Council Directive 93/85/EECShow full title

7. Isolation of C. sepedonicum

Diagnosis can only be confirmed if C. sepedonicum is isolated and so identified (8). Although C. sepedonicum is a fastidious organism, it can be isolated from symptomatic tissue. However, it may be outgrown by rapidly growing saprophytic bacteria and, therefore, isolations directly from the tuber tissue pellet (3.3) are not recommended. Eggplants provide an excellent selective enrichment medium for the growth of C. sepedonicum and also provide an excellent confirmatory host test.

Isolations should be made from all symptomatic potato tubers and eggplants (4, 6). Maceration of eggplant stems when necessary should be carried out as in 3. and 6.9.

7.1. Streak suspensions on to one of the following media: (formulae are given in Appendix 6):

nutrient dextrose agar (for subculture only),

yeast peptone glucose agar,

nutrient yeast dextrose agar,

yeast extract mineral salts agar.

Incubate at 21 ^oC for up to 20 days.

C. sepedonicum is slow-growing, usually producing pin-point, cream, domed colonies within 10 days.

Re-streak to establish purity.

Growth rates are improved with subculture. Typical colonies are creamy-white or ivory, rounded, smooth, raised, convex-domed, mucoid-fluidal, with entire edges and usually 1 to 3 mm in diameter.

Identification

Many Gram positive coryneform bacteria, with colonial characters similar to those of C. sepedonicum, may be isolated from healthy or diseased potatoes and eggplants. In this context C. sepedonicum must be identified by the following tests:

IF test (5.1),

eggplant test,

nutrition and physiological tests (Appendix 7),

• oxidation/fermentation test (O/F),

• oxidase test,

• growth at 37^oC,

• urease production,

• aesculin hydrolysis,

• starch hydrolysis,

• tolerance of 7 % sodium chloride solution,

• indole test,

• catalase test,

• H₂8 production,

• citrate utilization,

• gelatin hydrolysis

• acid from: glycerol, lactose, rhamnose and salicin,

• Gram stain.

All tests should include a known C. sepedonicum control. Nutritional and physiological tests should be made using inocula from nutrient agar subcultures. Morphological comparisons should be made from nutrient dextrose agar cultures.

For the IF test, cell populations should be adjusted to 10⁶ cells/ml. The IF titre should be similar to that of the known C. sepedonicum culture.

For the eggplant test cell populations should be adjusted to c 10⁷ cells/ml. Eggplant tests should be made using 10 plants for each of the test organisms, again using known C. sepedonicum culture and sterile water controls; with pure cultures typical wilting should be obtained within 20 days but plants not showing symptons after this time should be incubated for a total of 30 days at temperatures conducive to eggplant growth but not exceeding 30^oC (Appendix 5). If after 30 days symptoms are not present, the culture cannot be confirmed as being a pathogenic form of Corynebacterium sepedonicum.

Test	C. sepedonicum
O/F	Inert or weakly oxidative
Oxidase	-
Catalase	+
Nitrate reduction	-
Urease activity	-
H2S production	-
Indole production	-
Citrate utilization	-
Starch hydrolysis	- or weak
Growth at 37o	-
Growth in 7 % NaCl	-
Gelatin hydrolysis	-
Aesculin hydrolysis	+
Acid from:
— Glycerol	-
— Lactose	- or weak
— Rhamnose	-
— Salicin