Endophyte species influence the biomass production of the native grass Achnatherum sibiricum (L.) Keng under high nitrogen availability

Abstract Research on the interaction of endophytes and native grasses normally takes infection status into account, but less often considers the species of endophyte involved in the interaction. Here, we examined the effect of endophyte infection, endophyte species, nitrogen availability, and plant maternal genotype on the performance of a wild grass, Achnatherum sibiricum. Six different Epichloë‐infected maternal lines of A. sibiricum were used in the study; three lines harbored Epichloë gansuensis (Eg), while three lines harbored Epichloë sibirica (Es). These endophytes are vertically transmitted, while Eg also occasionally produces stromata on host tillers. We experimentally removed the endophyte from some ramets of the six lines, with the infected (E+) and uninfected (E−) plants grown under varying levels of nitrogen availability. Eg hosts produced more aboveground biomass than Es hosts only under high nitrogen supply. Endophyte species did not show any influence on the maximum net photosynthetic rate (P max), photosynthetic nitrogen use efficiency, or total phenolics of A. sibiricum under all nitrogen conditions. However, the plant maternal genotype did influence the P max and shoot biomass of A. sibiricum. Our results show that endophyte species influenced the shoot biomass of A. sibiricum, and this effect was dependent on nitrogen supply. As with most coevolutionary interactions, A. sibiricum that harbored Eg and Es may show pronounced geographic variation in natural habitats with increased nitrogen deposition. In addition, stroma‐bearing endophyte (Eg) provides positive effects (e.g., higher biomass production) to A. sibiricum plants during the vegetative growth stage.

