Improvements for better detection, isolation and characterization of X. fastidiosa

Since the first outbreak of Xylella fastidiosa (Xf) on Polygala myrtifolia in natural settings in 2015 in France, Xf has been detected on more than thirty plant species with a validated method based on Real-Time PCR (Harper et al, 2010) associated to DNA extraction with QuickPick™ Plant DNA kit (Bio-Nobile) and KingFisher™ automate (Thermo Fisher Scientific). The sample preparation and isolation performed on modified PWG medium (EPPO, 2016) have been optimized and more than 40 Xf strains were isolated from Coffee plants, various ornamentals and trees. Critical reagents used for characterization of isolates directly on plant and on isolated strains according to a multilocus sequence typing (MLST) (http://pubmlst.org/xfastidiosa/) following the amplification protocol of Yuan et al. (2010) were evaluated to optimize EPPO protocol PM 7/24. Thus isolates in France were mostly allocated to sequence types ST6 and ST7 (subspecies multiplex) although other subspecies were identified punctually (Denancé et al., 2017). Philaenus spumarius, known as vector in Apulia is widespread in mainland France and Corsica. A detection method of X f in insects was validated based on Real-Time PCR (Harper et al., 2010) performed in duplex with internal controls 18S (Ioos et al., 2009), after DNA extraction using the same commercial kit (Bio-Nobile). The rate of contaminated insects based on individual insect testing varied, according to the outbreak locations in Corsica, from 4% to 25%.This research is financially supported by the Project H2020 PONTE.