Fast detection of Plenodomus tracheiphilus, the causal agent of Citrus Mal Secco Disease, by real time loop-mediated isothermal amplification assay

Citrus mal secco is a destructive tracheomycotic disease caused by mitosporic fungus Plenodomus tracheiphilus (Petri) Gruyter, Aveskamp, and Verkley, severely affecting cultivated citrus species, particularly lemon trees in the Mediterranean region. A novel diagnostic assay based on real-time loop-mediated isothermal amplification (LAMP) was developed for the rapid detection of P. tracheiphilus. A set of six LAMP primers was designed to specifically target a 204-nucleotide sequence within the ITS1-5.8S-ITS2 region of nuclear rRNA genes. The assay’s sensitivity, specificity, exclusivity and inclusivity were evaluated on a panel of 21 isolates and compared with conventional PCR and the real-time quantitative PCR (qPCR). Results showed that the developed real-time LAMP assay could rapidly detect the pathogen genomic DNA within only 15 minutes, with no registered cross-reactivity with non-target fungal isolated from citrus plants or Citrus species. Moreover, reliable amplification was achieved using minimally processed samples. Specifically, two rapid extraction methods have been tested for an in-field plant diagnosis of P. tracheiphilus directly from the leaf surface. Overall, the developed real-time LAMP assay provides a potential tool for a quick, simple, specific and sensitive detection of P. tracheiphilus, allowing an early disease prediction and reducing the risk of epidemics.