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

Robène Isabelle, Maillot-Lebon Véronique, Chabirand Aude, Moreau Aurélie, Becker Nathalie, Moumène Amal, Rieux Adrien, Campos Paola, Gagnevin Lionel, Gaudeul Myriam, Baider Claudia, Chiroleu Frédéric, Pruvost Olivier. 2020. Development and comparative validation of genomic-driven PCR-based assays to detect Xanthomonas citri pv. citri in citrus plants. BMC Microbiology, 20:296, 13 p.

Journal article ; Article de recherche ; Article de revue à facteur d'impact Revue en libre accès total
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Url - éditeur : https://rdcu.be/b9LBi

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: LOD95% = 754 CFU ml− 1 (15 cells per reaction), 100% inclusivity, 97.2% exclusivity for XAC1051-2qPCR; LOD95% = 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.

Mots-clés Agrovoc : Xanthomonas campestris citri, Bacteria, Surveillance épidémiologique, Diagnostic, PCR

Mots-clés complémentaires : Xanthomonas citri pv. citri, test PCR en temps réel

Mots-clés libres : Asiatic citrus canker, Surveillance, Real-time quantitative PCR, Diagnostics, Cycle cut-off, Ancient DNA

Classification Agris : H20 - Plant diseases

Champ stratégique Cirad : CTS 4 (2019-) - Santé des plantes, des animaux et des écosystèmes

Agence(s) de financement européenne(s) : European Commission, European Regional Development Fund

Auteurs et affiliations

  • Robène Isabelle, CIRAD-BIOS-UMR PVBMT (REU) - auteur correspondant
  • Maillot-Lebon Véronique
  • Chabirand Aude, ANSES-LSV (REU)
  • Moreau Aurélie, ANSES-LSV (REU)
  • Becker Nathalie, MNHN (FRA)
  • Moumène Amal, Université de la Réunion (REU)
  • Rieux Adrien, CIRAD-BIOS-UMR PVBMT (REU)
  • Campos Paola, CIRAD-BIOS-UMR PVBMT (REU)
  • Gagnevin Lionel, CIRAD-BIOS-UMR IPME (FRA)
  • Gaudeul Myriam, MNHN (FRA)
  • Baider Claudia, MSIRI (MUS)
  • Chiroleu Frédéric, CIRAD-BIOS-UMR PVBMT (REU) ORCID: 0000-0002-4874-5357
  • Pruvost Olivier, CIRAD-BIOS-UMR PVBMT (REU)

Source : Cirad-Agritrop (https://agritrop.cirad.fr/596895/)

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