121

Bacteria and phytoplasmas / Bactries et phytoplasmes

Bacteriophages and the control of Erwinia carotovora

subsp. carotovora
M. Ravensdale, T.J. Blom, J.A. Gracia-Garza, A.M. Svircev, and R.J. Smith

Abstract: Fourteen bacteriophages of Erwinia carotovora subsp. carotovora, the causal agent of bacterial soft rot, were
isolated from samples of fertilizer solutions taken from a greenhouse producing calla lily (Zantedeschia spp.). To avoid
a single-host selection bias, a mixture of four bacterial hosts was used to enrich bacteriophages. Molecular characterization
of the phages with restriction endonuclease digestions indicated that two distinct types had been isolated. Representatives
from both molecular groups were studied by electron microscopy to determine their morphology. The phages possessed
icosahedral heads with long, flexible tails. On the basis of morphotypes, the bacteriophages of E. carotovora subsp.
carotovora were placed in the order Caudovirales. Limited host-range studies revealed that both phage groups would
lyse only E. carotovora subsp. carotovora isolates taken from calla tubers infected with bacterial soft rot. When assayed
for persistence in fertilizer solutions for calla lilies, phage titres were found to be stable in the solutions made with sterile
reverse-osmosis water, but not with nonsterile tap water. Phages did not persist in preplant-treatment solutions for calla
tubers. Biological control of E. carotovora subsp. carotovora by the bacteriophages was assessed in fertilizer solutions,
on the surface of plugs of calla tuber tissue, and under greenhouse conditions. Phage isolates were able to significantly
reduce bacterial populations in fertilizer solutions and on the surface of plugs of calla tuber tissue. The biocontrol ability
of the bacteriophages was inhibited by the presence of Fe ethylenediaminetetraacetic acid in fertilizer solutions. On plugs
of tuber tissue and in greenhouse trials, phages were able to reduce the incidence of soft-rot symptoms by 50% or more.
Key words: bacteriophages, Erwinia carotovora subsp. carotovora, calla lily, Zantedeschia sp., biological control,
bacterial soft rot.
Rsum : Quatorze bactriophages de lErwinia carotovora subsp. carotovora, lagent de la pourriture molle, furent isols
dchantillons de solutions nutritives utilises en serre de production de callas (Zantedeschia spp.). Afin dviter un biais
d un seul hte, un mlange de quatre bactries htes fut utilis pour lenrichissement des bactriophages. La caractrisation
molculaire des phages par la digestion laide dendonuclases de restriction indiqua que deux types avaient t isols.
Des reprsentants des deux groupes molculaires furent tudis par microscopie lectronique afin den dterminer la
morphologie. Les phages possdaient des ttes icosadriques et de longues queues flexibles. Selon leur morphotype, les
bactriophages de lE. carotovora subsp. carotovora furent classs dans lordre des Caudovirales. Des tudes limites sur
leur gamme dhtes rvlrent que les deux groupes ne lyseraient que les isolats dE. carotovora subsp. carotovora
provenant de bulbes de callas atteints de la pourriture molle. Lors dessais sur la persistance dans les solutions nutritives
pour callas, la teneur en phages fut stable dans les solutions nutritives labores partir dune eau strile produite par
osmose inverse, mais pas avec une eau du robinet non strile. Les phages ne survcurent pas dans des solutions pour le
traitement de prplantation de bulbes de calla. La lutte biologique contre lE. carotovora subsp. carotovora laide de
bactriophages fut value en solution nutritive, sur la surface de rondelles de tissu de bulbe de calla et lors dessais en serre.
Les isolats de phages ont rduit significativement les populations bactriennes dans les solutions nutritives et en surface
des rondelles de tissu de bulbe de calla. La capacit des bactriophages en matire de lutte biologique fut inhibe par la
prsence de Fe acide thylnediamine-ttractique dans les solutions nutritives. Sur les rondelles de bulbe et lors des
essais en serre, les phages ont pu rduire la frquence de la pourriture molle de 50% ou plus.
Mots-cls : bactriophages, Erwinia carotovora subsp. carotovora, calla, Zantedeschia sp., lutte biologique, pourriture molle.
Ravensdale et al.: calla lily / bacterial soft rot / Erwinia carotovora / bacteriophages / biological control 130
Accepted 30 March 2007.
M. Ravensdale1 and T.J. Blom.2 Department of Plant Agriculture, University of Guelph, Guelph, ON N1G 2W1, Canada.
J.A. Gracia-Garza and A.M. Svircev. Southern Crop Protection and Food Research Centre, Agriculture and Agri-Food Canada,
Vineland Station, ON L0R 2E0, Canada.
R.J. Smith. Department of Biology, University of Western Ontario, London, ON N6A 5B7, Canada.
1
2

Present address: Scottish Crop Research Institute, University of Dundee, Scotland.

Corresponding author (e-mail: tblom@uoguelph.ca).

