Bacterial Stem Rot in Greenhouse

Pepper (Capsicum annuum L.) in

Sardinia (Italy): Occurrence of Erwinia
carotovora subsp. carotovora
1. M. Fiori,
2. A. Schiaffino

Article first published online: 20 JAN 2004

DOI: 10.1046/j.1439-0434.2003.00796.x

Issue

Journal of Phytopathology
Volume 152, Issue 1, pages 28–33, January 2004

Additional Information(Show All)

How to CiteAuthor InformationPublication History

How to Cite

Fiori, M. and Schiaffino, A. (2004), Bacterial Stem Rot in Greenhouse Pepper

(Capsicum annuum L.) in Sardinia (Italy): Occurrence of Erwinia carotovora subsp.
carotovora. Journal of Phytopathology, 152: 28–33. doi: 10.1046/j.1439-
0434.2003.00796.x

Author Information

1. Authors’ address: Dipartimento di Protezione delle Piante, Università

degli studi di Sassari, Via E. De Nicola, 07100 Sassari, Italy (correspondence
to M. Fiori, E-mail: fiorim@uniss.it)

*Correspondence: Dipartimento di Protezione delle Piante, Università degli studi di

Sassari, 07100 Sassari, Italy
Publication History

1. Issue published online: 20 JAN 2004

2. Article first published online: 20 JAN 2004
3. Received March 24, 2003; accepted October 8, 2003

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Keywords:

• Capsicum annuum;
 • stem rot;
• Erwinia carotovora subsp. carotovora;
• Pectobacterium carotovorum subsp. carotovorum;
• polymerase chain reaction restriction fragment length polymorphism

Abstract

An unusual bacterial disease was observed in pepper plants during research carried
out in greenhouses in central-north Sardinia. The characteristics were: the presence of
lesions and exudates on stems, soft rot of the pith, and a brownish-black colour in the
petioles and leaf-veins. Only two isolates of 21 were pathogens. One was obtained
from exudate present on the stem and the other from pith. Experimental infections
revealed that the bacterial isolates were particularly aggressive in the stems and fruit
of pepper and tomato. Biochemical, physiological and serological tests in conjunction
with fatty acid profile analysis confirmed that they were Erwinia carotovora subsp.
carotovora (Jones) Bergey et al. The product of 434 bp polymerase chain reaction
(PCR) enabled a preliminary identification of isolates to be made. Restriction
fragment length polymorphism (RFLP) analysis of amplification products showed
that the isolates DPP 23ef and DPP 24m, strain type CFBP 2046 and DPP 281, isolated
from pepper fruit, belonged to the RFLP group 12, whereas DPP 29, also isolated
from pepper fruit, was included in RFLP group 1. Measures to prevent and control
this recently introduced disease are suggested in the conclusion of this paper.

..…………………………………………………………… .
World Journal of Microbiology and Biotechnology
Volume 8, Number 2, 115-120, DOI: 10.1007/BF01195828

Research Papers

Production of pectolytic enzymes

fromErwinia grown on different carbon
sources
V. E. Shevchik, A. N. Evtushenkov, H. V. Babitskaya and Y. K. Fomichev

Abstract
A quantitative analysis of pectolytic enzymes (polygalacturonase (PG), pectin methyl
esterase (PME) and six isoenzymes of pectate lyase (PL)) produced byErwinia
bacteria in the presence of diverse carbon sources was made by preparative
electrophoresis. Synthesis of each of these enzymes was regulated independently;
different induction and repression ratios (about 10- to 1000-fold) were observed for
diverse PL isoenzymes, PG and PME. The possibility of using specially constructed
media for the production of pectinase complexes with a specific spectra of pectolytic
enzymes has been demonstrated
European Journal of Plant Pathology
Volume 122, Number 4, 561-569, DOI: 10.1007/s10658-008-9325-y

Pectobacterium carotovorum subsp.

carotovorum can cause potato blackleg
in temperate climates
Eisse G. de Haan, Toos C. E. M. Dekker-Nooren, Gé W. van den Bovenkamp, Arjen
G. C. L. Speksnijder, Patricia S. van der Zouwen and Jan M. van der Wolf

Abstract
It is well established that the pectinolytic bacteria Pectobacterium atrosepticum (Pca)
and Dickeya spp. are causal organisms of blackleg in potato. In temperate climates,
the role of Pectobacterium carotovorum subsp. carotovorum (Pcc) in potato blackleg,
however, is unclear. In different western and central European countries plants are
frequently found with blackleg from which only Pcc can be isolated, but not Pca or
Dickeya spp. Nevertheless, tubers vacuum-infiltrated with Pcc strains have so far
never yielded blackleg-diseased plants in field experiments in temperate climates. In
this study, it is shown that potato tubers, vacuum-infiltrated with a subgroup of Pcc
strains isolated in Europe, and planted in two different soil types, can result in up to
50% blackleg diseased plants.

