Council Directive 93/85/EEC of 4 October 1993 on the control of potato ring rot (c. 85)

[F1 ANNEX I U.K. TEST SCHEME FOR DIAGNOSIS, DETECTION AND IDENTIFICATION OF THE RING ROT BACTERIUM, CLAVIBACTER MICHIGANENSIS (Smith) Davis et al. ssp. SEPEDONICUS (Spieckermann et Kotthoff) Davis et al. SCOPE OF THE TEST SCHEME

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.

Appendix 5 Media for isolation and culture of C. m. subsp. sepedonicus

(b) Validated selective growth media U.K.

MTNA medium U.K.

Unless otherwise stated all media components are from BDH.

Yeast extract (Difco)	2,0 g
Mannitol	2,5 g
K 2 HPO 4	0,25 g
KH 2 PO 4	0,25 g
NaCl	0,05 g
MgSO 4 .7H 2 O	0,1 g
MnSO 4 .H 2 O	0,015 g
FeSO 4 .7H 2 O	0,005 g
Agar (Oxoid no. 1)	16,0 g
Distilled water	1,0 L

Dissolve ingredients, adjust pH to 7,2. After autoclaving (at 121 °C for 15 minutes) and cooling down to 50 °C, add the antibiotics: trimethoprim 0,06 g, nalidixic acid 0,002 g, amphotericin B 0,01 g.

Stock antibiotic solutions: trimethoprim (Sigma) and nalidixic acid (Sigma) (both at 5 mg/ml), in 96 % methanol, amphotericin B (Sigma) (1 mg/ml) in dimethyl sulfoxide. Stock solutions are filter-sterilized.

Note: U.K.

Durability of basal medium is three months. After antibiotics are added durability is one month when stored refrigerated.

NCP-88 medium U.K.

Nutrient agar (Difco)	23 g
Yeast extract (Difco)	2 g
D-mannitol	5 g
K 2 HPO 4	2 g
KH 2 PO 4	0,5 g
MgSO 4 .7H 2 O	0,25 g
Distilled water	1,0 L

Dissolve ingredients, adjust pH to 7,2. After autoclaving and cooling down to 50 °C, add the following antibiotics: Polymyxin B sulphate (Sigma) 0,003 g, nalidixic acid (Sigma) 0,008 g, Cycloheximide (Sigma) 0,2 g.

Dissolve antibiotics in stock solutions as follows: nalidixic acid in 0,01 M NaOH, cycloheximide in 50 % ethanol, polymyxin B sulphate in distilled water. Stock solutions are filter-sterilized.

Note: U.K.

Durability of basal medium is three months. After antibiotics are added durability is one month when stored refrigerated.]