A portable fluorescence-based recombinase polymerase amplification assay for the detection of mal secco disease by Plenodomus tracheiphilus
Ermes Ivan Rovetto, Matteo Garbelotto, Salvatore Moricca, Marcos Amato, Federico La Spada, Santa Olga Cacciola
Abstract
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.