Ralstonia solanacearum Facing Spread-Determining Climatic Temperatures, Sustained Starvation, and Naturally Induced Resuscitation of Viable but Non-Culturable Cells in Environmental Water

Abstract

Ralstonia solanacearum is a bacterial phytopathogen affecting staple crops, originally from tropical and subtropical areas, whose ability to survive in temperate environments is of concern under global warming. In this study, two R. solanacearum strains from either cold or warm habitats were stressed by simultaneous exposure to natural oligotrophy at low (4 °C), temperate (14 °C), or warm (24 °C) temperatures in environmental water. At 4 °C, the effect of temperature was higher than that of oligotrophy, since R. solanacearum went into a viable but non-culturable (VBNC) state, which proved to be dependent on water nutrient contents. Resuscitation was demonstrated in vitro and in planta. At 14 °C and 24 °C, the effect of oligotrophy was higher than that of temperature on R. solanacearum populations, displaying starvation-survival responses and morphological changes which were stronger at 24 °C. In tomato plants, starved, cold-induced VBNC, and/or resuscitated cells maintained virulence. The strains behaved similarly regardless of their cold or warm areas of origin. This work firstly describes the natural nutrient availability of environmental water favoring R. solanacearum survival, adaptations, and resuscitation in conditions that can be found in natural settings. These findings will contribute to anticipate the ability of R. solanacearum to spread, establish, and induce disease in new geographical and climatic areas.

Keywords: VBNC; bacterial wilt; environmental stress; global warming; pathogenicity.

Figure 6

Influence of temperature and oligotrophy on Ralstonia solanacearum stress induction in environmental water and stress responses by the bacterium. Adapted to R. solanacearum from a proposed model by [67]. See text for details.

Figure 1

Effect of low temperature under nutrient-limiting conditions on survival of Ralstonia solanacearum strain IVIA-1602.1 during 40-day periods in water. Microcosms of: EW−4, environmental water at 4 °C; DW−4, distilled water at 4 °C; EW−24, environmental water at 24 °C, and DW−24, distilled water at 24 °C. Total (■), viable (●), and culturable cells on SMSA (▲) and YPGA (♦) media. Data from one representative environmental water sample have been plotted. Points are mean ± standard deviation of triplicate microcosms.

Figure 2

Similarity in trends of culturable cell counts from Ralstonia solanacearum strain IVIA-1602.1-inoculated environmental water microcosms throughout 40-day periods at 4 °C and 24 °C. Inc stands for Increments, which were calculated with the differences between mean values of culturable data at both temperatures with respect to the values at time zero (Inc zero). (Left): comparison of culturable data on SMSA medium among environmental water (EW)—1 (♦), EW—2 (●), EW—3 (▲), and EW—4 (■). (Right): comparison between culturable data on SMSA (■), and YPGA (▲) media for the four environmental water samples.

Figure 3

Proportions of coccoid cells appearing in Ralstonia solanacearum strain IVIA-1602.1 populations starved in the environmental water microcosms during 40-day periods at 24 °C, 14 °C, and 4 °C. (Left) Symbols for each temperature: 24 °C (●), 14 °C (■), and 4 °C (▲). Data from one representative environmental water sample have been plotted. Points are mean ± standard deviation of triplicate microcosms. (Right) Representative fluorescence microscopy images of proportions of coccoid cells (white arrows) of the bacterium at each temperature. Scale bars: 5 µm.

Figure 4

Pathogenicity of Ralstonia solanacearum strain IVIA-1602.1 previously starved in environmental water microcosms during 40 days, in tomato plants cv. ‘Roma’. Viable (red bars) and culturable (pink bars) cells per plant, and percentage of wilted plants (■). Only assays performed in at least weekly intervals from one representative environmental water (EW) at 4 °C (EW−4), 14 °C (EW−14) and 24 °C (EW−24) have been plotted. Points are mean ± standard deviation (SD) of triplicate microcosms. Absolute value for 100% wilting refers to 24 plants (6 × 4 sets). At the three temperatures, SD of wilting values for most of the points was zero, and ± 2.0% in some cases. Control plants inoculated with freshly grown R. solanacearum strain IVIA-1602.1 developed 100% wilting, while those inoculated with PBS were negative.

Figure 5

Viability of Ralstonia solanacearum strain IVIA-1602.1 populations at 40 days post-inoculation in the environmental water microcosms. (A,B) starved cells at 24 °C, and (C,D) starved and cold-induced VBNC cells at 4 °C. Viability was similar between starved (A,B) and VBNC (C,D) R. solanacearum cells, although in (C,D) cells were no culturable on solid media. For VBNC cells, this time was considered time zero of VBNC induction of the R. solanacearum populations in the resuscitation assays. Viability was measured by the DVC method and subsequent staining with acridine orange [41,42], and estimated in 10⁶ VBNC cells/mL. Scale bars: 5 µm.