Potato Research 44 (2001) 2 6 5 - 2 7 0

Modified crystal violet pectate medium (CVP) based on a

new polypectate source (Slendid) for the detection and
isolation of soft rot erwinias
L.J. H Y M A N , L. S U L L I V A N , I.K. T O T H and M.C.M. P E R O M B E L O N

Scottish Crop Research Institute, Invergowrie, Dundee DD2 5DA, Scotland, UK

Accepted for publication: 15 August 2001

Additional keywords: potato, bacteria, pectic enzymes, Erwinia carotovora, Erwinia
chrysanthemi, Solanum taberosum

Summary
The selective-diagnostic crystal violet pectate (CVP) medium for the detection and isolation of
soft rot erwinias was modified and improved to allow the use of a new source of sodium
polypectate (Slendid type 440), as the previous polypectate (Bulmer) is no longer available.
Two formulations were developed: CVP-S1 medium was less transparent but otherwise similar
to the Bulmer polypectate-based CVP medium (CVP-B) except that NaOH was added and
CaCI~ concentration reduced. CVP-S2 medium was prepared by mixing equal volumes of two
double strength preparations containing 1) polypectate and NaOH, and 2) all other
ingredients, both sterilised separately. Although erwinia cavity formation was slower, it
showed a number of advantages over CVP-B and CVP-S1 media, thereby facilitating
colony/cavity detection and enumeration. These included the absence of precipitate, greater
firmness and a reduced risk of liquefaction in the presence of large erwinia numbers, and a
reduction in the number of saprophytic bacteria.

Introduction

Crystal violet pectate medium (CVP) (Cuppels & Kelman, 1974) is the best and most
commonly used selective-diagnostic medium for the detection, isolation and
enumeration of soft rot erwinias, notably Erwinia carotovora subsp, carotovora
(Ecc), E. carotovora subsp, atroseptica (Eca) and E. chrysanthemi (Ech), in plants
and the environment. Detection of soft rot erwinias depends on the characteristic
deep pits or cavities formed by colonies of the bacteria in this medium, a feature
which is highly dependent on the type of polypectate used. Only a few preparations
are suitable for use in CVP: those which are not toxic and have the right gelling and
cavity formation characteristics.
The Sunkist sodium polypectate used in the original formulation of Cuppels &
Keiman (1974) was replaced when it became unavailable and the formulation was
modified to suit another polypectate preparation derived from citrus (Bulmer)
(P6rombelon & Burnett, 1991). This new medium was subsequently simplified by the
omission of the antibiotic novobiocin (Van der Wolf & P6rombelon, 1998).
Unfortunately, the Bulmer citrus polypectate has now become unavailable and an
alternative low ester citrus pectin (Slendid sodium polypectate type 440), which is
widely used in the fast-food industry in the USA, has been found as a replacement.
However, modifications to the formulation of the CVP medium given in Van der

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L.J. HYMAN, L. SULLIVAN, 1.K. TOTH AND M.C.M. PEROMBELON

Wolf & P6rombelon (1998) were necessary to allow erwinia growth and to maintain
the diagnostic character of the medium. Two new formulations of CVP based on
Slendid polypectate are described below.

