The decreasing trend of the competitive advantage of endophyte-infected Achnatherum sibiricum over endophyte-free plants under high nitrogen conditions was reversed by pathogenic fungi inoculation

According to the nitrogen-disease hypothesis, plant diseases will become more serious with the aggravation of global nitrogen (N) deposition. Numerous studies have indicated that Epichloë endophytes can enhance host plant resistance to pathogens. It is unclear how the competitive ability of endophyte-infected (EI) over endophyte-free (EF) plants changes under the interference of N deposition and plant disease. In this study, Achnatherum sibiricum, native to the Inner Mongolia steppe of China, was used as experimental material. We experimentally manipulated N addition and pathogen inoculation and examined the growth and competition between EI and EF A. sibiricum. The results showed that EI plants had a greater competitive advantage than EF plants under low N conditions, and this advantage decreased with the N supply. When high N and pathogens were both present, pathogen inoculation reversed the adverse effects of high N supply on the competitive advantage of EI A. sibiricum. Epichloë endophytes not only reduced the disease of EI plants but also reduced the disease of neighboring EF plants. Meanwhile, Epichloë endophytes changed the response of the host disease to N. The disease index of EF plants increased with increasing leaf N content, while that of EI plants did not change. This study highlights that under the dual factors of N deposition and pathogen infection, endophytic fungi improve the competitive ability of host plants. Our results show that considering both biotic and abiotic factors is crucial for predicting the dominance of plant-fungal symbionts in the community.


