Dataset Information

Detection and identification of Xanthomonas pathotypes associated with citrus diseases using comparative genomics and multiplex PCR.

ABSTRACT: Background:In Citrus cultures, three species of Xanthomonas are known to cause distinct diseases. X. citri subsp. citri patothype A, X. fuscans subsp. aurantifolii pathotypes B and C, and X. alfalfae subsp. citrumelonis, are the causative agents of cancrosis A, B, C, and citrus bacterial spots, respectively. Although these species exhibit different levels of virulence and aggressiveness, only limited alternatives are currently available for proper and early detection of these diseases in the fields. The present study aimed to develop a new molecular diagnostic method based on genomic sequences derived from the four species of Xanthomonas. Results:Using comparative genomics approaches, primers were synthesized for the identification of the four causative agents of citrus diseases. These primers were validated for their specificity to their target DNA by both conventional and multiplex PCR. Upon evaluation, their sensitivity was found to be 0.02 ng/µl in vitro and 1.5 × 104 CFU ml-1 in infected leaves. Additionally, none of the primers were able to generate amplicons in 19 other genomes of Xanthomonas not associated with Citrus and one species of Xylella, the causal agent of citrus variegated chlorosis (CVC). This denotes strong specificity of the primers for the different species of Xanthomonas investigated in this study. Conclusions:We demonstrated that these markers can be used as potential candidates for performing in vivo molecular diagnosis exclusively for citrus-associated Xanthomonas. The bioinformatics pipeline developed in this study to design specific genomic regions is capable of generating specific primers. It is freely available and can be utilized for any other model organism.

SUBMITTER: Fonseca NP

PROVIDER: S-EPMC6777491 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Detection and identification of Xanthomonas pathotypes associated with citrus diseases using comparative genomics and multiplex PCR.

Fonseca Natasha P NP Felestrino Érica B ÉB Caneschi Washington L WL Sanchez Angélica B AB Cordeiro Isabella F IF Lemes Camila G C CGC Assis Renata A B RAB Carvalho Flávia M S FMS Ferro Jesus A JA Varani Alessandro M AM Belasque José J Setubal Joao C JC Telles Guilherme P GP Aguena Deiviston S DS Almeida Nalvo F NF Moreira Leandro M LM

PeerJ 20191001

<h4>Background</h4>In Citrus cultures, three species of Xanthomonas are known to cause distinct diseases. X. citri subsp. citri patothype A, X. fuscans subsp. aurantifolii pathotypes B and C, and X. alfalfae subsp. citrumelonis, are the causative agents of cancrosis A, B, C, and citrus bacterial spots, respectively. Although these species exhibit different levels of virulence and aggressiveness, only limited alternatives are currently avail ...[more]

PMID: 31592342

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Project description:BackgroundAsiatic 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.ResultsOur 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.ConclusionsWe 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.

Dataset Information

Detection and identification of Xanthomonas pathotypes associated with citrus diseases using comparative genomics and multiplex PCR.

Publications

Detection and identification of <i>Xanthomonas</i> pathotypes associated with citrus diseases using comparative genomics and multiplex PCR.

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