Diversity of specialized metabolites in Phaeoacremonium species revealed by untargeted metabolomics and bioactivity assays.

Fungi belonging to the genus Phaeoacremonium are recognized as cross-kingdom pathogens, that affect both agricultural crops and human health. Here, we report the first comprehensive large-scale chemotaxonomic study based on untargeted MS-based metabolomics of 28 isolates representing 24 Phaeoacremonium species. The chemometric analysis was integrated with in vitro toxicity on human keratinocytes and cucumber (Cucumis sativus) cotyledons, serving as representative models for human and plant hosts, respectively. A targeted LC-MS/MS method validated the production of the naphthoquinones scytalone and isosclerone, previously associated with grapevine trunk diseases, but revealed high interspecific variability in their abundance. Chemometric analysis revealed that cultures of Phaeoacremonium species can be distinguished based on their chemical fingerprints. In total, 206 significant metabolic features were uncovered, spanning several chemical classes including prenol lipids, carboxylic acid derivatives, organooxygen compounds, and macrolides. Nevertheless, 36 metabolites were putatively identified. Under our experimental conditions, scytalone and isosclerone showed negligible cytotoxicity and only mild phytotoxicity. In contrast, several crude extracts gave strong toxic effects, suggesting that activity may depend on complex metabolite interactions rather than single compounds. Notably, extracts from grapevine-associated species displayed stronger phytotoxicity and cytotoxicity compared to those from olive or human sources, highlighting a more diverse or more potent array of bioactive metabolites. Together, these results expand the known chemical space of Phaeoacremonium and underscore the need for targeted in vivo studies to elucidate the ecological roles and potential health implications of their secondary metabolites.