Rapid screening tests for the assignment of Xylella fastidiosa genotypes to a subspecies cluster

Until now, different molecular tests can be used to assign novel Xf isolates to subspecies cluster, among which MLST/MLSA represents the most common method. Xf outbreaks in EU motivated the search for accurate and faster approaches to differentiate the isolates. Because MLST/MLSA requires PCR reactions and sequencing analyses, 2 independent approaches were recently developed and implemented for rapid taxonomic assignment of uncharacterized isolates: (1) single-nucleotide primer extension (SNuPE) method for the multiplex amplification of six Xylella DNA sequences (targeting all subspecies and three genotypes within Xf subsp. pauca including the type-isolate infecting olive in Italy); (2) high-resolution melting (HRM) analysis of the amplicon recovered from the gene encoding the conserved HL protein. Both assays were validated on a larger panel of isolates and proved to clearly differentiate Xf isolates currently known to occur in the Italian, France and Spain outbreaks. These rapid approaches could represent a useful tool for pre-screening of infected samples to be further analyzed by MLST or whole genome sequencing. In addition alternative genomic regions of Xf are going to be analyzed to implement approaches aimed to assign genotypes to a subspecies cluster, with the purpose to support a rapid identification of genotypes/subspecies at interception places or when new findings occur in a pest free area.