AUTHOR=Dupas Enora , Briand Martial , Jacques Marie-Agnès , Cesbron Sophie 

TITLE=Novel Tetraplex Quantitative PCR Assays for Simultaneous Detection and Identification of Xylella fastidiosa Subspecies in Plant Tissues

JOURNAL=Frontiers in Plant Science

VOLUME=10

YEAR=2019

URL=https://www.frontiersin.org/journals/plant-science/articles/10.3389/fpls.2019.01732

DOI=10.3389/fpls.2019.01732

ISSN=1664-462X

ABSTRACT=<p><italic>Xylella fastidiosa</italic> (<italic>Xf</italic>) is 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 subspecies <italic>fastidiosa</italic>, <italic>multiplex</italic>, and <italic>pauca</italic>. Initially limited to the Americas, <italic>Xf</italic> has been detected in Europe since 2013. As management of <italic>X. fastidiosa</italic> outbreaks 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 quantitative PCR (qPCR) assays for <italic>X. fastidiosa</italic> detection and subspecies identification <italic>in planta</italic> in a single reaction. We designed primers and probes using SkIf, a bioinformatics tool based on <italic>k</italic>-mers, to detect specific signatures of the species and subspecies from a data set of 58 genome sequences representative of <italic>X. fastidiosa</italic> diversity. 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 of <italic>X. fastidiosa</italic>, 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 allowed <italic>X. fastidiosa</italic> detection in all spiked matrices up to 10<sup>3</sup> cells.ml<sup>−1</sup>. 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 differentiating <italic>X. fastidiosa</italic> subspecies directly in plant samples.</p>