Comparative Genomics and Phylogenetic Analyses Suggest Several Novel Species within the Genus <i>Clavibacter</i> , Including Nonpathogenic Tomato-Associated Strains

Abstract

Members of the genus <i>Clavibacter</i> are economically important bacterial plant pathogens infecting a set of diverse agricultural crops (e.g., alfalfa, corn, potato, tomato, and wheat). Tomato-associated <i>Clavibacter</i> sp. strains account for a great portion of the genetic diversity of the genus, and <i>C. michiganensis</i><i>sensu stricto</i> (formerly <i>C. michiganensis</i> subsp. <i>michiganensis</i>), causing bacterial canker disease, is considered one of the most destructive seed-borne agents for the crop worldwide. However, current taxonomic descriptions of the genus do not reflect the existing diversity of the strains, resulting in unsatisfactory results in quarantine surveys for the pathogens. In this study, we used all the available genome sequences of <i>Clavibacter</i> sp. strains, including the type strains of newly described subspecies, to provide precise insight into the diversity of tomato-associated members of the genus and further clarify the taxonomic status of the strains using genotypic and phenotypic features. The results of phylogenetic analyses revealed the existence of nine hypothetical new species among the investigated strains. None of the three new subspecies (i.e., <i>C. michiganensis</i> subsp. <i>californiensis</i>, <i>C. michiganensis</i> subsp. <i>chilensis</i>, and <i>C. michiganensis</i> subsp. <i>phaseoli</i>) is included within the tomato-pathogenic <i>C. michiganensis</i><i>sensu stricto</i> lineage. Although comparative genomics revealed the lack of <i>chp</i> and <i>tomA</i> pathogenicity determinant gene clusters in the nonpathogenic strains, a number of pathogenicity-related genes were noted to be present in all the strains regardless of their pathogenicity characteristics. Altogether, our results indicate a need for a formal taxonomic reconsideration of tomato-associated <i>Clavibacter</i> sp. strains to facilitate differentiation of the lineages in quarantine inspections.<b>IMPORTANCE</b><i>Clavibacter</i> spp. are economically important bacterial plant pathogens infecting a set of diverse agricultural crops, such as alfalfa, corn, pepper, potato, tomato, and wheat. A number of plant-pathogenic members of the genus (e.g., <i>C. michiganensis</i><i>sensu stricto</i> and <i>C. sepedonicus</i>, infecting tomato and potato plants, respectively) are included in the A2 (high-risk) list of quarantine pathogens by the European and Mediterranean Plant Protection Organization (EPPO). Although tomato-associated members of <i>Clavibacter</i> spp. account for a significant portion of the genetic diversity in the genus, only the strains belonging to <i>C. michiganensis</i><i>sensu stricto</i> (formerly <i>C. michiganensis</i> subsp. <i>michiganensis</i>) cause bacterial canker disease of tomato and are subjected to the quarantine inspections. Hence, discrimination between the pathogenic and nonpathogenic <i>Clavibacter</i> sp. strains associated with tomato seeds and transplants plays a pivotal role in the accurate detection and cost-efficient management of the disease. On the other hand, detailed information on the genetic contents of different lineages of the genus would lead to the development of genome-informed specific detection techniques. In this study, we have provided an overview of the phylogenetic and genomic differences between the pathogenic and nonpathogenic tomato-associated <i>Clavibacter</i> sp. strains. We also noted that the taxonomic status of newly introduced subspecies of <i>C. michiganensis</i> (i.e., <i>C. michiganensis</i> subsp. <i>californiensis</i>, <i>C. michiganensis</i> subsp. <i>chilensis</i>, and <i>C. michiganensis</i> subsp. <i>phaseoli</i>) should be reconsidered.