[ANNEX II U.K. TEST SCHEME FOR DIAGNOSIS, DETECTION AND IDENTIFICATION OF RALSTONIA SOLANACEARUM (SMITH) YABUUCHI ET AL.
SECTION VI U.K. OPTIMISED PROTOCOLS FOR DETECTION AND IDENTIFICATION OF R. SOLANACEARUM
B. IDENTIFICATION TESTS U.K.
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).
1. Nutritional and enzymatic identification tests U.K.
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 | – |
2. IF test U.K.
2.1. Prepare a suspension of approximately 10 6 cells per ml in IF buffer (Appendix 4). U.K.
2.2. Prepare a twofold dilution series of an appropriate antiserum (see website http://forum.europa.eu.int/Public/irc/sanco/Home/main). U.K.
2.3. Apply the IF procedure (Section VI.A.5.). U.K.
2.4. A positive IF test is achieved if the IF titre of the culture is equivalent to that of the positive control. U.K.
3. ELISA test U.K.
Note: If performing only 2 identification tests, do not use another serological test in addition to this method. U.K.
3.1. Prepare a suspension of approximately 10 8 cells per ml in 1X PBS (Appendix 4). U.K.
3.2. Perform an appropriate ELISA procedure with a specific monoclonal antibody to R. solanacearum . U.K.
3.3. A positive ELISA test is achieved if the ELISA reading obtained from the culture is at least half that obtained for the positive control. U.K.
4. PCR tests U.K.
4.1. Prepare a suspension of approximately 10 6 cells per ml in molecular grade sterile water. U.K.
4.2. Heat 100 µl of the cell suspension in closed tubes in a heating block or boiling waterbath at 100 °C for four minutes. The samples may then be stored at -16 to -24 °C until required. U.K.
4.3. Apply appropriate PCR procedures to amplify R. solanacearum -specific amplicons (e.g. Seal et al. (1993); Pastrik and Maiss (2000); Pastrik et al. (2002); Boudazin et al. (1999); Opina et al. (1997), Weller et al. (1999). U.K.
4.4. A positive identification of R. solanacearum is achieved if the PCR amplicons are the same size and have the same restriction fragment length polymorphisms as for the positive control strain. U.K.
5. FISH test U.K.
5.1. Prepare a suspension of approximately 10 6 cells per ml in UPW. U.K.
5.2. Apply the FISH procedure (Section VI.A.7.) with at least 2 R. solanacearum -specific oligo-probes (Appendix 7). U.K.
5.3. A positive FISH test is achieved if the same reactions are achieved from the culture and the positive control. U.K.
6. Fatty acid profiling (FAP) U.K.
6.1. Grow the culture on trypticase soy agar (Oxoid) for 48 hours at 28 °C. U.K.
6.2. Apply an appropriate FAP procedure (Janse, 1991; Stead, 1992). U.K.
6.3. A positive FAP test is achieved if the profile of the presumptive culture is identical to that of the positive control. The presence of characteristic fatty acids are 14:0 3OH, 16:0 2OH, 16:1 2OH and 18:1 2OH and absence of 16:0 3OH is highly indicative of a Ralstonia sp. U.K.
7. Strain characterisation methods 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).
7.1. Biovar determination U.K.
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
| |
| 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 | low | low | high |
7.2. Genomic fingerprinting U.K.
Molecular differentiation of strains in the R. solanacearum complex can be achieved using several techniques, including: U.K.
7.2.1. Restriction fragment length polymorphism (RFLP) analysis (Cook et al. , 1989). U.K.
7.2.2. Repetitive sequence PCR using REP, BOX and ERIC primers (Louws et al. , 1995; Smith et al. , 1995). U.K.
7.2.3. Amplified fragment length polymorphism (AFLP) analysis (Van der Wolf et al. , 1998). U.K.
7.3. PCR methods 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).]