Development and analytical validation of a fluorescent recombinase polymerase amplification-assay for real-time detection of Phyllosticta citricarpa, the causative agent of Citrus Black Spot

Citrus Black Spot (CBS), caused by the fungus Phyllosticta citricarpa , is a disease of major diagnostic relevance in the citrus industry, as its causative agent is a regulated quarantine organism subjected to surveillance in the European and Mediterranean Plant Protection Organization (EPPO) Region. To develop a rapid molecular assay for pathogen detection, a real-time fluorescence recombinase polymerase amplification (RPA) assay targeting a 134 bp region of tef1 was developed through sequence alignment and in-silico specificity analysis. The assay showed analytical specificity for P. citricarpa , with no amplification from diagnostically relevant non-target citrus-associated pathogens, including Phyllosticta capitalensis and P. citriasiana . Analytical sensitivity, evaluated on plasmid and purified genomic DNA, enabled detection down to 1.0 × 10−7 ng/μL of plasmid ( ∼ 100 copies per reaction) and 3.5 × 10−3 ng of genomic DNA per reaction ( ∼ 113 genome copies). The workflow was further validated on crude citrus peel macerates spiked with P. citricarpa mycelium, with reliable detection to 1.0 mg/mL across matrices from Citrus sinensis , C. limon , and C. reticulata . Performance benchmarking against the EPPO-recommended TaqMan qPCR showed comparable sensitivity together with operational simplicity and tolerance to amplification inhibitors. The ability to detect P. citricarpa directly in crude citrus peel macerates, combined with rapid real-time fluorescent readout at low temperature and minimal equipment requirements, lays the foundation for the use of this assay as a simple and rapid molecular detection approach for citrus-associated plant matrices.