However, accumulating evidence suggests that the degree of the mutual benefit is conditional on nutrient factors, in particular nitrogen (N) availability in soils (Saikkonen, Lehtonen, Helander, Koricheva, & Faeth, 2006). As one of the most important limiting resources for plant growth, nitrogen affects all levels of plant function, from metabolism to biomass production and growth (Crawford, 1995;Marschner, 1995;Stitt & Krapp, 1999). Nitrogen is also a constituent of alkaloids in infected plants. For example, Epichloë endophytes may produce N-rich secondary metabolites, such as loline alkaloids that can constitute up to 2% of the dry weight of the plant (Blankenship et al., 2001;De-wen, Wang, Patchett, & Gooneratne, 2006). Elevated nitrogen supply can alter plant nutrient allocation and may affect nutrient exchange in ways that impact the production of N-based mycotoxins and carbon energy sources for endophytic growth. Höft, Verpoorte, and Beck (1996) also found a variable relationship between nitrogen and alkaloid production/accumulation. However, published reports of the effects of N availability on grass-endophyte associations are inconsistent. Arachevaleta, Bacon, Hoveland, and Radcliffe (1989) reported that the beneficial growth responses of tall fescue from one clone to endophyte depend on a high nitrogen supply in the environment. In another study using Lolium perenne seeds from a single plant (Ren, Gao, Wang, Wang, & Zhao, 2009), endophyte-infected (E+) plants maintained significantly higher above-and belowground biomass than endophyte-free (E−) hosts under high-nitrogen conditions, while the positive effect of endophyte infection disappeared under low-nitrogen conditions. These studies suggest that with nitrogen limitation, endophytes may have neutral or negative effects on the performance of host plants. However, several works have shown the opposite results. For example, in a low-nitrogen environment, the presence of an endophyte has been found to improve plant performance (such as vegetative growth and biomass accumulation) in three genotypes of L. perenne (Lewis, 2004;Ravel, Courty, Coudret, & Charmet, 1997).
In addition to environmental factors, Cheplick (1997) proposed that the benefits of infection may also depend on the genotype of the host plants. In the case of tall fescue and L. perenne, the effects of the plant genotype and interactions with endophyte infection were reported to have significant effects on the performance (such as tiller numbers, specific leaf area, nutrient content and biomass) of host plants (Belesky & Fedders, 1996;Cheplick, 1997Cheplick, , 1998Cheplick, , 2004Cheplick, , 2007Cheplick & Cho, 2003;Hesse et al., 2004;Rahman & Saiga, 2005).
In a 3-year common garden study of nine genotypes of L. perenne, Cheplick (2008) even found that the effects of the host genotype may outweigh the effects of fungal endophyte infection on the number of tillers and biomass of L. perenne. Nonetheless, many studies of host genotype interaction with endophyte infection only compared the performance of E+ and E− hosts, without considering endophyte species or genotypes (West, Popay, & Thom, 2007). When studying endophyte species of agronomic importance, researchers often use host plants inoculated with selected specific endophyte genotypes. Tall fescue infected by one of two distinct endophyte strains designated CTE and AR-584 were employed in a glasshouse experiment as reported in Rúa, McCulley, and Mitchell (2013). The total biomass of the host plants when infected with CTE was significantly greater than that of E− hosts, while the presence of AR-584 appeared to not be significant.
Another inoculation experiment employing two cultivars of tall fescue was performed using two endophyte genotypes (wild type, KY31, designated type, AR501) of Epichloë coenophiala. The results showed that there was a significant cultivar × endophyte strain interaction for most measured variables (such as the tiller number, growth rate, and photosynthesis; Assuero et al., 2000).
The endophyte utilizes photosynthetic carbon and additional elements, particularly nitrogen, from its host to carry out the metabolic processes necessary for its survival and growth (Christensen & Bennett, 2002). According to previous reports, endophyte infection can have significant effects on host photosynthetic parameters, and these effects are contradictory (Belesky Devine, Pallas, & Stringer 1987;Morse et al., 2002). Belesky et al. (1987) reported that E+ plants showed decreased rate of photosynthesis. On the other hand, Amalric, Sallanon, Monnet, Hitmi, Coudret, & Amalric (1999) demonstrated higher photosynthetic rates under drought stress, which is similar to the study that Marks and Clay (1996) conducted at high temperatures.
In addition to these results, the endophyte haplotype appears to override the infection status in some cases, at least in several physiological measures (e.g., Pn; Morse, Faeth, & Day, 2007). Thus, we expected that A. sibiricum infected with different endophyte species may have varied photosynthesis-related mechanisms.
