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

• DOI: 10.3389/fpls.2019.01732
• Publication Date: 2019-12-27T04:13:23Z
• Authors: Enora Dupas; Martial Briand; Marie-Agnès Jacques; Sophie Cesbron
• Keywords: Xylella fastidiosa; Subspecies; Multiplex

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