Can. J. Plant Pathol. 29: 121130 (2007)

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Can. J. Plant Pathol. Vol. 29, 2007

Introduction

Materials and methods

Bacteriophages as biocontrol agents have a number of

desirable characteristics that include their environmentally
friendly nature, since they are isolated from the same
natural environments as their bacterial hosts, and their highly
specific hostpathogen interaction (Flaherty et al. 2000,
2001; Jones et al. 1998; Balogh et al. 2003; Schnabel and
Jones 2001; Gill et al. 2003). Control of bacterial plant
diseases has traditionally relied on the use of antibiotics
and (or) copper compounds. Bacteriophages specific to
Erwinia carotovora subsp. carotovora (Jones) Bergey et
al. have been isolated from diseased plant materials and
their associated soils, fertilizer solutions, cull piles, sewage, and freshwater lakes and wells (Eayre et al. 1995;
Gross et al. 1991; Toth et al. 1993). Bacteriophages isolated from pectolytic Erwinia spp. have lower frequencies
of isolation and more restrictive host ranges when compared with phages whose bacterial hosts belong to other
enterobacteriacea (Eayre et al. 1995). Antibiotic resistance,
copper phytotoxicity, and the trend towards eliminating antibiotics in agriculture has revitalized research into phages
as biocontrol agents in agriculture (Balogh et al. 2003;
McKenna et al. 2001; Obradovic 2004).
Erwinia carotovora subsp. carotovora is the bacterial
pathogen associated with soft rot of calla. Calla tubers
(Zantedeschia spp.) are grown under field conditions in
milder climates, from seed or seed tubers. Greenhousegrown callas are traditionally forced from field-produced
rhizomes or tubers, allowing easy spread of bacterial and
viral pathogens (Kuehny 2000). Greenhouse losses from
bacterial soft rot due to Erwinia spp. may range from less
than 5% to complete crop losses in southern Ontario,
Canada (Blom and Brown 1999). Wright and Burge
(2000) reported that worldwide cultivation of this crop is
restricted because of the risks associated with the bacterial
soft rot. The disease-causing organism is currently controlled by stringent sanitation practices, precise environmental control, and preplant treatment of tubers with
bactericides and plant growth regulators (Blom and Brown
1999; Kuehny 2000; Wright and Burge 2000). Attempts to
biologically control epidemics of bacterial soft rot have
been undertaken (Costa and Loper 1994; Cronin et al.
1997; El-Hendawy et al. 1998; Kloepper 1983; Sharga and
Lyon 1998); however, there are no registered preplant
treatments with biocontrol agents available for use in
Ontario greenhouses (Blom and Brown 1999).
The objective of the present study was to determine
whether bacteriophages could control E. carotovora subsp.
carotovora in greenhouse-grown callas. Bacteriophages
of Erwinia spp. were collected from infested greenhouse soils and greenhouse water reservoirs. Phages
were isolated, enriched, purified, and characterized on
the basis of morphology, restriction fragment length
polymorphisms (RFLPs), and host-range studies. Single
phage isolates were assayed for their ability to persist
in nutrient-broth solutions and in preplant-treatment solutions for calla tubers. Finally, phage isolates were assayed
for their biocontrol efficacy in nutrient-broth solutions, on
the surface of calla tubers, and under greenhouse conditions.

Bacterial isolates and culture media

Isolates of E. carotovora subsp. carotovora, Erwinia
amylovora (Burrill) Winslow et al., Erwinia chrysanthemi
(Burkholder) Young et al., and Pantoea agglomerans
(Beijerinck) Gavini et al. were cultured on LuriaBertani
(LB) agar (Difco Laboratories, Detroit, Mich.) and incubated
at 26 C. Liquid cultures of E. carotovora subsp. carotovora
were grown in 250 mL Erlenmeyer flasks containing 100 mL
of LB broth and placed on an orbital shaker at 26 C. Isolates
of Escherichia coli (Migula) Castellani & Chalmers were
cultured on LB agar and incubated at 37 C. Liquid cultures
of Escherichia coli were grown in 250 mL Erlenmeyer flasks
containing 100 mL of LB broth and incubated at 37 C on an
orbital shaker. The bacterial isolates used in the present study
and their origins are listed in Table 1.
Phage isolation and culture media
Collections of bacteriophages were made in the summer
of 2001 and in the spring of 2003 from greenhouse operations in the Niagara region of southern Ontario. Samples of
infected plants, cull-pile runoff, fertilizer solutions, and
discharged fertilizer solutions were taken from each site.
Plants exhibiting symptoms of soft rot were taken in their
entirety and stored at 5 C in plastic bags. All solid and
liquid samples were enriched in a procedure modified from
Crosse and Hingorani (1958). Flasks containing 50 mL of
NBSYE + S (0.8% (m/v) nutrient broth, 0.5% food-grade
sucrose, and 0.25% yeast extract, supplemented with NaCl
(100 mmol/L) and MgCl2 (1 mmol/L)) were inoculated with
200 L from overnight cultures of each of the E. carotovora
subsp. carotovora hosts listed in Table 1. Either 1 g (fresh
mass) of plant sample or 1 mL of liquid sample was placed
into each flask and incubated for 20 h at 26 C on an orbital
shaker. Following incubation, 1 mL of chloroform was
added to kill the bacteria, and the mixture was placed on an
orbital shaker for 30 min. The suspension was decanted,
leaving the chloroform behind, and centrifuged at 9700g for
10 min. The resulting supernatant was decanted and centrifuged at 16 000 g for 45 min; the resulting pellet was dissolved in 5 mL of phosphate-buffered saline (PBS) and
stored at 4 C. Phage solutions from the enriched samples
were serially diluted and plated onto bacterial lawns prepared from propagation hosts listed in Table 1. Lawns were
checked for the formation of plaques after 24 h. Agar from
a single plaque was transferred from a lawn to a sterile
1.5 mL Eppendorf tube containing 1 mL NBSYE + S and
100 L overnight culture of the corresponding bacterial host,
using sterile pipette tips. These tubes were incubated on an
orbital shaker for 20 h. This single-plaque isolation procedure
was repeated 612 times to purify each isolate. Bacteriophages were enumerated on LB agar, using the soft-agar
overlay method described by Adams (1959). In between
experiments, phages were stored in PBS at 10 mmol/L,
NaCl (100 mmol/L) and MgCl2 (1 mmol/L) at 4 C.
Host-range analysis
Phage host ranges were tested against five E. carotovora
subsp. carotovora isolates and four unrelated bacterial species
(Table 1). Lawns of bacterial hosts were prepared by seeding

