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Open Access Research article

Multilocus Variable-Number-Tandem-Repeats Analysis (MLVA) distinguishes a clonal complex of Clavibacter michiganensis subsp. michiganensis strains isolated from recent outbreaks of bacterial wilt and canker in Belgium

Joanna Zaluga1*, Pieter Stragier1, Johan Van Vaerenbergh2, Martine Maes2 and Paul De Vos13

Author Affiliations

1 Laboratory of Microbiology, Department of Biochemistry and Microbiology, Ghent University, K.L. Ledeganckstraat 35, Gent, B-9000, Belgium

2 Plant-Crop Protection, Institute for Agricultural and Fisheries Research, ILVO, Burg. Van Gansberghelaan 96, Merelbeke, B-9820, Belgium

3 BCCM/LMG Bacteria collection - Laboratory of Microbiology Department of Biochemistry and Microbiology, Ghent University, K.L. Ledeganckstraat 35, Gent, B-9000, Belgium

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BMC Microbiology 2013, 13:126  doi:10.1186/1471-2180-13-126

Published: 5 June 2013

Abstract

Background

Clavibacter michiganensis subsp. michiganensis (Cmm) causes bacterial wilt and canker in tomato. Cmm is present nearly in all European countries. During the last three years several local outbreaks were detected in Belgium. The lack of a convenient high-resolution strain-typing method has hampered the study of the routes of transmission of Cmm and epidemiology in tomato cultivation. In this study the genetic relatedness among a worldwide collection of Cmm strains and their relatives was approached by gyrB and dnaA gene sequencing. Further, we developed and applied a multilocus variable number of tandem repeats analysis (MLVA) scheme to discriminate among Cmm strains.

Results

A phylogenetic analysis of gyrB and dnaA gene sequences of 56 Cmm strains demonstrated that Belgian Cmm strains from recent outbreaks of 2010–2012 form a genetically uniform group within the Cmm clade, and Cmm is phylogenetically distinct from other Clavibacter subspecies and from non-pathogenic Clavibacter-like strains. MLVA conducted with eight minisatellite loci detected 25 haplotypes within Cmm. All strains from Belgian outbreaks, isolated between 2010 and 2012, together with two French strains from 2010 seem to form one monomorphic group. Regardless of the isolation year, location or tomato cultivar, Belgian strains from recent outbreaks belonged to the same haplotype. On the contrary, strains from diverse geographical locations or isolated over longer periods of time formed mostly singletons.

Conclusions

We hypothesise that the introduction might have originated from one lot of seeds or contaminated tomato seedlings that was the source of the outbreak in 2010 and that these Cmm strains persisted and induced infection in 2011 and 2012. Our results demonstrate that MLVA is a promising typing technique for a local surveillance and outbreaks investigation in epidemiological studies of Cmm.

Keywords:
Clavibacter; MLVA; Bacterial typing; Epidemiology; Bacterial wilt and canker; Plant pathogen