Achnatherum sibiricum (L.) Keng, infected with two different endophytes, E. gansuensis and E. sibirica in its native populations, is a caespitose perennial grass distributed in northern China (Wei, Gao, Li, Xu, & Ren, 2006;Zhang et al., 2009). In previous studies by our laboratory, five A. sibiricum populations were sampled and the distribution and abundance of endophytes were documented. In total, 438 fungal isolates were obtained, but the phenomenon of double Epichloë infections in A. sibiricum has never been observed. The overall abundance of the two dominant endophyte species remained constant in all investigated populations (92%-95%; Zhang et al., 2009). Both E. gansuensis and E. sibirica can be vertically transmitted from the maternal host to the offspring by seed. However, conidia stromata have occasionally been observed on culms of A. sibiricum plants harboring E. gansuensis.
The conidia derived from the stromata can give rise to mycelia that in turn infect germinating endophyte-free seeds, which indicated its ability for horizontal transmission (Li et al., 2015). Furthermore, it has been reported that E. gansuensis naturally infects both A. sibiricum and A. inebrians (Li et al., 2004;Zhang et al., 2009), while E. sibirica naturally infects only A. sibiricum (Zhang et al., 2009). In contrast to A. inebrians, A. sibiricum plants have no obvious herbivore deterrence according to previous observations (Zhang et al., 2009). Given the above, A. sibiricum is considered to be an excellent research material to study the effect of endophyte species on host plants. In a previous study, we have compared the performance of E+ and E− A. sibiricum under different nitrogen availability levels. The results showed that A. sibiricum-Epichloë associations were conditional on both N and P availability, but more conditional on N than on P. The aim of this work was to characterize the effect of the endophyte species on the performance of A. sibiricum, and also to consider the effects of the maternal genotype of the host as well as the nitrogen availability in the soil. We addressed the following questions: (1) is the mutual effect between the endophyte and native grass related to the species of endophyte? If so, (2) does the influence of the endophyte species depend on the nitrogen availability? (3) Does the host maternal genotype affect the symbiosis of the endophyte and native grass?
Seeds of A. sibiricum in the present study were collected from natural populations in National Hulunber Grassland Ecosystem Observation and Research Station (119.67°E, 49.10°N) in northeast China. As A. sibiricum is a cross-pollinated grass, we hereafter refer to the selected maternal seed sources as maternal plant genotypes. Within this population, 200 maternal plants were randomly selected in 2012, and a distance of at least 5 m was maintained between the sampled plants.
Seeds were collected from the sampled plants and stored at 4°C. The infection rate was determined using the aniline blue staining method (Latch, Christensen, & Samuels, 1984). First, 100 seeds were randomly selected for the detection of the infection rate. Second, the other 100 randomly selected seeds were planted in 10 pots (1 m × 1 m) in the field. After 3 months, the infection rate was also determined. The results all showed that the endophyte infection frequency was 100%.
Five seeds from the same maternal plant were randomly used for the isolation and observation of endophytes. Seeds from each sample were surface-sterilized by placing them into a 50% sulfuric acid solution for 20 min, rinsing in sterile water, soaking for 20 min in a 3% sodium hypochlorite solution, and finally rinsing three times in sterile water. Then, the surface-sterilized seeds were placed on Petri dishes containing a potato dextrose agar (PDA) medium. The Petri dishes were incubated at 25°C for 3 weeks. After purification, two types of endophyte were observed: E. gansuensis and E. sibirica based on their colony morphology which had been reported by Zhang et al. (2009).
We used seeds from six naturally infected maternal plants (G1-G6) from the same population. Among them, three of the six naturally infected plants used in our experiments (G1-G3) harbored E. gansuensis (termed Eg) and the other three (G4-G6) harbored E. sibirica (termed Es). The seeds from G1 to G6 were all divided into two parts and stored at 4°C (endophyte-infected, E+) or underwent a 30-day 60°C heat treatment (endophyte-removed, E−). In our previous study, we found that this high temperature treatment had no significant effect on the germination rate, germination potential, or germination index of the seeds of A. sibiricum (Li, Han, Ren, & Gao, 2010). 0.4 mmol/L CuSO 4 ·5H 2 O, and pH 6.0 ± 0.1. Nitrogen was added in the form of NH 4 NO 3 , which was delivered as 0.1 m mol N (LN) or 1 m mol N (HN), respectively. The total set concentrations of the N treatments were applied according to the lower and upper limits in the natural habitat of A. sibiricum (Liu, 2004). During the experiment, 0.8 L of nutrient solution was added once a week to each pot, a total of 15 times. These pots were all located in the experimental field at Nankai University, with transparent flashing above them. Plants were subjected to ambient light and temperature regimes. The positions of the pots were randomly rotated each week to minimize location effects.

