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,Tomáš Kiss,Jana Čechová,Jakub Pečenka,Lucia Ragasová,Aleš Eichmeier,Ruslan Kalendar

doi:10.1371/journal.pone.0227559

Eliška Peňázová, Miloň Dvořák + Show 6 more

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https://doi.org/10.1371/journal.pone.0227559

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Journal: PloS one	Publication Date: Jan 7, 2020
Citations: 12	License type: CC BY 4.0

Affiliation: Mendel University in Brno

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.

Highlights

Clavibacter michiganensis subsp. michiganensis (Cmm), Pseudomonas syringae pv. tomato (Pst) and bacterial spot-causing xanthomonads (BSX) represent major bacterial pathogens of tomato [1, 2]
Reference strains of Cmm (NCPPB 2979), Pst (NCPPB 1106), X. euvesicatoria (Xe) (NCPPB 2968), X. vesicatoria (Xv) (NCPPB 422), X. gardneri (Xg) (NCPPB 881) and X. perforans (Xp) (NCPPB 4321) grown overnight in Mueller-Hinton Broth (HiMedia, Mumbai, India), and bacterial suspensions of approximately 108 CFU.ml-1 were prepared in 0.9% sterile physiological saline solution
Designed sequences were compared with sequences available in the GenBank database using a threshold of 95% similarity

Summary

Introduction

Clavibacter michiganensis subsp. michiganensis (Cmm), Pseudomonas syringae pv. tomato (Pst) and bacterial spot-causing xanthomonads (BSX) represent major bacterial pathogens of tomato [1, 2]. Tomato (Pst) and bacterial spot-causing xanthomonads (BSX) represent major bacterial pathogens of tomato [1, 2]. Cmm and BSX are quarantine organisms in the European Union (EPPO A2 list) and are subjected to strict international phytosanitary controls [3, 4]. Significant losses caused by Pst have been reported [5, 6]. Cmm (Actinobacteria; family, Microbacteriaceae; genus, Clavibacter), the causal agent of bacterial wilt and canker of tomato, is considered one of the most important bacterial pathogens in tomato plantings worldwide. The pathogen infects tomato plants epiphytically through.

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