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: 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.

Trends in Molecular Diagnosis and Diversity Studies for Phytosanitary Regulated Xanthomonas

“…Genome informed diagnostic protocols have been developed in recent years that could be further improved (e.g., [ 80 , 109 , 283 ]). Whole-genome sequences and pangenomic analyses of multiple strains belonging to all relevant xanthomonads could also facilitate the identification of new markers for the identification of different pathogens, at the species level but also markers of taxa below species and subspecies level (pathovars and races).…”

“…citri and X. citri pv. aurantifolii types B and C. Extensive work has been undertaken on citrus canker diagnosis to compare and set up the most appropriate protocols to detect and identify strains of the three citrus canker types [ 78 , 79 , 80 ]. Protocols were designed for targeting genes of ribosomal sequences or those identified to be factors involved in virulence such as rpf , hrpW, or pthA .…”

“…Protocols were designed for targeting genes of ribosomal sequences or those identified to be factors involved in virulence such as rpf , hrpW, or pthA . Besides, the precise identification of new targets was recently performed based on comparative genomic analysis of the different types of xanthomonads that cause disease in citrus [ 80 ]. Several protocols were initially developed on conventional PCR [ 81 , 82 , 83 , 84 , 85 , 86 ] or recently into droplet PCR [ 75 ].…”

“…Analysis of genome sequences has informed the development of assays for several Xanthomonas pathogens (regulated and non-regulated) based on PCR [ 80 , 86 , 267 , 283 , 284 , 285 ] or LAMP [ 63 , 109 , 113 , 123 , 134 , 286 , 287 ]. Genomics-informed assays have not always been entirely successful.…”

Diversity and Geographical Structure of Xanthomonas citri pv. citri on Citrus in the South West Indian Ocean Region

“…A subset of 42 strains ( Table S1 ), including strains assigned to all lineage 1 GCs, as well as the four pathotype A* strains, were assayed using the X. citri pv. citri -specific real-time quantitative assay XAC1051-qPCR [ 36 ]. Amplifications were performed with the Quantstudio 5 (QS5) (Applied Biosystems) real-time PCR system, using the GoTaq ® probe qPCR master mix kit (Promega, Charbonnières-les-Bains, France).…”