<i>Trachymyrmex septentrionalis</i> ants promote fungus garden hygiene using <i>Trichoderma</i>-derived metabolite cues.

Abstract

Fungus-growing ants depend on a fungal mutualist that can fall prey to fungal pathogens. This mutualist is cultivated by these ants in structures called fungus gardens. Ants exhibit weeding behaviors that keep their fungus gardens healthy by physically removing compromised pieces. However, how ants detect diseases of their fungus gardens is unknown. Here, we applied the logic of Koch's postulates using environmental fungal community gene sequencing, fungal isolation, and laboratory infection experiments to establish that <i>Trichoderma</i> spp. can act as previously unrecognized pathogens of <i>Trachymyrmex septentrionalis</i> fungus gardens. Our environmental data showed that <i>Trichoderma</i> are the most abundant noncultivar fungi in wild <i>T. septentrionalis</i> fungus gardens. We further determined that metabolites produced by <i>Trichoderma</i> induce an ant weeding response that mirrors their response to live <i>Trichoderma</i>. Combining ant behavioral experiments with bioactivity-guided fractionation and statistical prioritization of metabolites in <i>Trichoderma</i> extracts demonstrated that <i>T. septentrionalis</i> ants weed in response to peptaibols, a specific class of secondary metabolites known to be produced by <i>Trichoderma</i> fungi. Similar assays conducted using purified peptaibols, including the two previously undescribed peptaibols trichokindins VIII and IX, suggested that weeding is likely induced by peptaibols as a class rather than by a single peptaibol metabolite. In addition to their presence in laboratory experiments, we detected peptaibols in wild fungus gardens. Our combination of environmental data and laboratory infection experiments strongly support that peptaibols act as chemical cues of <i>Trichoderma</i> pathogenesis in <i>T. septentrionalis</i> fungus gardens.