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Biomedical and Life Sciences

European Journal of Plant Pathology
Volume 126, Number 2, 175-185, DOI: 10.1007/s10658-009-9531-2

Original Research
Pectobacterium carotovorum subsp.
brasiliensis causing blackleg on potatoes
in South Africa
Johanna J. van der Merwe, Teresa A. Coutinho, Lise Korsten and Jacqueline E. van
der Waals

Abstract
In South Africa during the 2006/2007 potato growing season, outbreaks of blackleg
occurred, causing severe economic losses in commercial potato production fields.
Symptoms were initially observed on only one stem per plant, on which the top leaves
rolled upwards, wilted and became necrotic. As symptoms progressed to the lower
leaves with subsequent leaf desiccation, a light to dark brown discolouration of the
vascular system at the stem base developed, followed by external darkening. Under
prevailing wet and humid conditions stems became slimy and pale. In the stems, the
pith became necrotic and hollow. These symptoms were similar to those described in
Brazil, where the causal agent was identified as a new subspecies, Pectobacterium
carotovorum subsp. brasiliensis (Pbcb). Isolations from plants showing typical
blackleg symptoms were made on CVP medium. Sequences and phylogenetic
analysis of the partial 16S–23S rDNA intergenic spacer region indicated that the
isolates were Pbcb. Comparison of PCR-RFLP patterns of the 16S–23S rDNA of
isolates to reference cultures confirmed the identity of the South African blackleg
strains as Pbcb, identical to strain 8 isolated in Brazil. This is the first report of Pbcb
in South Africa and it appears to be the most important causal agent of blackleg in
South Africa. The disease poses a major potential threat to the South African potato
industry especially in terms of seed exports, tuber quality and yield.

Differentiation of Erwinia carotovora

subsp. carotovora and Erwinia
carotovora subsp. atroseptica isolated
from potato by Western blot and
subsequent indirect ELISA
Journal of Applied Microbiology
Volume 69, Issue 1, pages 17–24, July 1990
Additional Information(Show All)

How to CiteAuthor InformationPublication History

How to Cite

Alarcon, B., Lopez, M. M., Cambra, M., Gorris, M. T. and Guerri, J. (1990),
Differentiation of Erwinia carotovora subsp. carotovora and Erwinia carotovora
subsp. atroseptica isolated from potato by Western blot and subsequent indirect
ELISA. Journal of Applied Microbiology, 69: 17–24. doi: 10.1111/j.1365-
2672.1990.tb02906.x

Author Information

1. Instituto Valenciano de Investigaciones Agrarias, Apartado Oficial,

46113, Moncada, Valencia, Spain

*Correspondence: M. M. Lopez,

*Correspondence: * Corresponding author.

Electrotransfer of protein bands from a polyacrylamide gel to a hydrophobic poly-

vinylidene difluoride (PVDF) membrane (Western blot) and their serological
determination by indirect ELISA (immunoblotting) were used to differentiate Erwinia
carotovora subsp. carotovora (Ecc) from Erwinia carotovora subsp. atroseptica
(Eca). Ninety strains: 69 Ecc, 19 Eca and two Erwinia chrysanthemi (Echr) were
examined. Eight polyclonal antisera against whole cells, glutaraldehyde fixed cells,
glycopro-teins, and somatic antigens were prepared. Antisera produced with
glutaraldehyde fixed cells did not recognize any band of the protein pattern. The
remaining antisera recognized a limited number of bands. Two protein bands allowed
differentiation of the two subspecies by the antisera against glycoproteins. One band
with an estimated molecular weight of 36000 Da was present in the 19 Eca strains
tested and another band with an estimated molecular weight of 35 000 Da was present
in the 69 Ecc strains, except for three cases. The strains of Echr showed a band with
an estimated weight of 33 000 Da.

