Inuence of Epichloë bromicola endophytic fungi on secondary metabolites following alkali tolerance improvement in host, Hordeum bogdanii

Purpose: The aim of this study was to investigate how endophytic fungi affect secondary metabolites of H. bogdanii under alkaline stress at different concentrations. It is currently unclear whether the mechanism via which endophytic fungi improve the alkali tolerance of Hordeum bogdanii affects secondary metabolites. Unveiling this knowledge is crucial for understanding the tolerance mechanism of H. bogdanii to alkaline stress. Methods: Endophyte-infected (E+) and endophyte-free (E-) individuals of H. bogdanii were used as materials in this study. Vermiculite was used for plant cultivation and was carried out in the laboratory. After mixed alkali stress treatment, the roots, stems, and leaves of the plants were collected to measure the indicators related to secondary metabolites. Results: The results showed that endophytic fungi signicantly increased the contents of phosphorus, polyphenols, and alkaloids, and the activities of polyphenol oxidase and acid phosphatase, and signicantly reduced avonoid content. The content of polyphenols and alkaloids in stems, polyphenol oxidase activity in stems and leaves, and acid phosphatase activity in leaves were signicantly affected. In general, endophytic fungi improved the alkali resistance of H. bogdanii by improving the related indicators of secondary metabolites. Conclusions: The ndings of this study may aid in amplifying the alkali resistance mechanism of endophytic fungi to H. bogdanii as well as provide insights into improving the alkali resistance of other plants. The results of the present


Introduction
Plant secondary metabolism is the result of plant adaptation to the ecological environment during long-term evolution (Wang et al. 2020). Plant secondary metabolites are diverse, with different properties. According to their chemical structures, they can be divided into phenols, terpenes, and nitrogen-containing organic compounds ). Secondary metabolites are natural compounds produced by plants and have a variety of physiological roles (Chen et al. 2009). Stress can signi cantly promote the synthesis of plant secondary metabolites, which participate in the coordination of the relationship between plants and their environments (Tu 2019). Secondary metabolites have been found to play an important role in drought and salt tolerance in plants ( Ahmed et al. 2015). Endophytic fungi can produce a variety of alkaloids, including organic amines, pyrrolizidines, and ergot alkaloids (Xu et al. 2010). Endophytic fungi in gramineous plants can produce four major alkaloids (i.e., organic amines such as peramine, pyrrolizidines such as loline, indole diterpene derivatives such as lolitrem B, and ergot alkaloids such as ergovaline). These have various biological activities, such as resistance to pathogenic bacteria, resistance to nematodes, and enhancement of plant allelopathy.
Hordeum bogdanii is a wild gramineous grass distributed in Xinjiang, Gansu, Qinghai, Inner Mongolia, and other regions of China. It is an important wild germplasm resource and is used as forage grass. It has strong cold tolerance and saltalkali resistance, and plays an important role in the improvement of deserti cation and saline-alkali land (Jia 1987; Ma et al. 1998). The endophytic fungi of grasses are a large group that grow and complete all or most of their life cycle within plants, while the grasses do not show external symptoms (Gao et al. 2007;Bongiornoet al. 2016). The endophytic fungus and host coevolve and have a mutually bene cial relationship (Chen et al. 2017; Tanaka et al. 2012). Endophytic fungi obtain nutrients from the host grass, while simultaneously enhancing the resistance of the grass to biological and abiotic stresses, thereby promoting its ability to adapt to the environment. Examples of this include promoting growth, resisting harm by herbivores and nematodes (Kuldau and Sun 1996, 1997;Nan 1996). In addition, a comparative study of H bogdanii plants with endophytic fungi (E+) and without endophytic fungi (E-) in the Altai area of Xinjiang, China, showed that endophytic fungi promoted the growth of host H. bogdanii and increased tiller number, biomass, and the forage yield of plants (Nan 1996).
Polyphenols are important, physiologically active, secondary metabolites involved in the regulation of growth, development, and response to adversity in plants. Studies have shown that the level of phenolic compounds in plants is signi cantly increased under a variety of biological or abiotic stresses (Chen 2020). Polyphenol oxidase activity is related to ear germination resistance (Huang et al. 2021). Flavonoids can be used as regulators in the process of plant growth and development and improve plant resistance to certain adverse stresses (Chu et al. 2007). Moreover, plant endophytic fungi can produce secondary metabolites, such as alkaloids and avonoids (Su 2018).
However, little information is available on the effects of endophytic fungi on secondary metabolites under alkaline stress. Therefore, it is unclear if the mechanism of endophytic fungi improving the alkali tolerance of the host H. bogdanii affects the secondary metabolites. This knowledge is greatly signi cant for understanding the tolerance mechanism of H. bogdanii to alkaline stress. In this study, we examined the content of secondary metabolites and related enzymes involved in the process. Our ndings will help to amplify the alkali resistance mechanism of endophytic fungi to H. bogdanii and supply a new eld for exploring effective ways to improve the alkali resistance of plants.