Introduction
Human activities over the last century, such as the excessive use of fertilizers and the combustion of fossil fuels, have released massive amounts of nitrogenous compounds, increasing the level of reactive nitrogen (N) on the Earth's land surface (Galloway et al. 2008;Greaver et al. 2016).On the one hand, increased atmospheric deposition of reactive N can promote the rapid growth of nitrophilous species, which thrive best in soils where N is plentiful, and make them obtain relative advantages in interspecific/intraspecific competition (Bai et al. 2015;Farrer and Suding 2016;Tian et al. 2016).On the other hand, increased leaf N concentration can increase the resources available to pathogens, resulting in plants that may be more susceptible to disease (Mitchell et al. 2003), thereby enabling disease-resistant species to have an advantage in competition.In natural ecosystems, the combination of biotic and abiotic factors leads to more complex intra-and interspecific interactions (Borer et al. 2014;Crawford et al. 2021).
Symbiotic microbes also affect the competitive ability of host plants (van der Heijden et al. 1998).
There is a symbiotic relationship between cool season grasses (subfamily Pooideae) and Epichloë (= Neotyphodium) fungal endophytes, and the fitness of these endophytes and their host plants are tightly coupled (Clay and Schardl 2002).Epichloë can promote host plant growth (Chen et al. 2020;Guo et al. 2015) and enhance host plant resistance to drought, herbivores, pathogens, etc. (Cibils-Stewart et al. 2022;Decunta et al. 2021;Graff et al. 2020;Rho et al. 2018).Epichloë may also influence the intraand interspecific competitive ability of host plants.Previous studies have demonstrated that the endophyte can enhance the intraspecific competitive ability of the hosts Festuca arundinacea (Hill et al. 1991(Hill et al. , 1998;;Marks and Cheplick 1991), Festuca pratensis (Malinowski et al. 1997), Bromus benekenii (Brem and Leuchtmann 2002), Leymus chinensis (Liu et al. 2020a) and Festuca rubra (Vázquez-de-Aldana et al. 2013).However, a few studies have yielded inconsistent findings.For instance, endophytic fungal infection had no effect on the intraspecific competitive ability of the host Lolium perenne (Cheplick et al. 2014) and a negative effect on the intraspecific competitive ability of the host Brachypodium sylvaticum (Brem and Leuchtmann 2002).
An increasing number of studies indicate that the impact of Epichloë endophytes on host grasses is related to N supply, and this effect varies with different grass-endophyte symbionts.For example, some studies indicated that Epichloë promoted the growth of the hosts Trisetum spicatum and Festuca pratensis under a relatively high N supply (Buckley et al. 2019;Dirihan et al. 2015), while others indicated that the endophyte had no effect on the growth of Achnatherum inebrians under high N supply but improved its growth under low N conditions (Hou et al. 2021;Wang et al. 2018).To date, no work has reported the effect of N supply on the intraspecific competitive ability of Epichloë plants.In a related study, Saari and Faeth (2012) found that the endophyte could enhance the intraspecific competitive ability of Festuca arizonica under low nutrient conditions, but this competitive advantage disappeared with increasing environmental nutrients.
According to the nitrogen-disease hypothesis, high N supply can directly increase foliar fungal disease severity by altering plant biochemical characteristics and pathogen resources (Mitchell et al. 2003).The nitrogen-disease hypothesis is widely known in agricultural ecosystems (Chen et al. 2007;Guo et al. 2021;Lecompte et al. 2013).In natural ecosystems, Ebeling et al. (2021) investigated plant leaf damage in 153 plant taxa from 27 grassland communities and found that pathogen damage increased with increasing the N content in grasses and legumes.Epichloë fungal endophytes are widely distributed in grasses (Leuchtmann et al. 2014;Song et al. 2016).And many published studies have shown that the endophyte may enhance the resistance of host grasses to disease (Clarke et al. 2006;Pérez et al. 2020;Zhang et al. 2022).When N addition and pathogens co-occur, how intraspecific competitive ability is affected by Epichloë remains unknown.Understanding the effects of multiple environmental changes on the intraspecific relationships of host plants associated with endophytes is critical to predicting the population dynamics and community diversity of host plants.
Achnatherum sibiricum is normally a subordinate plant species in grasslands and is usually colonized by Epichloë endophytes with high infection rates (86%-100%) in natural habitats (Wei et al. 2006).Previous studies have demonstrated that Epichloë endophytes can promote the growth of A. sibiricum (Li et al. 2016;Zhou et al. 2018).To explore the effect of Epichloë on the intraspecific competitive ability of host plants in multiple environmental factors, A. sibiricum was used as the target plant, and N addition and pathogen inoculation were used as experimental factors.Specifically, we aimed to address the following questions: (1) How does Epichloë affect the intraspecific competitive ability of the host A. sibiricum?(2) Is the effect of Epichloë on the intraspecific competitive ability of the host A. sibiricum related to N addition or pathogen infection?(3) When N addition and pathogen Vol.: (0123456789) infection co-occur, how does Epichloë affect the intraspecific competitive ability of A. sibiricum?

