RESUMO
The genus Clavibacter comprises one species and five subspecies of plant-pathogenic bacteria, four of which are classified as quarantine organisms due to the high economic threat they pose. Clavibacter michiganensis subsp. michiganensis is one of the most important pathogens of tomato, but the recommended diagnostic tools are not satisfactory due to false-negative and/or -positive results. To provide a robust analysis of the genetic relatedness among a worldwide collection of C. michiganensis subsp. michiganensis strains, relatives (strains from the four other C. michiganensis subspecies), and nonpathogenic Clavibacter-like strains isolated from tomato, we performed multilocus sequence-based analysis and typing (MLSA and MLST) based on six housekeeping genes (atpD, dnaK, gyrB, ppK, recA, and rpoB). We compared this "framework" with phenotypic and genotypic characteristics such as pathogenicity on tomato, reaction to two antisera by immunofluorescence and to five PCR identification tests, and the presence of four genes encoding the main C. michiganensis subsp. michiganensis pathogenicity determinants. We showed that C. michiganensis subsp. michiganensis is monophyletic and is distinct from its closest taxonomic neighbors. The nonpathogenic Clavibacter-like strains were identified as C. michiganensis using 16S rRNA gene sequencing. These strains, while cross-reacting with C. michiganensis subsp. michiganensis identification tools, are phylogenetically distinct from the pathogenic strains but belong to the C. michiganensis clade. C. michiganensis subsp. michiganensis clonal complexes linked strains from highly diverse geographical origins and also strains isolated over long periods of time in the same location. This illustrates the importance of seed transmission in the worldwide dispersion of this pathogen and its survival and adaptation abilities in a new environment once introduced.
Assuntos
Actinomycetales/classificação , Actinomycetales/genética , Sementes/microbiologia , Solanum lycopersicum/microbiologia , Actinomycetales/isolamento & purificação , Actinomycetales/patogenicidade , Proteínas de Bactérias/genética , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Ribossômico/química , DNA Ribossômico/genética , Dados de Sequência Molecular , Tipagem de Sequências Multilocus , Filogenia , Doenças das Plantas/microbiologia , RNA Ribossômico 16S/genética , Fatores de Virulência/genéticaRESUMO
During direct somatic embryogenesis in leaves of Cichorium hybrid clone '474', 38 kDa beta-1,3-glucanases are accumulated in the culture medium of the embryogenic hybrid to a higher level when compared with a non-embryogenic cultivar. In the same time, embryogenic cells were surrounded by a cell wall that was characterized by the presence of callose. This callosic deposition disappeared as embryos grew. Callose consisted of beta-1,3-glucan linkages and so represented a possible substrate for beta-1,3-glucanases. Using immunolocalization experiments, we demonstrated that from the three types of callose deposits observed during the culturing of Cichorium leaf explants, only the callose present in the walls surrounding reactivated cells seemed specifically related to somatic embryogenesis. Moreover, callose and the 38-kDa beta-1,3-glucanases were co-localized dispersed throughout the thick and swelled walls of reactivated cells and embryo cell walls. This suggests that callose and beta-1,3-glucanases are implicated in the process of somatic embryogenesis since they were always detected in or quite near embryogenic and embryo cell. This also suggested that beta-1,3-glucanases could be involved in the degradation of this callose.
