Commelinaceomycesaneilematis (formerly Ustilagoaneilematis) is a recently reclassified ascomycete that infects Murdanniakeisak and is phylogenetically related to the mycotoxin-producing fungus Villosiclava virens (= Ustilaginoidea virens). Because of this close relationship, C. aneilematis was hypothesized to produce cytotoxic secondary metabolites similar to those of V. virens. In this study, the cytotoxicity and secondary metabolites of C. aneilematis were investigated. The MeOH extract obtained from rice cultures was fractionated by solvent partitioning, and the resulting fractions were evaluated using the WST-1 assay. The CH3CN fraction exhibited marked cytotoxicity against several human cancer cell lines, particularly HT-29 cells. Chemical investigation of this active fraction led to the isolation of two known cytotoxic metabolites, isochaetochromin B2 (1) and ustilaginoidin D (2), together with a new isocoumarin glycoside (3). Compounds 1 and 2 showed cytotoxicity against PANC-1 and HT-29 cells, whereas 3 was inactive at concentrations up to 100 μM. These results indicate that 1 and 2 are the likely contributors to the cytotoxicity of the CH3CN extract. The isolation of ustilaginoidin D (2), a characteristic metabolite of V. virens, provides chemotaxonomic evidence supporting the recent phylogenetic reclassification of C. aneilematis. Furthermore, because the host plant of C. aneilematis, Murdanniakeisak, is a weed that can contaminate rice forage, the production of these cytotoxic metabolites suggests a potential toxicological risk to livestock. This study provides the first chemical and toxicological characterization of C. aneilematis, demonstrating that this reclassified fungus is a previously unrecognized producer of biologically active secondary metabolites.
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