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

Abstract

<jats:title>Abstract</jats:title><jats:p><jats:italic>Xylella fastidiosa</jats: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 <jats:italic>fastidiosa</jats:italic>, <jats:italic>multiplex</jats:italic> and <jats:italic>pauca</jats:italic>. Initially limited to the Americas, <jats:italic>Xf</jats:italic> has been detected in Europe since 2013. As management of <jats:italic>X. fastidiosa</jats: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 qPCR assays for <jats:italic>Xylella fastidiosa</jats:italic> detection and subspecies identification <jats:italic>in planta</jats:italic> in a single reaction. We designed primers and probes using SkIf, a bioinformatics tool based on <jats:italic>k</jats:italic>-mers, to detect specific signatures of the species and subspecies from a dataset of 58 genome sequences representative of <jats:italic>X. fastidiosa</jats: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 <jats:italic>X. fastidiosa</jats: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 <jats:italic>X. fastidiosa</jats:italic> detection in all spiked matrices up to 10<jats:sup>3</jats:sup> cells.mL<jats:sup>−1</jats: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 <jats:italic>X. fastidiosa</jats:italic> subspecies directly in plant samples.</jats:p>

Nanopore sequencing for the detection and the identification of Xylella fastidiosa subspecies and sequence types from naturally infected plant material

“…Although several diagnostic methods are available for the detection of Xf (EPPO, 2016b), the identification of subspecies and ST are currently laborious and time-consuming. Recently, a tetraplex qPCR assays was developed for simultaneous detection and identification of subspecies in plant tissues (Enora et al , 2019). The detection of Xf at subspecies level is fundamental and in case of new outbreaks or new plant hosts, the identification of the ST is strongly recommended (EPPO Standard PM7/24 (4)).…”

“…This latter approach was assessed in order to overcome the low throughput of the flowcells and to set up a more reliable and sensitive method, based on the amplification of two housekeeping genes followed by Nanopore sequencing. Sequencing of cysG and malF for subspecies discrimination is suggested by the EPPO Standard PM7/24 (4) and required, since 2018, in France (Enora et al , 2019). Nanopore amplicon sequencing was more efficacious than real-time PCR in Xf detection of the spiked samples with low concentration of Xf target DNA (dataset 3, Tab.…”

Novel Virulent Bacteriophages Infecting Mediterranean Isolates of the Plant Pest Xylella fastidiosa and Xanthomonas albilineans

“…fastidiosa (Xff )) were recently detected in Europe, in particular in the Mediterranean area, probably introduced through commercial exchanges and causing a variety of diseases such as the leaf scorch of olive trees in Italy [14]. Continuous monitoring using standardized real-time PCR protocols is now performed in southern Europe [15,16]. However, in the absence of any efficient and authorized chemical method to control Xf, a major challenge is to develop environmentally friendly biotechnologies to control this plant disease.…”

“…In this context, a group from Texas A&M University proposed the use of bacteriophages as a treatment for Pierce's disease [28][29][30]. Following the same line of investigation, we attempted to isolate Xf -specific phages and to focus on the three Xf subspecies present in the Mediterranean basin, since the strains currently spreading in Europe differ from the original Pierce's disease causative strain Xff [14,16]. In order to develop phage cocktails active against specific strains of interest, i.e., Xf strains endemic in Europe, one should first constitute a collection of dedicated and specific phages, determine their genome sequences and test them in vitro as well as in vivo for their lytic activity.…”

Quantification of Major Bacteria and Yeast Species in Kefir Consortia by Multiplex TaqMan qPCR

“…High costs for labeling of each end of the probes with different dyes is in comparison to the use of intercalating dyes is compensated by higher selectivity and a reduced process time in multiplexed assays. Multiplex TaqMan qPCR recently appeared to be a very useful tool for the identification and quantification of different microbial communities (Farkas et al, 2017;Baudy et al, 2019;Enora et al, 2019;Lochman et al, 2019). Accordingly, this study aims to establish a multiplex qPCR approach for the simultaneous detection and quantification of eleven frequently reported bacteria and yeast species of milk kefirs.…”