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

Abstract

BackgroundClavibacter 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.ResultsA 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.ConclusionsWe 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.

Clavibacter michiganensis subsp. michiganensis strains from central Chile exhibit low genetic diversity and sequence types match strains in other parts of the world

“…Eight primer pairs (Clav‐VNTR2, Clav‐VNTR5, Clav‐VNTR9, Clav‐VNTR13, Clav‐VNTR15, Clav‐VNTR16, Clav‐VNTR22 and Clav‐VNTR26) reported by Zaluga et al . () targeted VNTR loci of 20 to 45 bp tandem repeat (TR) units. In order to increase the probability of polymorphism, six primers targeting VNTR loci (MV‐VNTR1, MV‐VNTR2, MV‐VNTR7, MV‐VNTR9, MV‐VNTR10 and MV‐VNTR11) of 6 to 15 bp tandem repeat (TR) units were designed.…”

“…MLVA was carried out with four primer pairs designed by Zaluga et al . () and four primer pairs specifically designed in this study. The results of a MLVA typing assay depends on the quality of TR markers.…”

“…For MLVA analysis, primers to amplify eight Cmm VNTRs (Clav‐VNTR2, Clav‐VNTR5, Clav‐VNTR9, Clav‐VNTR13, Clav‐VNTR15, Clav‐VNTR16, Clav‐VNTR22 and Clav‐VNTR26) were tested as described by Zaluga et al . (). Additionally, new amplification primer pairs were designed to amplify other Cmm VNTRs.…”

“…Comparative analysis of genetic variation and population diversity of Cmm strains from different countries using diverse DNA‐based methods have been reported (Kleitman et al ., ; De León et al ., ; Jacques et al ., ; Milijašević‐Marcic et al ., ; Zaluga et al ., ; Tancos et al ., ; Croce et al ., ; Wassermann et al ., ). Such information is useful to establish efficient strategies for Cmm control.…”

Clostridium difficile infection: Early history, diagnosis and molecular strain typing methods

“…Multilocus variable-number tandem-repeat analysis (MLVA) has been suggested to have higher discriminatory power than other typing methods for investigating the relatedness between C. difficile strains [201]. It has been widely used in medical microbiology as an alternative or complement to other typing techniques such as PFGE, rep-PCR or MLST [202]. MLVA utilizes the naturally occurring variation in the number of tandem repeat DNA sequences found in many different loci in the genome.…”