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The Low-Temperature-Induced Viable-But-Nonculturable State Affects the Virulence of Ralstonia solanacearum Biovar 2

The physiology and virulence of Ralstonia solanacearum biovar 2 strain 1609, kept in water at 4 and 20°C, were studied. At 20°C, total cell and plate count (colony forming units; CFU) numbers were similar, between log 5.03 and log 5.55 CFU, and log 5.03 and log 5.51 cells per ml, at days 0 and 132, respectively. However, CFU in the cultures kept at 4°C dropped from log 6.78 CFU/ml at day 0 to below detection after 84 days. The presence of catalase in the agar resulted in higher CFU, and at day 84, log 1.95 CFU/ml still was detectable. No colonies were observed at day 125. The presence of viable-but-nonculturable (VBNC) cells in the 4°C cultures was confirmed using SYTO9 viability staining. Viable cell numbers were log 1.77 higher than CFU on plates with catalase. At day 84 and after 125 days, log 3.70 viable cells per ml still were present. Shifts in subpopulations differing in viability were found by flow cytometric sorting of 4°C-treated cells stained with SYTO9 (healthy) and propidium iodide (PI; compromised). The SYTO9-stained cell fractions dropped from 99 to 39%, and the PI-stained fractions increased from 0.7 to 33.3% between days 0 and 125. At 20°C, the SYTO9-stained fraction remained stable at 99% until day 132. SYTO9-stained cells sorted from 4°C cultures at day 100 were injected into tomato plants. Upon incubation for 30 days, these plants did not show wilting. However, more than log 4.19 CFU and log 8.17 cells were recovered from these plants. Cells from colonies isolated from the nonwilted plants did not regain their virulence as demonstrated by subsequent injection into several new sets of tomato plants. Cells from 4°C cultures injected at day 125 were not able to cause wilting of, or proliferate in, tomato plants. The threat posed by VBNC R. solanacearum cells upon incubation at 4°C was thus ephemeral because cells lost their capacity to cause disease after 125 days.