APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Apr. 2003, p.

21332138
0099-2240/03/$08.000 DOI: 10.1128/AEM.69.4.21332138.2003
Copyright 2003, American Society for Microbiology. All Rights Reserved.

Vol. 69, No. 4

Bacteriophages of Erwinia amylovora

J. J. Gill,1,2 A. M. Svircev,1* R. Smith,3 and A. J. Castle2
Southern Crop Protection and Food Research Centre, Agriculture and Agri-Food Canada, Vineland Station, Ontario, Canada L0R
2E01; Department of Biology, University of Western Ontario, London, Ontario, Canada N6A 5B73; and Department of
Biological Sciences, Brock University, St. Catharines, Ontario, Canada L2S 3A12
Received 14 October 2002/Accepted 20 January 2003

Fifty bacteriophage isolates of Erwinia amylovora, the causal agent of fire blight, were collected from sites in
and around the Niagara region of southern Ontario and the Royal Botanical Gardens, Hamilton, Ontario.
Forty-two phages survived the isolation, purification, and storage processes. The majority of the phages in the
collection were isolated from the soil surrounding trees exhibiting fire blight symptoms. Only five phages were
isolated from infected aerial tissue in pear and apple orchards. To avoid any single-host selection bias, six
bacterial host strains were used in the initial isolation and enrichment processes. Molecular characterization
of the phages with a combination of PCR and restriction endonuclease digestions showed that six distinct
phage types, described as groups 1 to 6, were recovered. Ten phage isolates were related to the previously
characterized E. amylovora PEa1, with some divergence of molecular markers between phages isolated from
different sites. A study of the host ranges of the phages revealed that certain types were unable to efficiently lyse
some E. amylovora strains and that some isolates were able to lyse the epiphytic bacterium Pantoea agglomerans.
Representatives from the six molecular groups were studied by electron microscopy to determine their
morphology. The phages exhibited distinct morphologies when examined by an electron microscope. Group 1
and 2 phages were tailed and contractile, and phages belonging to groups 3 to 6 had short tails or openings with
thin appendages. Based on morphotypes, the bacteriophages of E. amylovora were placed in the order Caudovirales, in the families Myoviridae and Podoviridae.
Erwinia amylovora, a member of the Enterobacteriaceae, is
the causal organism of fire blight, a serious disease of the pome
fruit (1921). The disease-causing organism is currently controlled by the antibiotic streptomycin and pruning. Another
control measure became apparent when bacteriophages that
were able to infect E. amylovora and an avirulent yellow bacterium commonly found in the orchard ecosystem were discovered (8, 12). The yellow bacterium was subsequently identified
as Pantoea agglomerans (Erwinia herbicola). Erskine (11) recognized that E. amylovora phages may play an important role
in the epidemiology of fire blight and proposed the use of
phages released from the yellow saprophytic bacterium (lysogenic antagonists) as biological control agents (11).
Richie (16, 17) isolated E. amylovora phages from aerial
portions of fire blight-infected trees by using as a host strain E.
amylovora 110 Rif (16). The phages, named PEa1 and PEa7,
belonged to two distinct groups based on chemical and physical
data. Recently, E. amylovora phages were collected from orchards with fire blight symptoms and were characterized by
plaque morphology, PCR, restriction fragment polymorphisms, pulse-field gel electrophoresis, and host range studies
(18).
The objective of this work was to estimate the diversity of
bacteriophages collected from orchards in southern Ontario
that had active fire blight disease symptoms. To overcome any

potential host-induced bias, the initial isolation and enrichment of the phages exploited a six-host system. The host ranges
were determined for each phage isolate. Each phage isolate
was examined under the electron microscope and was placed in
phage families based on its morphology or morphotype as
described by Ackermann and colleagues (16, 14). In addition,
the phages were all grown on a common host, E. amylovora
110R, and further characterized by PCR using the PEa1-specific primers and restriction fragment length polymorphisms
(RFLPs).
MATERIALS AND METHODS
Bacterial strains and media. Strains of E. amylovora and P. agglomerans were
cultured on nutrient agar (Difco Laboratories, Detroit, Mich.) amended with
0.25% yeast extract (Difco) and 0.5% food grade sucrose (NASYE) and incubated at 26C. Liquid culture was carried out with nutrient broth (Difco)
amended with 0.25% yeast extract and 0.5% food-grade sucrose (NBSYE) and
incubated at 26C in an orbital shaker. Strains of Pseudomonas spp. were grown
on pseudomonas agar F (Difco) and incubated at 26C. Escherichia coli was
grown on Luria-Bertani medium (Difco) and incubated at 37C. Bacteriophages
were plated using the soft agar overlay method described by Adams (7). Phages
were diluted and stored in sterile 0.4% nutrient broth at 4C. Strains of bacteria
used in the experiments and their origin are listed in Table 1.
Phage isolation. Collections were made from mid-June to late August 1998
from sites in and around the Niagara region and Hamilton, Ontario, Canada. At
each collection site, cuttings were taken from the aerial portions of trees and soil
samples were taken from the bases of trees by using a stainless-steel soil corer
(diameter, 2 cm; length, 35 cm) driven to a depth of 10 to 20 cm approximately
1 m from the base of the tree. All soil and aerial samples were enriched in liquid
cultures in a procedure modified from that of Crosse and Hingorani (10). Flasks
containing 60 ml of NBSYE were inoculated with 200-l overnight cultures of
each of the six E. amylovora propagation hosts listed in Table 1. Into each flask
was placed 50 to 60 g (wet weight) of soil or 10 to 20 g (fresh weight) of aerial
tissue and incubated for 18 to 20 h. The resulting slurry was agitated thoroughly
with 500 l of chloroform and centrifuged at 4C and 8,000 g for 20 min. The
supernatant was removed with a pipette and stored at 4C over chloroform. The

* Corresponding author. Mailing address: Agriculture and AgriFood Canada, Southern Crop Protection and Food Research Centre,
4902 Victoria Ave. N., P.O. Box 6000, Vineland Station, ON, Canada
L0R 2E0. Phone: (905) 562-4113, ext. 227. Fax: (905) 562-4335. Email: svirceva@agr.gc.ca.
Present address: Agriculture and Agri-Food Canada, Food Research Program, Guelph, ON, Canada N1G 5C2.
2133

2134

GILL ET AL.

