Metagenomic sequencing as a diagnostic tool for Xylella fastidiosa diseases such as olive quick decline syndrome and Pierce’s disease of grapes

The plant pathogenic bacterium Xylella fastidiosa causes Pierce’s disease of grapevine and olive quick decline syndrome. Devastating X. fastidiosa (Xf) outbreaks in Europe on olive highlight this pathogen’s capacity to expand its host range and geographical distribution. Primary control strategy for Xf diseases is whole crop eradication; therefore, faster and accurate detection tools are necessary to prevent pathogen dispersion. The current diagnostic approach requires amplification of one or two different Xf genome regions, but it does not consider new or emerging variants. To remove biases towards known sequences, we tested low-cost, user-friendly metagenomic nextgeneration sequencing as a molecular tool for Xf detection and diversity analysis. As a proof of concept, we identified Xf to strain level in artificially single- and mixed-infected plant samples at concentrations as low as one picogram per gram of tissue. We then tested naturally infected extracted DNA samples from France, Italy and the United States. We successfully identified Xf subspecies to subgroups in all samples. We classified these reads into core and unique Xf sequences based on average nucleotide identity variation. Interestingly, we detected Xf reads from samples that had tested negative with real-time PCR. We also used this approach to detect Xf in Italian mediterranean shrubs, which are considered potential pathogen reservoirs. Overall, we developed a diagnostics pipeline that could be easily extended to other pathogens and implemented for emerging plant threat surveillance.