Comparative genomics of the Gram-positive bacterial pathogen of maize, Clavibacter nebraskensis, reveals genomic variation between strains of varying aggressiveness

Background Clavibacter nebraskensis ( Cn ) is a Gram-positive bacterial plant pathogen that infects the vascular tissue of maize, causing Goss’s Wilt and Leaf Blight. The disease is characterized by tan, necrotic lesions, water-soaked freckles, and systemic wilt during early growth stages in susceptible varieties. It first emerged in Nebraska in 1969, but effective management strategies quelled the outbreak. However, since the mid-2000s, it has re-emerged as a particularly destructive maize pathogen, causing over 30% yield losses in susceptible fields. Results We sequenced 17 Cn strains of varying aggressiveness isolated from epidemics spanning their earliest detection to subsequent outbreaks in the mid-2010s across various US states and Manitoba to characterize the genomic diversity of the species. We found no strong population structure associated with geographic origin or year of isolation based on haplotype analysis, and genome wide variation in orthologous gene families content did not strongly associate with virulence. We detected five heterogeneous plasmids in our strains, whereas to date, only one plasmid sequence has been reported for a single Cn strain. Putative virulence genes, including the celA gene and other secreted CAZymes, are well conserved across all Cn genomes. Biofilm formation has been hypothesized to be important for infection, although analysis of in vitro biofilm formation did not correlate significantly with virulence in our study. However, we identified unique alleles involved in sugar transport in strains with significantly higher biofilm formation. Importantly, we identified large deletions in a secreted cellulase within the linker peptide region between the carbohydrate-binding domain and the cellulase domain in an avirulent strain. Cellulase activity testing in vitro revealed correlations between sequence variation at this locus and cellulase activity, as well as a strong, positive correlation between in vitro cellulase activity and the virulence phenotype in planta. Conclusion Our analyses provide insight into genomic diversity within the species, including the presence of novel plasmids, genomic islands, and conserved pathogenicity genes. Notably, deletions within a secreted cellulase are associated with avirulence in planta and in vitro cellulase activity, but further functional genetic characterization is necessary to fully elucidate the role of this cellulase in infection.