Council Directive 98/57/EC of 20 July 1998 on the control of Ralstonia solanacearum (Smith) Yabuuchi et al. (c. 57)

[F1 ANNEX II U.K. TEST SCHEME FOR DIAGNOSIS, DETECTION AND IDENTIFICATION OF RALSTONIA SOLANACEARUM (SMITH) YABUUCHI ET AL.

Textual Amendments

F1 Substituted by Commission Directive 2006/63/CE of 14 July 2006 amending Annexes II to VII to Council Directive 98/57/EC on the control of Ralstonia solanacearum (Smith) Yabuuchi et al..

SECTION VI U.K. OPTIMISED PROTOCOLS FOR DETECTION AND IDENTIFICATION OF R. SOLANACEARUM

A. DIAGNOSTIC AND DETECTION TESTS U.K.

4. Selective isolation U.K.

4.1. Selective plating U.K.

Note: Before using this method for the first time, perform preliminary tests to ensure reproducible detection of 10 ³ to 10 ⁴ colony-forming units of R. solanacearum per ml added to extracts from samples which previously tested negative. U.K.

Use an appropriately validated selective medium such as SMSA (as modified by Elphinstone et al. , 1996; see Appendix 2).

Care is required to differentiate R. solanacearum from other bacteria able to develop colonies on the medium. Furthermore, colonies of R. solanacearum may show atypical morphology if plates are overcrowded or antagonistic bacteria are also present. Where effects of competition or antagonism are suspected, the sample should be re-tested using a different test.

Highest sensitivity of detection by this method can be expected when using freshly prepared sample extracts. However, the method is also applicable for use with extracts which have been stored under glycerol at -68 to -86  °C.

As positive controls, prepare decimal dilutions from a suspension of 10 ⁶ cfu per ml of a virulent biovar 2 strain of R. solanacearum (e.g. NCPPB 4156 = PD 2762 = CFBP 3857). To avoid any possibility of contamination, prepare positive controls totally separately from samples to be tested.

For each newly prepared batch of a selective medium its suitability for growth of the pathogen should be tested before it is used to test routine samples.

Test control material in an identical manner as the sample(s).

4.1.1. Perform an appropriate dilution plating technique aiming to ensure that any background saprophytic colony-forming populations are diluted out. Spread 50 - 100 µl per plate of sample extract and each dilution. U.K.

4.1.2. Incubate plates at 28 °C. Read plates after 48 hours and daily thereafter up to six days. Typical R. solanacearum colonies on SMSA medium are milky white, flat, irregular and fluidal and after three days incubation develop pink to blood-red coloration in the centre with internal streaking or whorling. (see website http://forum.europa.eu.int/Public/irc/sanco/Home/main). U.K.

Note: Atypical colonies of R. solanacearum sometimes form on this medium. These may be small, round, entirely red in colour and non-fluidal or only partially fluidal and therefore difficult to distinguish from saprophytic colony-forming bacteria. U.K.

4.1.3. Purify presumptive R. solanacearum colonies after streaking or dilution plating onto a general nutrient medium to obtain isolated colonies (see Appendix 2). U.K.

4.1.4. Store cultures short-term in sterile water (pH 6 to 8, chlorine free) at room temperature in the dark, or long term in a suitable cryoprotectant medium at -68 to -86  °C or lyophilised. U.K.

4.1.5. Identify presumptive cultures (see Section VI.B.) and perform a pathogenicity test (see Section VI. C). U.K.

Interpretation of selective plating test results U.K.

The selective plating test is negative if no bacterial colonies are observed after six days or if no presumptive colonies typical of R. solanacearum are found, provided that no inhibition is suspected due to competition or antagonism by other bacteria and that typical R. solanacearum colonies are found in the positive controls.

The selective plating test is positive if presumptive R. solanacearum colonies are isolated.

4.2. Enrichment procedure U.K.

Use a validated enrichment medium such as modified Wilbrink broth (see Appendix 2). U.K.

This procedure can be used to selectively increase R. solanacearum populations in sample extracts and increase sensitivity of detection. The procedure also effectively dilutes inhibitors of the PCR reaction (1:100). It should be noted, however, that enrichment of R. solanacearum can fail due to competition or antagonism by saprophytic organisms which are often simultaneously enriched. For this reason, isolation of R.solanacearum from enriched broth cultures may be difficult. In addition, since populations of serologically related saprophytes can be increased, the use of specific monoclonal antibodies rather than polyclonal antibodies is recommended where the ELISA test is to be used.

4.2.1. For enrichment-PCR, transfer 100 µl of sample extract into 10 ml of enrichment broth (Appendix 2) previously aliquoted into DNA-free tubes or flasks. For enrichment-ELISA, higher proportions of sample extract to broth can be used (e.g. 100 µl in 1,0 ml of enrichment broth). U.K.

4.2.2. Incubate for 72 hours at 27 to 30 °C in shaking culture or static culture with caps loosely- fitted to permit aeration. U.K.

4.2.3. Mix well before using in ELISA or PCR tests. U.K.

4.2.4. Treat enriched broth in an identical manner as the sample(s) in the above tests. U.K.

Note: If inhibition of enrichment of R. solanacearum is anticipated, due to high populations of certain competing saprophytic bacteria, enrichment of sample extracts before any centrifugation or other concentration steps may give better results.] U.K.