Council Directive 93/85/EECShow full title

[F1 9. IDENTIFICATION U.K.

Identify pure cultures of presumptive C. m. subsp. sepedonicus 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.

9.1. Nutritional and enzymatic identification tests U.K.

Determine the following phenotypic properties which are universally present or absent in C. m. subsp. sepedonicus , according to the methods of Lelliott and Stead (1987), Klement et al. (1990), Schaad (2001), Anonymous (1987).

All media should be incubated at 21 °C and examined after six days. If no growth has occurred, incubate for up to 20 days.

All tests must include a known C. m. subsp. sepedonicus control. Nutritional and physiological tests must be made using inocula from nutrient agar subcultures. Morphological comparisons must be made from nutrient dextrose agar cultures.

Tests	Expected result
Oxidation/Fermentation (O/F) test	Inert or weakly oxidative
Oxidase activity	–
Growth at 37 °C	–
Urease activity	–
Aesculin hydrolysis	+
Starch hydrolysis	– or weak
Tolerance of 7 % NaCl	–
Indole production	–
Catalase activity	+
H 2 S production	–
Citrate utilization	–
Gelatin liquefaction	–
Acid glycerol	–
Acid from lactose	– or weak
Acid from rhamnose	–
Acid from salicin	–
Gram stain (Appendix 9)	+

9.2. IF-test U.K.

(a)

Prepare a suspension of approximately 10 ⁶ cells per ml in IF buffer (Appendix 3).

(b)

Prepare a 2-fold dilution series of an appropriate antiserum.

(c)

Apply the IF procedure (section 4).

(d)

A positive IF test is achieved if the IF titre of the culture is equivalent to that of the positive control.

9.3. PCR test U.K.

(a)

Prepare a suspension of approximately 10 ⁶ cells per ml in ultra pure water (UPW).

(b)

Heat 100 µl of the cell suspension in closed tubes in a heating block or boiling waterbath at 100 °C for four minutes. If required, addition of freshly-prepared NaOH to a final concentration of 0,05M may assist cell lysis. The samples may then be stored at -16 to -24  °C until required.

(c)

Apply appropriate PCR procedures to amplify C. m. subsp. sepedonicus specific amplicons (e. g. Pastrik, 2000; see Appendix 4; Li and de Boer, 1995; Mills et al. , 1997; Pastrik and Rainey, 1999; Schaad et al. , 1999.

(d)

A positive identification of C. m. subsp. sepedonicus is achieved if the PCR amplicons are the same size and have the same restriction fragment length polymorphisms as for the positive control strain.

9.4. FISH test U.K.

(a)

Prepare a suspension of approximately 10 ⁶ cells per ml in UPW.

(b)

Apply the FISH procedure (section 5).

(c)

A positive FISH test is achieved if the same reactions are achieved from the culture and the positive control.

9.5. Fatty acid profiling (FAP) U.K.

(a)

Grow the culture on trypticase soy agar (Oxoid) for 72 hours at 21 °C (+/– 1°).

(b)

Apply an appropriate FAP procedure (Janse, 1991; Stead, 1992).

(c)

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 15:1 Anteiso A, 15:0 Iso, 15:0 Anteiso, 16:0 Iso, 16:0 and 17:0 Anteiso is highly indicative of C. m. sepedonicus . Other genera such as Curtobacterium, Arthrobacter and Micrococcus also have some of these acids but 15:1 Anteiso A is a rare acid in these bacteria but occurs in all Clavibacter spp. at between 1 to 5 %. In C. m. sepedonicus the value is usually around 5 %.

9.6. BOX-PCR U.K.

(a)

Prepare a suspension of approximately 10 ⁶ cells per ml in UPW.

(b)

Apply the test according the procedure (Smith et al. , 2001).]

Textual Amendments

F1 Substituted by Commission Directive 2006/56/EC of 12 June 2006 amending the Annexes to Council Directive 93/85/EEC on the control of potato ring rot.