Materials and methods

Selective media
(a) Bulmer polypectate-based CVP medium (CVP-B) was prepared as in Van der
Wolf & P6rombelon (1998).
(b) Slendid polypectate-based one-step CVP medium (CVP-S1) contained: 0.5 g
tryptone (Oxoid L42), 2.5 g tri-sodium citrate, 1.0 g NaNO 3, 5.1 ml 10% aqueous
solution CaCI2.2H20, 1.0 ml crystal violet (0.075% aqueous solution), 1.3 ml 5 M
NaOH, 2.0 g agar (BDH Agar Powder), 9.0 g sodium polypectate (Slendid type 440;
M. Burger Enterprises, 2225 Eton Ridge, Madison, Wisconsin 53705, USA) and 500
ml cold distilled water. The ingredients, except the agar and polypectate, were
dissolved in water. The polypectate was then added slowly, using a magnetic stirrer to
produce a deep vortex in order to avoid the formation of lumps, followed by the agar.
The medium was autoclaved for 15 min at 120 ~ and the pressure restored slowly to
avoid bubbles within the medium. As the medium cannot be re-melted readily, ca. 18
ml per 9 cm Petri dish was poured immediately after autoclaving, while gently
rotating the flask by hand.
(c) Slendid polypectate-based two-step CVP medium (CVP-S2): The media were
prepared in batches not exceeding 500 ml for ease of mixing and pouring. Mix A
contained the ingredients, with the exception of polypectate and NaOH, at the
amounts given in (b) but in half the volume of water (250 ml). Mix B contained the
same amounts of polypectate and NaOH as in (b) also in 250 ml water. The
ingredients were dissolved, while taking the same precautions as in (b). After
autoclaving and while still hot, Mix A was poured into Mix B by gently rotating the
flask during pouring. The final pH was ca. 7.0. Plates were chilled for at least 2 h at
4 ~ after pouring if they were to be used immediately. Otherwise, plates were stored
at 4 ~ for up 2 months in a sealed polythene bag. Before use, the plates were
thoroughly dried with the lids ajar for 1 h at 45-50 ~ in a ventilated oven or in a
laminar flow cabinet at room temperature for 4 h.

Effects of formulation changes on erwinia growth and recovery

Substituting Bulmer by Slendid polypectate in the CVP formulation (Van der Wolf &
P6rombelon, 1998) required adjustment of the pH by the addition of 5 M NaOH prior
to autoclaving to allow erwinia growth. The effects of different proportions of
Slendid polypectate, agar and CaCI 2 on the medium firmness and cavity shape were
examined in an attempt to reduce the formation of a fine precipitate affecting the
transparency of the medium. For the same reason, a two step formulation was
developed by mixing, after autoclaving, equal volumes of double strength
preparations of 1) Slendid polypectate/NaOH and 2) all other ingredients but with
different concentrations of CaC12.

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The requirement for tryptone, which affects erwinia growth, cavity formation and
cavity size at 27, 33.5 and 37 ~ in the CVP-B medium, was checked in the CVP-S1
and CVP-S2 media. The suitability of five different commonly available commercial
agar sources for use in the new CVP formulations was also assessed. Recovery
efficiency of two Eca strains (SCRI 1024, SCRI 1039), two Ecc strains (SCRI 193,
SCRI 258) and one Ech strain (SCRI 4065) obtained from the Scottish Crop
Research Institute (SCRI) culture collection was determined on nutrient agar (NA)
and the three pectate-based media (CVP-B, CVP-S1 and CVP-S2). Overnight shaken
cultures of the bacteria in nutrient broth were diluted in sterile water to ca. 103 cells
ml l and 100 lal spread on duplicate plates of the media before incubation at 27 ~ for
48 h. Growth of saprophytic bacteria on Bulmer and Slendid polypectate-based
media was examined by plating 10-2 dilutions of potato tuber peel extract containing
ca. 106 bacteria ml l , supplemented with 0.075% dithiothreitol as anti-oxidant
(P6rombelon & Van der Wolf, 1998), onto duplicate plates of the media and
incubating at 27 ~ for up to 72 h. The peel extract was prepared as described by Van
der Wolf & P6rombelon (1998) using two certified erwinia-free seed stocks. The
effect of the different media on the mean of the colony counts for each strain was
subjected to ANOVA.