Experimental materials
Achnatherum sibiricum is a common perennial grass in the Inner Mongolia grassland of China.It is normally a subordinate species in grasslands but sometimes may become dominant (Ma et al. 1985).The mature seeds of A. sibiricum and planting soil used in this study were collected from natural populations in the National Hulunber Grassland Ecosystem Observation and Research Station in northeast China (119.67°E,49.10°N).At the collection site, 300 A. sibiricum plant individuals were selected at random.The harvested seeds were naturally dried and stored at 4 °C.One hundred seeds from different plant individuals were randomly selected to analyse the infection frequency by Epichloë based on the aniline blue-lactic acid staining method (Latch et al. 1984).All seeds were colonized by Epichloë, and the infection rate of seeds was 100%.Half of the collected endophyteinfected (EI) seeds were randomly selected and placed in a convection drying oven for 30 d at 60 °C to obtain endophyte-free (EF) seeds.This method has been shown to have no significant effect on the seed germination rate, germination potential and germination index (Li et al. 2012;Ren et al. 2011).A previous investigation by Zhang et al. (2009) found that different plant individuals of A. sibiricum in the same geographic population were infected with Epichloë sibirica (Es) and Epichloë gansuensis (Eg), respectively.However, different species of Epichloë endophytes had no significant effect on the growth and pathogen resistance of A. sibiricum (Shi et al. 2020;Zhou et al. 2019).Therefore, this study only considered whether the plants were infected by Epichloë and did not distinguish the endophyte species.
Soil from the collection site was sieved (2 mm), homogenized, and then autoclaved twice at 0.11 MPa and 121 °C for 30 min, with a 24 h resting period in between.Both EI and EF A. sibiricum seeds were surface sterilized with 0.5% NaClO to remove any potential pathogens and cleared with sterile distilled water.The surface sterilized seeds were planted into a plastic pot (20 cm top diameter and 22 cm deep) that received 2.5 kg of sterilized soil.Two weeks after germination, eight plant individuals were placed in each pot (four EI A. sibiricum + four EF A. sibiricum individuals in mixtures).Before the experiment, the endophyte infection status of each plant was checked based on the aniline blue-lactic acid staining method to ensure that the plants were infected with Epichloë (Latch et al. 1984).
Rhizoctonia solani used as pathogen in this study was obtained from the Agricultural Culture Collection of China (ACCC).R. solani, a necrotrophic fungus, is able to infect plant species from many monocot and dicot families (Anderson 1982).It is a common soilspread pathogen in grassland communities, resulting in brown or tan color lesions on the leaf sheath and leaves (Ampt et al. 2022).The R. solani strain was activated on potato dextrose agar (PDA).After three days, mycelium was cut with a sterile 6 mm diameter punch and cultured in liquid medium (PDA medium without agar) with shaking at 120 r/min and 25 °C for 3 days.The cultured fungal mass was washed with sterile water three times and homogenized for 30 s using a homogenizer to prepare a suspension.The absorbance of this suspension was measured at a wavelength of 600 nm (OD 600 = 2.3) and stored at 4 °C in a refrigerator until use (Zang et al. 2010).

Experimental design
A four-factor random block design was adopted.The first factor was N treatment with two levels: low nitrogen (LN) and high nitrogen (HN).The second factor was pathogen inoculation treatment with two levels: pathogen inoculation (P +) and pathogen non-inoculated (P-).The third factor was the endophyte infection status of A. sibiricum with two levels: endophyte-infected (EI) and endophyte-free (EF).The fourth factor was mixture type: monoculture of EI A. sibiricum, monoculture of EF A. sibiricum, mixtures of EI A. sibiricum and EF A. sibiricum.Each combination was replicated six times, resulting in a total of 72 pots.Different N treatments were controlled by changing the concentration of NH 4 NO 3 in the nutrient solution.The nutrient solution included NH 4 NO 3 (HN: 10 mM, LN: 2 mM), 5.0 mM CaCl 2 , 5.0 mM KCl, 2.5 mM MgSO 4 CuSO 4 •5H 2 O, pH 6.0 ± 0.1.After 40 days of germination, 300 mL of nutrient solution was applied every two weeks, four times in total.Ten weeks after germination, the pathogen suspension was inoculated.The pathogen suspension (5 mL) was injected into the rhizosphere of each plant with a sterile syringe.For the pathogen non-inoculated group, 5 mL of sterile water was injected into the rhizosphere of each plant.This experiment began on 10 May 2021 and was conducted in the campus experimental field at the College of Life Sciences, Nankai University, Tianjin, China, and lasted for 110 days.During the whole experiment, plants were watered 2-3 times per week to meet the normal growth of plants.The plants were harvested at the end of the experiment.As the native soil used in this study was sandy soil, it was possible to harvest each plant in the same pot, separately.Harvested plants were dried for 48 h at 65 °C to obtain biomass.

