In nature, the symbiotic relationship between plants and microorganisms is crucial for ecosystem balance and plant growth. This study investigates the impact of Epichloë endophytic fungi, which are exclusively present aboveground, on the rhizosphere microbial functions of the host Melica transsilvanica. Using metagenomic methods, we analyzed the differences in microbial functional groups and functional genes in the rhizosphere soil between symbiotic (EI) and non-symbiotic (EF) plants. The results reveal that the presence of Epichloë altered the community structure of carbon and nitrogen cycling-related microbial populations in the host’s rhizosphere, significantly increasing the abundance of the genes (porA, porG, IDH1) involved in the rTCA cycle of the carbon fixation pathway, as well as the abundance of nxrAB genes related to nitrification in the nitrogen-cycling pathway. Furthermore, the presence of Epichloë reduces the enrichment of virulence factors in the host rhizosphere microbiome, while significantly increasing the accumulation of resistance genes against heavy metals such as Zn, Sb, and Pb. This study provides new insights into the interactions among endophytic fungi, host plants, and rhizosphere microorganisms, and offers potential applications for utilizing endophytic fungi resources to improve plant growth and soil health.

Epichloë 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, Epichloë bromicola was successfully inoculated into Bromus inermis 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 (p < 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.