Morphological characterization and genetic diversity of Clavibacter michiganensis subsp. michiganensis in tomato-producing areas in Mexico (Atena Editora)

Abstract

Clavibacter michiganensis subsp. michiganensis (Cmm) is the bacterium that causes bacterial canker in tomato (Solanum lycopersicum L.). This disease is one of the main factors limiting the production of this crop both in open fields and in greenhouses. The objective of this research was to determine the phenotypic and genotypic variability in different strains of Cmm isolated from the main tomato-producing areas of Mexico. A descriptive study was carried out from October 2015 to April 2019 and included samplings from different tomato-producing areas as well as the inclusion of Cmm strains from the Phytopathology Laboratory of the Center for Research in Food and Development, AC, collected from 2015-2016. The samples obtained were isolated in artificial culture medium; subsequently, they were phenotypically characterized by colour, size, mucus type, pathogenicity and virulence, and finally, the genes associated with the pathogenicity of the bacteria and that are located in the plasmids (CelA and Pat-1) were amplified by PCR of the pathogenicity island in the chromosome (tomA, chpC, ppaA). In addition, sequencing of the ITS region of the 16S rRNA gene of the isolated strains was performed to carry out phylogenetic analysis. In the study period, 60 strains of Cmm that showed diversity in colour, size and colonial mucus type were isolated. The strains were classified as orange yellow (5 strains), yellow (30 strains) and cream yellow (25 strains).They were classified as small (8 strains), medium (27 strains) or large (25 strains); 50 strains presented a nonmucoid consistency and 10 presented a mucoid consistency. All the strains in the study were pathogenic but with different degrees of virulence. The Cmm9 and Cmm68 strains were highly virulent. Meanwhile, the Cmm84 and Cmm98 strains showed a lower degree of virulence, presenting a delay of approximately 7 days before the appearance of the first symptoms of bacterial canker in the tomato plants. For all the strains except for Cmm84 and Cmm98, all the genes associated with pathogenicity were amplified; for the Cmm84 and Cmm98 strains, the Pat-1 gene (located in the pCM2 plasmid), which is directly associated with the induction of disease symptoms, was not amplified. This finding could be associated with the reduction in the virulence of these strains. The phylogenetic analysis of the ITS region of the 16S rRNA gene of the Cmm strains shows the formation of 8 groups, corroborating the genetic diversity of this bacterium. The results of this research provide information about the phenotypic and genotypic variability in Cmm, which could mean that bacterial canker outbreaks can be caused by a complex of clones introduced to Mexico from different geographical locations over time.