount of total soil volume consisting of water. Control plants were watered to maintain field capacity, i.e. soil moisture readings of about 20–25%. For plants in the drought treatment, sufficient water was added to keep the soil moisture within 5–10%. Different nutrient levels were achieved by the addition of different volumes of complete Hoagland nutrient solution with composition as follows: 5.0 mM Ca(NO3 )2 , 5.0 mM KNO3 , 2.5 mM MgSO4 ·7H2 O, 2.0 mM KH2 PO4 , 29 mM Na2 -EDTA, 20 mM FeSO4 ·7H2 O, 45 mM H3 BO3 , 6.6 mM MnSO4 , 0.8 mM ZnSO4 ·7H2 O, 0.6 mM H2 MoO4 , 0.4 mM CuSO4 ·5H2 O and pH 6.0 ± 0.1. During the experiment, 200, 25 and 0 mL of nutrient solution was added once a week per pot for HF, LF and NF treatments, respectively. Nutrient addition was

L. chinensis is a perennial rhizomatous grass. Due to its excellent stress tolerance, grasslands dominated by L. chinensis are widely distributed at the eastern end of the Eurasian steppe, from North Korea westward to Mongolia and northern China, and northwestward to Siberia. Early spring emergence and rapid growth, high palatability and herbage production make these grasslands ideal for grazing and forage production (Li et al., 1983). L. chinensis usually shows a high level of vegetative propagation but low sexual reproduction (Huang et al., 2004). In the middle and northeast parts of the Inner Mongolia steppe, L. chinensis is one of the most important dominant species. During the summer of 2009, we examined 12 populations from the middle to the north-east – covering the main distribution area of L. chinensis in the Inner Mongolia

2.2. Experimental design For the experiment, we cloned material from the plants mentioned above. In November 2010, we transplanted seven tillers of approximately equal size into a white plastic pot (23-cm diameter and 25-cm depth) filled with 5 kg of sand. Here water and nutrients availability were considered simultaneously. Water treatment included two levels: well watered and drought. In our previous study, we found that a high fertilizer level was detrimental to grass growth under drought conditions (Ren et al., 2011). So under the well-watered conditions, high and low fertilizer levels (HF and LF, respectively) were applied; while under drought, LF and no fertilizer levels (NF) were applied. EI and EF plants were separately grown in four treatment combinations: well watered/HF; well watered/LF; drought/LF and drought/NF. Each treatment was replicated five times. The experiment lasted 50 d, from 22 November 2010 to 11 January 2011. The experiment was carried out in a greenhouse at Nankai University, Tianjin, China. All ramets were examined for endophyte status following staining with lactophenol aniline blue (Latch et al., 1984) at the beginning and end of the experiment.

Please cite this article in press as: Ren, A., et al., Benefits of a fungal endophyte in Leymus chinensis depend more on water than on nutrient availability. Environ. Exp. Bot. (2013), http://dx.doi.org/10.1016/j.envexpbot.2013.11.019

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ARTICLE IN PRESS 0.029 1.412 0.253 0.003 0.172 0.031 <0.01 0.101 0.939 11.85 3.034 0.006 29.40 7.528 0.015 0.037 0.651 0.700 5.152 0.212 0.154 9.730 0.400 0.290 0.010 0.894 0.656 8.568 0.018 0.206 15.29 0.033 0.367 Significant P-values are in bold.

72.14 4.811 25.99 0.620 0.041 0.223 0.279 <0.01 0.687 1.254 70.79 0.168 6.844 386.3 0.916 0.684 0.600 0.027 0.171 0.286 5.927 0.477 0.796 16.51 0.355 0.696 0.012 0.908 0.159 8.062 1.873 0.328 16.64 0.072 <0.01 <0.01

0.037 0.053 0.686 5.183 4.354 0.169 5.304 4.456 0.173 0.783 <0.01 0.756 0.078 13.73 0.101 11.88 2078 15.23 0.884 0.490 0.380 0.022 0.502 0.818 Under drought conditions Endophyte (E) 1 0.317 1 7.258 Fertilizer (F) 1 11.83 E×F

F

3.706 351.9 9.645 0.400 3.707 <0.01 352.0 0.029 9.647 0.749 77.15 5.792

MS F F MS

Total root

P MS

F Fine root

MS

P Shoot MS F

P MS

F

P

Biomass Leaf no.