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123

Table 1. Description of phenotypes, origin, and sources of bacteria and bacteriophage

isolates used in the present study.

Bacterial isolates
Erwinia carotovora subsp. carotovora
Ecc1a
Ecc26a
Ecc48a
Ecc71a
Ecc83
Escherichia coli DH5
Erwinia chrysanthemi EchS80
Erwinia amylovora Ea 6-4
Pantoea agglomerans

Phenotype

Origin

Source

Wild
Wild
Wild
Wild
Wild

Calla
Potato
Calla
Potato
Calla

J. Gracia-Garzab
D. Cupplesc
J. Gracia-Garza
D. Cupples
J. Gracia-Garza
R. Lod
A.M. Svircevb
A.M. Svircev
ATCC 33243e

type
type
type
type
type

Wild type

Bacteriophage isolates
Ecc1Ecc12 (group 1)
Ecc13Ecc14 (group 2)

Pear

CFSf
FSRg

This study
This study

Bacterial isolates used as hosts during the initial isolation of the bacteriophages.
Southern Crop Protection and Food Research Centre, Agriculture and Agri-Food Canada (AAFC),
Vineland Station, Ontario.
c
Southern Crop Protection and Food Research Centre, AAFC, London, Ontario.
d
Department of Microbiology, University of Guelph, Guelph, Ontario.
e
American Type Culture Collection.
f
Circulating fertilizer solution from greenhouse, 2001.
g
Fertilizer-solution reservoir from greenhouse, 2003.
b

5 mL of molten agar with 100 L of 1 108 colony-forming

units (CFU) per millilitre of host suspension. Phage solutions were diluted to 1 107 phage-forming units (PFU)/mL,
and 10 L aliquots were spotted onto the lawns. Plates were
incubated at room temperature in a laminar-flow bench for
10 min prior to incubation at 26 C for 1820 h. Phage
treatments that produced plaques were scored as positive
whereas treatments with no discernable effect were scored
as negative. Experiments were repeated once.
DNA extraction
DNA was isolated and purified using the mini -DNA
preparation kit (Qiagen, Mississauga, Ont.). DNA pellets
were dried and resuspended in 50 L of sterile reverse-osmosis
(RO) water, and DNA concentrations were determined by
using the Beckman DU640 spectrophotometer (optical
density at 260 nm) and DNA/oligo Quant software.

Transmission electron microscopy

High-titre phage solutions were prepared and centrifuged
at 8000g for 45 min and the supernatant with the phages
was decanted. The supernatant was centrifuged at 16 000 g
for 1 h and the phage pellet was resuspended in sterile
distilled water containing MgCl2 at a concentration of
2 mmol/L. Drops (20 L) of phage solution were placed on
carbon-coated Formvar grids (200 nm mesh) for negative
staining. The phage solution was allowed to sit for 45 s,
then the solution was wicked off with filter paper by
capillary action and replaced with a drop of 2% (m/v) uranyl
acetate for 1 min. The uranyl acetate was drawn off with
filter paper, and the grid with phages was allowed to air-dry.
The electron microscope was calibrated by using catalase
crystals at the same magnification than for viewing the
phages. Specimens were examined in an electron microscope (Philips CM10) operating at 80 kV and were photographed using Kodak 4489 electron-microscopy film.

Restriction fragment length polymorphism

Analyses of RFLPs were conducted using the enzymes
EcoRI, BamHI, and Bsh1236I (MBI Fermentas, Burlington,
Ont.) according to the suppliers instructions and using 0.5 g
of DNA plus 6 L of enzyme bovine serum albumin
mixture per 50 L of reaction solution. Digestion in each
enzyme phage DNA combination was conducted separately, overnight at 37 C. Fragments were run on a 1%
agarose gel in TAE buffer (Tris, 4.84 g/L; glacial acetic
acid, 1.14 mL/L; ethylenediaminetetraacetic acid (EDTA),
1 mmol/L) and stained with ethidium bromide (1g/mL) for
11 min. Bands were visualized and photographed under UV
light (geldoc 1000 camera, Quantity One Software; Bio-Rad,
Hercules, Calif.) and compared with 1-kb and 100-bp ladders
or -phage HindIII fragments (Invitrogen, Carlsbad, Calif.).

