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

ACCESS DATA

Publications

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

Similar Datasets

Project description:Xylella fastidiosa is an economically important bacterial plant pathogen. With insights gained from 72 genomes, this study investigated differences among the three main subspecies, which have allopatric origins: X. fastidiosa subsp. fastidiosa, multiplex, and pauca The origin of recombinogenic X. fastidiosa subsp. morus and sandyi was also assessed. The evolutionary rate of the 622 genes of the species core genome was estimated at the scale of an X. fastidiosa subsp. pauca subclade (7.62 × 10-7 substitutions per site per year), which was subsequently used to estimate divergence time for the subspecies and introduction events. The study characterized genes present in the accessory genome of each of the three subspecies and investigated the core genome to detect genes potentially under positive selection. Recombination is recognized to be the major driver of diversity in X. fastidiosa, potentially facilitating shifts to novel plant hosts. The relative effect of recombination in comparison to point mutation was calculated (r/m = 2.259). Evidence of recombination was uncovered in the core genome alignment; X. fastidiosa subsp. fastidiosa in the United States was less prone to recombination, with an average of 3.22 of the 622 core genes identified as recombining regions, whereas a specific clade of X. fastidiosa subsp. multiplex was found to have on average 9.60 recombining genes, 93.2% of which originated from X. fastidiosa subsp. fastidiosa Interestingly, for X. fastidiosa subsp. morus, which was initially thought to be the outcome of genome-wide recombination between X. fastidiosa subsp. fastidiosa and X. fastidiosa subsp. multiplex, intersubspecies homologous recombination levels reached 15.30% in the core genome. Finally, there is evidence of X. fastidiosa subsp. pauca strains from citrus containing genetic elements acquired from strains infecting coffee plants as well as genetic elements from both X. fastidiosa subsp. fastidiosa and X. fastidiosa subsp. multiplex In summary, our data provide new insights into the evolution and epidemiology of this plant pathogen.IMPORTANCEXylella fastidiosa is an important vector-borne plant pathogen. We used a set of 72 genomes that constitutes the largest assembled data set for this bacterial species so far to investigate genetic relationships and the impact of recombination on phylogenetic clades and to compare genome content at the subspecies level, and we used a molecular dating approach to infer the evolutionary rate of X. fastidiosa The results demonstrate that recombination is important in shaping the genomes of X. fastidiosa and that each of the main subspecies is under different selective pressures. We hope insights from this study will improve our understanding of X. fastidiosa evolution and biology.

Project description:BackgroundThe 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.ResultsSkIf (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.ConclusionsSkIf 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.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets