Institut für nationale und internationale Angelegenheiten der Pflanzengesundheit
Phyllosticta citricarpa, a fungal pathogen included in the EPPO A1 list (EPPO, 2022), is a quarantine pest not present in the EU countries. It is the causal agent of “citrus black spot” disease (CBS), affecting citrus plants throughout subtropical climates and causing a reduction in both fruit quantity and quality. When introduced into a new area, early detection is crucial to start adequate disease control programmes. The genus Phyllosticta includes numerous phytopathogenic, endophytic and saprophytic species that are closely similar based on molecular characteristics. For this reason, P. citricarpa represents one of the most complex microorganisms for the development of specific molecular identification tools. The major issues in the molecular identification of P. citricarpa are related with the possibility to clearly distinguish this species from the closely related taxa, such as P. paracitricarpa and P. citriasiana. Interestingly, both P. citricarpa and P. paracitricarpa cause on citrus fruits disease symptoms attributable to CBS (Guarnaccia et al., 2017; EFSA, 2019). In view of the high genetic similarity among P. citricarpa, P. paracitricarpa, and P. citriasiana and the need for early detection of CBS, a specific, rapid, in situ diagnostic tool is being developed by UNICT (IT) and Agdia (FR) under the BiOrangePack project, funded by PRIMA Med (https://mel.cgiar.org/projects/biorangepack). This diagnostic tool is based on Recombinase Polymerase Amplification (RPA), an isothermal amplification technique that can be used in situ by growers and inspectors, and could be considered as a screening test. Molecular methods, such as conventional PCR, real-time PCR and other isothermal amplification-based tests, are already available to detect and identify P. citricarpa (Ioos et al., 2023). Some of them are included in the EPPO Diagnostic Protocol PM 7/17(3) 2020, but distinction between P. citricarpa and P. paracitricarpa requires phylogenetic marker sequencing, such as the translation elongation factor 1-alpha (TEF1) gene, after isolation of the fungus in pure culture. Recently, Zajc and colleagues (2023) developed a TEF1-based qPCR assay for P. citricarpa detection that does not cross-react with the closely related Phyllosticta species. In the framework of the proposed Euphresco project (TPS for CBS) a collaborative test performance study (TPS) will be organised, to verify the robustness and the reproducibility of the RPA-based method in different laboratories. The capability of detection and identification of P. citricarpa will be validated and its performance will be compared with other molecular diagnostic protocols currently recommended. The ability of the method to distinguish P. citricarpa from the closely related species will be also analysed.
Federal Ministry of Food and Agriculture
