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Multiplex real-time PCR for the detection of Clavibacter michiganensis subsp. michiganensis, Pseudomonas syringae pv. tomato and pathogenic Xanthomonas species on tomato plants.

ABSTRACT: A multiplex real-time PCR method based on fluorescent TaqMan® probes was developed for the simultaneous detection of the tomato pathogenic bacteria Clavibacter michiganensis subsp. michiganensis, Pseudomonas syringae pv. tomato and bacterial spot-causing xanthomonads. The specificity of the multiplex assay was validated on 44 bacterial strains, including 32 target pathogen strains as well as closely related species and nontarget tomato pathogenic bacteria. The designed multiplex real-time PCR showed high sensitivity when positive amplification was observed for one pg of bacterial DNA in the cases of Clavibacter michiganensis subsp. michiganensis and Pseudomonas syringae pv. tomato bacteria and 100 pg for bacterial spot-causing xanthomonads. The reliability of the developed multiplex real-time PCR assay for in planta detection was verified by recognition of the target pathogens in 18 tomato plants artificially inoculated by each of the target bacteria and tomato samples from production greenhouses.

SUBMITTER: Penazova E

PROVIDER: S-EPMC6946519 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Multiplex real-time PCR for the detection of Clavibacter michiganensis subsp. michiganensis, Pseudomonas syringae pv. tomato and pathogenic Xanthomonas species on tomato plants.

Peňázová Eliška E Dvořák Miloň M Ragasová Lucia L Kiss Tomáš T Pečenka Jakub J Čechová Jana J Eichmeier Aleš A

PloS one 20200107 1

A multiplex real-time PCR method based on fluorescent TaqMan® probes was developed for the simultaneous detection of the tomato pathogenic bacteria Clavibacter michiganensis subsp. michiganensis, Pseudomonas syringae pv. tomato and bacterial spot-causing xanthomonads. The specificity of the multiplex assay was validated on 44 bacterial strains, including 32 target pathogen strains as well as closely related species and nontarget tomato pathogenic bacteria. The designed multiplex real-time PCR sh ...[more]

PMID: 31910230

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Project description:Tomato bacterial canker caused by Clavibacter michiganensis subsp. michiganensis is one of the most important seed-borne tomato diseases around the globe. The disease was initially reported in 1993 in Iran, and it became a rising threat for the multibillion dollar tomato industry of the country during the last decade. In this study, using phylogeographic analyses, we determined genetic diversity and geographic distribution of C. michiganensis subsp. michiganensis in Iran. Our field surveys showed that the pathogen is expanding into the southern and eastern areas of the country. Furthermore, multilocus sequence analysis and typing (MLSA/MLST) using the sequences of five housekeeping genes (atpD, gyrB, ppk, recA, and rpoB) revealed that 37 C. michiganensis subsp. michiganensis strains isolated in Iran had high genetic diversity and placed in 15 sequence types (STs), while all the available 184 worldwide C. michiganensis subsp. michiganensis sequences were placed in 43 STs. MLSA divided the worldwide C. michiganensis subsp. michiganensis strains into two phylogroups (I and II). Among the 37 strains isolated in Iran, 30 strains clustered in phylogroup I, while 7 strains clustered in phylogroup II. Phylogeographic data inferred from the allelic profile of the five housekeeping genes suggested multiple introductions of C. michiganensis subsp. michiganensis inoculum into Iran, while the geographic origin of the Iranian C. michiganensis subsp. michiganensis strains remains undetermined. Further analyses using higher numbers of strains are warranted to decipher the evolutionary history of C. michiganensis subsp. michiganensis in Iran. Additionally, stricter seed/transplant inspections are recommended to reduce the risk of pathogen expansion to areas with no history of the disease.IMPORTANCEClavibacter michiganensis subsp. michiganensis, the causal agent of tomato bacterial canker disease, is one of the economically important pathogens of solanaceous crops (e.g., eggplant, pepper, and tomato) around the world. The disease occurs in many countries, with a particular importance in regions characterized by high precipitation and humid environmental conditions. As a seed-borne pathogen, C. michiganensis subsp. michiganensis is included in the A2 (high risk) list of quarantine pathogens by the European and Mediterranean Plant Protection Organization (EPPO). Bacterial canker disease was reported for the first time in 1993 in Iran, while the geographic distribution, genetic diversity, and phylogenetic position of the causal agent remain undetermined. In this study, using the multilocus sequence analysis and typing (MLSA/MLST) approach, we provided a phylogeographic scheme for the C. michiganensis subsp. michiganensis strains isolated in Iran. Furthermore, global-scale phylogenetic analyses led to determination of phylogenetic position of Iranian C. michiganensis subsp. michiganensis strains among worldwide population of the pathogen. Based on diversity parameters and population structure, we suggest relatively higher genetic diversity of the bacterial canker pathogen in Iran than has so far been observed in the other areas of the world. Results obtained in this study provide a novel insight into the genetic diversity and population structure of the bacterial canker pathogen on a global scale.