Results and discussion

The main features required of CVP medium for the detection, isolation and
quantification of soft rot erwinias are a) firmness to allow plating and streaking
without damaging the medium, b) formation of the characteristic deep cavities by soft
rot erwinia colonies, c) adequate transparency to facilitate detection of the cavities,
especially in the presence of large numbers of non-erwinia colonies, d) non-toxic,
hence high erwinia recovery efficiency, and e) selectivity to reduce non-erwinia
bacterial growth that would otherwise inhibit or mask the growing erwinia colonies.
CVP medium made from the new polypectate using the previous formulation had a
sufficiently firm surface for plating and streaking. However, the low pH (ca. 5.5)
prevented growth and cavity formation by soft rot erwinias. Although it was not
necessary to adjust the pH using the Bulmer polypectate, 2.6 ml 11 of 5 M NaOH was
required to obtain a final pH of ca. 7 with the Slendid polypectate. Moreover,
addition of tryptone was found to be necessary for the rapid growth of soft rot
erwinia colonies and the formation of deep cavities, as with the Bulmer polypectate-
based formulation. It is likely that tryptone chelates some toxic contaminant present
in the polypectate preparation. Lastly, the source of agar used in the Slendid
polypectate-based CVP formulations could affect firmness and erwinia cavity shape.
Whereas BDH Agar Powder, Difco Agar Granulated and Oxoid L28 preparations
were equally satisfactory, Gibco-BRL Select Agar and Difco Agar Noble were not.
Although erwinia cavity shape was satisfactory for diagnostic purposes, the
presence of a fine precipitate in the Slendid-based medium made it less transparent,
and thus hampered detection of cavities, especially in the presence of large numbers
of saprophytic bacterial colonies. This is especially important when detecting and

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L.J. HYMAN, L. SULLIVAN~ I.K. TOTH AND M.C.M. PEROMBELON

enumerating erwinia cavities by holding the Petri dish upside down to best visualise
the characteristic bright halo around the cavities. The precipitate appeared to be
formed as a result of CaCI, reacting with the polypectate on autoclaving. When
different concentrations of CaCI. (3.4-13.6 ml ll), polypectate (16-20 g 1-I) and agar
(3-5 g 11) in different combinations were tested, precipitate formation was reduced
progressively only by lowering the CaCI, concentration. Gelling of the media and
cavity size was optimal at levels of polypectate and agar as given in the CVP
formulation given in Van der Wolf & P6rombelon (1998). Although transparency
was not as good with this medium (CVP-S1) as with CVP-B, reducing the
concentration of CaC12 by 25% to 10.2 ml 1-1, while maintaining those of polypectate
and agar, was a satisfactory compromise (Fig. 1). Precipitate formation was

Fig. 1. Cavity formation in three different crystal violet pectate (CVP) media inoculated with
100 pl suspensions of E. carotovora subsp, carotovora strain SCR1258 (Ecc), E. carotovora
subsp, atroseptica strain SCRI 1039 (Eca) and E. chrysanthemi strain SCR14065 (Ech) and
incubated at 27 ~ for 48 h.
CVP media: CVP-B, CVP prepared with Bulmer polypectate; CVP-SI, one step CVP
prepared with Slendid polypectate; CVP-S2, two-step CVP prepared with Slendid polypectate.

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completely eliminated and a clear medium obtained when equal volumes of double
strength preparations of 1) polypectate/NaOH and 2) the rest of the CVP ingredients,
separately autoclaved, were mixed (CVP-S2). Equally, as reducing the level of CaCIo
by 25% had no adverse effect, it was also adopted for the CVP-S2 formulation.
Growth and cavity formation on the CVP-S1 medium by soft rot erwinias was
comparable to that on CVP-B (Fig. 1). However, cavity size varied within and
between the three erwinias; some strains of Ecc (3/10), Eca (3/13) and Ech (1/5)
formed smaller cavities on both media incubated at 27 ~ for 48 h, reflecting what was
probably lower growth rate and/or lower pectic enzyme production. In contrast, the
CVP-S2 set more rapidly and was firmer than the other two CVP media. More
importantly, it had no precipitate, which facilitated cavity detection, especially in the
presence of large numbers of saprophytic bacterial colonies. However, cavity size
tended to be smaller, and strains that formed smaller cavities on CVP-S1 required 72
h incubation at 27 ~ for good cavity formation. Nevertheless, this could be
advantageous, as the risk of liquefaction of the medium, a problem that can occur
with the two other CVP media, is reduced when erwinia populations are high.
Recovery rate of the three erwinias on the CVP-B, CVP-S1 and CVP-S2 was
similar to N A when using pure cultures (Table 1). However, when inoculated with
dilutions of erwinia-free tuber peel extracts, significantly fewer saprophytic bacterial
colonies grew on the two CVP-S media than on CVP-B, with CVP-S2 medium being
the most selective (Table 1).
The differential effect of incubation temperatures (27, 33.5 and 37 ~ on the
pattern of cavity formation in the two CVP-S media, caused by the three soft rot

