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Applied and Environmental Microbiology, January 2005, p. 140-148, Vol. 71, No. 1
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.1.140-148.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

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

Paola Caruso,¹ Jose Luis Palomo,² Edson Bertolini,¹ Belén Álvarez,¹ María M. López,¹ and Elena G. Biosca^1,3*

Instituto Valenciano de Investigaciones Agrarias (IVIA),¹ Departamento de Microbiología y Ecología, Universidad de Valencia, Valencia,³ Centro Regional de Diagnóstico, Salamanca, Spain²

Received 7 March 2004/ Accepted 17 August 2004

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.

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* Corresponding author. Present address: Departamento de Microbiología y Ecología, Universidad de Valencia, Av. Dr. Moliner 50, Burjassot 46100, Valencia, Spain. Phone: 34 96 354 3194. Fax: 34 96 354 3202. E-mail: elena.biosca{at}uv.es.

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Applied and Environmental Microbiology, January 2005, p. 140-148, Vol. 71, No. 1
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.1.140-148.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

This article has been cited by other articles:

• Alvarez, B., Lopez, M. M., Biosca, E. G. (2008). Survival strategies and pathogenicity of Ralstonia solanacearum phylotype II subjected to prolonged starvation in environmental water microcosms. Microbiology 154: 3590-3598 [Abstract] [Full Text]  
• Alvarez, B., Lopez, M. M., Biosca, E. G. (2007). Influence of Native Microbiota on Survival of Ralstonia solanacearum Phylotype II in River Water Microcosms. Appl. Environ. Microbiol. 73: 7210-7217 [Abstract] [Full Text]