Evaluation of disease symptoms
Three weeks after inoculating R. solani, we assessed the disease severity degree at the base of the pseudostem of each plant individual (IRRI 1980) in all pathogen inoculation treatments.Grade 0: no disease spot; Grade 1: the disease spot area is less than 25% of the leaf sheath area.Grade 3: the disease spot area accounts for 25 to 50% of the leaf sheath area (excluding 50%); Grade 5: the disease spot area accounts for 50 to 75% of the leaf sheath area (excluding 75%); Grade 7: the disease spot is greater than or equal to 75% of the leaf sheath area; Grade 9: the disease spot symptoms penetrate the leaf sheath.The disease index (DI) was calculated as follows: where DI represents the disease index, x represents the degree of disease severity, t represents the total number of leaves at each degree of disease severity, and s represents the highest disease severity observed.

Competitiveness
The aggressivity index (AGR) of EI A. sibiricum versus EF A. sibiricum was calculated according to McGilchrist and Trenbath (1971): where RY is the relative yield of species EI or EF, using the ratio of the dry weight of a species grown in the mixture (DM EI,EF or DM EF,EI ) to the dry weight in the respective monoculture (DM EI,EI or DM EF,EF ).AGR EI,EF = 0 indicates that the two species have the same competitive ability; AGR EI,EF > 0 indicates a higher competitive ability of species EI than that of species EF; and AGR EI,EF < 0 indicates a lower competitive ability of species EI than that of species EF.
Leaf nitrogen and total phenolic content Compared to root N, leaf N use efficiency was more sensitive to soil available N and biomass (Fu 2020).Here, leaf N concentration was determined using an elemental analyzer (Vario EL/micro cube, Elementar, Hanau, Germany).The total phenolic concentration of A. sibiricum was determined by the Folin-Ciocalteu colorimetric method.Gallic acid was used as a standard to calculate the total phenolic content (Chen et al. 2015).

Statistical analyses
All statistical analyses were performed with R version 4.1.1(R Core Team 2021).To examine the effects of the endophyte, N addition and plant mixture type on the disease index of A. sibiricum, we used three-way analysis of variance (ANOVA).A four-way analysis of variance (ANOVA) was used to analyze the effects of the endophyte, N addition, pathogen infection and mixture type on the biomass and leaf N content of plants.To examine the effects of N addition and pathogen inoculation on the AGR of A. sibiricum, we used two-way analysis of variance (ANOVA).A one-sample t test was used to compare the difference between the AGR and zero.To evaluate whether Epichloë endophytes can affect disease resistance and the trade-off between the growth and defence of the host grass with N addition, we used regression models to analyze the relationship between the biomass/disease index and leaf N content for EI and EF, respectively.To investigate whether the phenolic compounds were related to N content, we used regression models to analyze the relationship between the phenolic compounds and leaf N content for EI and EF, respectively.To investigate whether the disease index was related to N content or phenolic compounds, we used regression models to analyze the relationship between disease index and leaf N content/phenolic compounds for EI and EF, comprehensively.For regression analysis, we used the data of pathogen inoculation treatments.Shapiro-Wilk and Levene's tests were performed to determine data distribution and homogeneity.

Results
Disease index caused by R. solani Epichloë endophytes significantly reduced the disease index of the host A. sibiricum, while the degree of reduction was related to N supply and plant mixture type.In different N treatments, the disease index of EF A. sibiricum increased significantly with the N supply, while the disease index of EI A. sibiricum was not significantly affected by N levels.Therefore, the beneficial effect of Epichloë on the disease resistance of host grasses was more obvious under high N supply (Fig. 1a).For different plant mixture types, compared with monoculture, the disease index of both EI and EF A. sibiricum significantly decreased in the mixture (Fig. 1b), and the decline in EF A. sibiricum was greater; that is, Epichloë may not only reduce the disease resistance of host A. sibiricum but also reduce the disease resistance of non-host plants with host neighbors.

Plant growth performance
The biomass of A. sibiricum was significantly affected by the endophyte, N addition, pathogen infection and mixture type (Table 1).Under high N conditions, Epichloë endophytes significantly increased the biomass of the host plants in both monoculture and mixture, regardless of whether pathogen was inoculated or not.Under low N conditions,  however, Epichloë endophytes increased the biomass of the host only in pathogen non-inoculated mixture (Fig. 2).

