Dataset Information

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

ABSTRACT: Xylella fastidiosa (Xf) 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 fastidiosa, multiplex, and pauca. Initially limited to the Americas, Xf has been detected in Europe since 2013. As management of X. fastidiosa 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 X. fastidiosa detection and subspecies identification in planta in a single reaction. We designed primers and probes using SkIf, a bioinformatics tool based on k-mers, to detect specific signatures of the species and subspecies from a data set of 58 genome sequences representative of X. fastidiosa 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 X. 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 allowed X. fastidiosa detection in all spiked matrices up to 103 cells.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 differentiating X. fastidiosa subspecies directly in plant samples.

SUBMITTER: Dupas E

PROVIDER: S-EPMC6951419 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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

Dupas Enora E Briand Martial M Jacques Marie-Agnès MA Cesbron Sophie S

Frontiers in plant science 20191227

Xylella fastidiosa (Xf) 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 fastidiosa, multiplex, and pauca. Initially limited to the Americas, Xf has been detected in Europe since 2013. As management of ...[more]

PMID: 31956326

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Project description:BACKGROUND:The phytopathogenic bacterium Xylella fastidiosa was thought to be restricted to the Americas where it infects and kills numerous hosts. Its detection worldwide has been blooming since 2013 in Europe and Asia. Genetically diverse, this species is divided into six subspecies but genetic traits governing this classification are poorly understood. RESULTS:SkIf (Specific k-mers Identification) was designed and exploited for comparative genomics on a dataset of 46 X. fastidiosa genomes, including seven newly sequenced individuals. It was helpful to quickly check the synonymy between strains from different collections. SkIf identified specific SNPs within 16S rRNA sequences that can be employed for predicting the distribution of Xylella through data mining. Applied to inter- and intra-subspecies analyses, it identified specific k-mers in genes affiliated to differential gene ontologies. Chemotaxis-related genes more prevalently possess specific k-mers in genomes from subspecies fastidiosa, morus and sandyi taken as a whole group. In the subspecies pauca increased abundance of specific k-mers was found in genes associated with the bacterial cell wall/envelope/plasma membrane. Most often, the k-mer specificity occurred in core genes with non-synonymous SNPs in their sequences in genomes of the other subspecies, suggesting putative impact in the protein functions. The presence of two integrative and conjugative elements (ICEs) was identified, one chromosomic and an entire plasmid in a single strain of X. fastidiosa subsp. pauca. Finally, a revised taxonomy of X. fastidiosa into three major clades defined by the subspecies pauca (clade I), multiplex (clade II) and the combination of fastidiosa, morus and sandyi (clade III) was strongly supported by k-mers specifically associated with these subspecies. CONCLUSIONS:SkIf is a robust and rapid software, freely available, that can be dedicated to the comparison of sequence datasets and is applicable to any field of research. Applied to X. fastidiosa, an emerging pathogen in Europe, it provided an important resource to mine for identifying genetic markers of subspecies to optimize the strategies attempted to limit the pathogen dissemination in novel areas.

Dataset Information

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

Publications

Novel Tetraplex Quantitative PCR Assays for Simultaneous Detection and Identification of <i>Xylella fastidiosa</i> Subspecies in Plant Tissues.

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