Phage viability in fertilizer solutions

Two fertilizer stock solutions (m/v) for callas were prepared with autoclaved RO water. The stock solutions were:
(A) Ca(NO3)2, 8.86%; NH4NO3, 0.62%; FeEDTA (13%
Fe), 0.11%; and (B) KNO3, 4.05%; MgSO4, 1.36%; KH2PO4,
0.24%; Mg(NO3)2, 3.29%; MnSO4, 0.011%; ZnSO4, 0.006%;
Borax, 0.015%; CuSO4, 0.002%; Na2MoO4, 0.001%. The
stock solutions A and B were autoclaved separately and
then combined and diluted 100-fold in water to obtain the
final fertilizer solutions employed in all subsequent studies.
Water used for dilution of the final fertilizer solutions consisted of autoclaved and nonautoclaved RO water and of
autoclaved and nonautoclaved tap water. For phage Ecc3,
100 L of 1 1010 PFU/mL was added to 900 L of the
fertilizer solutions in sterile plastic 1.5 mL Eppendorf tubes

124

and incubated at 26 C for 5 weeks. Phage Ecc14 could

not be consistently enriched to high titres; therefore, solutions for this isolate were tested at a concentration of 1
107 PFU/mL. The positive control was constituted of 100 L
of phage solution mixed with 900 L of sterile PBS. Surviving phages were enumerated as PFU per millilitre on a
weekly basis for 5 weeks by spotting sample dilutions on an
Ecc1 bacterial lawn. Treatments were replicated 3 times,
and the experiment was repeated 2 times.
Preplant-treatment solutions for calla tubers
Preplant-treatment solutions consisted of: (A) Promalin,
6 mL/L (1.8% (m/v) benzyladenine and 1.8% (m/v) gibberellins (GA4+7); Valent BioSciences Canada, Toronto, Ont.);
(B) fixed copper, 3 g/L (50% (m/m) copper oxychloride;
United Agri-products, Dorchester, Ont.); (C) Silwet 77,
0.13 mL/L (United Agri-products); or (D) a mixture of all
three previous compounds at the same final concentrations
as described above, in nonsterile RO water. The positive
control consisted of 100 L of 1 109 PFU/mL added to
10 mL of sterile PBS and to 10 mL of nonsterile RO water.
Phage Ecc3 was diluted to 1 109 PFU/mL, and 100 L
of the solution was added to 10 mL of preplant-treatment
solutions for calla tubers in a 14 mL sterile plastic culture
tube and incubated for 48 h at 26 C. Surviving phages
were enumerated as PFU per millilitre.
Biological control in liquid media
Phages were assayed for in-vitro biocontrol activity, in
fertilizer solutions prepared from: (1) combined stocks A
and B (as previously described), (2) stock A without FeEDTA,
and stock B (as previously described), or (3) NBSYE + S.
Media (10 mL) containing Ecc1 at 1 103 CFU/mL were
inoculated with 100 L of 1 107 PFU/mL (multiplicity of
infection (MOI), 100:1) in a 14 mL culture tube. PBS inoculated with 100 L of Ecc1 at 1 103 CFU/mL served as a
positive control, and media without bacteria treated with
either 100 L of 1 107 PFU/mL or 100 L of PBS served
as negative controls. These suspensions were incubated for
24 h at 26 C on an orbital shaker at 200 r/min (1 r = 2 rad),
serially diluted in sterile phosphate buffer (PB), and 100 L
portions were streaked onto plates containing MMS + A
(modified MillerSchroth medium, 0.8% (m/v) nutrient
broth, 5% food-grade sucrose, 2% agar, 0.9% (v/v) bromothymol blue, 0.25% neutral red, supplemented with antibiotics, erythromycin (50 mg/L) and cyclohexamide (50 mg/L)
(Sigma, Oakville, Ont.)). The addition of antibiotics to MSS
produced a selective medium for E. carotovora subsp.
carotovora. Surviving bacteria were incubated for 24 h at
26 C and enumerated as CFU per millilitre when dilutions
yielded 30300 CFU per plate. All treatments were replicated 3 times and the entire experiment was repeated once.
Biocontrol assay on calla tuber plugs
Plugs (11 mm in diameter) from calla tubers (Zantedeschia elliottiana (W. Wats.) Engl. Zantedeschia albomaculata (Hook.) Baill. hybrid yellow, Golden State Bulb
Growers, Moss Landing, Calif.) were taken with a sterile
cork borer and sliced transversely to a thickness of 24 mm
while eliminating the outer suberized layer. The slices were
immersed in 70% (v/v) ethanol for 10 s, placed in a 1%

