Discovery of novel antibiotics and other biologically active secondary metabolites from plant-associated fungi: pharmaceutical and biocontrol potential

Ash dieback, a severe disease caused by the invasive fungus Hymenoscyphus fraxineus, poses a significant threat to common ash (Fraxinus excelsior) populations and associated ecosystems across Europe. The disease causes significant twig and branch necroses, as well as root collar infections, which eventually result in wood discoloration, structural instability, and a drastic decline in ash populations. To combat this devastating pathogen, endophytic fungi associated with ash trees offer a promising biocontrol strategy. Aside from their potential for disease management, these endophytes are also a rich source of bioactive compounds, making themvaluable for drug discovery and biotechnological applications. The main objectives of this dissertation were to screen endophytic fungi for bioactive compounds that could serve as lead compounds in drug development and to investigate the potential of these organisms as biocontrol agents against Hymenoscyphus fraxineus. To achievethese goals, endophytic fungi were cultivated in various media, followed by the extraction and isolation of secondary metabolites using classical chromatographic methods. The purified compounds were structurally elucidated and evaluated for bioactivity through a series of bioassays, including antimicrobial, antifungal, biofilm inhibition, cytotoxicity, and phytotoxicity tests. Furthermore, their antifungal activity against H. fraxineus was specifically assessed, and the effects of cytochalasins on actin dynamics were also investigated. The study identified several compounds with diverse and significant bioactivities, highlighting their potential for further development. Notably, PF1140, CJ-17,572, and mycorrhizin A exhibited strong antifungal activity against H. fraxineus. Additionally, cryptosporioptides, particularly cryptosporioptide C, displayed potent biofilm dispersal activity against S. aureus and effectively inhibited C. albicans biofilm formation, underscoring their potential as antibiofilm agents. Furthermore, several cytochalasins were examined for their role as irreversible actin modulators.A complementary study was conducted to investigate the factors contributing to variations in virulence between two H. fraxineus strains (Strain 7 and 13), with a particular focus on their secondary metabolite profiles and the potential effects of these differences on phytotoxicity. The results showed that the two strains have different secondary metabolite profiles. Extracts from Strain 7, which exhibited higher virulence in planta, also showed enhanced phytotoxicity in the leaf puncture assay. In a follow-up to this study, secondary metabolites of H. fraxineus DSM 117123 were isolated, leading to the first identification of cytochalasins in thispathogen. Given their known phytotoxic properties, these findings suggest that cytochalasins may contribute to the virulence of H. fraxineus.Finally, co-cultivation experiments were performed to assess the antagonistic potential of endophytic fungi against H. fraxineus and their impact on secondary metabolite production. Several endophytes with strong antagonistic activity were identified. While the overall secondary metabolite profiles remained largely unchanged, production levels varied depending on the specific H. fraxineus strain and endophyte involved.Overall, this research enhances our understanding of endophytic fungi as sources of novel bioactive compounds and provides insights into their interactions with the plant pathogenic fungus H. fraxineus. These findings may facilitate the development of new strategies for sustainable disease management and natural product discovery for pharmaceutical and agricultural applications.