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 X. fastidiosa isolates to
subspecies clusters, among which MLST/MLSA represents the most common method. X. fastidiosa
outbreaks in EU motivated the search for accurate and faster approaches to differentiate the X.
fastidiosa 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 that allows to
differentiate all subspecies and three genotypes within X. fastidiosa subsp. pauca including the typeisolate
infecting olive in Italy and (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 X. fastidiosa isolates currently known to occur in
the Italian, France and Spain outbreaks. These rapid approaches could represent a useful tool for prescreening
of infected samples to be further analyzed by MLST or whole genome sequencing. In
addition alternative genomic regions of X. fastidiosa 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