Detection, quantification and

characterization of Erwinia carotovora
ssp. carotovora contaminating pepper
seeds

Plant Pathology
Volume 50, Issue 1, pages 117–123, February 2001

Abstract

Erwinia carotovora ssp. carotovora (Ecc) was isolated from commercial pepper
(Capsicum annuum) seed lots and identified according to biochemical and
pathogenicity tests, cellular fatty acid composition, and PCR reaction with primers
based on the pel gene sequence. RAPD-PCR analysis using nine arbitrary primers
showed similarity between Ecc isolates obtained from pepper. Ecc isolates from
pepper, tomato, potato and cabbage formed four distinct groups, according to the
original host. A natural Ecc population higher than 100 CFU g−1 seed significantly
affected seed germination. A significant correlation between the level of Ecc on the
seeds and the percentage of diseased seedlings was also observed. Surface
sterilization of the seeds with sodium hypochlorite eliminated the pathogen, indicating
that the bacteria were located on the surface of the seeds. Contaminated pepper seeds
can be a primary and important source of inoculum

Erwinia carotovora on Potatoes

Media and Enrichment Techniques

Anilkumar, T. B. and B. P. Chakravarti. Survival of Pseudomonas lapsa and

Erwinia carotovora, stalk rot pathogens on maize in seed and a medium for
isolation and detection of Erwinia carotovora in soil. Proc. Indian Natl. Sc.
Acad. 37:322-325.
Ech survived for 3 to 5 months in seeds inoculated by puncturing them with a
needle and soaking them in a bacterial suspension. The medium developed
for isolate from soil is:

First Layer
magnesium
0.2 g
sulfate
mono
ammonium 2.0 g
phosphate
potassium
0.2 g
chloride
sodium citrate 2.0 g
sodium 5.0 g
 chloride
calcium
3.0 g
chloride
sodium
5.0 g
taurocholate
bactobrom-
0.08 g
thymol blue
crystal violet 0.001 g
actidione 100 ppm
agar 20 g
water 1000 ml

Second Layer
sodium
1g
polypectate
EDTA 0.1 g
water 100 ml

Burr, T. J. and M. N. Schroth. 1977. Occurrence of soft-rot Erwinia spp. in soil

and plant material. Phytopathol. 67:1382-1387.

PT Medium g/L
polygalacturonic acid 5
NaNO3 1
K2HPO4 4
MgSO4-6H2O 0.2
sodium heptadecy sulfate
0.1 ml
(tergitol anionic 7)
1N NaOH 1 ml
agarose 18 g

Add all ingredients prior to autoclaving. Erwinia is white with scalloped edges
on this medium. It can be differentiated from other bacteria by flooding the
plates with 1% cetyltrimethylammonium bromide (cetrimide). Centrimide is
toxic, so the cells must be transferred to a new plate within 5 min. of flooding
the PT medium. For samples with low levels of Erwinia, the samples may be
added to PT broth and incubated for 24 hrs at 28C aerobically or for 48 hrs
anaerobically prior to plating on PT agar.

Cuppels, D. and A. Kelman. 1974. Evaluation of selective media for isolation

of soft rot bacteria from soil and plant tissue. Phytopathol. 64:468-475.

CVP per 500

 ml water
1 N NaOH 4.5 ml
10% CaCl2-2H2O 3.0 ml
NaNO3 1.0 g
Agar 1.5 g
Sodium Polypectate 10 g
10% SDS 0.5 ml
0.075% crystal violet 1.0 ml

Pour 300 ml of boiling water into a preheated blender jar, then add the first 4
ingredients. Blend at high speed for 15 seconds while adding the polypectate.
Blend for another 15 seconds while adding 200 ml of hot water. Pour the
medium into a 1 L bottle and add the SDS and crystal violet. Autoclave for 25
min.

The medium will be very soft and translucent with a brown-violet tint. Great
care must be used to not disturb the medium when spreading or streaking
bacteria onto CVP plates.

Friedman, B. A. 1964. Carbon source and tetrazolium agar to distinguish

virulence in colonies of Erwinia carotovora. Phytopathol. 54:494-495.

Kado, C. I., and M. G. Heskett. 1970. Selective media for isolation of

Agrobacterium, Corynebacterium, Erwinia, Pseudomonas, and Xanthomonas.
Phytopathol. 60:969-976.

Kerr, A. 1953. A method for isolating soft-rotting bacteria from soils. Nature
172:1155.

Lee, Y.-A. and C.-P. Yu. A differential medium for the isolation and rapid
identification of a plant soft rot pathogen, Erwinia chrysanthemi. J. Microbiol.
Methods 64:200-206.

