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Characterization of Clavibacter michiganensis subsp. michiganensis strains from recent outbreaks of bacterial wilt and canker in Serbia

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Abstract

Sixty-eight Clavibacter michiganensis subsp. michiganensis (Cmm) strains from recent outbreaks of bacterial wilt and canker in Serbia were collected from several tomato growing regions during a three-year period. The pathogen was identified based on bacteriological characteristics and pathogenicity tests and the identity of strains was confirmed by DAS ELISA and PCR amplification using primers CMM5/6 and PSA4/R. The strains showed homogeneity in biochemical and physiological properties. However, pathogenicity tests revealed differences in virulence that are presumably due to a loss of the pat-1 gene. Further strain characterization using DNA-based methods revealed a high diversity of the Serbian Cmm strains. Based on multi-locus sequence typing (MLST) analyses of five genes, Cmm strains were divided into seven groups. The pulsed-field gel electrophoresis (PFGE) pattern of a selection of strains supported the groupings based on trees of the kdpA/sdhA sequences. On the other hand, groupings made according to PFGE and MLST were not correlated to plasmid content in all cases. This study suggested that high genetic variability of the Serbian Cmm strains was detected both in MLST and PFGE analyses, and could have resulted either from new Cmm strains being introduced by seeds from different origins or as a consequence of an intraspecific hybridization process. In addition, this study proposed MLST as an efficient tool in epidemiological studies, population biology investigations and tracking the routes of transmission of pathogens. Four of the five house-keeping genes (kdpA, sdhA, ligA and gyrB) selected to characterize Cmm strains proved to be suitable for the MLST analysis. This is the first study carried out on the characterization of Cmm using MLST.

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Abbreviations

Cmm :

Clavibacter michiganensis subsp. michiganensis

DAS ELISA:

Double Antibody Sandwich ELISA

MLST:

Multi-locus sequence typing

NJ:

Neighbor joining

PFGE:

Pulsed-field gel electrophoresis

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Acknowlegdements

This research was carried out as a collaboration of the Faculty of Biology, University of Bielefeld, Germany, Department of Genetechnology/Microbiology and the Institute of Pesticides and Environmental Protection, Belgrade, Serbia, Department of Plant Pathology. A segment of this study was conducted as a part of the Project TR 34043, which is financially supported by the Ministry of Education and Science of the Republic of Serbia. The sequencing of the PCR products was conducted by IIT Biotech GmbH (Bielefeld, Germany).

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Authors and Affiliations

  1. Institute of Pesticides and Environmental Protection, Banatska 31b, P.O. Box 163, 11080, Belgrade, Serbia

    Svetlana Milijašević-Marčić, Biljana Todorović, Emil Rekanović & Ivana Potočnik

  2. Department of Genetechnology/Microbiology, Faculty of Biology, University of Bielefeld, 33501, Bielefeld, Germany

    Karl-Heinz Gartemann, Jonas Frohwitter & Rudolf Eichenlaub

Authors
  1. Svetlana Milijašević-Marčić
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  2. Karl-Heinz Gartemann
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  3. Jonas Frohwitter
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  4. Rudolf Eichenlaub
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  5. Biljana Todorović
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  6. Emil Rekanović
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  7. Ivana Potočnik
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Corresponding author

Correspondence to Svetlana Milijašević-Marčić.

Electronic supplementary material

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Supplementary Fig. 1

Alignment of the 693-bp kdpA amplicons. (PDF 72 kb)

Supplementary Fig. 2

Alignment of 700 bp of overlapping sequence from the sdhA amplicons (PDF 75 kb)

Supplementary Fig. 3

Alignment of the 606-bp gyrB PCR products. (PDF 69 kb)

Supplementary Fig. 4

Alignment of the 544-bp ligA PCR products. (PDF 69 kb)

Supplementary Fig. 5

Alignment of the 529-bp tomA PCR products. (PDF 69 kb)

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Milijašević-Marčić, S., Gartemann, KH., Frohwitter, J. et al. Characterization of Clavibacter michiganensis subsp. michiganensis strains from recent outbreaks of bacterial wilt and canker in Serbia. Eur J Plant Pathol 134, 697–711 (2012). https://doi.org/10.1007/s10658-012-0046-x

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