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

Please note that the date you requested in the address for this web page is not an actual date upon which a change occurred to this item of legislation. You are being shown the legislation from , which is the first date before then upon which a change was made.

[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

B. IDENTIFICATION TESTS 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

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

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).]