Competitiveness
The aggressivity index (AGR) was used to evaluate the competitive ability of EI A. sibiricum relative to EF A. sibiricum.One-sample t test results showed that the AGR of A. sibiricum was significantly greater than zero under both LNP-and HNP + treatments; that is, the competitive ability of EI A. sibiricum was significantly higher than that of EF A. sibiricum.The AGR of A. sibiricum did not differ from zero under both HNP-and LNP + treatments; that is, EI A. sibiricum and EF A. sibiricum had the same competitive ability.The competitive advantages of EI A. sibiricum compared to EF A. sibiricum were affected by the interaction between N and pathogens (Fig. 3).On the one hand, the effect of N supply on the competitiveness of EI A. sibiricum was related to pathogen inoculation.pathogen non-inoculated conditions, high N addition significantly reduced the competitive advantage of EI A. sibiricum; under pathogen inoculated conditions, high N addition significantly improved the competitive advantage of EI A. sibiricum; that is, the negative effect of EI A. sibiricum over EF plants on the competitive ability with N addition was reversed by fungal pathogen inoculation.
On the other hand, the effect of pathogen inoculation on the competitiveness of EI A. sibiricum was related to N supply.Pathogen inoculation reduced the competitive advantage of EI A. sibiricum under low N addition; however, pathogen inoculation enhanced the competitive advantage of EI A. sibiricum under high N addition (Fig. 3).

Nitrogen content in plants
The leaf N content of A. sibiricum was significantly affected by the interaction of the N addition and pathogen infection (Table 1).Under LNPconditions, EI plants had higher leaf N content than EF plants.However, the leaf N content of EI and EF plants was similar under the other treatments (Fig. 4a).The leaf N content of A. sibiricum was also affected by the interaction between the endophyte and mixture type (Table 1).Epichloë significantly increased the leaf N content of A. sibiricum in the mixture, but had no significant effect in monoculture (Fig. 4b).
The relationships between plant growth, disease index and total phenolic content of A. sibiricum The biomass of EI A. sibiricum increased significantly with increasing leaf N content, while the disease index of EI A. sibiricum did not change (Fig. 5a); that is, EI A. sibiricum does not compromise its defense against pathogens with the growth.The biomass and disease index of EF A. sibiricum increased significantly with increasing leaf N content; that is, the defenses against pathogens of EF A. sibiricum decreased with the growth (Fig. 5b).The total phenol content of EI A. sibiricum increased significantly with increasing N content (p = 0.017), while the total phenol content of EF A. sibiricum did not change (p = 0.843) (Fig. 5d).
When the data of EI and EF A. sibiricum were analyzed comprehensively, linear regression results showed that the disease index of A. sibiricum showed no significant change with increasing N (Fig. 5c), but decreased significantly with increasing total phenol content (Fig. 5e).

