Influence of native microbiota on survival of Ralstonia solanacearum phylotype II in river water microcosms

Abstract

Ralstonia solanacearum phylotype II biovar 2 causes bacterial wilt in solanaceous hosts, producing severe economic losses worldwide. Waterways can be major dissemination routes of this pathogen, which is able to survive for long periods in sterilized water. However, little is known about its survival in natural water when other microorganisms, such as bacteriophages, other bacteria, and protozoa, are present. This study looks into the fate of a Spanish strain of R. solanacearum inoculated in water microcosms from a Spanish river, containing different microbiota fractions, at 24 degrees C and 14 degrees C, for a month. At both temperatures, R. solanacearum densities remained constant at the initial levels in control microcosms of sterile river water while, by contrast, declines in the populations of the introduced strain were observed in the nonsterile microcosms. These decreases were less marked at 14 degrees C. Lytic bacteriophages present in this river water were involved in the declines of the pathogen populations, but indigenous protozoa and bacteria also contributed to the reduced persistence in water. R. solanacearum variants displaying resistance to phage infection were observed, but only in microcosms without protozoa and native bacteria. In water microcosms, the temperature of 14 degrees C was more favorable for the survival of this pathogen than 24 degrees C, since biotic interactions were slower at the lower temperature. Similar trends were observed in microcosms inoculated with a Dutch strain. This is the first study demonstrating the influence of different fractions of water microorganisms on the survival of R. solanacearum phylotype II released into river water microcosms.

FIG. 1.

Dynamics of R. solanacearum ph II strain IVIA-1602.1: total (⋄), viable (□), and culturable cell counts on YPGA (▵) and SMSA (○) plates at 24°C (left) and 14°C (right) in river water microcosms. Results are shown for sterile control water (A, E), 0.2-μm-filtered water (B, F), 0.8-μm-filtered water (C, G), and untreated water (D, H). For 0.2-μm-filtered water microcosms, dashed lines refer to a phage-resistant variant of the inoculated strain of R. solanacearum. The detection thresholds of the techniques were ∼10² cells/ml for IF and ∼10 CFU/ml for YPGA and SMSA. Data points for values below the detection limit are shown as faded lines. Points are the means for two separate assays performed in duplicate, and error bars indicate variation as the standard deviation for each point. Similar results were obtained for the other two water samples.

FIG. 2.

Dynamics of a selected phage-resistant variant of R. solanacearum ph II strain IVIA-1602.1: total (⋄), viable (□), and culturable (○) cell counts on SMSA plates at 14°C in river water microcosms. Results are shown for sterile control water (A), 0.2-μm-filtered water (B), 0.8-μm-filtered water (C), and untreated water (D). Points are the means for two separate assays performed in duplicate, and error bars indicate variation as the standard deviation for each point.

FIG. 3.

Time course of the interaction between R. solanacearum ph II strain IVIA-1602.1 and a selected river water phage coinoculated at different ratios in sterile natural water at 24°C (A, B, C, D) and at 14°C (E, F). Total R. solanacearum cell counts (⋄) are shown only in panels B, C, E, and F. Culturable R. solanacearum cells (▵) and plaque counts of the phage (▪) were both on YPGA. The bacterium (CFU/ml)-to-phage (PFU/ml) ratios were 10⁶ to 2 × 10² (A), 10⁶ to 2 × 10³ (B, E), 10⁶ to 7 × 10³ (C, F), and 10⁶ to 2 × 10⁴ (D). Dashed lines refer to a phage-resistant variant of the inoculated strain of R. solanacearum. Points are the means for two separate assays performed in duplicate, and error bars indicate variation as the standard deviation for each point.