Biology subjects

Dupas, E.

Publications and source records attributed to Dupas, E..

6 recordsLinked to original sources

bioRxiv2023

Analysis of the worldwide diversity of Xanthomonas hortorum pv. carotae, the agent of bacterial blight of carrot, reveals two distinct populations.

Xanthomonas hortorum pv. carotae is a pathogen responsible for the bacterial blight of carrot. While it infects carrot fields worldwide, its diversity remains unknown. Here, we validated by PCR the identification as X. hortorum pv. carotae of most of the strains isolated from carrot symptoms. We study their diversity by sequencing seven housekeeping genes. The analysis confirmed the identity of most of the strains previously identified as X. hortorum pv. carotae and highlighted the presence of two clusters inside the pathovar carotae. The 18 non-X. hortorum pv. carotae have been identified as Xanthomonas from other species. As the X. hortorum pv. carotae strains clustered only in two groups by this analysis, a new MLVA scheme was developed. It allowed to validate the subclusteration in two groups and observe diversity among them. At the same times based on this study and data from the literature, we proposed the strain CFBP 7900 as the neopathotype strain of the pathovar. The strain should replacement the holopathotype strain CFBP 4997, as it is no longer available in international collection.

pathology↗

bioRxiv2022

Two bridgehead invasions of Xylella fastidiosa subsp. multiplex in France

Of American origin, a wide diversity of Xylella fastidiosa strains belonging to different subspecies have been reported in Europe since 2013 and its discovery in Italian olive groves. Strains from the subspecies multiplex (ST6 and ST7) were first identified in France in 2015 in urban and natural areas. To trace back the most probable scenario of introduction in France, the molecular evolution rate of this subspecies was estimated at 3.2165 x 10-7 substitutions per site per year, based on heterochronous genome sequences collected worldwide. This rate allowed the dating of the divergence between French and American strains in 1987 for ST6 and in 1971 for ST7. The development of a new VNTR-13 scheme allowed tracing the spread of the bacterium in France, hypothesizing an American origin. Our results suggest that both sequence types were initially introduced and spread in Provence-Alpes-Cote dAzur (PACA); then they were introduced in Corsica in two waves from the PACA bridgehead populations.

microbiology↗

bioRxiv2021

Metagenomic sequencing for rapid identification of Xylella fastidiosa from leaf samples

Xylella fastidiosa (Xf) is a globally distributed plant pathogenic bacterium. The primary control strategy for Xf diseases is eradicating infected plants; therefore, timely and accurate detection is necessary to prevent crop losses and further pathogen dispersal. Conventional Xf diagnostics primarily relies on quantitative PCR (qPCR) assays. However, these methods do not consider new or emerging variants due to pathogen genetic recombination and sensitivity limitations. We developed and tested a metagenomics pipeline using in-house short-read sequencing as a complementary approach for affordable, fast, and highly accurate Xf detection. We used metagenomics to identify Xf to strain level in single and mixed infected plant samples at concentrations as low as one picogram of bacterial DNA per gram of tissue. We also tested naturally infected samples from various plant species originating from Europe and the United States. We identified Xf subspecies in samples previously considered inconclusive with real-time PCR (Cq > 35). Overall, we showed the versatility of the pipeline by using different plant hosts and DNA extraction methods. Our pipeline provides taxonomic and functional information for Xf diagnostics without extensive knowledge of the disease. We hope this pipeline can be used for early detection of Xf and incorporated as a tool to inform disease management strategies. IMPORTANCEXylella fastidiosa (Xf) destructive outbreaks in Europe highlight this pathogens capacity to expand its host range and geographical distribution. The current disease diagnostic approaches are limited by a multiple-step process, biases to known sequences, and detection limits. We developed a low-cost, user-friendly metagenomic sequencing tool for Xf detection. In less than three days, we were able to identify Xf subspecies and strains in field-collected samples. Overall, our pipeline is a diagnostics tool that could be easily extended to other plant-pathogen interactions and implemented for emerging plant threat surveillance.

