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 the 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 real-time quantitative PCR. Results showed that the developed real-time LAMP assay could rapidly detect the pathogen genomic DNA within only 15 min, with no registered cross-reactivity with nontarget fungal isolates 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.