Table 1. Mean number of colony forming units (cfu) of five strains of Erwinia spp., and of
saprophytic bacteria in 10 .2 dilution of tuber peel extract from two potato stocks, on four
media: Nutrient agar (NA), Bulmer polypectate CVP medium (CVP-B). one step (CVP-S 1)
and two step (CVP-S2) Slendid polypectate-based CVP at 27 ~ and 48 h.

Media Mean cfu 100/aln bacterial suspension/peel extract

Erwinia spp. strain no. Saprophytic bacteria

SCRI SCRI SCRI SCRI SCRI Stock A Stock B

1024a 1039~l 193h 258b 4065c

NA 195.0 303.5 368.0 209.0 189.0

CVP-B 199.5 301.0 322.5 214.0 169.5 107.0 343.0
CVP-SI 207.0 276.0 320.5 249.0 161.5 76.0 260.0
CVP-S2 194.0 305.0 367.5 252.5 157.0 50.5 185.0

SEM 10.7 16.5 13.8 19.4 10.0 5.5* 13.9"*

a Erwinia carotovora subsp, atroseptica.

h E. carotovora subsp, carotovora.
c E. chrvsanthemi.
* P<0J35
** P<0.01

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L.J. HYMAN, L. SULLIVAN, I.K. TOTH AND M.C.M. PEROMBELON

erwinias, was similar to that on CVP-B (P6rombelon & H y m a n , 1986). Therefore, the
two CVP-S media could also be used in population dynamics studies of the bacteria in
pathogenesis by jointly inoculating strains with known contrasting cavity formation
pattern, followed by dilution-plating on CVP and incubating at the differential
temperatures to determine numbers.
In conclusion, CVP-S1 m e d i u m could be used satisfactorily for erwinia detection
and enumeration. However, although cavity formation is slower, CVP-S2 medium is
firmer and more transparent and, coupled with a reduced risk of both liquefaction in
the presence of large numbers of erwinias and of overcrowding by saprophytic
bacteria, makes it the medium of choice.

Acknowledgement

S C R I is grant-aided by the Scottish Executive Environment and Rural Affairs

D e p a r t m e n t ( S E E R A D ) . The authors thank A. Kelman, North Carolina State
University, U S A for suggesting the use of Slendid polypectate and for arranging its
marketing by M. Burger Associates, USA.

References
Cuppels, D. & A. Kelman, 1974. Evaluation of selective media for isolation of soft rot bacteria
from soil and plant tissue. Phytopathology 64: 468--475.
P6rombelon, M.C.M. & 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.
P6rombelon, M.C.M. & L.J. Hyman, 1986. A rapid method to identify and quantify soft rot
erwinias directly from plant material based on their temperature tolerances and sensitivity to
erythromycin. Journal of Applied Bacteriology 60: 61-67.
P6rombelon, M.C.M. & J.M. Van der Wolf, 1998. Potato tuber peel extract preparation and
detection probes. In: M.C.M. P6rombelon & J.M. Van der Wolf (Eds), Methods for the
detection and quantification of Erwhlia carotovora subsp, atroseptica on potatotes: a
laboratory manual. Scottish Crop Research Institute Occasional Publication No. 10, pp.
3-10.
Van der Wolf, J.M. & M.C.M. P6rombelon, 1998. Immunomagnetic separation-colony count
on CVP medium (IMS-CVP). In: M.C.M. P6rombelon & J.M. Van der Wolf (Eds), Methods
for the detection and quantification of Erwinia carotovora subsp, atroseptica on potatotes: a
laboratory manual. Scottish Crop Research Institute Occasional Publication No. 10, pp.
11-18.

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