Plasmid copy number variation impacts pathogenicity and quantification of Curtobacterium flaccumfaciens pv. flaccumfaciens infecting mung bean

In bacteria, plasmids can confer the ability to cause disease. Although they can potentially vary in copy number, little has been reported on the dynamics of plasmids in plant pathogenic bacteria. Pathogenicity of the bacterium Curtobacterium flaccumfaciens pv. flaccumfaciens (Cff), which causes foliar disease on leguminous crops, including mung bean (Vigna radiata), has previously been linked to a plasmid. This study explored the variation in plasmid copy number among a genetically diverse collection of 25 Cff isolates using purposely designed quantitative PCR assays for chromosomal and plasmid DNA targets. Pathogenicity and virulence of six Cff isolates, including one plasmid-free isolate, were assessed on the susceptible mung bean cultivar Opal-AU using visual symptoms, trifoliate dry weights and Cff DNA quantities following stem inoculation. Plasmid copy numbers varied significantly (p <0.001) among plasmid-carrying isolates, ranging from 1.37 to 2.74. For subsequent quantification in planta, the single-copy chromosomal PCR target gene, gyrase b, was selected. Mung beans inoculated with plasmid-carrying isolates exhibited significant variation in visual symptoms (p <0.001), while trifoliate dry weights and Cff DNA quantities did not significantly differ. The treatment using a plasmid-free isolate was not significantly different from the negative control for each of the disease traits. Knowledge of plasmid dynamics in Cff populations lays a foundation for improved understanding of the inheritance and impact of plasmid-related traits. The quantification assays will be useful for monitoring Cff populations and the demonstrated variation in pathogenicity and virulence can assist efforts to breed host plant germplasm with reduced susceptibility to Cff.