Analysis of plant and environmental samples for the quarantine fungus Phyllosticta citricarpa in four European citrus‐growing areas

Abstract Phyllosticta citricarpa is the causal agent of citrus black spot (CBS), a disease affecting most citrus species and cultivars, particularly lemons ( Citrus limon ) and sweet oranges ( Citrus sinensis ). The pathogen causes fruit blemishes and premature fruit drop, leading to substantial yield and quality losses. Phyllosticta citricarpa is of global economic importance and is regulated by several citrus‐growing countries, including those in the European Union (EU). The aim of this study was to develop a novel approach for early detection of P. citricarpa in environmental samples and to assess the status of P. citricarpa in selected citrus‐growing areas of the EU where this species had previously been reported from leaf litter. Between 2018 and 2021, citrus plant material, air and rainwater samples were collected and analysed for the presence of Phyllosticta spp. in orchards in Greece, Italy, and Malta. Molecular analyses using qPCR assays targeting P. citricarpa were employed for the analysis of air and rainwater samples. The performance of the testing protocol was evaluated prior to sample analysis, and 1000 conidia were determined to be the minimum number of spores required for reliable detection. No CBS symptoms were observed, and no evidence of P. citricarpa was found in collected citrus plant material, air, or rainwater samples. The only Phyllosticta species detected during this study was the endophyte P. capitalensis , isolated from citrus leaf litter in Greece. These findings support the results of official EU surveys, which indicate the current absence of P. citricarpa in the surveyed areas and demonstrate the value of integrating environmental monitoring with molecular diagnostics for plant health surveillance. The methodologies developed here offer a robust framework for early warning and rapid response, supporting efforts to prevent CBS outbreaks in Europe and can be adapted for other fungal pathogens.