Plant material:
The seeds of H. bogdanii were collected from Wensu County, Aksu District, Xinjiang Province, China (80°76 E, 41°58 N, H 1514 m). A E+ plant and half of its tillers were sterilized with fungicide to obtain homogenous plants without endophytic fungi (E-). The seeds of the E+ and E-plants bred in the eld were used in this experiment. In the greenhouse of Tarim University (daytime temperature 25 °C, night temperature 15 ℃, 14 h light per day), E+ and E-H. bogdanii grass were planted in plastic owerpots (pot diameter 13 cm, bottom diameter 8 cm, height 12 cm). Every week, 200 ml of Hoagland nutrient solution was poured in each pot and plants were watered regularly. After the plants were cultured for 6 weeks, the endophytic fungi were detected under a microscope using the sheath aniline blue test method (Li et al. 2008), and the E+ and E-plants were determined again.

Alkali stress treatment:
With a Na 2 CO 3 :NaHCO 3 ratio of 1:1, ve concentrations of mixed alkali (25,50,100,150, and 200 mmol/L) were used to treat H. bogdanii plants, with no added alkali as the control. Each treatment had ve replicates. After 21 days of treatment, fresh plant root, stem, and leaf samples were collected and immediately frozen in a refrigerator at −80 °C. The remaining samples were dried, crushed, sieved, and stored at room temperature to determine the relevant indicators.   (4) The contents of avonoids and alkaloids, and acid phosphatase activity were measured using a kit from Suzhou Keming Biotechnology Co., Ltd.

Data processing and statistical analysis methods
All data in this paper were calculated by Excel 2016, mapped by Sigmaplot12.5, and Spss19 was used to analyze the signi cance of the difference. The effects of alkali treatment and endophytic fungi on the contents of phosphorus, polyphenols, avonoids, and alkaloids, and the activities of polyphenol oxidase and acid phosphatase in H. bogdanii were detected by two-way ANOVA The effects of different alkali treatment concentrations on the contents of phosphorus, polyphenols, avonoids, and alkaloids, and the activities of polyphenol oxidase and acid phosphatase in H. bogdanii were detected by one-way ANOVA. An independent sample t-test was used to detect the differences in the contents of E+ and Ephosphorus, polyphenols, avonoids, and alkaloids, and the activities of polyphenol oxidase and acid phosphatase at the same alkali concentration.

