Fungal endophytes for biocontrol of ash dieback: The antagonistic potential of Hypoxylon rubiginosum

Abstract Our goal is to develop a biocontrol agent using endophytic fungi to control ash dieback, a disease that endangers the very existence of the European ash, Fraxinus excelsior. Such a protective endophyte should produce metabolites toxic to the invasive fungus Hymenoscyphus fraxineus, the causal agent of ash dieback. From a total of 340 fungal endophytes from F. excelsior, the interactions with 13 exemplary isolates are presented here. In co-culture between H. fraxineus and endophyte, there was almost always reciprocal inhibition between pathogen and endophyte. Endophytes that inhibited H. fraxineus more than they were inhibited by the pathogen were inoculated into axenically cultured F. excelsior to select those that grew asymptomatically in the host. Most of the endophytes caused disease symptoms in axenically cultured F. excelsior seedlings, highlighting the importance of not only relying on co-culture experiments, but also using in planta experiments. By means of a multi-locus genealogy in conjunction with morphological studies and a comparison with the ex-epitype culture, one of the most promising isolates was identified as Hypoxylon rubiginosum, an ascomycete fungus that is closely associated with Fraxinus and frequently produces its sexual morph on dead wood of the host. Using preparative HPLC, metabolites were isolated from mono- and co-cultures of H. fraxineus and H. rubiginosum and their structures elucidated by NMR spectroscopy and HR mass spectrometry. The known phytotoxin viridiol was detected as a major metabolite of the ash pathogen in co-culture and the previously known antifungal metabolite, phomopsidin, as the major secondary metabolite of H. rubiginosum. In addition, the new metabolite, 10-hydroxyphomopsidin, was obtained from H. rubiginosum. Phomopsidin, a known inhibitor of beta-tubulin that targets the eukaryotic cytoskeleton, inhibited the pathogen, but also showed mild cytotoxic effects. The fact that H. rubiginosum grows asymptomatically in planta and that it produces the antifungal metabolite phomopsidin qualifies it as a promising candidate for further development as a biocontrol agent for ash dieback.