I need tips for isolating Ralstonia solanacearum from eucalyptus. I have used some methods, such as isolating from root exudates, but it didn't work. I also tried indirect isolation from macerated infected tissue using Kelman, NYDA, and TTC media. I know it is Ralstonia based on the agglutination test results. I would like to know if there are alternative methods.
Alternative Methods
Media and Growth Conditions
Sample Handling
Molecular Techniques
Additional Tips
- Sample Collection: Collect samples from different parts of the plant, including roots, stems, and leaves, to increase the likelihood of detecting the pathogen.
- Mixed Infection: Be aware of the possibility of mixed infections and consider using multiple isolation techniques to ensure R. solanacearum is isolated.
Debora Santos Ralstonia, in plants with symptoms of wilting, is often found in combination with secondary pathogens and saprophytes. Some of them have similar phenotype on TCC, and such as Advenella sp. can give you positive PCR reaction with many reported Rs-specific primers . To isolate the pathogen, you should take the youngest infected shoots and cut the stems along the xylem vessels. Using a microbiological metallic loop, gently tear the vessel tissue and spread the inoculum on petri plates containing general-purpose agar (YDC or King's B). Use RS-selective media to culture single suspected colonies. For comparison and confirmation of colony identity, use a reference strain of R. solanacearum and perform an agglutination test. It is important to note that polyclonal antibodies may not always be specific enough for practical purposes.
SMSA medium is probably the best for Ralstonia isolation from mixed populations and verification of isolates.
Imazaki, I., Nakaho, K. Pyruvate-amended modified SMSA medium: improved sensitivity for detection of Ralstonia solanacearum . J Gen Plant Pathol 76, 52–61 (2010). https://doi.org/10.1007/s10327-009-0208-7
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