Early detection of Plenodomus tracheiphilus by a fluorescence-portable RPA assay: a new smart solution in citriculture

Mal secco, caused by the mitosporic fungus Plenodomus tracheiphilus, is a significant vascular disease affecting citrus crops, particularly in the Mediterranean region. Current diagnostic methods, such as PCR-based assays, though reliable and sensitive, are limited by their complexity and unsuitability for field use. This study presents a novel, portable, fluorescence-based recombinase polymerase amplification (RPA) assay for the early detection of P. tracheiphilus. RPA technology, which operates at constant low temperatures, was chosen for its simplicity, rapidity, and high sensitivity. The developed assay was evaluated for its ability to detect P. tracheiphilus in non-purified DNA samples and compared with the EPPO standard SYBR® Green I Real Time-PCR assay. The RPA assay demonstrated high sensitivity and complete specificity, confirming its potential as a field-deployable diagnostic tool. Tests on lemon plant samples, both symptomatic and asymptomatic for mal secco, further validated the assay's effectiveness in diverse conditions. The implementation of this RPA assay could significantly enhance disease management in citriculture by enabling timely and accurate in-field diagnosis.