Epichloë endophytes effect on host competitive ability depended on nitrogen supply
The relationship of Epichloë-grass mutualism was related to nutrient supply, especially N supply (Saikkonen et al. 2006).For example, Epichloë endophytes are more favorable for the growth of L. perenne and A. inebrians under low N conditions (Hou et al. 2021;Lewis 2004;Ravel et al. 1997;Wang et al. 2018), while it is more favorable for the growth of F. arundinacea under high N conditions (Arachevaleta et al. 1989).To date, no research has discussed the effects of N on the intraspecific competition of endophyte-infected plants.The present study found that the intraspecific competitive ability of EI over EF A. sibiricum was related to N supply.Specifically, EI plants had a greater competitive advantage than EF plants under low N conditions, and this advantage decreased with N supply.Saari and Faeth (2012), who controlled the amount of NPK compound fertilizer, found that endophytic fungi could improve the intraspecific competition ability of F. arizonica under low nutrient conditions, and this competitive advantage disappeared with the increase in nutrients, which was similar to the results in this study.The possible reasons why Epichloë improve the competitive ability of host plants under low N conditions are as follows: on the one hand, Epichloë may increase root absorption area (Malinowski et al. 1998b;Wang et al. 2017) and root metabolic activity (Chen et al. 2020) of hosts to promote N absorption by host plants; on the other hand, compared to EF plants, EI plants may improve the N use efficiency of hosts by allocating more N to the photosynthetic system (Li et al. 2012) and increasing the activities of nitrate reductase, nitrite reductase, and glutamine synthetase (Wang et al. 2018).Under high N conditions, the N advantage of EI plants relative to EF plants decreased, and as a result, the competitive advantage also decreased.
Epichloë endophytes changed the response of plant disease to nitrogen Numerous studies on croplands have found that the N addition significantly aggravates crop diseases (Guo et al. 2021;Lecompte et al. 2013).In grassland ecosystems, Liu et al. (2020b) observed that N Fig. 5 The relationship between disease index and/ or biomass and leaf nitrogen content in endophyteinfected Achnatherum sibiricum (a), endophytefree (b), endophyte-infected and endophyte-free (c), the relationship between total phenolic content of Achnatherum sibiricum (endophyte-infected or endophyte-free) and leaf nitrogen content (d), and the relationship between the disease index of Achnatherum sibiricum (endophyte-infected and endophyte-free) and total phenolic content (e).EI: endophyte-infected plants; EF: endophyte-free plants addition increased fungal diseases in grassland communities, especially in grasses, and fungal diseases caused by both necrotrophic and biotrophic pathogens were aggravated with increasing N levels.Ebeling et al. (2021) found that the fungal disease severity of grasses increased with increasing the N by conducting a survey of 27 grassland communities in 10 countries.A previous study on A. sibiricum reported that the endophyte may improve host resistance to necrotrophic and biotrophic pathogens (Shi et al. 2020).However, there are currently no studies reporting whether the effect of Epichloë on host resistance to diseases changes with increasing N.In this study, we found that the disease index of EF plants increased with increasing N content, which supports the nitrogen disease hypothesis, but the disease index of EI plants did not change significantly with increasing N content, thereby failing to support the nitrogen disease hypothesis.Our results further found that Epichloë endophytes significantly increased the total phenolic content of host plants, and the total phenolic content of A. sibiricum leaves was significantly negatively correlated with the disease index.Phenolic compounds, an important class of secondary metabolites, play a crucial role in plant disease resistance (Cheynier et al. 2013).The results of this study indicate that Epichloë endophytes may enhance the disease resistance of host plants by increasing the content of phenolic compounds with increasing soil available N.

