<scp>FISH</scp> Illumination of the Oak Wilt Pathogen, <i>Bretziella fagacearum</i> , on Vectoring Insects and in the Red Oak Host Tissues

ABSTRACT Oak wilt, caused by Bretziella fagacearum , is a destructive vascular disease of oaks in North America, yet fine‐scale spatial localisation of the pathogen in host tissues and on insect vectors remains poorly characterised. In this study, we developed and validated a species‐specific fluorescence in situ hybridisation (FISH) probe targeting the 28S rDNA of B . fagacearum and evaluated its capacity to detect and localise the pathogen in naturally infected red oak ( Quercus rubra ) tissues and on sap beetle vectors (Nitidulidae). Probe specificity was confirmed through fluorescence in situ hybridisation (FISH) and confocal laser scanning microscopy (CLSM), which showed strong hybridisation to B . fagacearum and no detectable signal in closely related Ceratocystidaceae. FISH successfully visualised ascospores on Carpophilus sayi collected from sporulating mats, with consistent localisation on terminal abdominal sternites and occasionally on mesoventral grooves, legs and antennae. Within oak twig tissues, FISH detected B . fagacearum hyphae predominantly in vessel elements, fibres, and occasionally in axial and ray parenchyma. In infected petioles, hyphae occurred in xylem vessels, collenchyma, and parenchyma, demonstrating pathogen presence beyond midrib tissues. Overall, this study establishes CLSM–FISH as a powerful visualisation tool for investigating the host–pathogen–vector interactions of the oak wilt disease.