Hordeum vulgare is an important source of feed and forage for livestock, and of food and drink for humans, but its utilization rate is lower than that of other cereal crops, thus it is crucial to improve barley agronomic traits and production. Epichloë bromicola is an endophyte that was isolated from wild barley (Hordeum brevisubulatum). Previous studies have found that Epichloë can indirectly influence the growth of host plants by affecting soil chemical characteristics, the microbial community, and by producing a range of secondary metabolites. However, underlying effects of Epichloë on the abundance and diversity of soil and root microbes have not been well-studied. In addition, there is a question regarding the relationship between endophyte-produced alkaloids and effects on the root and rhizosphere microbial communities. The objective of this study was to investigate changes in agronomic traits, nutritional properties, peramine, soil chemical and microbial community in the fourth generation of new barley symbionts EI (E. bromicola-infection) and EF (E. bromicola-free) in LQ+4 and LZ+4. We understand the plant height and biomass of EI in LZ+4 were significantly higher than those of EF. The HPLC analysis showed that the peramine content of EI in LQ+4 and LZ+4 was 0.085 and 0.1 mg/g, respectively. We compared the bacterial and fungal communities by analyzing the 16s rRNA (for bacteria) and ITS rDNA regions (for fungi). Our data revealed that the composition of fungal communities in rhizosphere soil of LZ+4 EI are higher than EF. In addition, the diversity and richness of fungal communities in root and rhizosphere soil of LQ+4 EI and LZ+4 EI are significantly higher than EF. Rhizosphere soil microbial community composition was higher than that in roots in LQ+4 and LZ+4. Peramine was significantly and positively correlated with the richness of the soil fungal community. Moreover, the principal component analysis (PCoA) results indicated that E. bromicola significantly influenced the community composition of root and rhizosphere soil microbes in both LQ+4 and LZ+4. Our results illustrate that E. bromicola can influence barley growth, peramine production and microbial communities associated with barley.

Epichloë endophytes have been found in cool-season grasses and can produce alkaloids that are toxic to vertebrates and insects. Due to their beneficial effects, Epichloë can provide plants with resistance to some abiotic and biotic stresses. The biological and physiological characteristics of the endophytic strains XJE1, XJE2, and XJE3 isolated from wild barley were measured across a range of pH, salt concentrations, and growth values. The phylogenetic position of the Epichloë isolates was examined using the tefA and actG genes. The optimal pH values for mycelial growth of XJE1, XJE2, and XJE3 were 7–8, 6–7, and 8–9, respectively. The isolates grew significantly better at 0.3 mol/L NaCl than at 0.5 mol/L and 0.1 mol/L NaCl. Based on the conidiophore and conidia morphology, growth characteristics, and phylogenetic relationships, the endophyte isolated from wild barley is likely Epichloë bromicola. These isolates exhibited differences in mating types and alkaloid biosynthesis genes. Screening for salt tolerance and alkaloid biosynthetic genes in endophytic strains will provide new insights into useful traits to breed into new forage germplasms.

Epichloë endophytes enhance plant defense and biotic stress resistance through mutualistic interactions in natural hosts. However, whether these protective effects are retained in annual non-native hosts such as barley (Hordeum vulgare L.) remains unclear. An integrated multi-omics approach was used to characterize defense responses in Epichloë bromicola-infected barley (LD1) and endophyte-free barley (CK) following pathogen challenge. The results show that LD1 had 20.0% lower disease incidence and 24.1% lower disease index than CK. LD1 also exhibited 21.1% lower malondialdehyde (MDA) content and higher antioxidant enzyme activities, indicating more effective control of oxidative damage and improved redox homeostasis. Integrated transcriptomic and metabolomic analyses revealed distinct defense responses between LD1 and CK. LD1 showed stronger activation of glutathione metabolism and antioxidant pathways than CK after 6 h of endophyte inoculation. This was consistent with more efficient control of early reactive oxygen species (ROS) dynamics. By 48 h, LD1 preferentially enriched phenylpropanoid biosynthesis and mitogen-activated protein kinase (MAPK) signaling, accompanied by increased accumulation of defense-related metabolites and enhanced structural and chemical defenses. Collectively, the study demonstrates that Epichloë bromicola enhances disease resistance in barley by reprogramming host defense dynamics from early redox regulation to late structural and chemical reinforcement.

Background: Artificial inoculation of Epichloë endophytes into elite forage germplasm aims to establish beneficial symbioses for developing high-yield, high-quality, and stress-tolerant cultivars, but host specificity of the fungi often causes compatibility issues in non-natural hosts. Methods: The E. bromicola isolated from native wild barley was inoculated into cultivated wild barley (Hordeum brevisubulatum) and cultivated barley (Hordeum valgare), forming Hb+Eb and Hv+Eb. The NHb+Eb (native wild barley naturally infected with E. bromicola) served as a control. We analyzed fungal colonization patterns and symbiotic gene regulation to clarify the compatibility between E. bromicola and non-natural hosts. Results: Compared with NHb+Eb and Hb+Eb, E. bromicola in Hv+Eb showed obvious hyphal vacuolization. E. bromicola colonization altered host trichome morphology and induced stomatal closure. Correspondingly, expression of the siderophore biosynthesis gene sidN and the NADPH oxidase complex genes (NoxA, NoxB, NoxR, RacA) was significantly lower (p < 0.05) in Hv+Eb than in Hb+Eb and NHb+Eb. Conclusions: This study reveals that the incompatibility between cultivated barley and E. bromicola is characterized by altered hyphal morphology, which is linked to the downregulation of sidN and Nox. These findings provide a critical theoretical foundation for developing highly compatible cereal-Epichloë germplasms.