Genome-barcoding (NGS) of quarantine bacterial pathogens relevant to the European Union: development and application with Nanopore methodology

Globalization and climate change are accelerating the spread of plant pathogens in new areas. Keeping the expansion of these pathogens under control is fundamental because they impact both the conservation of ecosystems and nations’ economy. Their spread often comes from asymptomatic plant materials so, to prevent it, it is necessary to improve detection and identification methodologies. In this study, quarantine relevant pathogens Xylella fastidiosa, Xanthomonas citri and Pantoea stewartii have been considered. It is key to identify these pathogens at sequence-type, pathovar and subspecies levels, as these provide insight on their host specificity as well as potential damage. Therefore, this work aims at developing a sensitive and specific diagnostic system for a precise identification even in asymptomatic conditions. We decided to rely on MinION from Oxford Nanopore Technology, a portable, fast, and easy to use device, paired with an ad hoc bioinformatics pipeline. An amplicon- Nanopore sequencing, based on selected housekeeping genes (e.g. MLST), has been developed. These selected genes allow the distinction of the pathovar, subspecies or ST of the different tested bacteria. Several plant species have been spiked with different bacterial suspensions at known concentrations. Then, a multiplex- (for Xylella fastidiosa) and duplex- (Xanthomonas citri) PCR amplification of housekeeping genes has been developed. The prepared samples were finally sequenced with Nanopore to test the workflow. Preliminary results indicate that this approach is promising for the detection and identification of these priority pathogens. Further study will be done to determine the limit of detection and specificity.