Can. J. Plant Pathol. Vol. 29, 2007

(v/v) HClO solution for 10 min, and rinsed 3 times in sterile

RO water. A minimum of five tubers were used in each trial
to randomize variations caused by individual tubers. Tuber
slices were placed on a sterile Whatman No. 1 filter paper
in a Petri plate (9 cm in diameter) and moistened with
sterile RO water. The upper surface of the plug was inoculated with 10 L of Ecc1 at 1 105 CFU/mL in PB, followed
by 10 L of 1 107 PFU/mL for each of the single-phage
isolates or by 10 L of a mixture of phage isolates (MOI,
100:1). The phage mixture was produced by mixing equal
amounts of each phage isolate, yielding a solution of 1
107 PFU/mL. Positive and negative controls were performed
by inoculation as previously described. Plates were sealed
with parafilm to prevent drying and incubated for 5 d at
26 C. After incubation, plugs were assayed for disease
incidence by applying pressure to the surface of the plug
with a metal spatula. If the tissue readily collapsed, the plug
was scored as diseased; if the tissue was turgid, no incidence was recorded. Following this assay, each sample was
transferred to 10 mL of sterile PB. The resulting suspension
was vortexed twice and sonicated for 30 s. The suspension
was then serially diluted in sterile PB and streaked onto
MMS agar. Colonies were counted after 24 h of incubation
at 26 C. All treatments were replicated 3 times, and the
entire experiment was repeated once.
Greenhouse trials
Single-phage isolates and a mixture of five phage isolates
were selected for further study regarding their in-vivo biocontrol activities. Plastic pots (10 cm in diameter) were
filled full with Sun Gro aggregate No. 4 (Sun Gro
Horticulture, Maisonnette, N.B.) soilless mix. Untreated calla
tubers were planted directly in the soil medium, with the
top parts being exposed and the pots were watered lightly to
ensure capillary action. The exposed parts of the tubers
were treated with 25 mL of Ecc1 at 1 104 CFU/mL
suspended in tap water. Immediately following inoculation
with the bacteria, tubers were treated with 25 mL of singlephage isolates or a mixture of phage isolates in tap water at
1 106 PFU/mL (MOI, 100:1). Additionally, one treatment
consisted of 25 mL of phage mixture applied at time 0 and
7 d (2 mixture). The positive control consisted of tubers
treated with Ecc1 followed by 25 mL of tap water; the negative controls consisted of tubers treated with 25 mL of tap
water or with 25 mL of phage mixture. After the treatments,
the pots were topped up with the soilless mix and lightly
watered. Each treatment replication consisted of 9 pots
placed in a tray (30 cm 40 cm) lined with clean, opaque
plastic and subirrigated with calla fertilizer solution on a
regular basis. Treatments were replicated 3 times, in a
randomized complete block design and the experiment was
repeated twice. Average 24 h greenhouse temperatures were
maintained at ~21 C. The tubers and roots were visually
scored as either positive or negative for maceration at 7 weeks
post inoculation.
Statistical methods
When appropriate, data were log10-transformed and the
significance of the main factors and interactions were determined by general linear model (GLM) procedures, using
SAS (Windows version 8.0, SAS Institute Inc., Cary, N.C.).

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125

Table 2. Host-range patterns for bacteriophages on Erwinia carotovora subsp. carotovora

and selected nonhost species.
Group 1
Bacterial isolates

Ecc1

Erwinia carotovora subsp. carotovora

Ecc1a
(+)
Ecc26

Ecc48
(+)
Ecc71

Ecc83

Escherichia coli DH5

Erwinia chrysanthemi EchS80

Erwinia amylovora

Pantoea agglomerans

Group 2
Ecc3

Ecc5

Ecc13

Ecc14

(+)

(+)

(+)

(+)

Note: (+), weak plaque formation; +, plaque formation; , no plaque formation.

a
Bacterial isolate on which the phages were initially isolated and propagated.

When possible, data from each repeated experiment were

pooled. Where significant main-treatment effects occurred,
differences among means were determined by Students t
test (P 0.05).
Regression coefficient analysis was conducted using the
GLM procedures of SAS; differences between regression
coefficients were determined by a StudentsNewmann
Keuls test.

Results
Phage isolation
A total of 14 phages were isolated from greenhouse
fertilizer solutions and diseased calla tubers (Table 1). The
fertilizer solutions were obtained from commercial greenhouses that had infected calla lilies. Four isolates of E. carotovora subsp. carotovora, Ecc1, Ecc26, Ecc48, and Ecc71,
were used in the enrichment process. However, the only
host suitable for the isolation and enrichment of phages was
isolate Ecc1 of E. carotovora subsp. carotovora.
Host range
Erwinia carotovora subsp. carotovora isolates Ecc1, Ecc26,
Ecc48, Ecc71, and Ecc83 were used to establish host ranges
for the phages (Table 2). Single-phage isolates Ecc1, Ecc3,
Ecc5, Ecc13, and Ecc14 produced plaques only on Ecc1
and Ecc48. On bacterial hosts Ecc1 and Ecc48, the plaques
produced by phages Ecc1, Ecc3, and Ecc5 belonging to
group 1 (Ecc1Ecc12), as defined in the following section
Restriction fragment length polymorphism, were weaker than
the plaques produced by the two phages of group 2 (Ecc13
and Ecc14). Phages were unable to produce plaques on isolates Ecc26, Ecc71, and Ecc83 of E. carotovora subsp.
carotovora and selected non E. carotovora hosts.
Restriction fragment length polymorphism
On the basis of RFLP patterns, the bacteriophages of
E. carotovora subsp. carotovora were separated into two groups
(data not shown). Group 1 consisted of 12 phage isolates from
the 2001 sampling, and group 2 consisted of two phage isolates
from the 2003 sampling. All phage isolates from group 1 produced identical banding patterns when digested with restriction