microbiology↗

bioRxiv2020

Development of a Nested-MultiLocus Sequence Typing approach for a highly sensitive and specific identification of Xylella fastidiosa subspecies directly from plant samples

Different sequence types (ST) of Xylella fastidiosa were already identified in France and Spain based on direct MultiLocus Sequence Typing (MLST) of plant DNA samples. However, direct typing of plant DNA is partly efficient. In order to improve the sensitivity of X. fastidiosa identification, we developed a direct nested-MLST assay on plant extracted DNA. This method was performed based on a largely used scheme targeting seven housekeeping gene (HKG) loci (cysG, gltT, holC, leuA, malF, nuoL, petC). Nested primers were designed from multi-sequence alignments of 38 genomes representing all subspecies and one genome of Xylella taiwanensis. Sequences obtained were long enough to be used for BLAST comparison in PubMLST database. No nonspecific amplification products were observed in these samples. Efficiency of the nested-MLST was tested on extracted DNA from 106 samples proven positive (Cq<35) or equivocal (35[≤]Cq[≤]40) using the Harpers qPCR test. Samples analyzed included 49 plant species and two insect species (Philaenus spumarius, Neophilaenus campestris) that were collected in 2017 (106 plant samples in France), in 2018 (162 plant samples in France, 40 plant samples and 26 insect samples in Spain), and in 2019 (30 plant samples in Spain). With the conventional-MLST assay, no complete MLST profile was obtained for any of the samples from France and for most samples (59/66) from Spain. Conversely, with the nested approach, complete profiles were obtained for six French plant samples, 55 Spanish plant samples and nine Spanish insect samples. The threshold was improved by 100 to 1000 times compared to conventional PCR and was between 22 pg.mL-1 to 2.2 pg.mL-1 depending on the HKG. Using nested-MLST assay, plants that were not yet considered hosts tested positive and revealed novel alleles in France, whereas for Spanish samples it was possible to assign the subspecies or ST to samples considered as new hosts in Europe. Direct typing by nested-MLST from plant material has an increased sensitivity and may be useful for epidemiological purposes.

microbiology↗

bioRxiv2019

Novel tetraplex qPCR assays for simultaneous detection and identification of Xylella fastidiosa subspecies in plant tissues

Xylella fastidiosa 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 qPCR assays for Xylella 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 dataset 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.

molecular biology↗

bioRxiv2019

Comparison of real-time PCR and droplet digital PCR for the detection of Xylella fastidiosa in plants

Xylella fastidiosa (Xf) is a quarantine plant pathogen bacterium originating from the Americas and that has emerged in Europe in 2013. Xf can be detected directly on plant macerate using molecular methods such as real-time PCR, which is a sensitive technique. However, some plants may contain components that can act as PCR reaction inhibitors, which can lead to false negative results or an underestimation of the bacterial concentration present in the analyzed plant sample. Droplet digital PCR (ddPCR) is an innovative tool based on the partitioning of the PCR reagents and the DNA sample into thousands of droplets, allowing the quantification of the absolute number of target DNA molecules present in a reaction mixture, or an increase of the detection sensitivity. In this study, a real-time PCR protocol, already used for Xf detection in the framework of official surveys in the European Union, was transferred and optimized for Xf detection using ddPCR. This new assay was evaluated and compared to the initial real-time PCR on five plant matrices artificially inoculated and on naturally infected plants. In our conditions, this new ddPCR enabled the detection of Xf on all artificially inoculated plant macerates with a similar limit of detection, or a slight benefit for Quercus ilex. Moreover, ddPCR improved diagnostic sensitivity as it enabled detection of Xf in samples of Polygala myrtifolia or Q. ilex that were categorized as negative or close to the limit of detection using the real-time PCR. Here, we report for the first time a ddPCR assay for the detection of the bacterium Xf.

molecular biology↗