Chemical Characterization of Secondary Metabolites of Escovopsioides nivea and Their Antagonistic Activity Against the Leaf-Cutter Ant Fungal Mutualist Leucoagaricus gongylophorus

Microfungi of the genus Escovopsioides are antagonists within the obligate symbiosis between fungus-growing ants (Tribe Attini) and their fungal cultivars, yet the chemical basis of their interactions remains largely unexplored. Similar to species of the closely related genus Escovopsis, Escovopsioides species frequently exhibit antagonistic interactions with the basidiomycete Leucoagaricus gongylophorus, the fungal cultivar maintained by attine ants. In this study, Escovopsioides nivea strains were isolated from nests of the higher attine ants Acromyrmex octospinosus and Mycetomoellerius mikromelanos collected in central Panama. One strain isolated from an A. octospinosus nest was selected for chemical investigation and evaluation of its antagonistic activity against L. gongylophorus. Chemical analysis using one- and two-dimensional NMR spectroscopy in combination with electrospray ionization mass spectrometry (ESIMS) led to the identification of five known secondary metabolites from Escovopsioides nivea: rugulotrosin A (1), microthecin (2), 2-(1-oxo-2-hydroxyethyl)furan (3), chrysophanol (4), and physcion (5). Bioassays against L. gongylophorus revealed that rugulotrosin A (1), chrysophanol (4), and physcion (5) exhibited moderate antagonistic activity at 100 μg concentrations. To the best of our knowledge, this is the first report describing secondary metabolites produced by a member of the genus Escovopsioides and demonstrating their antagonistic activity against the mutualistic fungal cultivar of fungus-growing ants. These findings broaden the known chemical diversity of Escovopsioides and provide new insights into the ecological functions and evolutionary significance of microfungal antagonists associated with the fungus-growing ant symbiosis.