All statistical analyses were performed with SPSS 10.0 (SPSS, Chicago). For some variables (tiller number, leaf number and biomass allocation), natural log transformation was used to homogenize variance and to obtain a normal distribution of residuals. Analysis of covariance (ANCOVA) was used to compare tiller and leaf number of EI and EF plants, and the tiller and leaf number at the beginning of the experiments were used as covariates, respectively. Nutrient availability and endophyte infection were analyzed using a two-way analysis of variance (ANOVA). We also performed a two-way ANOVA with factor of endophyte infection and environment treatment with 4 levels: well watered/HF, well watered/LF, drought/LF and drought/NF). Differences between the means were compared using Duncan’s multiple-range tests at P < 0.05. Principal component analysis (PCA) was performed with growth and physiological traits in order to reduce the variables to the ones explaining best the effects.

Tiller no.

2.7. Statistical analyses

df

Leaf chlorophyll concentration was measured according to the procedure of Lin et al. (1999). To determine leaf N concentration, leaf samples were dried and ground in a mortar to pass through a 0.15-mm sieve. Total N was determined by Kjeldahl digestion. To examine potential mechanisms of drought tolerance or avoidance, we also measured the contents of free proline and soluble sugars in leaves; however, there were no significant differences between EI and EF plants in all treatments and therefore these data are not presented.

Table 1 Two-way ANOVA for vegetative growth of endophyte-infected (EI) or endophyte free (EF) Leymus chinensis.

2.6. Other response variables

P

At the end of the treatments, gas exchange measurements were made on the youngest fully expanded attached leaf with a LI-COR 6400 infrared gas analyzer (LI-COR, Lincoln, NE, USA). The same leaf was also used for measurements of SLA and N content. In this way, differences among leaves of the same plant were avoided when the relationships among the variables were analyzed. Photosynthesis–light responses of plants were assessed under 400 ␮mol mol−1 CO2 . Maximum photosynthetic rate (Pmax ) and saturation photosynthetic photon flux density (PPFD) were determined from the Pn (net photosynthetic rate) – PPFD curve. Photosynthesis–CO2 responses of plants were assessed under saturation PPFD of 1200 ␮mol m−2 s−1 . The leaf temperature was held constant at 25 ◦ C. Using the data for photosynthesis, chlorophyll, SLA and N content, the N allocation to photosynthesis was calculated using the methods described by Ren et al. (2011) and Li et al. (2012). PNUE was calculated as the ratio of Pmax to N content.

2.944 0.107 463.7 27.42 <0.01 47,748 9.72 <0.01 3584

2.5. Gas exchange

MS

Total biomass

P

Measurements of tiller and leaf numbers were made on all pots at the beginning and end of the experiment. At the end of the experiment, leaves, fibrous roots and rhizomes were harvested separately. Ten fully expanded leaves growing on vegetative tillers per pot were chosen for measuring of leaf area and they were weighed separately for the determination of specific leaf area (SLA). Then all plant parts were oven-dried at 60 ◦ C and weighed.

F

Root:shoot

2.4. Growth and biomass

Under well watered conditions Endophyte (E) 1 105.9 1 986.2 Fertilizer (F) 1 349.6 E×F

P

applied 15 times in total. The positions of the pots were randomly rotated each week to minimize location effects.

0.868 0.252 0.622

3

<0.01 0.043 <0.01

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Fig. 1. Mean (±SD) tiller number and leaf number of endophyte-infected (EI) or endophyte-free (EF) Leymus chinensis under various conditions of water and nutrient availability. An asterisk denotes significance between EI and EF at P < 0.05.

3. Results 3.1. Shoot growth Under well-watered conditions, tiller and leaf number were significantly affected by an interaction of fertilizer level and endophyte status (Table 1). At the end of the experiment, EI plants produced significantly more tillers and leaves than EF plants in the HF treatment, while in the LF treatment there were no significant differences between EI and EF plants (Fig. 1). Drought stress significantly reduced tiller and leaf growth compared with well-watered HF treatment. However, endophyte infection had no significant effect on the number of tillers and leaves under drought conditions (Table 1 and Fig. 1).

