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

Eliška Peňázová; Miloň Dvořák; Lucia Ragasová; Tomáš Kiss; Jakub Pečenka; Jana Čechová; Aleš Eichmeier

doi:10.1371/journal.pone.0227559

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

PLoS One. 2020 Jan 7;15(1):e0227559. doi: 10.1371/journal.pone.0227559. eCollection 2020.

Authors

Eliška Peňázová¹, Miloň Dvořák², Lucia Ragasová³, Tomáš Kiss¹, Jakub Pečenka¹, Jana Čechová¹, Aleš Eichmeier¹

Affiliations

¹ Mendeleum-Department of Genetics, Faculty of Horticulture, Mendel University in Brno, Lednice, Czech Republic.
² Department of Forest Protection and Wildlife Management, Faculty of Forestry and Wood Technology, Mendel University in Brno, Brno, Czech Republic.
³ Department of Vegetable Sciences, Faculty of Horticulture, Mendel University in Brno, Lednice, Czech Republic.

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.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Actinobacteria / genetics
Actinobacteria / isolation & purification*
Actinobacteria / physiology
Clavibacter
Environment, Controlled
Pseudomonas syringae / genetics
Pseudomonas syringae / isolation & purification*
Pseudomonas syringae / physiology
Real-Time Polymerase Chain Reaction*
Solanum lycopersicum / growth & development
Solanum lycopersicum / microbiology*
Xanthomonas / genetics
Xanthomonas / isolation & purification*
Xanthomonas / physiology

Supplementary concepts

Clavibacter michiganensis

Grants and funding

The work was supported from ERDF "Multidisciplinary research to increase application potential of nanomaterials in agricultural practice" (No. CZ.02.1.01/0.0/0.0/16_025/0007314). This research was also supported by the project no. TJ01000274. The real-time instrument was provided by Phytophthora Research Centre. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.