Detection of Erwinia spp.
Sylvie Priou & Liliam Gutarra
International Potato Center (CIP), Lima, Perú
1. Detection from stems exhibiting blackleg
Cut a fragment of the stem exhibiting blackleg and take with a flame sterilized blade
(scalpel) the vascular tissue just above the visible rotted tissue. Squash with 0.5 ml of
sterile distilled water. Dilute 1:100 (100l of stem extract + 9.9 ml sterile distilled water),
agitate and streak 100l on CVP medium. After 48 h incubation at 25-27C the
characteristic pits formed by the pectinolytic erwinias are observed (see picture and
paper from Perombelon and Burnett, 1991).
2. Detection from water
The irrigation water can be directly streaked on CVP medium with a glass bar (Drigalski
spatula; 100l / plate or four drops with the Pasteur pipette), analyze several samples.
After 48 h incubation at 25-27C the characteristic pits formed by the pectinolytic
erwinias are observed (see picture). The detection limit is 104 bact./l of water.
The bacteria can also be concentrated by centrifuging 200 ml of water at 8,000-10,000
rpm. The pellet is resuspended in water and streaked on CVP medium.
3. Detection from soil
The screenhouse soil can be analyzed by mixing 3 g with the Pectate enrichment
medium in order to complete to a volume of 20 ml (analyze several soil samples) in a
20 ml-beaker or other flask. If you don’t have a 20 ml beaker add more soil and the
corresponding volume of medium in order to fill the flask completely. Cover with a
parafilm paper or a cap and incubate 48 h at 22-25C. Streak 50 l (or two drops) on
CVP medium.
4. Latent infection in tubers
1. Method from De Boer and Kelman (1975), but without wounding the tubers.
A water film is maintained on the tuber surface, by wrapping the tubers in wet paper
and then in saran wrap (thin plastic film). The wrapped tubers are then put inside a
sealed plastic bag and incubated for 4 days at 22-25C, depending of the
environmental conditions where occur the Erwinia diseases. Small cream colored
rotting lesions form around the wounds. When sliced out and exposed to the air, the
rots are brown discolored. To confirm that the rotting is not due to other bacterial
saprophytes, the rotted tissue should be dispersed in sterile water and streaked on CVP
to isolate the Erwinia spp as detailed above.
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2. Enrichment procedure of Perombelon et al. (1987).
Latent contamination of tubers with small numbers of erwinias may be detected by
incubating the tubers (as previously described) for only 2 days at 22-25C, depending of
the environmental conditions where occur the Erwinia diseases. The tubers are then
peeled and fragments around the lenticels are squashed with a hammer in plastic bags
by adding 0.5 ml of the extraction buffer (sodium sulfite 0.2 % in sterile distilled water)
per g of peel. The peel extract can be directly streaked on CVP medium or after a 10 -3
dilution in water.
5. Selective growth media:
The crystal violet pectate (CVP) medium from Perombelon and Burnett (1991) is highly
suitable for isolation of Erwinia spp. from plants, soil and water.
Ingredients:
10% CaCl2.2H2O
NaNO3
Agar (Difco)
Sodium polypectate (Bulmer)
0.075% Crystal violet
10% Sodium Lauryl Sulfate (=SDS)
Trisodium citrate dihydrate
Tryptone
Distilled water
Procedure:
6.8 ml
1.0 g
2.0 g
9.0 g
1.0 ml
0.5 ml
2.5 g
0.5 g
500 ml
a) Preheat a blender by rinsing with hot water,
b) Place 500 ml boiling distilled water in the blender,
c) Blend at low speed the following ingredients together: CaCl2.2H2O,
aNO3, trisodium citrate, tryptone, crystal violet and agar,
d) Slowly add the polypectate,
e) Blend at high speed,
f) Place the medium in a 2-liter flask containing the SDS,
g) Adjust the pH at 7.5 with NaOH 1N,
h) Autoclave for 25 min at 120C,
i) Do not allow to cool before pouring,
j) Allow the surface of the medium to dry before use (one day),
k) Streak 50 l or two drops of the extracts with a Drigalski spatula and
incubate 48 h at 25-27C.
Soft rot erwinias form easily-recognized colonies in steep-sided pits (see picture),
although care is needed so as not confuse them with the shallower concave
depressions formed by the pectinolytic pseudomonads.
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6. Enrichment procedure
Enrichment of small populations of erwinias in soil, water or plant material in Pectate
enrichment medium (Meneley and Stanghellini, 1976) facilitates their detection.
Ingredients:
MgSO4.7H2O
(NH4)2 SO4
K2HPO4
Sodium polypectate (Bulmer)
Distilled water
Procedure:
a)
b)
c)
d)
e)
f)
0.32 g
1.08 g
1.08 g
1.62 g
1000 ml
Dissolve each salt in 1/3 of the distilled water;
add MgSO4 solution to (NH4)2 SO4 solution and mix well;
add K2HPO4 solution and mix well;
add the sodium polypectate and steam for 1 h;
adjust pH to 7.2;
autoclave for 15 min at 120C.
Erwinia spp. can be detected after anaerobic incubation for 48 h at 25-27C, by
streaking 50 l or two drops of the enriched extracts on CVP.
7. References
De Boer, S. H. and A. Kelman 1975. Evaluation of procedures for detection of
pectinolytic Erwinia spp. on potato tubers. Am. Potato J. 52:117-123.
Meneley, J. C. and M. Stanghellini 1976. Isolation of soft rot Erwinia spp. from
agricultural soils using an enrichment technique. Phytopathology 66: 367-370.
Perombelon, M. C. M., V. M. Lumb and L. J. Hyman 1987. A rapid method to identify
and quantify soft rot erwinias on potato seed tubers. EPPO Bul. 17:25-35.
Perombelon, M. C. M. and E. M. Burnett 1991. Two modified crystal violet pectate
(CVP) media for the detection , isolation and enumeration of soft rot erwinias. Potato
research 34:79-85.
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