A Validated LNA ‐Modified Probe‐Based qPCR Assay for the Fast and Accurate Detection of the Root Rot Pathogen Phytophthora ×

ABSTRACT

Phytophthora × cambivora is a destructive, hybrid pathogen that has plagued European forests and agroforestry systems for at least two centuries. This polyphagous oomycete, known as the causal agent of ink disease, is responsible for the decline of chestnut forests and orchards, as well as beech and oak stands. Its widespread occurrence in nurseries, traded propagation material, soil, irrigation basins, puddles along forest tracks, and watercourses increases the risk of local and long‐distance spread. Traditional identification methods based on symptom assessment, baiting and isolation on selective media no longer meet modern diagnostic requirements because they are time‐consuming, require specialised mycological expertise and often lack sufficient sensitivity and specificity. Currently, there are few methods available that use real‐time PCR to detect and quantify the pathogen. A new quantitative real‐time PCR (qPCR) assay that uses a locked nucleic acid (LNA) probe has been developed and validated on artificially infected chestnut tissue to address this issue. Our assay targeted a mitochondrial intergenic region between the NAD7 and COX3 genes. The primers and probe were designed using the OligoArchitect software. Specificity assays tested the target against a wide range of oomycetes, with validation carried out according to EPPO standard PM7/98. This method detects low levels of P. × cambivora DNA in infected chestnut tissue with a detection limit of 600 fg/μL, close to that of purified DNA. This qPCR‐LNA assay offers a fast, specific and robust tool for pathogen detection in a variety of applications, including routine diagnostics in nurseries and other vulnerable settings.