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Fungus gardens of attine ants in Brazil were surveyed to determine the presence of killer toxin-producing yeasts and to define their antifungal activities and ecological importance. Our results indicate that 10 out of 59 yeast species isolated from fungal gardens are killer yeasts. Killer yeasts were less likely to inhibit the growth of yeasts isolated from the same environment but more effective at inhibiting yeasts isolated from other environments, supporting a role for killer yeasts in shaping the community composition. All killer yeasts had genome-encoded killer toxins and lacked cytoplasmic toxin-encoding elements (i.e., double-stranded RNA satellites and linear double-stranded DNAs). Of all the killer yeasts associated with attine ants,\n                      <jats:italic toggle=\"yes\">Candida sinolaborantium<\/jats:italic>\n                      (strain LESF 1467) showed a broad spectrum of antifungal activities against 39 out of 69 (57%) of yeast strains tested for toxin susceptibility. The complete genome sequence of\n                      <jats:italic toggle=\"yes\">C. sinolaborantium<\/jats:italic>\n                      LESF 1467 identified a new killer toxin, Ksino, with similarities in the primary sequence and tertiary structure to the\n                      <jats:italic toggle=\"yes\">Saccharomyces cerevisiae<\/jats:italic>\n                      killer toxin named Klus. Surveys of publicly available genome databases identified homologs of Ksino in the genomes of yeast strains of\n                      <jats:italic toggle=\"yes\">Saccharomycetes<\/jats:italic>\n                      and\n                      <jats:italic toggle=\"yes\">Pichiomycetes<\/jats:italic>\n                      , as well as other species of Ascomycota and Basidiomycota filamentous fungi. This demonstrates that killer yeasts can be widespread in attine ant fungus gardens, possibly influencing the fungal community composition and the importance of these complex microbial communities in the discovery of novel antifungal molecules.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>IMPORTANCE<\/jats:title>\n                    <jats:p>\n                      Attine ants perform essential ecosystem services through the harvesting of substrates for fungiculture. The cultured fungi are a food source for attine ants. Characterizing antifungal toxin-producing yeasts (killer yeasts) is vital to understanding how they might protect gardens from invasion by unwanted fungal species. This study also describes a new toxin named Ksino from the yeast\n                      <jats:italic toggle=\"yes\">Candida sinolaborantium<\/jats:italic>\n                      , a member of a new group of toxins found across many different species of fungi. This work supports the role of killer yeasts in the ecology of fungicultures and competition between fungi. The observed high prevalence of killer yeasts in fungal gardens also enables the discovery of novel antifungal molecules with the potential to be applied against disease-causing fungi.\n                    <\/jats:p>\n                  <\/jats:sec>","DOI":"10.1128\/aem.02246-25","type":"journal-article","created":{"date-parts":[[2026,1,21]],"date-time":"2026-01-21T14:00:26Z","timestamp":1769004026000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":2,"title":["Novel killer yeasts and toxins from the gardens of fungus-growing ants"],"prefix":"10.1128","volume":"92","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-4293-5886","authenticated-orcid":false,"suffix":"Jr.","given":"Rodolfo","family":"Bizarria","sequence":"first","affiliation":[{"id":[{"id":"https:\/\/ror.org\/03hbp5t65","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Biological Sciences, University of Idaho","place":["Moscow, USA"]},{"id":[{"id":"https:\/\/ror.org\/00987cb86","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of General and Applied Biology, S\u00e3o Paulo State University (UNESP), Institute of Biosciences","place":["Rio Claro, Brazil"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jack W.","family":"Creagh","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/03hbp5t65","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Biological Sciences, University of Idaho","place":["Moscow, USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Renato A.","family":"Corr\u00eaa dos Santos","sequence":"additional","affiliation":[{"name":"Laboratory of Computational, Evolutionary, and Systems Biology, Center for Nuclear Energy in Agriculture, University of S\u00e3o Paulo","place":["Piracicaba, Brazil"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lily L.","family":"Givens","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/03hbp5t65","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Biological Sciences, University of Idaho","place":["Moscow, USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sara A.","family":"Coss","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/03hbp5t65","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Biological Sciences, University of Idaho","place":["Moscow, USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tanner J.","family":"Badigian","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/03hbp5t65","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Biological Sciences, University of Idaho","place":["Moscow, USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Adrian V.","family":"Chavez","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/03hbp5t65","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Biological Sciences, University of Idaho","place":["Moscow, USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Rim T.","family":"Tekle","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/03hbp5t65","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Biological Sciences, University of Idaho","place":["Moscow, USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Noah","family":"Fredstrom","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/00cvxb145","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Genome Sciences, University of Washington","place":["Seattle, USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"F. 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