| Photosynthesis and gas exchange parameters
At the end of the treatments, gas exchange measurements were conducted on sunny and windless days. The second youngest fully expanded leaf in a pot was measured with a LI-COR 6400 infrared gas analyzer (LI-Cor, Lincoln, NE, USA). The same leaf was also used to measure the specific leaf weight and nitrogen content (N%). We developed light response curves for three randomly selected A. sibiricum leaves and determined the light saturation point (LSP) per plot using a LI-COR 6400. Under 400 μmol mol −1 CO 2 , the maximum net photosynthetic rate (P max ) was then measured at a PPFD (1,000 μmol m −2 s −1 ) of the LSP in the plot (Chen, Yu, Chen, & Xu, 2006).
The nitrogen content in the leaves was measured using a vario MACRO CHN analyzer. The photosynthetic nitrogen use efficiency (PNUE) was calculated as the ratio of P max to the area-based leaf nitrogen concentration (Hikosaka, 2004).

| Leaf mass per area (LMA) and total phenolic concentration
On 5th August, four fully expanded leaves per pot, growing on vegetative tillers, were oven-dried at 60°C. They were weighed separately to determine the LMA. At the same time, three fully expanded leaves per pot were randomly collected and placed into an ice box. The total concentration of phenolics was immediately analyzed according to the method of Malinowski, Belesky, Hill, Baligar, and Fedders (1998).

| Biomass production
At the end of the period, all of the tillers in each pot were cut off at ground level on 10th September. No reproductive shoots or stromata were observed on the tillers of the plant. The tillers were placed in an oven at 105°C for 30 min, oven-dried at 80°C for 24 h, and then weighed to determine the aboveground biomass.

| Endophyte alkaloid analysis
The endophyte alkaloid concentration was measured in leaf sheaths at the middle of the trial. Two tillers per plant in each pot were cut at ground level on 7th August 2013. The leaf sheaths collected from each pot were combined into a bulk sample and temporarily stored at −20°C. Prior to endophyte alkaloid analysis, bulk samples were ground in liquid nitrogen and then immediately freeze-dried for 24 hr. Aliquots of 50 mg of the dried and ground material were analyzed for ergovaline and peramine using the methods described by Moore, Pratley, Mace, and Weston (2015). The limit of quantitation for ergovaline and peramine is 0.1 μg g −1 .

| Stromata observation
After they were cut off, the host plants in each pot were watered every 2 weeks until winner irrigation. Then, the pots were buried in the earth at ground level and covered with plastic transparent film.
The next spring, 800 ml of distilled water was added into each pot on 12th March. After 2 weeks, 0.5L of LN and HN nutrient solutions were added into each pot. The formation of stromata was recorded from 1st April to 30th May.

| Data analysis
Data analysis was conducted according to Morse et al. (2007). First, an initial multivariate analysis of variance (MANOVA) was used to examine the effect of infection (E+ or E−), maternal plant genotype, and nitrogen availability treatment effects on growth parameters and biomass production of A. sibiricum (SYSTAT 13.0). The plant maternal genotype was a nested factor within endophyte species because each plant half-sib genotype was associated with only one of the two endophytes. As to whether the endophyte effect depends on nitrogen availability, we expect a significant interaction between the nitrogen treatment and endophyte presence. As to whether the host maternal genotype affects the symbiosis of the endophyte and native grass, we expect a significant infection of the host maternal genotype. Second, under question 1, to assess the effect of endophyte species on growth parameters and biomass production, we conducted ANOVA (SPSS 21.0) with only E+ plants because endophyte-removed plants had no endophyte species associated with them.

| Photosynthesis and Photosynthetic nitrogen use efficiency (PNUE)
There was a significant two-way interaction between the nitrogen treatment and endophyte infection for the photosynthetic parameters of A. sibiricum (Table 1, Figure 1) Figure 4). However, this significant effect of the endophyte species did not appear under LN conditions (p = .309; Table 2, Figure 4).

| Total phenolic concentration and leaf mass per area (LMA)
The total phenolic concentration and LMA of host leaves were significantly affected by endophyte infection, but not by endophyte species (Tables 1, 2). Under both HN and LN treatments, the total phenolic content ( Figure 5) and LMA ( Figure 6) of E+ plants were significantly higher (p < .001, p < .001) than those of E− plants. Meanwhile, these two parameters did not vary among the six maternal plant genotypes (Table 1).

| Endophyte alkaloid production
No ergovaline was detected in any of the sampled plants. Some samples (3 of 27) showed low (<0.6 mg g −1 ) peramine production with no peramine detected in any other samples.

