Development of a qPCR assay for the rapid detection of the avocado scab fungus Elsinoe perseae

Abstract Elsinoe perseae , the causal agent of avocado scab disease, is a high-priority biosecurity threat to the Australian avocado industry. Symptom-based diagnosis is unreliable, and morphological identification is hindered by the slow growth of Elsinoe species on artificial media and the absence of a sexual reproductive stage in the field and in axenic culture. Consequently, a rapid and reliable molecular diagnostic tool capable of detecting the pathogen directly from infected fruit or leaf tissue is essential. In this study, we developed a quantitative PCR (qPCR) assay targeting two genomic regions: the internal transcribed spacer 1 (ITS1) region of the rDNA and the RNA polymerase II subunit B ( rpb2 ) gene. The assay can be applied in either singleplex or duplex formats. Both the ITS- and rpb2 -targeted assays consistently detected E. perseae DNA from pure cultures. However, detection in infected avocado leaf and fruit tissues was primarily driven by the ITS assay, whereas the rpb2 assay exhibited reduced sensitivity, likely reflecting differences in copy number between multicopy rDNA regions and the single-copy rpb2 gene. No cross-reactivity was observed against other Elsinoe species or common fungal pathogens of avocado. High analytical sensitivity was achieved, with reliable detection of both targets at concentrations of 10³ copies/µL of synthetic gBlocks dsDNA and 0.1 ng/µL of genomic DNA, using a conservative threshold of Ct ≤ 30. Assay performance was unaffected by minor variations in qPCR reagents and instrument platforms. This assay provides a rapid, sensitive, and highly specific diagnostic tool for biosecurity applications, supporting accurate detection, surveillance, and early response to potential incursions of E. perseae.