Metagenomic sequencing for rapid identification of Xylella fastidiosa from leaf samples

Xylella fastidiosa (Xf) is a globally distributed plant pathogenic bacterium. To date, 595 plant species have been reported as Xf hosts (EFSA 2020). Because the primary control strategy for Xf diseases is the eradication of infected plants, timely and accurate detection is necessary to prevent crop losses and further pathogen dispersal. Current standard Xf diagnostics primarily relies on a quantitative PCR (qPCR) assays. Nevertheless, these methods do not consider new or emerging variants due to pathogen genetic recombination and are limited by their sensitivity. In this study, we developed and tested a metagenomics pipeline using in-house short-read sequencing as a complementary approach for affordable and accurate Xf detection. We were able to use metagenomics to identify Xf to strain level in single and mixed infected plant samples, at concentrations as low as one picogram of bacterial DNA per gram of tissue. We also tested naturally infected samples from a variety of plant species originating from France, Italy and the United States. We identified Xf subspecies in samples that were previously considered inconclusive (Cq > 35) with real-time PCR (EPPO 2019). Overall, we showed the flexibility of the pipeline by using seven different plant hosts and DNA extraction methods. Our pipeline provides taxonomic and functional information for Xf diagnostics without extensive knowledge of the disease. We hope this pipeline can be used for early detection of Xf and other plant pathogens and incorporated as a tool to inform disease management strategies.