Ralstonia solanacearum is a soilborne plant pathogen that normally invades hosts through their roots and then systemically colonizes aerial tissues. Previous research using wounded stem infection found that the major factor in causing wilt symptoms was the high-molecular-mass acidic extracellular polysaccharide (EPS I), but the beta-1,4-endoglucanase (EG) also contributes to virulence. We investigated the importance of EPS I and EG for invasion and colonization of tomato by infesting soil of 4-week-old potted plants with either a wild-type derivative or genetically well-defined mutants lacking EPS I, EG, or EPS I and EG. Bacteria of all strains were recovered from surface-disinfested roots and hypocotyls as soon as 4 h after inoculation; that bacteria were present internally was confirmed using immunofluorescence microscopy. However, the EPS-minus mutants did not colonize stems as rapidly as the wild type and the EG-minus mutant. Inoculations of wounded petioles also showed that, even though the mutants multiplied as well as the wild type in planta, EPS-minus strains did not spread as well throughout the plant stem. We conclude that poor colonization of stems by EPS-minus strains after petiole inoculation or soil infestation is due to reduced bacterial movement within plant stem tissues.

One susceptible and two resistant cultivars of tomato were tested for differences in infection by Ralstonia solanacearum and for the subsequent multiplication, colonization, and production of the wilt-inducing virulence factor, exopolysaccharide I (EPS I). Bacterial ingress into the taproot was fastest in the susceptible cv. Marion, followed by the resistant cvs. L285 (fivefold slower) and Hawaii 7996 (15-fold slower). Once inside the taproot, R. solanacearum colonized, to some extent, almost all regions of the resistant and susceptible plants. However, colonization occurred sooner in the susceptible than in the resistant cultivars, as measured by viablecell counts of bacteria in the midstems. Rates of multiplication and maximum bacterial cell densities were also greater in the susceptible than in the resistant cultivars. Growth experiments utilizing xylem fluid from infected and uninfected plants indicated that neither antimicrobial activities nor reduced levels of growth-supporting nutrients in the xylem fluids were responsible for the reduced bacterial multiplication in the resistant cultivars. Quantification of EPS I in the infected plants, using an enzyme-linked immunosorbent assay, revealed that the bacterial populations in the susceptible cultivar produced greater amounts of EPS I per plant than those in the resistant cultivars. Immunofluorescence microscopy using antibodies against either EPS I or R. solanacearum cells revealed that bacteria and EPS I were distributed throughout the vascular bundles and intercellular spaces of the pith in the susceptible cultivar, whereas in the resistant cultivars, bacteria and EPS I were restricted to the vascular tissues.

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