Understanding the potential origin and epidemiological consequences of the Spanish outbreaks caused byXylella fastidiosa subspecies multiplex

Outbreaks in Europe associated with Xylella fastidiosa (Xf) subspecies multiplex are the most frequent, accounting for the larger number of susceptible plant species (more than 70%), with some host overlap among the different regions. Diverse Sequence Types (ST) have been detected across distinct geographical regions: ST6, ST7 and ST79 in Corsica and PACA region (France), ST7 and ST81 in the Balearic Islands (Spain), ST6 in the province of Alicante and Madrid (mainland Spain), ST7 in the Douro Littoral region (Portugal), and ST87 in the region of Tuscany (northern Italy). In recent years, genetic analysis and pathogenicity tests have provided evidence of biological, ecological and host range diversity among strains of the same subspecies and STs. Draft genomes of 12 Spanish isolates of Xf subsp. multiplex (ST6 and ST81) were used for comparative genomic studies with currently available genomes of the same subspecies from France and Italy. Phylogenetic analysis based on core genomes, accessory genomes and single nucleotide polymorphisms indicate that: (i) European outbreaks associated with strains of Xf subsp. multiplex most likely result from distinct independent introductions; (ii) ST6-strains recovered from Spain and France, although sharing the same ST, fell in distinct phylogenetic subgroups; (iii) ST81 strains from the Balearic Islands and ST6 strains from Alicante differentiated in distinct phylogenetic groups (i.e. ST81 isolates are closer to ST6 isolates from California and France than ST6 isolates from Alicante, which in turn are closer to ST7 isolates from the USA and France); (iv) a low number of SNPs are detected among the strains recovered in Alicante, suggesting a recent introduction in the area. Additionally, whereas some recombination events were found among ST81 isolates from Mallorca and ST6 isolates from France with isolates of Xf subsp. fastidiosa ST1 from Mallorca, no evidence of recombination among ST6 isolates from Alicante with other European Xf isolates was found. Pathogenicity tests on the three main Spanish olive cultivars are being conducted with isolates belonging to ST6 and ST81 and compared to Xf subsp. pauca ST53 from Italy and ST80 from Ibiza. This work highlights that although the use of MLST approach is a powerful tool for resolving genetic relationships among isolates, the exploration of the whole genomes brings to more comprehensive information that in the future may help to retrieve more robust correlations with the biology and host range of the sequenced isolates. Furthermore, since current European regulation of Xf is based on the subspecies present in each outbreak, these results combined with further pathogenicity tests on main crops may help to establish management and regulation policy standards for the affected areas in Europe.