Dataset Information

Development and comparative validation of genomic-driven PCR-based assays to detect Xanthomonas citri pv. citri in citrus plants.

ABSTRACT:

Background

Asiatic Citrus Canker, caused by Xanthomonas citri pv. citri, severely impacts citrus production worldwide and hampers international trade. Considerable regulatory procedures have been implemented to prevent the introduction and establishment of X. citri pv. citri into areas where it is not present. The effectiveness of this surveillance largely relies on the availability of specific and sensitive detection protocols. Although several PCR- or real-time PCR-based methods are available, most of them showed analytical specificity issues. Therefore, we developed new conventional and real-time quantitative PCR assays, which target a region identified by comparative genomic analyses, and compared them to existing protocols.

Results

Our assays target the X. citri pv. citri XAC1051 gene that encodes for a putative transmembrane protein. The real-time PCR assay includes an internal plant control (5.8S rDNA) for validating the assay in the absence of target amplification. A receiver-operating characteristic approach was used in order to determine a reliable cycle cut-off for providing accurate qualitative results. Repeatability, reproducibility and transferability between real-time devices were demonstrated for this duplex qPCR assay (XAC1051-2qPCR). When challenged with an extensive collection of target and non-target strains, both assays displayed a high analytical sensitivity and specificity performance: LOD_95% =?754?CFU?ml^-?1 (15 cells per reaction), 100% inclusivity, 97.2% exclusivity for XAC1051-2qPCR; LOD_95% =?5234?CFU?ml^-?1 (105 cells per reaction), 100% exclusivity and inclusivity for the conventional PCR. Both assays can detect the target from naturally infected citrus fruit. Interestingly, XAC1051-2qPCR detected X. citri pv. citri from herbarium citrus samples. The new PCR-based assays displayed enhanced analytical sensitivity and specificity when compared with previously published PCR and real-time qPCR assays.

Conclusions

We developed new valuable detection assays useful for routine diagnostics and surveillance of X. citri pv. citri in citrus material. Their reliability was evidenced through numerous trials on a wide range of bacterial strains and plant samples. Successful detection of the pathogen was achieved from both artificially and naturally infected plants, as well as from citrus herbarium samples, suggesting that these assays will have positive impact both for future applied and academic research on this bacterium.

SUBMITTER: Robene I

PROVIDER: S-EPMC7528614 | biostudies-literature | 2020 Oct

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Development and comparative validation of genomic-driven PCR-based assays to detect Xanthomonas citri pv. citri in citrus plants.

Robène Isabelle I Maillot-Lebon Véronique V Chabirand Aude A Moreau Aurélie A Becker Nathalie N Moumène Amal A Rieux Adrien A Campos Paola P Gagnevin Lionel L Gaudeul Myriam M Baider Claudia C Chiroleu Fréderic F Pruvost Olivier O

BMC microbiology 20201001 1

<h4>Background</h4>Asiatic Citrus Canker, caused by Xanthomonas citri pv. citri, severely impacts citrus production worldwide and hampers international trade. Considerable regulatory procedures have been implemented to prevent the introduction and establishment of X. citri pv. citri into areas where it is not present. The effectiveness of this surveillance largely relies on the availability of specific and sensitive detection protocols. Although several PCR- or real-time PCR-based methods are av ...[more]

PMID: 33004016

Similar Datasets

Project description:BackgroundXanthomonas axonopodis pv. citri (Xac) is an obligate aerobic phytopathogen constantly exposed to hydrogen peroxide produced by normal aerobic respiration and by the plant defense response during plant-pathogen interactions. Four putative catalase genes have been identified in silico in the Xac genome, designated as katE, catB, srpA (monofunctional catalases) and katG (bifunctional catalase).Methodology/principal findingsXac catalase activity was analyzed using native gel electrophoresis and semi-quantitative RT-PCR. We demonstrated that the catalase activity pattern was regulated in different growth stages displaying the highest levels during the stationary phase. KatE was the most active catalase in this phase of growth. At this stage cells were more resistant to hydrogen peroxide as was determined by the analysis of CFU after the exposition to different H(2)O(2) concentrations. In addition, Xac exhibited an adaptive response to hydrogen peroxide, displaying higher levels of catalase activity and H(2)O(2) resistance after treatment with sub-lethal concentrations of the oxidant. In the plant-like medium XVM2 the expression of KatE was strongly induced and in this medium Xac was more resistant to H(2)O(2). A XackatE mutant strain was constructed by insertional mutagenesis. We observed that catalase induction in stationary phase was lost meanwhile the adaptive response to peroxide was maintained in this mutant. Finally, the XackatE strain was assayed in planta during host plant interaction rendering a less aggressive phenotype with a minor canker formation.ConclusionsOur results confirmed that in contrast to other Xanthomonas species, Xac catalase-specific activity is induced during the stationary phase of growth in parallel with the bacterial resistance to peroxide challenge. Moreover, Xac catalases expression pattern is modified in response to any stimuli associated with the plant or the microenvironment it provides. The catalase KatE has been shown to have an important function for the colonization and survival of the bacterium in the citrus plant during the pathogenic process. Our work provides the first genetic evidence to support a monofunctional catalase as a virulence factor in Xac.