23 g nutrient agar
10 ml glycerol
0.4 g MnCl2-4H2O

Logan, C. 1963. A selective medium for the isolation of soft-rot coliforms from
soil. Nature 199:263.

Meneley, J. C. and M. E. Stranghellini. 1976. Isolation of soft-rot Erwinia spp.

from agricultural soils using an enrichment technique. Phytopathol. 66:367-
370.
A soil enrichment technique was used to isolate Ec from various soils. Most
isolates were Ecc or Eca, but all were able to cause blackleg of potato
(defined as blackening, wilting, and soft rot of potato stems). The enrichment
medium consisted of 225 ml distilled water, 0.625 g sodium polypectate, 2.5
ml 10% (NH4)2SO4, 2.5 ml 10% K2HPO4, and 1.5 ml 5% MgSO4-7H2O.
This medium was added to 25 g soil in a 250 ml flask and incubated
anaerobically for 48 hours. Cultures were serially diluted and plated on
pectate. Ec was isolated from 6 of 9 fields tested. Of 34 strains isolated, 2
were Eca and 23 were Ecc. Nine of the strains could not be identified to
subspecies.

Miller, T. D. and M. N. Schroth. 1972. Monitoring the epiphytic population of

Erwinia amylovora on pear with selective medium. Phytopathol. 62:1175-
1182.
MS medium has also been used to isolate E. chrysanthemi. The colonies are
described as white with orange centers on this medium.

Naumann, K. and W. Ficke. 1972. Salmonellen-Shigellen-Agar, ein im

vergleich zu anderen spezialnahrboden einfaches selektivsubstrat zum
nachweis von Pectobacterium carotovorum var. atrosepticum (val Hall)
Dowson. Zbl. Bakteriol., Parasitenkd., Infekt. Krankh. u. Hygiene 2:180-189.

Noble, M., and D. C. Graham. 1956. A selective medium for the isolation of
coliform soft-rot bacteria from plant tissue. Nature 178:1479-1480.

Paton, A. M. 1959. An improved method for preparing pectate gels. Nature

183:1812.

Pérombelon, M. C. M. 1971. A semi-selective medium for estimating

population densities of pectolytic Erwinia spp. in soil and plant material.
Potato Res. 14:158.

Pérombelon, M. C. M. 1971. A quantal method for determining numbers of

Erwinia carotovora var. carotovora and E. carotovora var. atroseptica in soils
and plant material. J. Appl. Bact. 34:793-798.
This method combines the maximum likelihood method with a spot-plate
method to estimate Ecc and Eca populations. It can be used to estimate
populations that are much smaller than can be detected by dilution plating.

Pérombelon, M. C. M. and R. Lowe. 1971. The effects of bile salts media and
the age of inocula in quantitative studies of populations of Erwinia carotovora
var. carotovora and E. carotovora var. atroseptica. J. Appl. Bact.

Sands, D. C. and R. S. Dickey. 1978. Palatinose utilization as a differential

test for Erwinia species. Proc. 4th Int. Conf. Plant Path. Bact. 555-559.
Eca produces palatinose and one other reducing sugar from sucrose. Eca, but
not Ecc, is also able to use palatinose as a sole carbon source. The authors
attempted to use a minimal medium with palatinose as the sole carbon source
to recover Eca from spiked soil samples, but had very low recovery rates.
They suggest that recovery rates could be improved by addition of other
components while still maintaining selectiveness for Eca over Ecc.
Segall, R. H. 1971. Selective medium for enumerating Erwinia species
commonly found in vegetable packinghouse waters. Phytopathol. 61:425-426.

g/L
Raffinose 10
dibasic potassium phosphate 2
ammonium sulfate 5
eosin yellow 0.4
methylene blue 0.065
actidine 250 ppm
neomycin 40 ppm
novobiocin 40 ppm
agar 15 g
up to one
water
liter
*add the raffinose after
autoclaving. This can be
done by making a filter-
sterilized concentrated stock
solution.

E. carotovora and E. chrysanthemi form red, raised colonies with a purple

center. The colonies are surrounded by a white halo with an irregular margin.
The authors used this media to estimate Erwinia CFU present in vegetable
packinghouse wash water. They found levels between 10E4 and 10E5 in
carrot, radish, celery, tomato, and pepper water water. They found that the
percent of produce that decayed was higher after washing in packinghouse
water than if the produce was not washed.