under well-watered conditions; however, under drought conditions PNUE was significantly increased by endophyte infection but not by fertilizer levels (Table 3 and Fig. 3). 3.5. Principal component analysis Principal component analysis, performed with growth and physiological traits, identified two factors: component 1, explaining 46.2% of the variance, and component 2 explaining 31.3%. For component 1, well watered/HF/EI (WHFEI) and well watered/HF/EF (WHFEF) were separated significantly with drought treatments

3.2. Biomass allocation Under well-watered conditions, there were no significant differences in shoot, root and total biomass between EI and EF plants for the HF treatment (Table 2). LF treatment induced significant decreases in shoot biomass in both EI and EF plants, but EI decreased less than EF shoot biomass, and thus EI shoot biomass was higher than EF shoot biomass. There were no differences in root biomass between EI and EF. Therefore, the root:shoot ratio was significantly lower in EI than EF plants for the LF treatment (Table 2). Drought stress significantly inhibited plant growth, but this inhibition was significantly mitigated by endophyte infection. EI plants had significantly more shoot biomass, more fibrous root biomass and thus higher total biomass. Fertilizer level had no significant effect on biomass under drought (Table 1 and Fig. 2). 3.3. N concentration and allocation

Fig. 2. Mean (±SD) biomass of endophyte-infected (EI) or endophyte-free (EF) Leymus chinensis under drought stress. An asterisk denotes significance between EI and EF at P < 0.05.

Area-based leaf N concentration tended to be lower for EI than EF plants, although differences were only significant under drought conditions (Tables 3 and 4). However, similar fractions of N were allocated to photosynthetic machinery under well-watered HF conditions. When nutrients and/or water were limited, however, significantly higher fractions of N were allocated to photosynthetic machinery for EI compared to EF plants (Table 4). 3.4. Pmax (maximum net photosynthetic rate) and PNUE (photosynthetic N use efficiency) Lower N concentration of EI leaves did not result in lower Pmax compared with EF leaves. In contrast, Pmax of EI was similar to that of EF for all water and fertilizer treatments (Table 4). PNUE was significantly improved by fertilizer supply and endophyte infection

Fig. 3. Mean (±SD) photosynthetic nitrogen use efficiency (PNUE) of endophyteinfected (EI) and endophyte-free (EF) Leymus chinensis under various water availabilities. An asterisk denotes significance between EI and EF at P < 0.05.

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Table 2 Biomass allocation of endophyte-infected (EI) or endophyte-free (EF) Leymus chinensis under well-watered conditions. Treatment

Biomass (g)

Root:shoot

Shoot

Fine root

Rhizome

Total root

Total biomass

High fertilizer (HF) EI EF

19.3a 19.8a

10.1a 7.6b

0.8a 1.1a

10.8a 8.7a

30.1a 28.5a

0.561c 0.441c

Low fertilizer (LF) EI EF

12.3b 10.0c

8.0b 9.2ab

0.6a 0.9a

8.6a 10.1a

20.9b 20.2b

0.702b 1.004a

Note: Significant differences (P < 0.05) for each variable are indicated by lowercase for variables where fertilizer treatment and endophyte infection were analyzed together.

Table 3 Two-way ANOVA for photosynthetic parameters and nitrogen allocation of endophyte-infected (EI) or endophyte-free (EF) Leymus chinensis. df

NA MS

PT

Pmax

PNUE

F

P

MS

F

P

MS

F

P

MS

F

P

Under well watered conditions 1 0.064 Endophyte (E) 1 0.527 Fertilizer (F) E×F 1 0.002

1.893 15.48 0.062

0.188 <0.01 0.807

0.028 0.005 0.001

11.95 2.338 0.581

<0.01 0.165 0.468

0.107 197.6 2.234

0.015 27.56 0.307

0.906 <0.01 0.595

38.41 22.93 0.009

9.378 5.600 0.002

0.016 0.046 0.964

Under drought conditions 1 Endophyte (E) 1 Fertilizer (F) 1 E×F

32.69 18.57 1.131

<0.01 <0.01 0.303

0.088 0.000 0.011

10.73 0.000 1.311

0.011 0.989 0.285

2.775 0.575 0.497

0.390 0.081 0.070

0.550 0.783 0.798

36.42 3.200 0.948

5.634 0.495 0.147

0.045 0.502 0.712

0.729 0.414 0.025

Note: NA , area-based leaf nitrogen concentration; PT , the fraction of leaf nitrogen allocated to the photosynthetic machinery; Pmax , maximum net photosynthetic rate; PNUE, photosynthetic nitrogen use efficiency. Significant P-values are in bold.