Effects of endophytic fungi on phosphorus content in roots of H. bogdanii under different concentrations of alkali stress
Under the same treatment, the phosphorus content in the roots of plants with endophytic fungi (E+) was higher than that in the roots of plants without endophytic fungi (E-) ( Figure 1). Although the phosphorus content in the roots of E+ and Eplants was not signi cantly different under the 200 mmol/L alkali treatment, the phosphorus content of E+ plants was signi cantly higher than that of E-plants in the other alkali treatments (P < 0.05). The phosphorus content in the roots of H. bogdanii plants reached the highest content at an alkali treatment concentration of 25 mmol/L, which was signi cantly higher than the phosphorus content in the roots of the control plants and showed a signi cant decreasing trend as the alkali concentration increased (P < 0.05). From the two-factor ANOVA table (Table 1), both endophytic fungi and alkali treatment signi cantly affected the phosphorus content in the roots of H. bogdanii, and there was an interaction between the endophytic fungi and alkali treatment. Under the same treatment, except for the high alkali concentration treatment of 200 mmol/L, the content of avonoids in the stems of E+ plants was signi cantly lower than that of E- (Figure 2). With an increase in alkali stress, avonoid contents increase in E+ plants. The avonoid contents in the E+ plants in the 150 mmol/L and 200 mmol/L treatments were the highest, and were signi cantly higher than that in the control. (P < 0.05). From the two-factor ANOVA (Table 1), endophytic fungi and alkali treatment signi cantly affected the avonoid content in H. bogdanii stems, and there was an interaction between endophytic fungi and alkali treatment (P < 0.05).
Under the same treatment, the polyphenol content in E+ H. bogdanii stems was higher than that of E-, and the difference was signi cant when alkali treatments were 100 mmol/L, 150 mmol/L, and 200 mmol/L (P < 0.05) ( Figure 3A). The polyphenol content increased with an increase in the alkali treatment concentration, and the polyphenol content in the stem was the highest under the 200 mmol/L alkali treatment. Except for the 25 mmol/L treatment and the control, treatments were not signi cantly different. The polyphenol content in the stems of the other treatments were signi cantly higher than that of the control treatment.
Although there was no signi cant difference in polyphenol content between E + and E-roots in the 25 mmol/L treatment, in the other treatments, polyphenol content in E + plant roots was signi cantly higher than that in E-plants.(P < 0.05) ( Figure 3B). The effect of alkali concentration stress in E+ roots rst lead to an increase and then a decrease in polyphenol content. The polyphenol content was the highest at 150 mmol/L, and the control was the lowest; the polyphenol content in E-root was also the lowest in the control treatment. Therefore, alkali stress also signi cantly affected the polyphenol content in the roots and stems of H. bogdanii, and the results of the two-way ANOVA showed that both the endophytic fungi and alkali treatment signi cantly affected the content of polyphenols in the roots and stems of H. bogdanii, and that there was an interaction between the two (P < 0.05).

Effects of endophytic fungi on alkaloids in the stems of H. bogdanii under different concentrations of alkali stress
Under the same treatment, the alkaloid content of E+ plants was signi cantly higher than that of E-plants ( Figure 4). With an increase in the alkali stress treatment concentration, the content of alkaloids in the stems of H. bogdanii showed a signi cant downward trend. With an increase in the alkali treatment concentration, the alkaloid content of E+ stems was 22.9%, 24.6%, 20.8%, 20.1%, 9.7%, and 8.8% higher than that of the E-stem under the same alkali stress. From the twofactor statistical analysis table (Table 1), both endophytic fungi and alkali treatment signi cantly affected the alkaloid content in stems, and there was an interaction between endophytic fungi and alkali treatment (P < 0.05). Under the same treatment, the acid phosphatase activity in the leaves of E+ H. bogdanii plants was higher than that of E-( Figure 5A). Except for the insigni cant difference in the control, in all other treatments E+ were signi cantly higher than E-(P < 0.05). Acid phosphatase activity showed a decreasing trend with increasing alkali stress concentration, and the alkali treatment was signi cantly lower than that of the control. From the two-factor analysis table (Table 2), it can be concluded that both endophytic fungi and alkali treatment signi cantly affected the acid phosphatase activity in H. bogdanii leaves (P < 0.05), and there was an interaction between the two (P < 0.05).