We have successfully isolated Ecc from potato tubers with soft rot symptoms
using this medium. In our experience, both Ecc and Ech form red, raised
colonies within 1 to 2 days after streaking the plates. We also found that we
could leave the actidine, neomycin, and novobiocin out of the medium and still
easily isolate Ecc from diseased potato tubers. The centers of the colonies
turned purple after 5-7 days. Two other colony types were common when
isolating Ecc from diseased potato tubers (metallic green and flat red with a
dark red halo); neither were Erwinia. The metallic green colonies are probably
a Klebsiella species based on 16S rDNA sequence.
Streak from a potato tuber with soft rot. The red Ecc colonies are the most
numerous on the plate.

Stewart, D. J. 1962. A selective-diagnostic medium for the isolation of

pectinolytic organisms in the Enterobacteriaceae. Nature 195:1023.

Thorne, S. N. 1972. A pectate gel medium for the isolation and diagnosis of
pectinolytic bacteria. J. Appl. Bact. 35:357-358.

Low methoxy pectin 25g

Peptone 10g
Lab-Lemco 10g
Gluconic acid d-lactone 7.5g
Calcium orthophosphate 5g
up to 1 L with distilled water

Make a thin paste of pectin in ethanol to facilitate its solution in water.

Dissolve the peptone and Lab-Lemco into 800 ml of water and add it to the
pectate paste. Heat the medium to 95C, then cool it to 30C and adjust to pH
8.0 with c. 25 ml of 3N NaOH.

Dissolve the gluconic acid d-lactone in 50 ml of water, then add the calcium
orthophosphate to this solution. Stir this solution into the above medium. Let
the medium stand for 10 min, then adjust the pH to 8.5 and the volume to 1 L.
Sterilize the medium for 5 min at 109C. The medium will gel in about 3 hrs.

Tsuyama, H. and M. Sakamoto. 1952. Isolation methods of the soft-rot

causing bacteria from the soil. Sci. Rep. Res. Inst. Tohoku, Univ. 3:29-34.
This paper describes media and a carrot slice method used in several papers
published in the 1970s concerning Ecc infection of cabbage.

The authors used Drigalski’s medium, which was developed for isolation of
intestinal bacteria:
Bouillon Agar 1 liter
Lactose 10 g
Crystal violet (0.1% aq. sol.) 5 ml
Bromothymol blue (0.2% sol.) 40 ml
adjust the pH to 7.0-7.2
Crystal violet inhibits growth of gram-positive bacteria. Ecc colonies are yellow
or green during the first five days on this medium, but may eventually turn
blue. In this paper, only about 17% of these colonies caused soft rot.

Webb, L. E. 1974. A study of test media used for the identification of soft-
rotting Erwinia species. I. Utilization of carbohydrates. Phytopathol. Z. 80:267-
278.
The authors noted that carbon utilization studies often used different basal
media and pH indicators, which is likely to affect the results of these studies.
The authors surveyed the literature and tested several media. They note that
several of the media gave confusing results because they caused production
of alkaline products that masked the pH change required for the pH indicator
to show that a carbohydrate was used.

The most reliable medium found was a modified Collins and Lyne medium
(Collins, C. H. and P. M. Lyne. 1970. Microbiological Methods. Butterworths,
London):
MgSO4-7H2O 0.02%
KCl 0.02%
NH4H2PO4 0.1%
Yeast Extract 0.1%
Bromothymol blue 8 ml of 0.1% aqueous solution
Carbohydrate 1%

Webb, L. E. 1976. A study of test media used for the identification of soft-
rotting Erwinia species. II. Utilization of organic acids. Phytopathol. Z. 85:26-
34.
The authors noted that fewer studies have looked at organic acid utilization
than at carbon utilization. The tested several media and found that the
addition of yeast extract to these media was important for accurate results.

The most reliable medium found was a modified Collins and Lyne medium
(Collins, C. H. and P. M. Lyne. 1970. Microbiological Methods. Butterworths,
London):
MgSO4-7H2O 0.1%
NaCl 0.1%
(NH4)2HPO4 0.1%
KH2PO4 0.05%
Yeast Extract 0.08%
Agar 1.2%
Phenol Red 0.4 ml of 0.2% solution in 50% ethanol per 100 ml of medium
Organic acids 0.2% (filtered solutions of sodium salts of the organic acids
were added after autoclaving)

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