Pathogenicity Assessment of Fungi Isolated From <i>Curculio glandium</i> ‐Infested and Uninfested Acorns of Persian Oak ( <i>Quercus brantii</i> ) in the Zagros Forests of Iran

ABSTRACT Forest regeneration hinges on seed health. Here, we investigated factors influencing Zagros oak forest regeneration by isolating fungi from acorns at 14 forest sites in Kermanshah province in western Iran that were either uninfested or infested with larvae of Curculio glandium Marsham (Coleoptera: Curculionidae). To identify C. glandium larvae, a PCR‐based diagnostic assay was developed using species‐specific primers FCuGl and RCuGl, designed from the cytochrome c oxidase I ( COI ) gene. Nested PCR enhanced sensitivity, detecting 20 pg. of genomic DNA by re‐amplifying direct PCR products. Primer specificity was confirmed via PCR on C. glandium larval DNA from acorns, with no cross‐reactivity observed in other insects. This method enabled rapid, reliable identification of C. glandium in oak acorns. Fungal taxa were identified via morphological traits and sequencing of the internal transcribed spacer (ITS) and a portion of the translation elongation factor 1‐alpha (TEF‐1α) gene regions, revealing 23 species. Pathogenicity was assessed by inoculating oak acorns and two‐year‐old oak seedlings with these fungi. Most isolates had minimal impact on acorn germination, but some species induced canker formation in seedlings. Given the presence of these fungi in weevil‐free acorns, transmission by C. glandium appears unlikely. About 90% of infested acorns germinated in the same year that they matured under greenhouse conditions, but dropped to 30% after 1 year of storage. Overall, this study provides critical insights into the key biotic agents impairing oak regeneration, highlighting the need for integrated management of both weevils and pathogenic fungi to support the health and sustainability of Zagros forests.