R. solanacearum / Culture media
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| Author: | Patrice G. Champoiseau of University of Florida |
| Reviewers: | Caitilyn Allen of University of Wisconsin; Jeffrey B. Jones, Carrie Harmon and Timur M. Momol of University of Florida |
| Publication date: | September 12, 2008 |
| Project title: | Ralstonia solanacearum race 3 biovar 2: Detection, exclusion, and analysis of a select agent pathogen |
| Supported by: | The United States Department of Agriculture - National Research Initiative Program (2007-2010) |
| Non-selective media | |||||||||
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Non-selective media are generally used for growth of pure culture of R. solanacearum. (e.g. to retrieve cultures from frozen stocks or for successive plating of cells). These media can also be used for isolation of R. solanacearum from fresh, symptomatic plants, due to the high density of the pathogen in the tissues. | |||||||||
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- Casamino acid-Peptone-Glucose (CPG) medium <See reference at the bottom of this page>
Adjust pH to 6.5-7.0 if necessary. Autoclave at 121˚C for 20 minutes. On solid medium, colonies of R. solanacearum usually are visible after 48-72 hours of incubation at 28ºC. Colonies of the normal (or virulent type) are white or cream-colored, irregularly-round, fluidal, and opaque; and colonies of the mutant (or non-virulent) type are uniformly round, smaller, and butyrous (dry). This shift from virulent to non-virulent bacterial cells occurs during storage or under oxygen stress in liquid media. |
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- Tripheny tetrazolium chloride (TTC or TZC) medium <See reference at the bottom of this page> Prepare 1 liter of CPG medium:
Adjust pH to 6.5-7.0 if necessary. Autoclave at 121˚C for 20 minutes. After autoclaving cool the medium to 55˚C and add 5 ml of a 1% stock solution of 2, 3, 5-tripheny tetrazolium chloride. The stock can be filter sterilized or autoclaved for 5 minutes at 121˚C, and stored at 4˚C or frozen. On solid medium, colonies of R. solanacearum usually are visible after 48-72 hours of incubation at 28ºC. This medium was developed to differentiate between the two colony types: virulent colonies appear white with pink centers and non-virulent colonies appear dark red. |
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| Semi-selective media | |||||||||
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Semi-selective media can be used for isolation of the pathogen from nonsymptomatic plants or from water and soil samples. Although widely used, they do not support growth of all R. solanacearum strains and/or do not suppress growth of all related or unrelated Gram-negative bacteria. | |||||||||
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- SM-1 medium <See reference at the bottom of this page> Prepare 1 liter of TTC (or TZC) medium:
Adjust pH to 6.5-7.0 if necessary. Autoclave at 121˚C for 20 minutes. After autoclaving cool the medium to 55˚C and add 5 ml of a 1% stock solution of 2, 3, 5-tripheny tetrazolium chloride. The stock can be filter sterilized or autoclaved for 5 minutes at 121˚C, and stored at 4˚C or frozen.
* Merthiolate tincture contains 1 part merthiolate per 1000 parts of 50% alcohol. Determine the best concentration to suppress local microflora, as suggested <See reference at the bottom of this page>. ** Dissolve in 5 ml of 70% ethanol 30 minutes prior to use. |
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- SMSA medium <See reference at the bottom of this page> Prepare 1 liter of TTC (or TZC) medium, except substitute glycerol (5 ml per liter) for the glucose:
Adjust pH to 6.5-7.0 if necessary. Autoclave at 121˚C for 20 minutes. After autoclaving cool the medium to 55˚C and add 5 ml of a 1% stock solution of 2, 3, 5-tripheny tetrazolium chloride. The stock can be filter sterilized or autoclaved for 5 minutes at 121˚C, and stored at 4˚C or frozen.
* Dissolve in 5 ml of 70% ethanol 30 minutes prior to use. On solid medium, colonies of R. solanacearum usually are visible after 2-5 days of incubation at 28ºC. Typical bacterial colonies appear fluidal, irregular in shape, and white with pink centers. |
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| Reference source Denny, TP; Hayward, AC. 2001. Gram-negative bacteria: Ralstonia. Pages 151-174 in: Laboratory guide for identification of plant pathogenic bacteria, 3rd ed. Schaad, NW; Jones, JB; Chun, W, eds. APS Press, St. Paul, M. N. |
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| Text references - Casamino acid-Peptone-Glucose (CPG) medium Kelman, A. 1954. The relationship of pathogenicity in Pseudomonas solanacearum to colony appearance on a tetrazolium medium. Phytopathology 44:693-695. - Tripheny tetrazolium chloride (TTC or TZC) medium Kelman, A. 1954. The relationship of pathogenicity in Pseudomonas solanacearum to colony appearance on a tetrazolium medium. Phytopathology 44:693-695. - SM-1 medium Granada, GA; Sequeira, L. 1983. A new selective medium for Pseudomonas solanacearum. Plant Disease 67:1084-1088. - Modified SMSA medium Elphinstone, JG; Hennessy, J; Wilson, JK; Stead, DE. 1996. Sensitivity of different methods for the detection of Pseudomonas solanacearum in potato tuber extracts. EPPO/OEPP Bulletin 26:663-678. French, ER; Gutarra, L; Aley, P; Elphinstone, J. 1995. Culture media for Pseudomonas solanacearum isolation, identification and maintenance. Fitopatologia 30:126-130. |
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