endonucleases BamHI, EcoRI, or Bsh1236I. Phage isolates of

group 2 produced identical RFLP banding patterns when digested with BamHI, EcoRI, and Bsh1236I.
Transmission electron microscopy
Phage isolates specific to Erwinia carotovora subsp. carotovora used in the present study were characterized by icosahedral heads and long, flexible tails (Fig. 1). Phage Ecc5
was seen as a tailed and contracted phage; however, all
other phages were never observed in a contractile state.
Bacteriophage viability in fertilizer solutions and in
preplant-treatment solutions for calla tubers
Selected phages were evaluated for their ability to survive
over a period of 45 weeks in calla fertilizer solutions prepared with nonautoclaved, RO and local tap water (Fig. 2).
Linear regression analysis indicated that phage Ecc3 (group
1) titres remained constant (the rate of decay was not significantly different from zero (P 0.05)) at 1 109 PFU/mL in
the PBS control. Phage titres in fertilizer solution made
with autoclaved tap water declined at a rate of 0.033(log10
PFU/mL)/d, which was similar to that of the control. Phage
titres in both fertilizer solutions made with nonautoclaved
water (tap or RO) declined at significantly higher rates (approximately 3 times the above stated rate) than in the other
treatments. A similar pattern of phage persistence in the
above media was observed for phage Ecc13 (group 2; data
not shown).
Preplant treatment solutions for calla tubers initially contained 1 107 PFU/mL. After 48 h of incubation, phage
Ecc3 (group 1) could not be recovered from preplant-treatment
solutions for calla tubers containing Silwet 77, Promalin, fixed
copper, or a mixture of all three compounds. Phages were
recovered from both nonsterile RO water and sterile PBS controls. Survival in PBS control was approximately 12 times
higher than in RO water control (data not shown).
Biological control in liquid media
Phages did not significantly (P 0.05) reduce the density
of E. carotovora subsp. carotovora in the fertilizer solution
containing FeEDTA (data not shown). In fertilizer solutions
without FeEDTA, Ecc3, Ecc13, and Ecc14 signifi-

126
Fig. 1. Transmission electron micrographs of Erwinia
carotovora subsp. carotovora bacteriophages. Isolate Ecc5 was
a contractile phage (Fig. 1a) consisting of icosahedral heads and
long tails (Fig. 1b). Isolate Ecc11 was characterized by a
slight bend in the tail and prominent tail appendages (Fig. 1c).
The tails of isolate Ecc3 may or may not have a slight bend
(Fig. 1d). Scale bars represent 100 nm.

cantly reduced CFU density of E. carotovora subsp. carotovora in two trials (data not shown). The fertilizer solution
containing FeEDTA supported significantly higher numbers of E. carotovora subsp. carotovora than fertilizer solutions without FeEDTA.
Biocontrol assay on calla tuber plugs
Selected phage isolates were investigated for activity
against E. carotovora subsp. carotovora, as estimated by
inhibition of tissue maceration in assays on calla tuber plugs.
Single-phage isolates Ecc3, Ecc5, and Ecc12 (group 1)
exhibited significant (P 0.05) levels of biocontrol activity
(Fig. 3). Single-phage isolates Ecc13 and Ecc14 (group 2)
or a mixture of five phages did not exhibit significant
biocontrol activity. We could not correlate bacterial reductions with symptoms of soft rot (data not shown).
Greenhouse trials
Under greenhouse conditions, single-phage isolates Ecc2,
Ecc3, Ecc9, and Ecc14 or a mixture of all phage isolates, applied once and twice during the course of the experiment, exhibited significant (P 0.05) biocontrol activity
when evaluated for their ability to inhibit maceration of
tuber tissue (Fig. 4).

Can. J. Plant Pathol. Vol. 29, 2007

Discussion
Phage isolation and characterization
Fertilizer solutions from commercial greenhouses where
diseased callas were present were an ideal source of E. carotovora subsp. carotovora phages. The bacterial populations
in fertilizer solutions originated from infected tubers; therefore, bacterial diversity should be relatively narrow. In the
present study, E. carotovora subsp. carotovora phages were
enriched by only one of the four hosts used, and the host of
the phage isolate came from an infected calla tuber. Phages
in groups 1 and 2 had identical host ranges. They produced
plaques only on two of five E. carotovora subsp. carotovora
isolates (Table 2), these two isolates originating from infected calla tubers (Table 1). Phages were unable to lyse related bacteria of the same family or the same genus. While
these results indicate that the host specificity of these
E. carotovora subsp. carotovora phages is high and intrasubspecific, the number of bacterial hosts tested in the present
study was small; further host-range analyses are required to
conclusively determine the relative promiscuity of these
phages. Additionally, the phages in this study originated
from a single site; therefore, this small sample may not be
representative of the host range of E. carotovora subsp.
carotovora phages in general. Comparatively, Eayre et al.
(1995) isolated 24 E. carotovora subsp. carotovora phages
from a freshwater lake and surface waters. The phages also
exhibited a very limited host range when tested on 62 isolates of E. carotovora subsp. carotovora. Considering that
all of the phages in our work were isolated from the same
site, it is reasonable to assume that phages exhibiting identical restriction patterns and host ranges may be the same
phage (Gill et al. 2003).
An enrichment system involving four isolates of E. carotovora subsp. carotovora as hosts was utilized specifically
to prevent the selection of phages specific to any one bacterial
isolate, thereby increasing the diversity of the phages in the
collection. However, the two groups of E. carotovora subsp.
carotovora phages isolated in our study were highly host
specific (Table 2). Phages of group 1 were possibly brought
to the greenhouse with calla tubers infected with E. carotovora subsp. carotovora for summer crop of 2001, and
phages of group 2 were possibly imported with the tubers
for spring crop of 2003. The Californian fields producing
the seed tubers for these crops may be a source of genetic
diversity for E. carotovora subsp. carotovora adapted to
calla and their associated phages. Moreover, it is likely that
the viral and bacterial microflora of these fields varies from
location to location and from year to year.
Restriction fragment length polymorphisms of digested
phage DNA confirmed the separation of the isolated phages
into two groups (data not shown). Isolates of group 1 displayed identical RFLP patterns consistent with the fact that
they were all taken from the same location at the same time.
While group 2 was taken from the same location as group
1, the nature of the bacteriophages inhabiting the greenhouse had changed over time.
Transmission electron micrographs of phages in groups 1
and 2 indicated that they possessed icosahedral heads with
long tails. This places these phages into morphotypes A1
(Myoviridae) or B1 (Siphoviridae) of the system refined by

