Trachymyrmex septentrionalis ants promote fungus garden hygiene using Trichoderma-derived metabolite cues

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 garden 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 genomic sequencing, fungal isolation, and in vitro infection experiments to establish Trichoderma spp. as pathogens of Trachymyrmex septentrionalis fungus gardens. Our environmental data showed that Trichoderma are the most abundant non-cultivar fungi in wild T. septentrionalis fungus gardens. We further determined that metabolites produced by Trichoderma induce an ant weeding response that mirrors their response to live Trichoderma. Combining ant behavioral experiments with bioactivity-guided fractionation and statistical prioritization of metabolites in Trichoderma extracts demonstrated that T. septentrionalis ants weed in response to peptaibols, a specific class of secondary metabolites known to be produced by Trichoderma fungi. Similar assays conducted using purified peptaibols, including the two new 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 in vitro experiments strongly support that peptaibols act as chemical cues of Trichoderma pathogenesis in T. septentrionalis fungus gardens. ### Competing Interest Statement K.E.K., S.P.P., A.M.C.R., J.R.-C., R.M.S., C.E.E., M.E., M.E.A., J.L.K, and M.J.B declare no competing interest. N.H.O., C.J.P. and H.A.R are members of the Scientific Advisory Board of Clue Genetics, Inc. N.H.O. is also a member of the Scientific Advisory Board of Mycosynthetix, Inc. J.J.J.v.d.H. is a member of the Scientific Advisory Board of NAICONS Srl. P.C.D. is a scientific co-founder and advisor to Ometa and Enveda with prior approval by UC-San Diego and an advisor to Cybele.