Use of a new SNP-based assay and multilocus SSR markers to assess the genetic diversity in Xylella fastidiosa subsp. pauca and its potential application to the study of the olive quick decline syndrome in Italy

Trabajo presentado en el International Symposium on the European outbreak of Xylella fastidiosa in olive, celebrado en Bari del 21 al 24 de octubre de 2014.Xylella fastidiosa is a Gram-negative, xylem-inhabiting phytopathogenic bacterium that is insect-transmitted and has very slow growth in vitro. This bacterium causes severe diseases and great yield losses in a number of very important crops, including almond, citrus, coffee, grapevine, and peach, and other disorders of perennial crops and landscape plants such as oleander. Several pathogenic variants of the bacterium have been described, which are often host-specific and taxonomically have been raised to the subspecies level: Xylella fastidiosa subsp. fastidiosa, X. fastidiosa subsp. multiplex, X. fastidiosa subsp. pauca, and X. fastidiosa subsp. sandyi. We have identified two haplotypes of X. fastidiosa subsp. pauca (Xfp) based in the gyrB sequence of moe than 90 bacterial isolates infecting citrus and coffee plants in different states in Brazil. The haplotypes were identified based on a single-nucleotide polymorphism (SNP) and correlated with host of origin. From that SNP, a mini-sequencing protocol wa s designed to differentiate haplotypes according to the host source that showed robustness for predicting Xfp host source in blind assays using DNA extracted from cultures of Xfp-infected plants, and bacterial-fed insect vectors. Combined use of the newly developed mini-sequencing protocol and of three previously developed multilocus SSR markers indicated that two haplotypes and distinct isolates of Xfp infect citrus and coffee in Brazil, and that multiple isolates genetically different may be present in a single orchard or infecting a single tree. The combined use of the SNP-based protocol and SSR analyses can be very useful for the spatio-temporal epidemiological studies of Xfp in Brazil and for the recent outbreak of X. fastidiosa in olive in Apulia (Italy) aimed to determining host specificity of bacterial isolates, existence of host jumping, vector feeding habits, etc. Indeed, X. fastidiosa isolates from Italy are phylogenetically close to Xfp based on gyrB sequences and also sho w a new SNP that differentiates them from the citrus and coffee isolates from Brazil.N