Metabolic Regulation and Saline–Alkali Stress Response in Novel Symbionts of Epichloë bromicola-Bromus inermis

<i>Epichloë</i> endophytic fungi are important microbial resources in agriculture and animal husbandry. Because of their stable symbiosis, species transmission, and positive effects on host plants, the use of endophytic fungi in grass breeding is of great significance. In this study, six inoculation methods were used, including the sterile seedling slit inoculation method, sterile seedling cut inoculation method, sterile seedling injection inoculation method, seed soaking inoculation method, seed piercing and then soaking inoculation method, and seed slit inoculation method. Spectrometry was used to construct new symbionts, and Liquid Chromatography-mass spectrometry was used to analyze the effects of endophytic fungi on the metabolism of new hosts. The physiological response of the new symbionts to salt and alkali stress was studied using a pot experiment. The results were as follows: In this study, <i>Epichloë bromicola</i> was successfully inoculated into <i>Bromus inermis</i> via the sterile seedling slit inoculation method, and new symbionts (EI) were obtained; the vaccination rate was 2.1%. Metabolites up-regulated by EI are significantly enriched in citrate cycle and ascorbate and aldarate metabolism, suggesting that the symbiosis of endophytic fungi indirectly triggers the production of reactive oxygen species (ROS) through multiple metabolic pathways. The saline-alkali stress test showed that the host antioxidant system was active after inoculation, and the total antioxidant capacity was significantly increased compared with non-symbionts (EF) under mild stress (<i>p</i> < 0.05), which provided important clues to reveal the complex mechanism of plant-fungus symbiosis. This study provides practical guidance and a theoretical basis for plant adaptation under climate change, health management of grass seeds, and soil improvement through endophytic fungi.