(DEI and DEF), and well watered/LF/EI and EF (WLF) was in the middle. For component 2, under both well watered/HF and drought treatments, EI and EF were differently grouped, while under well watered/LF treatment, EI and EF were grouped together (Fig. 4). The loading factors of each variable for each component are shown in Fig. 5. Growth traits including tiller number, leaf number and total biomass had higher factor weights in component

1. For component 2, PNUE and PT had higher statistical factor weights. 4. Discussion We showed that a beneficial interaction between the native grass L. chinensis and its associated fungal endophyte was

Fig. 4. Principal component analysis of growth and physiological traits. WHFEI, well watered high fertilizer treated endophyte-infected plants; WHFEF, well watered high fertilizer treated endophyte-free plants; WLF, well watered low fertilizer treated endophyte-infected and endophyte-free plants; DEI, drought low fertilizer and no fertilizer treated endophyte-infected plants; DEF, drought low fertilizer and no fertilizer treated endophyte-free plants.

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Fig. 5. Statistical factor weights of growth and physiological traits. Tiller, tiller number; Leaf, leaf number; Biomass, total biomass; PT , the fraction of leaf nitrogen allocated to the photosynthetic machinery; Pmax , maximum net photosynthetic rate; PNUE, photosynthetic nitrogen use efficiency.

conditional on available resources, especially water supply. Under well-watered conditions, total biomass was not affected by endophyte infection. Under drought conditions, however, there was significantly more total biomass in EI than EF plants. These findings are in agreement with previous reports on an agronomically important turf grass, tall fescue (Arachevaleta et al., 1989; Malinowski and Belesky, 2000), and other native grasses (Kannadan and Rudgers, 2008; Saona et al., 2010). In contrast, the results of the present study differ from those of previous studies of Achnatherum sibiricum and in Elymus virginicus, in which EI plants benefited more from the endophyte under daily watering than under drought (Rudgers and Swafford, 2009; Ren et al., 2011). We suggest that these differences in responses to infection may be mainly due to the different natural habitat from which these grass species originated. Gibert et al. (2012) also reported that plants collected from the most xeric sites demonstrated increased drought tolerance compared with individuals from mesic populations. Our data support the Table 4 Nitrogen allocation and maximum photosynthetic rate of endophyte-infected (EI) or endophyte-free (EF) Leymus chinensis under various conditions of water and fertilizer availability. Treatment

NA

PT

Pmax

EI EF EI EF

1.575ab 1.668a 1.230c 1.364bc

0.450ab 0.376b 0.514a 0.397b

28.87a 29.92a 21.62ab 20.94b

EI EF EI EF

1.555b 2.008a 1.338c 1.649b

0.590a 0.359bc 0.531a 0.419b

18.63a 19.18a 17.78a 19.15a

Well-watered conditions High Fertilizer (HF) Low fertilizer (LF) Drought conditions Low fertilizer (LF) No fertilizer (NF)

Note: NA , area-based leaf nitrogen concentration in g m−2 ; PT , the fraction of leaf nitrogen allocated to the photosynthetic machinery in g g−1 ; Pmax , maximum net photosynthetic rate in ␮mol m−2 s−1 . Significant differences (P < 0.05) for each variable are indicated by lowercase for variables where fertilizer treatment and endophyte infection were analyzed together.

