Fungal endophytes with anti-fungal metabolites reduce symptoms of ash dieback in Fraxinus excelsior in a greenhouse experiment

European ash trees (Fraxinus excelsior) are threatened by the non-native fungal pathogen Hymenoscyphus fraxineus, which causes crown dieback and root collar necroses. The exploitation of fungal endophytes, which naturally colonise trees asymptomatically and can produce bioactive metabolites, may provide opportunities as biocontrol agents to reduce symptom development in F. excelsior. We focused our investigations on isolates of four genera which fulfil these criteria and selected six promising candidates for greenhouse experiments: Diaporthe oncostoma (DSM 116298), Pezicula abietina (DSM 5141), Pezicula cf. ericae (DSM 110620), Nemania diffusa (DSM 116299), Hypoxylon perforatum (MUCL 54174) and Hypoxylon rubiginosum (DSM 106870). A detailed analysis of the secondary metabolomes by NMR (nuclear magnetic resonance) and HRMS (high resolution mass spectrometry) data identified known metabolites from these endophytes: mycorrhizin A produced by P. abietina, CJ-17,572 from P. cf. ericae, phomopsidin from Hyp. rubiginosum and cytochalasin E from N. diffusa as key anti-fungal agents. Besides detecting multiple potential cytochalasins, the new compound 9-epi-xylaolide A was isolated from D. oncostoma. A greenhouse experiment was conducted to determine whether the endophytes could reduce symptoms of ash dieback in planta. When two-year old F. excelsior saplings were artificially-inoculated with each of the six endophytes alone, no significant symptoms of disease developed. For two of the six endophytes, we observed significantly reduced necrotic lesion development when saplings were subsequently inoculated with a H. fraxineus strain of low virulence, compared to saplings inoculated only with the pathogen. In combinations of the six biocontrol candidates and a H. fraxineus strain of higher virulence, lesion development was initially inhibited, however inhibition was not significant and decreased at different rates over the monitoring period. Mortality was delayed in dual-inoculated saplings compared to saplings inoculated with either of the H. fraxineus strains alone. These results indicate symptoms and mortality associated with H. fraxineus infection can be reduced by endophytes.