Project description:Comparative studies in Xanthomonas have provided a vast amount of data that enabled to deepen in the knowledge of those factors associated with virulence and Xanthomonas plant interaction. The species of this genus present a wide range of host plants and a large number of studies have been focused to elucidate which mechanism are involved in this characteristic. In this study, comparative genomic and phenotypic analysis were performed between X. citri subsp. citri (Xcc), one of the most studied pathogens within Xanthomonas, and X. arboricola pv. pruni (Xap), a pathogen which has aroused great interest in recent time. The work was aimed to find those elements that contribute to their host divergence despite the convergence in the symptoms that each species cause on Citrus spp. and Prunus spp., respectively. This study reveals a set of genes that could be putatively associated with the adaptation of these pathogens to their hosts, being the most remarkable those involved in environmental sensing systems such as the case of the TonB-dependent transporters, the sensors of the two-component system and the methyl accepting chemotaxis proteins. Other important variants were found in processes related to the decomposition of the cell wall as could be appreciated by their dissimilar set of cell-wall degrading enzymes. Type three effectors, as one of the most important factors in delineating the host specificity in Xanthomonas, also showed a different array when comparing both species, being some of them unique to each pathogen. On the other hand, only small variations could be connected to other features such as the motility appendages and surface adhesion proteins, but these differences were accompanied by a dissimilar capacity to attach on host and non-host leaf surface. The molecular factors found in this work provide the basis to perform a more in-depth functional analyses that unveil those actual factors associated with pathogenesis and host specificity in Xcc and Xap.

Project description:The Xanthomonas citri pv. citri (X. citri) is a phytopathogenic bacterium that infects different species of citrus plants where it causes canker disease. The adaptation to different habitats is related to the ability of the cells to metabolize and to assimilate diverse compounds, including sulfur, an essential element for all organisms. In Escherichia coli, the necessary sulfur can be obtained by a set of proteins whose genes belong to the cys regulon. Although the cys regulon proteins and their importance have been described in many other bacteria, there are no data related to these proteins in X. citri or in the Xanthomonas genus. The study of the relevance of these systems in these phytopathogenic bacteria that have distinct mechanisms of infection is one essential step toward understanding their physiology. In this work, we used bioinformatics, molecular modeling and transcription analysis (RT-PCR) to identify and characterize the putative cys regulon genes in X. citri.We showed that the ATP Binding Cassette Transporter (ABC transporter) SbpCysUWA for sulfate uptake is conserved in X. citri and translated in presence of sulfate. On the other hand, differently from what is predicted in databases, according molecular modeling and phylogenetic analysis, X. citri does not show a proper taurine transporter, but two different ABC systems related to the alkanesulfonate/sulfonate transport that were recently acquired during evolution. RT-PCR analysis evidenced that these genes and their putative transcriptional regulator CysB are rather transcripted in XAM1, a medium with defined concentration of sulfate, than LB.The presence of at least three distinct systems for sulfate and sulfonates assimilation in X. citri evidenced the importance of these compounds for the bacterium. The transcription of genes involved with alkanesulfonate/sulfur compounds in XAM1 along to CysB suggests that despite the differences in the transporters, the regulation of these systems might be similar to the described for E. coli. Altogether, these results will serve as a foundation for further studies aimed to understanding the relevance of sulfur in growth, virulence and pathogenesis of X. citri and related bacteria.

Dataset Information

Development and comparative validation of genomic-driven PCR-based assays to detect Xanthomonas citri pv. citri in citrus plants.

Background

Results

Conclusions

Publications

Development and comparative validation of genomic-driven PCR-based assays to detect Xanthomonas citri pv. citri in citrus plants.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets