Development of a Simplified <scp>PCR</scp> ‐Nucleic Acid Lateral Flow Immunoassay ( <scp>PCR</scp> ‐ <scp>NALFIA</scp> ) for <i>Cryptostroma corticale</i> , the Causal Agent of Sooty Bark Disease

ABSTRACT Sooty bark disease (SBD), caused by the fungus Cryptostroma corticale , is causing a widespread outbreak on sycamore maple in Europe and is presently emerging as a threat to urban and native maple trees in the Pacific Northwest. Rising temperatures and prolonged drought conditions probably exacerbate SBD disease severity. Current detection methods for C . corticale rely on laboratory‐based PCR and morphological identification, which require specialised equipment and are not suited for rapid field deployment. We developed a portable, species‐specific PCR‐nucleic acid lateral flow immunoassay (PCR‐NALFIA) for point‐of‐care detection of C . corticale . The assay can be performed in 90 min by incorporating a 5‐min crude DNA extraction using cellulose dipsticks, a species‐specific ITS1‐targeted PCR with internal probe labelling on a portable PCR machine, and a lateral flow dipstick for visual detection. The method yielded accurate detection from infected wood, bark and conidia, with detection thresholds as low as 10 conidia or 0.1 mg of stroma in 0.5 mL buffer. The PCR‐NALFIA achieved 100% specificity when tested on 87 fungal samples, outperforming other available PCR assays. Furthermore, lyophilised reaction mixes were stable for 60 days at room temperature, making the assay viable for field applications. This tool offers a rapid and reliable diagnostic solution for managing SBD in forestry and urban environments.