| Does the mutual effect between endophyte and native grass relate to the species of endophyte?
Previous studies of the effects of endophytes on native grasses mostly considered the infection status-whether host grasses are infected or not. The existing results of the relationship are highly variable, ranging from mutualism (Brem & Leuchtmann, 2001;Crawford, Land, & Rudgers, 2010;Iannone et al., 2012;Nan, 1996;Ren et al., 2011;Rudgers & Swafford, 2009) to parasitism or commensalism (Faeth & Sullivan, 2003;Faeth et al., 2004;Hamilton & Faeth, 2005;Jani, Faeth, & Gardner, 2010;Morse et al., 2002;Saikkonen, Helander, Faeth, Schulthess, & Wilson, 1999). Generally, native grasses harbor a wider range of endophyte species compared with agronomic grasses (Iannone et al., 2012;Leuchtmann & Oberhofer, 2013;Shymanovich et al., 2015;Sullivan & Faeth, 2004;Zhang et al., 2009). For example, Leuchtmann and Oberhofer (2013) found that H. europaeus can harbor one of six endophyte species. Sullivan and Faeth (2004) also indicated that Festuca arizonica can naturally host one of at least two types of endophyte. It has been reported that the genetic variation in Epichloë endophytes can potentially influence the outcomes of interaction. Different Epichloë strains that have been intentionally inoculated into agronomic grasses may alter the growth and physiological properties of the host (Assuero et al., 2000). In that case, we may wonder whether the endophyte species will influence the outcome of the interactions of the endophytes with the host grasses. A previous study was conducted by Iannone et al. (2012) , 1990;Latch, Hunt, & Musgrave, 1985) produced or induced by the endophytes.
The symbiotic relationships between plants and fungi have a long evolutionary history. Illuminating what determines the diversity and structure of natural communities has long been a research hot spot (Jani et al., 2010). In the present study, plants harboring Eg showed significantly higher aboveground biomass than Es-infected plants under HN conditions, but not under LN conditions. Phoenix et al. (2012) proposed that nitrophilous species may be more competitive with increased nitrogen deposition and are then able to exclude other plant species in the habitat. As with most coevolutionary interactions, A. sibiricum that harbored Eg and Es may show pronounced geographic variation in natural habitats in the future and may change the diversity of natural communities (Cheplick & Faeth 2009).

| Does the host maternal genotype affect the symbiosis of endophytes and native grass?
The genetic variation of the host plant, even in cultivated grasses, where host genetic variation is reduced relative to wild grasses because of cultivation and selective breeding, can also result in different growth performances (Cheplick & Cho, 2003). Studies using L. perenne suggested that the plant genotype and its interaction with endophyte infection influences many traits of host plants, such as vegetative growth (Cheplick, 2004;Cheplick & Cho, 2003), physiological characteristics of photosynthesis (Spiering, Greer, & Schmid, 2006), biomass production (Cheplick, 2004(Cheplick, , 2007Hesse et al., 2004), and reproductive growth (Hesse et al., 2004). Research on native grass-endophyte symbiosis also supported this view. Faeth and Fagan (2002) conducted a field experiment using Festuca arizonica and suggested that the plant maternal genotype interacted with endophyte infection to affect the growth rate of host plants. After a growing season, two genotypes of E+ plants showed a greater growth rate than that of E− plants, while the others were not affected by endophyte infection. In another study of the same grass species (the same four genotypes) lasting 2 years in the field, the genotype × endophyte interaction also had significant effects on the growth of the host plants. However, in this study, endophyte infection showed negative effects in three genotype hosts . Similarly, in the present study, the plant maternal genotype significantly influenced the P max and shoot biomass of A. sibiricum. In addition, endophyte infection appears to override the host genotype, at least in determining the total phenolic concentration, LMA, and several photosynthetic parameters of A. sibiricum plants.