Ravensdale et al.: calla lily / bacterial soft rot / Erwinia carotovora / bacteriophages / biological control

127

Fig. 2. Surviving phage-forming units (PFU) of the bacteriophage Ecc3 of Erwinia carotovora subsp. carotovora isolated from calla
fertilizer solutions prepared in reverse osmosis (RO) and tap water over 36 d. Bars indicate the standard error of the mean (P 0.05).

Fig. 3. Effect of bacteriophage isolates on the development of symptoms of soft rot [Erwinia carotovora subsp. carotovora] on plugs
of calla tubers. Percentages represent proportions of 30 plugs showing symptoms of soft rot across two trials. Asterisks indicate
statistically significant (P 0.05) reductions in comparison with the control.

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Can. J. Plant Pathol. Vol. 29, 2007

Fig. 4. Effect of bacteriophages on incidence of soft rot caused by Erwinia carotovora subsp. carotovora in greenhouse-grown callas
after 7 weeks. Data from 54 plants across two trials are presented as the mean disease incidence of phage-treated plants. Asterisks
indicate statistically significant (P 0.05) reductions in comparison with the control. 2 mixture consisted of a phage mixture applied
at time 0 and 7 d.

Ackermann (2001). Except for Ecc5, the phages were not

observed in a contracted state. It is possible that variation in
phage preparation times caused us to miss observing other
phages in a contracted state; however, this is unlikely as
observations under transmission electron microscopy were
made on three separate occasions.
Phage viability in fertilizer solutions and preplanttreatment solutions for calla tubers
The persistence of bacteriophages of groups 1 and 2 in
fertilizer solutions over 45 weeks depended on the source
of water used, and whether or not this water was autoclaved
(Fig. 2). In fertilizer solutions made from tap water, the
initial decreases in phage titre may be attributed to chlorination of the municipal water. Chlorinated drinking water has
been shown previously to inactivate viruses (Engelbrecht et
al. 1980; Berg et al. 1989). In the case of autoclaved tap
water, the continued decline in viable phages over the
following 45 weeks can be attributed to the continued
action of this inorganic antiviral factor. Phage titres decreased rapidly in solutions made with nonsterilized water
compared with titres in sterile water (Fig. 2). Microbial
antagonism and viral survival in fresh water has been documented (Yates et al. 1985; Ward et al. 1986). In general,
viruses are less persistent in solutions made with fresh water
that has not been autoclaved than in the same solutions
made with autoclaved fresh water (Ward et al. 1986). Ward
et al. (1986) attributed this virucidal activity to the presence
of a heat-labile factor of microbial origin (likely an enzyme
or protein) rather than direct microbial attack. Furthermore,
many bacteria are capable of producing proteolytic enzymes
that exhibit antiviral activity (Cliver and Herrmann 1972;
Nasser et al. 2002).
Phage Ecc3 was unrecoverable after incubation for 48 h
in any of the preplant-treatment solutions for calla tubers.

This finding is consistent with previous reports of 100%

inactivation of viruses after a 30 min treatment with CuCl2
(copper II) at 100 mg/L (Sagripanti 1992), and of an
adverse effect of nonionic surfactants on bacteriophage survival (Chattopadhyay et al. 2002). However, Chattopadhyay
et al. (2002) did not observe complete inactivation of viral
activity as a result of nonionic surfactant treatment. While
there are no reports of antiviral activity by plant growth
regulators such as benzyladenine or gibberellic acid specifically, many plant metabolites are known to have antiviral
properties (Jassim and Naji 2003). Additionally, these plant
hormones were applied using a commercial formulation
(Promalin) and may contain compounds such as surfactants
that possess antiviral activity. The inability to recover phages
from any of the preplant-treatment solutions for calla tubers
could also be associated with the lack of sensitivity of the
spot-lysis method to detect phages (detection threshold, 1
102 PFU/mL).
Biocontrol assays
In the in-vitro biocontrol assays, bacteriophages of both
groups 1 and 2 reduced the number of CFU of E. carotovora subsp. carotovora in fertilizer solutions made without FeEDTA and in nutrient-broth solutions (data not
shown). However, reductions in bacterial numbers were not
observed in the fertilizer solution containing FeEDTA.
Romeo et al. (2001) reported that iron chelators inhibit the
replication in DNA viruses because of the iron dependency
of viral ribonucleotide reductase, an enzyme essential for
viral DNA synthesis. In regards to the bacteriophages in the
present study, it is possible that FeEDTA in the fertilizer
solution was taken up by E. carotovora subsp. carotovora,
and that intracellular iron was then sequestered away from
the bacteriophage by unbound EDTA, thereby inhibiting viral
replication. Alternatively, it is possible that EDTA inhibits