Project description:We have previously demonstrated that inoculation of tomato plants with 2,4-diacetylphloroglucinol (DAPG)- and hydrogen cyanide (HCN)-producing Pseudomonas brassicacearum LBUM300 could significantly reduce bacterial canker symptoms caused by Clavibacter michiganensis subsp. michiganensis In this study, in order to better characterize the population dynamics of LBUM300 in the rhizosphere of tomato plants, we characterized the role played by DAPG and HCN production by LBUM300 on rhizosphere colonization of healthy and C. michiganensis subsp. michiganensis-infected tomato plants. The impact of C. michiganensis subsp. michiganensis presence on the expression of DAPG and HCN biosynthetic genes in the rhizosphere was also examined. In planta assays were performed using combinations of C. michiganensis subsp. michiganensis and wild-type LBUM300 or DAPG (LBUM300ΔphlD) or HCN (LBUM300ΔhcnC) isogenic mutant strains. Populations of LBUM300 and phlD and hcnC gene expression levels were quantified in rhizosphere soil at several time points up to 264 h postinoculation using culture-independent quantitative PCR (qPCR) and reverse transcriptase quantitative PCR (RT-qPCR) TaqMan assays, respectively. The presence of C. michiganensis subsp. michiganensis significantly increased rhizospheric populations of LBUM300. In C. michiganensis subsp. michiganensis-infected tomato rhizospheres, the populations of wild-type LBUM300 and strain LBUM300ΔhcnC, both producing DAPG, were significantly higher than the population of strain LBUM300ΔphlD A significant upregulation of phlD expression was observed in the presence of C. michiganensis subsp. michiganensis, while hcnC expression was only slightly increased in the mutant strain LBUM300ΔphlD when C. michiganensis subsp. michiganensis was present. Additionally, biofilm production was found to be significantly reduced in strain LBUM300ΔphlD compared to the wild-type and LBUM300ΔhcnC strains.IMPORTANCE The results of this study suggest that C. michiganensis subsp. michiganensis infection of tomato plants contributes to increasing rhizospheric populations of LBUM300, a biocontrol agent, as well as the overexpression of the DAPG biosynthetic operon in this bacterium. The increasing rhizospheric populations of LBUM300 represent one of the key factors in controlling C. michiganensis subsp. michiganensis in tomato plants, as DAPG-producing bacteria have shown the ability to decrease bacterial canker symptoms in tomato plants.

Dataset Information

Multiplex real-time PCR for the detection of Clavibacter michiganensis subsp. michiganensis, Pseudomonas syringae pv. tomato and pathogenic Xanthomonas species on tomato plants.

Publications

Multiplex real-time PCR for the detection of Clavibacter michiganensis subsp. michiganensis, Pseudomonas syringae pv. tomato and pathogenic Xanthomonas species on tomato plants.

Similar Datasets

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