| Does the influence of endophyte species depend on nitrogen availability?
Recent studies showed that the potential effect of endophyte infection on host performance can also be influenced by the nitrogen conditions in the environment (Ahlholm, Helander, Lehtimäki, Wäli, & Saikkonen, 2002;Cheplick, 1998;Saikkonen et al., 2006 is consistent with the results obtained in tall fescue and L. perenne Saikkonen et al., 2006). Under LN conditions, this positive effect was weaker, while E+ plants did show a higher aboveground biomass than E− plants. This was similar to a previous study using A. sibiricum (Li Ren, Han, Yin, Wei, & Gao 2012). Under nitrogen deficiency, the N concentration was lower for E+ compared to E− plants; however, E+ plants had an elevated photosynthetic capacity.
Achnatherum sibiricum plants with a higher PNUE could be the result of investing relatively more of their N in photosynthetic machinery (Li et al. 2012). Previous reports have also indicated that organisms with a greater growth advantage in nutrient-poor conditions are those that are able to modify their body nutrient content and increase the efficiency of nutrient use without major decreases in their growth rates (Elser et al., 2003;Mulder & Bowden, 2007 increased significantly with higher rates of nitrogen addition. The frequency of Epichloë endophyte infections in natural grass populations has often been used to infer relative fitness advantages of harboring the endophytes (Cheplick, 1998;Clay, 1998

| Is the effect of stroma-bearing endophytes on host plants is negative?
It has been reported that endophytes that reproduce solely by vertical transmission should have reduced virulence to maintain the productivity of the host plants (Ewald, 1987(Ewald, , 1993. Therefore, symbioses of grasses with vertically transmitted endophytes may be highly mutualistic because both host and endophyte contribute to the persistence of the association (Ewald, 1987(Ewald, , 1993. However, endophyte species with a sexual cycle can produce stromata on flowering tillers, which prevents the floral development and seed set of the plant. These endophytes develop disease symptoms during their sexual cycles, and negative effects on host plants may predominate (Clay & Schardl, 2002;Ewald, 1987Ewald, , 1993Herre, 1993;Toft, Aeschlimann, & Bolis, 1991 (Li et al., 2015). The results in our study showed that Eg-infected plants had significantly higher biomass production than Es-infected hosts. Using A. sibiricum plants harboring either a vertically transmitted or vertically and horizontally transmitted endophyte, the results in our study confirmed the positive effects of a stroma-bearing endophyte on host plants during their vegetative growth stage.

| Possible evolutionary development of E. gansuensis and E. sibirica
In natural grass populations, the presence of E. gansuensis in both A. sibiricum and Achnatherum inebrians is common (Li et al., 2004;Zhang et al., 2009). Achnatherum sibiricum and A. inebrians belong to the same genus, are distributed in adjacent regions, and even share certain areas (Ma, 1985;Shi, 1997). However, E. gansuensis exhibits different abilities for alkaloid production in these two hosts. Achnatherum inebrians has been reported to possess ergonovine and ergine, which can cause toxicity in livestock (Li et al., 2004). In contrast, A. sibiricum has no obvious herbivore deterrence (Jin & Han, 2010). In the present study, the lack of any significant concentrations of ergovaline and peramine was observed in A. sibiricum that harbored Eg. For that matter, Eg exhibits greater beneficial effects in A. inebrians than in A. sibiricum.
In addition, Eg inhibits a different transmission mode in these two hosts. Achnatherum sibiricum-and A. inebrians-harboring Eg can be vertically transmitted, while A. sibiricum plants harboring Eg can occasionally produce conidia stromata and may be horizontally transmitted (Li et al., 2015). Many mutualistic interactions are thought to undergo the evolutionary pathway from pathogenic toward mutualistic symbioses. In this regard, the coevolution of endophytes with host plants may include the complete loss of horizontal transmission and sexual reproduction as well as the complete dependency of the host (Gundel, Omacini, Sadras, & Ghersa, 2010). It is possible that A. sibiricum may have been infected by Eg earlier than Es, and the beneficial effect of Eg to A. sibiricum was mainly in growth enhancement. When the host grass was exposed to a stressful environment such as low precipitation, infection with Es might be improved (Zhang et al., 2009 from studies using agronomic grass relative to natural populations and communities may be too simplistic (Saikkonen et al., 2006). With the greater genetic diversity in both the endophyte (Faeth & Sullivan, 2003) and its grass host (Saikkonen et al., 2006), natural populations should exhibit much greater variation. In the present study, we found the outcomes of endophyte A. sibiricum symbiosis in terms of host biomass and photosynthesis rates greatly depend on both environmental conditions and host endophyte genotypic combinations.