Seasonal Variation of Ralstonia solanacearum Biovar 2 Populations in a Spanish River: Recovery of Stressed Cells at Low Temperatures

Abstract

The presence of Ralstonia solanacearum biovar 2 in the watercourses of European countries is increasing, but little is known about its ecology in aquatic habitats. The detection of this pathogen in 2000 in one Spanish river led us to study its population density at different locations on the river over a period of 3 years. During 2000 and 2001, the pathogen was recovered at low densities (10 to 80 CFU/ml) by direct plating on modified SMSA agar from water samples at 14°C or higher, but its isolation was usually unsuccessful at temperatures below 9°C. To monitor the pathogen's abundance in winter, we used two liquid selective media for enrichment (at 29 and 35°C) and compared them by using spiked river water samples: modified Wilbrink broth (MWB) was more efficient than modified SMSA broth for double-antibody-sandwich indirect enzyme-linked immunosorbent assay (DASI-ELISA) detection of R. solanacearum. Enrichment in MWB at both temperatures allowed us to recover R. solanacearum cells that were nonculturable on solid media up to 25 days after their entry into the viable but nonculturable state. When we applied this technique to water samples during the cold months of 2001 and 2002, we obtained the best detection results by the most-probable-number method after enrichment at 35°C with MWB. The enrichment protocol was combined with DASI-ELISA and validated by Co-PCR to detect both naturally and artificially starved and cold-stressed cells in water, which were still infective. Overall, the data from this study demonstrate the effects of temperature variation on the population and culturability of R. solanacearum cells on solid media and their survival at low temperatures.

Survival strategies and pathogenicity of Ralstonia solanacearum phylotype II subjected to prolonged starvation in environmental water microcosms

“…Survival experiments were conducted similarly to Caruso et al (2005). For each of the four water samples, three microcosms were prepared.…”

“…Four river water samples were collected from different sites in Spain (Caruso et al, 2005), and each of them was separately used for survival experiments. Mineral nutrient concentrations ranged as follows, depending on the water sample (values per litre): Na, 5.7-12.2 mg; K, 1.4-1.6 mg; Mg, 3.5-3.8 mg; Ca, 10.3-12.2 mg; Fe, 0.21-0.25 mg; Mn, 0.04-0.06 mg; NO 3 2 , 4.5-6.8 mg, and P 2 O 5 , 0.24-0.36 mg. Organic matter contents estimated by biochemical oxygen demand were ,1.8 mg O 2 l 21 ; pH values of the different water samples were around neutrality, from 6.5 to 7.3.…”

“…After destruction of the plant, an inter-host life period has been proposed for this pathogen (Denny et al, 1994), where it would have to cope with the stress of oligotrophic habitats. Survival studies in sterile water have reported persistence of the bacterium for variable periods (Kelman, 1956;van Elsas et al, 2001;van Overbeek et al, 2004;Wakimoto et al, 1982), and occasional capacity to wilt susceptible hosts (Caruso et al, 2005;van Overbeek et al, 2004). Although some environmental factors affecting R. solanacearum physiology have been reviewed (Hayward, 1991;Kelman, 1953;van Elsas et al, 2005), knowledge of the survival strategies used by this pathogen under prevailing starvation in aquatic habitats is still scarce .…”

“…The occurrence of disease outbreaks linked to the presence of R. solanacearum phylotype (ph) II biovar (bv) 2 in European waterways (Caruso et al, 2005;van Elsas et al, 2000van Elsas et al, , 2001, together with the existence of European Union (EU) Directives banning irrigation of host plants with contaminated water (Anonymous, 1998(Anonymous, , 2000(Anonymous, , 2006, makes the establishment of R. solanacearum in non-host environments a matter of concern. As the life of R. solanacearum in water is still little known, this work aimed to investigate the survival mechanisms exhibited by ph II bv 2 of this pathogen in response to nutrient deprivation in environmental water microcosms over 4 years.…”

“…Under prolonged oligotrophy, reduction in cell size and change to a round cell shape are also considered as bacterial strategies to survive in the environment (Novitsky & Morita, 1976;Rollins & Colwell, 1986;Ruiz et al, 2001), as well as the induction of a viable but non-culturable (VBNC) state (Oliver, 2005;Roszak & Colwell, 1987) that constitutes a distinct physiological state from that of the starvation response (Heim et al, 2002). R. solanacearum becomes VBNC by exposure to copper (Grey & Steck, 2001) and after incubation at low temperature in soil and water (Caruso et al, 2005;van Elsas et al, 2000van Elsas et al, , 2001van Overbeek et al, 2004). Adaptations to oligotrophy have not previously been described for R. solanacearum.…”