Recombinase Polymerase Amplification Assay for the Field Detection of Mal Secco Disease by Plenodomus tracheiphilus

In this study, we developed a new diagnostic assay based on the recombinase polymerase amplification (RPA) technology to detect Plenodomus tracheiphilus, the anamorphic fungus responsible for the destructive vascular disease of lemon named mal secco, in infected tissues of host plants. A 142 bp RPA-compatible barcode was sought within the 544 bp Internal Transcriber Spacer (ITS) fragment identified in a previous study and its P. tracheiphilus-specificity was confirmed by BLAST in the NCBI database. This was the premise to design an RPA probe (RPA_Ptrach_Probe). The specificity and inclusivity of the RPA assay were tested on gDNA isolated from tissues of C. limon, isolates of P. tracheiphilus of various origins and axenic cultures of non-target organisms, including fungal and oomycete pathogens typically associated to citrus trees, such as Alternaria spp., Colletotrichum spp., Phyllosticta spp., Penicillium spp., Phytophthora spp. With a detection threshold of 1.0 pg of gDNA the RPA assay proved to be as sensitive as the SYBR(R) Green I Real Time-PCR test included in the diagnostic protocol for P. tracheiphilus of the European and Mediterranean Plant Protection Organization. RPA assay was even more sensitive than Real Time-PCR in tests on DNA samples obtained through a rapid extraction method. In tests, on naturally infected lemon twigs, molecular approaches were comparable to each other and performed better than conventional isolation method. Overall, results of this study demonstrate the potential of RPA for rapid, easy to handle and cost effective in-field diagnosis of mal secco.