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Council Directive 98/57/EC of 20 July 1998 on the control of Ralstonia solanacearum (Smith) Yabuuchi et al.
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Identify pure cultures of presumptive R. solanacearum isolates using at least two of the following tests based on different biological principles. U.K.
Include known reference strains where appropriate for each test performed (see Appendix 3).
Determine the following phenotypic properties, which are universally present or absent in R. solanacearum , according to the methods of Lelliott and Stead (1987), Klement et al. (1990), Schaad (2001).
Test | Expected result |
---|---|
Fluorescent pigment production | – |
Poly-ß-hydroxybutyrate inclusions | + |
Oxidation/fermentation (O/F) test | O+/F– |
Catalase activity | + |
Kovac’s oxidase test | + |
Reduction of nitrate | + |
Utilisation of citrate | + |
Growth at 40 °C | – |
Growth in 1 % NaCl | + |
Growth in 2 % NaCl | – |
Arginine dihydrolase activity | – |
Gelatine liquefaction | – |
Starch hydrolysis | – |
Aesculin hydrolysis | – |
Levan production | – |
Note: If performing only 2 identification tests, do not use another serological test in addition to this method. U.K.
Strain characterisation using one of the following methods is recommended for each new case of isolation of R. solanacearum . U.K.
Include known reference strains where appropriate for each test performed (see Appendix 3).
R. solanacearum is separated into biovars on the basis of the ability to utilise and/or oxidise three disaccharides and three hexose alcohols (Hayward, 1964 and Hayward et al. , 1990). Growth media for the biovar test is described in Appendix 2. The test can be successfully performed by stab inoculating the media with pure cultures of R. solanacearum isolates and incubating at 28 °C. If the media are dispensed into sterile 96 well cell culture plates (200 µl per well) colour change from olive green to yellow can be observed within 72 hours, indicating a positive test result.
Biovar | |||||
---|---|---|---|---|---|
1 | 2 | 3 | 4 | 5 | |
Utilisation of: | |||||
Maltose | – | + | + | – | + |
Lactose | – | + | + | – | + |
D (+) Cellobiose | – | + | + | – | + |
Mannitol | – | – | + | + | + |
Sorbitol | – | – | + | + | – |
Dulcitol | – | – | + | + | – |
Additional tests differentiate biovar 2 sub-phenotypes
a See Lelliott and Stead (1987) | |||
Biovar 2A (Worldwide distribution) | Biovar 2A (Found in Chile and Colombia) | Biovar 2T (Found in tropical areas) | |
---|---|---|---|
Utilisation of trehalose | – | + | + |
Utilisation of meso -inositol | + | – | + |
Utilisation of D ribose | – | – | + |
Pectolytic activity a | low | low | high |
Molecular differentiation of strains in the R. solanacearum complex can be achieved using several techniques, including: U.K.
Specific PCR primers (Pastrik et al , 2002; see Appendix 6) can be used to differentiate strains belonging to division 1 (biovars 3, 4 and 5) and division 2 (biovars 1, 2A and 2T) of R. solanacearum , as originally defined by RFLP analysis (Cook et al. , 1989) and 16S rDNA sequencing (Taghavi et al. , 1996).]
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