Novel tetraplex qPCR assays for simultaneous detection and identification ofXylella fastidiosasubspecies in plant tissues

Abstract

AbstractXylella fastidiosais an insect-borne bacterium confined to the xylem vessels of plants. This plant pathogen has a broad host range estimated to 560 plant species. Five subspecies of the pathogen with different but overlapping host ranges have been described, but only three subspecies are widely accepted, namely subspeciesfastidiosa,multiplexandpauca. Initially limited to the Americas,Xfhas been detected in Europe since 2013. As management ofX. fastidiosaoutbreaks in Europe depends on the identification of the subspecies, accurate determination of the subspecies in infected plants as early as possible is of major interest. Thus, we developed various tetraplex and triplex qPCR assays forXylella fastidiosadetection and subspecies identificationin plantain a single reaction. We designed primers and probes using SkIf, a bioinformatics tool based onk-mers, to detect specific signatures of the species and subspecies from a dataset of 58 genome sequences representative ofX. fastidiosadiversity. We tested the qPCR assays on 39 target and 30 non-target strains, as well as on 13 different plant species spiked with strains of the different subspecies ofX. fastidiosa, and on samples from various environmental and inoculated host plants. Sensitivity of simplex assays was equal or slightly better than the reference protocol on purified DNA. Tetraplex qPCR assays had the same sensitivity than the reference protocol and allowedX. fastidiosadetection in all spiked matrices up to 103cells.mL−1. Moreover, mix infections of two to three subspecies could be detected in the same sample with tetraplex assays. In environmental plant samples, the tetraplex qPCR assays allowed subspecies identification when the current method based on multilocus sequence typing failed. The qPCR assays described here are robust and modular tools that are efficient for differentiatingX. fastidiosasubspecies directly in plant samples.