A portable fluorescence-based recombinase polymerase amplification assay for the detection of mal secco disease by Plenodomus tracheiphilus

In this study, a new diagnostic assay to detect Plenodomus tracheiphilus, the causative agent of mal secco of citrus, was developed based on the recombinase polymerase amplification (RPA) technology. Mal secco is a well-known and damaging vascular disease, affecting primarily lemon (Citrus limon) and, to a lesser extent, other citrus species, including those in the genera Citrus, Fortunella, Poncirus and Severina. The disease poses a considerable threat to lemon production in most of the citrus-producing countries of the Mediterranean region and in the Black Sea area. RPA primers and probes were designed to amplify a 142 bp amplicon from the ITS regions of P. tracheiphilus. The inclusivity and specificity of the RPA assay were tested on gDNA isolated from a panel including 29 strains of various origin of P. tracheiphilus and 18 non-target fungal and oomycete plant pathogens typically isolated from citrus trees. The assay was specific to P. tracheiphilus and had a detection threshold of 1.0 pg of gDNA. Preliminary tests carried out on plant crude extract highlighted RPA's potential for the rapid, user-friendly, and cost-effective field diagnosis of mal secco.