Effects and Mechanisms of Sodium Nitroprusside, Spermidine, and Coumarin Addition In Vitro on Epichloë sinensis

Epichloë sinensis forms mutualistic symbiosis with the Chinese native grass Festuca sinensis. However, the mechanisms by which E. sinensis responds to host-derived defense signals remain unclear. To explore these responses, three compounds, sodium nitroprusside, spermidine, and coumarin, were added to a potato dextrose broth (PDB) medium to mimic host defense-related signals. Their effects on the growth, antioxidant capacity, and gene expression of three E. sinensis strains (1, 2, and 84F) isolated from different ecotypes of F. sinensis were identified. The results showed that in PDB, certain concentrations of sodium nitroprusside, spermidine, and coumarin treatments significantly promoted the growth of the three E. sinensis strains (P < 0.05) and significantly increased the total antioxidant capacity, superoxide anion scavenging ability, and hydroxyl radical scavenging ability of culture filtrate (P < 0.05). Most of the sodium nitroprusside and spermidine treatments significantly increased the nitric oxide (NO) concentration in the mycelia of these three E. sinensis strains (P < 0.05), except for the 1.0 mM spermidine treatment, which significantly reduced the NO concentration of strain 84F (P < 0.05). Three coumarin treatments significantly increased the NO concentration in the mycelia of strain 2 (P < 0.05) but significantly reduced the NO concentration of strain 84F (P < 0.05), and 0.68 mM coumarin treatment significantly reduced the NO concentration of strain 1 (P < 0.05). Structural equation modeling supported the hypothesis that exogenous additives affect mycelial biomass through the superoxide anion radical scavenging ability and provided moderate support for additives affecting growth through hydroxyl radical scavenging ability. These three compounds also affected the gene expression of E. sinensis strain 84F, with 135 differentially expressed genes (DEGs) detected in all of the comparisons. Functional annotation revealed that these DEGs were significantly enriched in "amino sugar and nucleotide sugar metabolism," "biosynthesis of antibiotics," "biosynthesis of amino acids," "sulfur metabolism," and "cellular iron ion homeostasis." In addition, these three compounds regulated the expression of 59 antioxidant-related genes and 31 NO synthesis-related genes of E. sinensis. These results suggest that E. sinensis is sensitive to host defense-related signals and can adjust its antioxidant capacity and key metabolic pathways in response, reflecting its physiological adaptability under in vitro conditions.