Ravensdale et al.: calla lily / bacterial soft rot / Erwinia carotovora / bacteriophages / biological control

the biocontrol ability of E. carotovora subsp. carotovora

phages by other means. For example, EDTA could bind
cations (other than iron) that serve to stabilize phages, such
as Ca2+ or Mg2+, or EDTA could interact with cations
bound to the capsid of the phage, as suggested by Adams
(1959). While reductions in bacterial populations in fertilizer
solutions without FeEDTA were significant, large numbers
of bacteria survived the bacteriophage treatments. The number of E. carotovora subsp. carotovora that survived the phage
treatment ranged from 101.3 to 103.6 CFU/mL. Thus it appears that the lower limit of biocontrol ability for this system is ~103.3 CFU/mL. This lower limit is most likely due
to physical escape of the pathogen from the phage. Some researchers (Wiggins and Alexander 1985; Payne and Jansen
2001) have reported that a minimal bacterial density of
~1 104 CFU/mL is required for bacteriophage replication.
This replication threshold could result from a requirement
by the phage for its host to be in a particular metabolic state,
which does not occur until populations of the host reach a
specific population density (Kasman et al. 2002). Alternatively, Kasman et al. (2002) suggested that the replication
threshold does not exist and that a mathematical model accounting for bacterial-cell density as well as phage adsorption time must be considered, when dealing with cell
densities less than 1 107 CFU/mL, to ensure accurate
MOI calculations.
In the bioassay on calla tuber plugs, phages of group 1
inhibited the ability of E. carotovora subsp. carotovora to
macerate plugs of calla tuber tissue, whereas phages of
group 2 and a mixture of both groups could not (Fig. 3). It
was unexpected that the mixture would prove ineffective,
considering that phages of group 1 proved effective. This
may have occurred as the overall concentration of phages
from group 1 in the mixture treatment would have been
60% (3 20%) of the concentration of phages from group 1
applied individually. The ability to suppress tissue maceration did not directly predict the ability of phages to control
bacterial populations on the surface of calla tuber plugs.
Phages from group 1 reduced tissue maceration by 50% or
more, while reducing bacterial growth by 70% or more,
whereas phages from group 2 did not significantly reduce
tissue maceration, even though they reduced bacterial growth
by 95% or more. While significant reductions in bacterial
growth did occur, the population surviving the phage treatment was large, numbering from 107.5 to 108.4 CFU/mL.
Thus, the lower limit of biocontrol ability for this system
appears to be ~108 CFU/mL. Utilizing bacteriophages to
control E. amylovora growth on the surface of pear plugs,
Gill (2002) found a similar lower limit in his system. Because
of the high bacterial populations on the plug surface, it is
unlikely that phages were unable to overcome a replication
threshold. Possibly, this lower limit is due to the physical
escape of the pathogen from the phage. In agreement with
this notion, Johnson (1994) suggested that a proportion of
pathogens remain inaccessible to the activity of the biocontrol agents. This may have occurred as phages would
have diffused away from the plug surface, or phages may
have been unstable on the surface of the plug. While conditions on the plug surface were within the limits tolerated by
most phages, it is possible that compounds of microbial or
plant origin contributed to the degradation of phages. The

129

mechanism(s) by which E. carotovora subsp. carotovora

can exist in high numbers on the plug surface and not cause
tissue maceration is not known.
In greenhouse trials, single-phage treatments reduced disease incidence by 40%70% while phage mixtures provided
significant reductions in disease incidence (Fig. 4).
This work represents important initial steps towards the
development of a bacteriophage-based biocontrol agent
against soft rot of calla caused by E. carotovora subsp.
carotovora, and provides a platform of knowledge for further
investigation. Characterization of the 14 phage isolated in
the present study allowed for subsequent comparison and
classification of E. carotovora subsp. carotovora phages.
Phages of E. carotovora subsp. carotovora in this collection
exhibited highly restricted host ranges, suggesting that the
use of a group of diverse isolates of E. carotovora subsp.
carotovora hosts would be advantageous for future phage
isolation procedures.
The persistence of E. carotovora subsp. carotovora phages
in fertilizer solutions exhibited a pattern that is consistent
with their putative roles as augmentative biocontrol agents.
Once the phage inhibiting or inactivating the FeEDTA
component was removed, phages exhibited the ability to reduce bacterial numbers in fertilizer solutions by up to 99%.
On the surface of calla tuber plugs, E. carotovora subsp.
carotovora phages reduced bacterial numbers by up to 97%
and disease symptoms up to 50%. However, in both of these
systems, a large number of bacteria escaped the phages,
leading to a lower limit of biocontrol efficacy. Most significantly, isolates of E. carotovora subsp. carotovora phages
reduced soft-rot incidence in greenhouse-grown calla lilies
by up to 70%. These results are encouraging and should
provide the impetus for further biocontrol evaluations, particularly in greenhouse environments.

Acknowledgements
This work was supported by special grants from the
Ontario Ministry of Agriculture and Food and Flowers
Canada, Ontario. We thank Ed Barszcz and Karin Schneider
for their assistance in the preparation of this manuscript.

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