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.

4.2. 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.