A fungal endophyte originating from the Canary Islands was identified as a potent antagonist against the fungal phytopathogen Hymenoscyphus fraxineus, which causes the devastating ash dieback disease. This endophyte was tentatively identified as Pezicula cf. ericae, using molecular barcoding. Isolation of secondary metabolites by preparative high-performance liquid chromatography (HPLC) yielded the known compounds CJ-17,572 (1), mycorrhizin A (3) and cryptosporioptides A–C (4–6), besides a new N-acetylated dihydroxyphenylalanin derivative 2, named peziculastatin. Planar structures were elucidated by NMR and HRMS data, while the relative stereochemistry of 2 was assigned by H,H and C,H coupling constants. The assignment of the unknown stereochemistry of CJ-17,572 (1) was hampered by the broadening of NMR signals. Nevertheless, after semisynthetic conversion of 1 into its methyl derivatives 7 and 8, presumably preventing tautomeric effects, the relative configuration could be assigned, whereas comparison of ECD data to those of related compounds determined the absolute configuration. Metabolites 1 and 3 showed significant antifungal effects in vitro against H. fraxineus. Furthermore, 4–6 exhibited significant dispersive effects on preformed biofilms of S. aureus at concentrations up to 2 µg/mL, while the biofilm formation of C. albicans was also inhibited. Thus, cryptosporioptides might constitute a potential source for the development of novel antibiofilm agents.

The secondary metabolome of Nemania diffusa, isolated as an ash endophytic fungus, was analyzed in detail. From its cultures, a previously undescribed cytochalasin 1 was isolated using preparative HPLC, together with six known congeners: 18-dehydroxy-cytochalasin E (2), cytochalasins Z₇ (3), Z₈ (4), and E (5), 18-dehydroxy-17-didehydro-cytochalasin E (6), and K Steyn (7). The structures of these compounds were determined using data from high-resolution mass spectrometry (HR-MS), in combination with 1D and 2D nuclear magnetic resonance (NMR) spectroscopy. Metabolites 1–4 share a characteristic 12-membered lactone moiety, placing them within a rarely examined cytochalasin subclass. Thus, the compounds were incorporated into our ongoing screening campaign to study the structure–activity relationship of this metabolite family. We initially determined their cytotoxicity in eukaryotic mouse fibroblast L929 cells using an MTT-based colorimetric assay, and further investigated their effect on the cellular actin dynamics of the human osteosarcoma cell line U-2OS in detail. Unexpectedly, we discovered a high number of irreversible compounds (1, 2, and 4). Additionally, we highlighted specific structural features within the 12-membered cytochalasin subclass that may play a role in directing the reversibility of these compounds.

Ash dieback is a tree disease caused by the fungal pathogen Hymenoscyphus fraxineus. Since its introduction into Europe, it has caused widespread and significant losses of the European ash, Fraxinus excelsior. Inoculations of F. excelsior with a low virulent H. fraxineus isolate were assessed as a promising method for reducing symptoms associated with ash dieback, presumably by triggering systemic induced resistance. Two strains of H. fraxineus were chosen based on observations of high and low in planta virulence. Crude extracts obtained from cultures of the highly virulent strain were more phytotoxic in a leaf puncture assay than ones obtained from the low virulent strain. UHPLC-DAD-MS/MS data identified the phytotoxin viridiol and the potential phytotoxin hyfraxin A in both cultures. However, the production of these compounds in vitro did not correspond with virulence in planta. To test the effects of priming, saplings of F. excelsior were first inoculated with the low virulent strain and subsequently with the highly virulent strain. On average, necrosis expansion on the stems was reduced by 53% in primed saplings at the end of the 14-week monitoring period, thus providing proof of the priming concept. These results contribute to our understanding of a possible integrated biological disease control approach for increasing resistance in saplings and reducing potential damages associated with pathogens, particularly during nursery propagation, out-planting and through the establishment phase. We discuss results in the context of relevant literature and summarise the limited availability of literature on priming and underlying principles in trees.

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.

This study deals with the elucidation of the life cycle of an inoperculate discomycete that was eventually collected from conifer wood in South Germany and has become famous for the extraordinary diversity of its secondary metabolites when it was studied extensively during the 1990s. It had then been identified as Lachnum papyraceum (Lachnaceae, Helotiales) based on morphological traits, and extracts from its mycelial cultures were found to possess extraordinary nematicidal and antibiotic activities. Over 60 different secondary metabolites were finally identified from this fungus after extensive variation of culture media and scale-up of production up to 100-L scale. Among the main active principles were mycorrhizin A and chloromycorrhizin A, which had first been reported in 1977 from an unnamed “mycorrhizal” fungus of the hemiparasitic plant, Monotropa hypopitys (pinesap), that was isolated in Sweden. We noted that both the Lachnum strain and the original mycorrhizin producer were still available in the public domain and decided to study them for comparison using a multilocus phylogeny and also generated secondary metabolite profiles of both strains using analytical high performance liquid chromatography coupled to diode array and mass spectrometric detection (HPLC–DAD/MS). Surprisingly, the sequence data as well as the secondary metabolite profiles of both strains were highly similar, and it was also confirmed by phylogenetic methods that the strains are indeed nested within the genus Lachnum by comparison of their ITS, LSU, and RPB2 sequences. The specimen called L. papyraceum in the old publications was tentatively re-identified by Hans-Otto Baral as L. cf. subvirgineum, but substantial further work on the taxonomy of the genus remains to be done, anyway. We conclude that some Lachnum species have a highly complex but all the more interesting life cycle, and the mycorrhizal symbiont partner may invade the host plant, where it may persist as an endophyte and finally turn saprotrophic on the wood of the senescent pine tree. The taxonomy of these fungi should also be further resolved in the future, using a polythetic concept that includes chemotaxonomic data and a multi-locus genealogy.