hypothesis that water availability is one such factor balancing the antagonist–mutualist continuum (Saikkonen et al., 1998; Müller and Krauss, 2005). In contrast to expectations that there might be a ‘metabolic cost’ to the host of harboring the fungal endophyte because of the limited supply of available photosynthate under low soil nutrient availability (Cheplick et al., 1989), the beneficial interaction between L. chinensis and its associated fungal endophyte was less conditional on nutrients. Under well-watered conditions, fertilizer treatments did not result in a difference in total biomass between EI and EF plants. Endophyte infection only changed resource distribution of the host grass. EI plants produced significantly more tillers and leaves with high nutrient supply and produced a higher shoot biomass and shoot:root ratio with low nutrient supply compared with their EF counterparts. Under drought conditions, EI produced significantly more biomass than EF plants regardless of fertilizer levels. To explain this difference we provide the following explanations combining previous studies and our present data. First, under nutrient-limited conditions endophyte infection tended to lower N concentration of the host leaf, but allocated significantly higher fractions of N to photosynthetic machinery, and thus EI had higher PNUE and shoot biomass than did EF plants. This finding suggests that it is N allocation to photosynthetic machinery instead of leaf N concentration itself that is more highly correlated with plant growth (Jeyasingh et al., 2009; González et al., 2010). Low N concentration of EI plants has also been reported in tall fescue by Rogers et al. (2011) and our previous study of A. sibiricum (Li et al., 2012). It has been reported that organisms with a greater growth advantage in nutrient-poor environments are those able to modify their body nutrient content and increase efficiency of nutrient use without major decreases in their growth rates (Elser et al., 2003; Mulder and Bowden, 2007). Moreover, under wellwatered LF conditions, EI plants increased shoot allocation and had lower root:shoot ratio than EF plants. Similar results were reported in L. perenne by Lewis et al. (1996) and Cheplick (2007) and in meadow fescue by Lehtonen et al. (2005). Higher shoot biomass might lead to higher levels of photosynthates and thus cover the

Please cite this article in press as: Ren, A., et al., Benefits of a fungal endophyte in Leymus chinensis depend more on water than on nutrient availability. Environ. Exp. Bot. (2013), http://dx.doi.org/10.1016/j.envexpbot.2013.11.019

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‘metabolic cost’ of harboring endophytes, considering that endophytes only constitute ≤0.2% of infected plant biomass (Tan et al., 2001). Finally, in the present study under drought stress, although no fertilizer was applied, there was no ‘metabolic cost’ in EI plants, suggesting that the relationship between endophyte effect and nutrient supply depends on both the specific host–endophyte associations and their environmental requirements (Ahlholm et al., 2002; Granath et al., 2007). Our findings may explain the pattern observed in the Inner Mongolia steppe, i.e. higher prevalence only occurs in the Abaga Banner, where water content and nutrients are more limited than in other areas where L. chinensis is prevailing. The results presented here are in accordance with our hypothesis that infections were more advantageous under drought. Prior studies suggest this would be due to a combination of drought stress avoidance and drought tolerance mechanisms. In drought stress avoidance, water deficits may be reduced in EI plants by faster stomatal closure (Malinowski et al., 1997; Elmi et al., 2000) and increased root absorption (Malinowski et al., 1999; Crush et al., 2004) in response to water stress. Drought tolerance mechanisms may include osmotic adjustment through increased accumulation of solutes (Richardson et al., 1992; West, 1994). In the present study, we found EI plants allocated more resources to fine roots under drought stress, which might enhance drought avoidance. Meanwhile, higher PT and PNUE of EI than those of EF plants most probably contribute to its drought tolerance. Endophyte presence did not appear to alter the concentrations of soluble sugars and free proline and so had no effect on osmotic adjustment; however, other solutes might be changed by endophyte infection (Richardson et al., 1992; Malinowski and Belesky, 2000). Since we did not measure osmotic potential in the base of the tillers, such assays would be useful next steps for understanding mechanisms of drought tolerance. The results presented here agreed with our hypothesis that infections were more advantageous under drought stress; however, the beneficial effects were independent of fertilizer supply. Admittedly, the duration of the pot experiment was short in comparison with the natural life span of the grass host and these results should be interpreted with caution. Nonetheless, it is important to note that even this short duration is relevant considering that the higher endophyte infection rate of L. chinensis only occurred in the driest area, Abaga Banner, throughout the north-east of the Inner Mongolia steppe. Fungal endophytes could play a significant role in management of environmental stress by the host species. By understanding how complex interactions between plants and their associated symbionts vary with stress, we can better predict how climate change will influence species distributions and abundances in the future (Kannadan and Rudgers, 2008). For L. chinensis, we predict that reduced water availability will result in greater benefits of the symbiosis for plant growth.

Author contributions AR conceived and designed the experiments. AR and YG wrote the paper. MW and LY performed the experiments. MW, YZ and XL analyzed the data.

Acknowledgements This research was funded by the National Natural Science Foundation (31270463) and Doctoral Program Foundation of Institutions of Higher Education of China (20130031110023).

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