ANNEX

“Appendix 2 Media for isolation and culture of R. solanacearum

(a) General growth media

Nutrient Agar (NA)

Nutrient Agar (Difco)	23,0 g
Distilled water	1,0 L

Dissolve ingredients and sterilise by autoclaving at 121 °C for 15 min.

Yeast Peptone Glucose Agar (YPGA)

Yeast extract (Difco)	5,0 g
Bacto-Peptone (Difco)	5,0 g
D(+) Glucose (monohydrate)	10,0 g
Bacto-Agar (Difco)	15,0 g
Distilled water	1,0 L

Dissolve ingredients and sterilise by autoclaving at 121 °C for 15 minutes.

Sucrose Peptone Agar (SPA)

Sucrose	20,0 g
Bacto-Peptone (Difco)	5,0 g
K2HPO4	0,5 g
MgSO4.7H2O	0,25 g
Bacto-Agar (Difco)	15,0 g
Distilled water	1,0 l
pH 7,2 – 7,4

Dissolve ingredients and sterilise by autoclaving at 121 °C for 15 minutes.

Kelman’s Tetrazolium Medium

Casamino acids (Difco)	1,0 g
Bacto-Peptone (Difco)	10,0 g
Dextrose	5,0 g
Bacto-Agar (Difco)	15,0 g
Distilled water	1,0 l

Dissolve ingredients and sterilise by autoclaving at 121 °C for 15 minutes.

Cool to 50 °C and add a filter-sterilised solution of 2,3,5-triphenyl tetrazolium chloride (Sigma) to obtain a final concentration of 50 mg per l.

(b) Validated selective growth media

SMSA medium (Englebrecht, 1994 as modified by Elphinstone et al., 1996)

Basal medium
Casamino acids (Difco)	1,0 g
Bacto-Peptone (Difco)	10,0 g
Glycerol	5,0 ml
Bacto-Agar (Difco); see Note 2.	15,0 g
Distilled water	1,0 L

Dissolve ingredients and sterilise by autoclaving at 121 °C for 15 minutes.

Cool to 50 °C and add filter-sterilised aqueous stock solutions of the following ingredients to obtain the specified final concentrations:

Crystal Violet (Sigma)	5 mg per l
Polymixin-B-Sulphate (Sigma P-1004)	600 000 U (approximately 100 mg) per l
Bacitracin (Sigma B-0125)	1 250 U (approximately 25 mg) per l
Chloramphenicol (Sigma C-3175)	5 mg per l
Penicillin-G (Sigma P-3032)	825 U (approximately 0,5 mg) per l
2,3,5-triphenyl tetrazolium chloride (Sigma)	50 mg per l

Note:

1. Use of reagents other than those specified above may affect growth of R. solanacearum.

2. Oxoid Agar #1 can be used in place of Bacto-Agar (Difco). In this case growth of R. solanacearum will be slower, although growth of competing saprophytes may also be reduced. Typical colonies of R. solanacearum may take 1 to 2 days longer to form and the red colouration may be lighter and more diffuse than on Bacto-Agar.

3. Increasing bacitracin concentration to 2 500 U per l may reduce populations of competing bacteria without affecting growth of Ralstonia solanacearum.

Store media and stock solutions of antibiotics at 4 °C in the dark and use within one month.

Plates should be free from surface condensation before use.

Avoid excess drying of plates.

Quality control should be performed after preparation of each new batch of medium by plating a suspension of a reference culture of R. solanacearum (see Appendix 3) and observing formation of typical colonies after incubation at 28 °C for two to five days.

(c) Validated enrichment media

SMSA Broth (Elphinstone et al., 1996)

Prepare as for SMSA selective agar medium but omit Bacto-Agar and 2,3,5-tetrazolium chloride.

Modified Wilbrink broth (Caruso et al., 2002)

Sucrose	10 g
Proteose peptone	5 g
K2HPO4	0,5 g
MgSO4	0,25 g
NaNO3	0,25 g
Distilled water	1 l

Sterilise by autoclaving at 121 °C for 15 minutes and cool to 50 °C

Add antibiotic stock solutions as for SMSA broth.”