Effects of endophytic fungi on polyphenol oxidase activity in stems and leaves of H. bogdanii under different levels of alkali stress
Under the same treatment, the polyphenol oxidase activity in the stems and leaves of the E+ plants was signi cantly higher than that of the E-plants (P < 0.05) ( Figure 5B and 5C). With an increase in the alkali treatment concentration, the activity of polyphenol oxidase in the stems and leaves of H. bogdani gradually decreased, and the difference between treatments was signi cant. As shown in Table 2, both endophytic fungi and alkali treatment signi cantly affected the polyphenol oxidase activity in the stems and leaves of H. bogdani, and there was an interaction between endophytic fungi and alkali treatment.  and was signi cantly higher than that of the control. The reason for this may be due to the growth-promoting effect of low alkali concentration, which caused the phosphorus content at this treatment to be signi cantly higher than that of the control. Li and Tian (2014) also showed that appropriate salt stress can promote the absorption of phosphorus by plants. Malinowski et al (2000) found that the accumulation of phosphorus in the roots and stems of tall Festuca (genotype DN2) E+ plants was signi cantly higher than that of E-plants. Acid phosphatase is an important enzyme that regulates phosphorus metabolism in organisms. Acid phosphatase not only participates in the growth and metabolism of organisms and signal transduction pathways, but also enhances plant resistance to phosphorus de ciency, drought, low and that the total phosphorus content also increased to a greater extent. The ndings of this study also showed that endophytic fungi promoted acid phosphatase activity in H. bogdanii leaves, which promoted an increase in phosphorus content. This difference was more obvious under alkaline stress; as the alkali concentration increased both the phosphorus content and acid phosphatase activity showed a downward trend.
Plant polyphenols are an inherent component of many plants. Under normal conditions, their content in plants is very low, but when plants are stimulated by foreign factors, the content of these substances will increase signi cantly to enhance the resistance to abiotic stress (Zhao 2004). Under alkaline stress, the polyphenol oxidase activity of H. bogdanii gradually decreased with the increase in alkali concentration. However, the E+ plant was signi cantly higher than E-, and the control E+ was also signi cantly higher than E-. Previous studies have shown that low concentrations of saline alkali stress can promote polyphenol oxidase activity. Yan et al (2021) and Zhao et al (2005) believe that the more alkaline the environment, the faster the enzyme activity decreases. This is consistent with the results of the trend of polyphenol oxidase activity in the stems and leaves of H. bogdanii obtained in the present study. Moreover, studies have also shown that when the activity of polyphenol oxidase decreases, the enzymatic reaction is inhibited, leading to an increase in polyphenol content (Zhang et al. 2013). The results of the present study also showed that under high alkali concentrations, the activity of polyphenol oxidase decreased and polyphenol content increased.
Under different concentrations of alkali stress, endophytic fungi had a signi cant effect on the alkaloid content in the stems of H. bogdanii. Alkali treatment reduced the alkaloid content of H. bogdanii. It has been reported that the total alkaloid content of plants decreases with an increase in pH (Tang and Chen 2011), which is consistent with the results of the present study. The alkaloid content of E+ plants was signi cantly higher than that of E-plants. Gao and Nan (2007) believed that endophytic fungi can signi cantly increase the alkaloid content of plants, which is consistent with this result.
Under different concentrations of alkali stress, endophytic fungi had a signi cant effect on the avonoid content in the stems of H. bogdanii. The avonoid content in E+ plants was signi cantly lower than that in E-plants, and the content of avonoids in high alkali concentrations was signi cantly higher than that in low alkali concentrations. Chen (2019) suggested that endophytic fungal infection signi cantly reduced the content of avonoids in H. bogdanii, and the results are similar to those of the present study; salt stress increases the total avonoid content in roots, stems, leaves, and owers (Yan 2011;Hou et al. 2016). Other studies (Zhou et al. 2004) showed that avonoids accumulate under stress. In this experiment, as the alkali concentration increased, the avonoid content increased, and the high alkali concentration avonoid content was signi cantly higher than the low alkali concentration. This result is similar to that of salt stress, indicating that both salt and alkali stress can increase avonoid content. Latch et al (1985) suggested that the increase in plant biomass of ryegrass infected with endophytic fungi may be related to gibberellin (GA). Wang et al (2007) reported that gibberellin can inhibit the synthesis of avonoids by reducing the activity of chalcone synthase (CHS). In the present study, we measured the hormones of E + and E-plants of Burton barley and found that the content of gibberellin GA3 in E + leaves of H. bogdanii, planted indoors and outdoors, was higher than that of E -(data to be published). The higher the content of GA3, the more it inhibited the content of avonoids, which was consistent with the result that the content of avonoids in E + plants was signi cantly lower than that of E-.
Alkaloids are N-based secondary metabolites, and avonoids are C-based secondary metabolites. According to the hypothesis of "carbon nutrient balance" proposed by Bryant et al (1983), there is a balance between C-based secondary metabolites (such as terpenes and phenols) and N-based secondary metabolites (such as alkaloids). In the present study, endophytic fungi signi cantly promoted the content of N-based secondary metabolites alkaloids, but signi cantly reduced the content of C-based secondary metabolites avonoids to maintain the nutrient balance in the plant. The experimental results also align with the "carbon nutrient balance" hypothesis.   Polyphenol oxidase activity and acid phosphatase activity in stems and leaves of E+ and E-Hordeum bogdanii under different alkali treatments (picture A is polyphenol oxidase activity in stem, picture B is polyphenol oxidase activity in leaves, picture C is acid phosphatase activity)