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Fungal farming ambrosia beetles cultivate a consortium of fungal partners that include obligate filamentous members and yeasts. These fungi are maintained in highly specialised insect organs, termed mycangia, and are cultivated as food along the beetle galleries elaborated within host trees. Here, we isolated fungi from the mycangia of\n                    <jats:styled-content style=\"fixed-case\">\n                      <jats:italic>Xyleborus affinis<\/jats:italic>\n                    <\/jats:styled-content>\n                    ambrosia beetles using both standard ethanol\u2010wash and ethanol\u2010free protocols. Omitting the ethanol wash significantly increased fungal recovery and diversity. We identify two previously described filamentous species,\n                    <jats:italic>Raffaelea arxii<\/jats:italic>\n                    and\n                    <jats:styled-content style=\"fixed-case\">\n                      <jats:italic>R. fusca<\/jats:italic>\n                    <\/jats:styled-content>\n                    , and the yeast,\n                    <jats:italic>Ambrosiozyma monospora<\/jats:italic>\n                    , together with two new filamentous fungi,\n                    <jats:italic>Neocosmospora affinis<\/jats:italic>\n                    and\n                    <jats:italic>Graphium ambrosium<\/jats:italic>\n                    , and two novel yeasts,\n                    <jats:italic>Alloascoidea xylebori<\/jats:italic>\n                    and\n                    <jats:italic>Wickerhamomyces ambrosius<\/jats:italic>\n                    , from both gallery walls and beetle mycangia. To meet Koch\u2010like postulates, using mycangial colonisation assays, we demonstrate that all seven fungal species were individually competent at colonising aposymbiotic\n                    <jats:styled-content style=\"fixed-case\">\n                      <jats:italic>X. affinis<\/jats:italic>\n                    <\/jats:styled-content>\n                    mycangia, demonstrating each as viable symbionts. Our results show that ethanol protocols can bias recovery of mycangial fungi, leading to underestimation of fungal diversity associated with ambrosia beetles. These findings provide a framework for improved characterisation of ambrosia beetle\u2010fungal mutualisms and experimental validation of fungal symbionts.\n                  <\/jats:p>","DOI":"10.1111\/1462-2920.70364","type":"journal-article","created":{"date-parts":[[2026,6,24]],"date-time":"2026-06-24T08:01:51Z","timestamp":1782288111000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Fulfilling Koch\u2010Like Postulates for Animal Fungal Mutualists: New Fungal Symbionts and Gallery and Mycangial Colonisation by\n                    <scp>\n                      <i>Xyleborus affinis<\/i>\n                    <\/scp>\n                    Ambrosia Fungi"],"prefix":"10.1111","volume":"28","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-5402-1315","authenticated-orcid":false,"given":"Abolfazl","family":"Masoudi","sequence":"first","affiliation":[{"name":"Department of Biological Sciences University of Illinois  Chicago Illinois USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Esther","family":"Tirmizi","sequence":"additional","affiliation":[{"name":"Department of Biological Sciences University of Illinois  Chicago Illinois USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mateo J.","family":"Valdiviezo","sequence":"additional","affiliation":[{"name":"Department of Biological Sciences University of Illinois  Chicago Illinois USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8986-5729","authenticated-orcid":false,"given":"Ross A.","family":"Joseph","sequence":"additional","affiliation":[{"name":"Department of Biological Sciences University of Illinois  Chicago Illinois USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2855-5158","authenticated-orcid":false,"given":"Nemat O.","family":"Keyhani","sequence":"additional","affiliation":[{"name":"Department of Biological Sciences University of Illinois  Chicago Illinois 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