Pathogen inoculation effects on the competitiveness of EI relative to EF plants depended on nitrogen
Epichloë endophytes can enhance the resistance of plants to diseases (Iannone et al. 2017;Pérez et al. 2020;Wang et al. 2016).However, it is still unclear whether Epichloë endophytes can affect disease resistance in neighboring plants.In this study, when EI and EF plants were grown separately, EI plants showed a stronger disease resistance capability.When they were grown together, Epichloë may not only reduce the disease of EI plants but also reduce the disease of neighboring EF plants, and the beneficial effect on EI plants was higher.This result suggests that Epichloë endophytes can not only enhance the host plant's own protection against pathogens but also, more importantly, improve the protection of other non-host neighboring plants in the community.
As previously stated, the reason why Epichloë endophytes increase the disease resistance of host plants may be related to the increase in total leaf phenolic content.It has been reported that Epichloë endophytes can not only increase the total phenolic content in host plants (Malinowski et al. 1998a;Pánka et al. 2013), but also increase the phenolic compound content in root exudates of host plants (Guo et al. 2015;Malinowski et al. 1998a).In this study, Epichloë endophytes may reduce the abundance of soil-borne pathogenic microorganisms by promoting the secretion of more phenolic compounds in the root (Rojas et al. 2016), thereby reducing the disease severity in both EI and neighboring EF plants.Regarding whether pathogen inoculation affects the competitive advantage of EI over EF plants, we found that the effects of pathogen inoculation on the competition between EI and EF plants were related to N supply.Although Epichloë endophytes significantly reduced the disease index of host plants, they had no significant effect on the competitive ability of host plants under low N supply.However, Epichloë endophytes not only significantly reduced the disease index of host plants but also enhanced the competitive ability of host plants under high N supply.This may be related to the effect of low N supply on plant resource allocation (Hahn et al. 2021;Sampedro et al. 2011).In this study, compared to EF plants, EI plants did not exhibit a significant growth advantage under low N supply, but had a lower disease index, indicating that resource allocation was prioritized for defense rather than growth under N limitation.Meanwhile, all N for Epichloë growth is derived from plants, and competition for limited resources between Epichloë endophytes and the host may result in a balanced cost-benefit relationship in the Epichloë-grass symbiosis, ultimately having no effect on the competitive ability of the host.Upon nutrient limitation removal, Epichloë endophytes not only increase plant resistance against pathogens but also promote growth, thereby enhancing the competitive ability of infected plants under high N supply.
Epichloë endophytes change the growth-defense trade-off of the host grass According to a trade-off between growth and defense theory, plants tend to divert more resources to defense at the expense of normal growth when encountering biotic stresses such as pathogens (Lind et al. 2013).Although the trade-off between growth and defense seems to be ubiquitous in plants (Kneitel and Chase 2004), the uncoupling of growth and defense functions has also been reported.For instance, Cao et al. (1998) found that overexpression of NPR1 (nonexpressor of pathogenesis-related gene 1) enhanced the resistance of Arabidopsis thaliana to pathogens with no obvious detrimental effect on the plants.In Epichloë-grass symbiosis, Bastías et al. (2021) found that when plant leaves were damaged by insect herbivores, Epichloë endophytes promoted the growth of host plants and improved host plant resistance to herbivores by producing alkaloids.In the present study, Epichloë endophytes enhanced host plant resistance to pathogens and promoted the growth of host plants under high N conditions.The promotion of Epichloë endophytes on the growth of A. sibiricum may be related to the upregulated expression of the auxin signaling pathway and photosynthesis-related genes (Shi et al. 2022), and the improvement of Epichloë endophytes on the resistance of the host A. sibiricum may be related to the increase in phenolic compounds.

Conclusion
In conclusion, the impact of Epichloë endophytes on the intraspecific competitive ability of host plants was affected not only by N supply but also by pathogens.The competitive advantage of A. sibiricum relative to endophyte-free A. sibiricum decreased with N supply, while pathogen inoculation reversed the adverse effects of high N supply.In grassland ecosystems, on the one hand, N supply will increase with global N deposition (Crawford et al. 2021).On the other hand, plant diseases have always been one of the important reasons for the degradation of grasslands (Cappelli et al. 2020;Mitchell et al. 2003), and the disease severity will become more serious with the increase in N deposition.The results of this study suggest that the advantage of endophyte-infected over endophytefree grasses would increase in the future.

Fig. 1
Fig. 1 Effects of the interaction between endophyte infection (E) × nitrogen addition (N) (a), and E × plant mixture type (C) (b) on the disease index for Achnatherum sibiricum.Different letters indicate significant differences (p < 0.05).EI:

Fig. 2 Fig. 3
Fig. 2 Effects of the endophyte, nitrogen addition, pathogen infection and mixture type on the biomass per plant for Achnatherum sibiricum.Different letters indicate significant differences (p < 0.05).EI: endophyte-infected plants; EF: endophyte-free plants; P-: pathogen non-inoculated; P + : pathogen inoculation; LN: low nitrogen treatment; HN: high nitrogen treatment; Monoculture, EI and EF A. sibiricum grow separately in pots; mixture, EI and EF A. sibiricum grow together in pots

Table 1
Analyses of variance (ANOVA) for plant biomass and leaf N content of Achnatherum sibiricum Vol:. (1234567890)