APPL. ENVIRON. MICROBIOL.

TABLE 1. Phenotypes, origin, and sources of E. amylovora isolates

used in this studya
Isolate

E. amylovora
Ea6-4*
Ea17-1-1*
EaD-7*
EaG-5*
Ea 29-7*
110R*
Ea 1-97
Ea 4-96
Ea 6-96b
BC20A
BC29
BC34A
BC1280
P. agglomerans
31420
49018
P. fluorescens A506
P. syringae MB-4
E. coli DH5-

Phenotype

Origin

wt
wt
wt
wt
wt
Rifr
wt
wt
wt
wt
Strr
wt
Strr

Pear
Pear
Pear
Pear
Apple
Apple
Raspberry
Raspberry
Raspberry
Crabapple
Pear
Apple
Apple

Source

D. Hunterb
D. Hunter
D. Hunter
D. Hunter
A. M. Svircevb
A. L. Jonesc
G. Braund
G. Braun
G. Braun
P. Sholberge
P. Sholberg
P. Sholberg
P. Sholberg
ATCCf
ATCC
Plant Health
Technologiesg
D. Cupplesh
A. Castlei

a
Asterisk denotes host strain during phage isolation and propagation. wt,
wild-type isolate. Rifr, rifampicin-resistant isolate. Strr, streptomycin-resistant
isolate.
b
Agriculture and Agri-Food Canada, Southern Crop Protection and Food
Research Centre, Vineland Station, Canada.
c
Department of Botany and Plant Pathology, Michigan State University, East
Lansing, Mich.
d
Agriculture and Agri-Food Canada, Atlantic Food and Horticulture Research Centre, Kentville, Canada.
e
Agriculture and Agri-Food Canada, Pacific Agri-Food Research Centre,
Summerland, Canada.
f
ATCC, American Type Culture Collection.
g
BlightBan A506; Boise, Id.
h
Agriculture and Agri-Food Canada, Southern Crop Protection and Food
Research Centre, London, Canada.
i
Department of Biological Sciences, Brock University, St. Catharines, Canada.

supernatant was diluted and plated onto six lawns each, seeded with one of the
propagation hosts listed in Table 1. Lawns were checked for the formation of
plaques after 24 and 48 h. Single plaques were picked from these lawns and
placed into microcentrifuge tubes containing 1 ml of NBSYE and 2% (vol/vol)
chloroform. Tubes were centrifuged at 8,000 g and stored at 4C. Bacteriophage isolates were purified by passage through this single-plaque isolation
procedure three times. Bacteriophage PEa1(h) (ATCC 29780-B1) was obtained
from the American Type Culture Collection. Representative phages in the collection are available upon request.
Host range analysis. The host ranges of all phage isolates were tested against
13 E. amylovora strains. Host ranges of a limited number of phages were also
tested against five bacterial strains representing four species other than E. amylovora. Bacterial lawns were prepared by seeding 3 ml of top agar with 107 CFU
bacteria suspended in 10 mM sodium phosphate buffer (pH 6.8). Phage lysates
were diluted to a concentration of 107 PFU/ml, and 10 l was spotted onto lawns.
Plates were dried in a laminar flow hood for 10 min and incubated at 26C for 18
to 20 h. Areas of clearing under points of phage application were scored as
positive, while areas which looked no different than the surrounding untreated
lawn were scored as negative. Experiments were repeated three times.
DNA extraction. Phage DNA was extracted using a method modified from that
of Manfioletti and Schneider (13). To each 10-ml volume of lysate was added
DNase I (Boehringer Mannheim, Laval, Canada) to a final concentration of 20
g/ml and RNase A (Boehringer Mannheim) to a final concentration of 100
g/ml. After incubation at room temperature for 15 min, 0.8 ml of 0.5 M EDTA
(Sigma) (pH 8) and proteinase K (Boehringer Mannheim) to a final concentration of 50 g/ml were added, followed by incubation at 45C for 15 min. DNA
was precipitated with 0.2% (wt/vol) hexadecyl trimethyl ammonium bromide
(Sigma) and 20 mM NaCl and incubated at 65C for 3 min, followed by cooling
on ice. The DNA-hexadecyl trimethyl ammonium bromide complex was pelleted
by centrifugation at 4C and 8,000 g for 10 min. The pellet was resuspended in

a minimal volume (usually 1 ml) of 1.2 M NaCl, and the DNA was precipitated
with two volumes of 95% ethanol. DNA was resuspended in a minimal volume
of sterile distilled water and stored at 20C.
PCR. Primer sequences specific for bacteriophage PEa1 were generously donated by A. L. Jones of Michigan State University. Primer sequences were 5
AATGGGCACCGTAAGCAGT 3 for PEa1-A and 5 TAATGGGTATGATA
GAAGGCAGAC 3 for PEa1-B. Primers were expected to amplify a 304-bp
product.
Reactions were run in 50-l volumes using a 0.2 M concentration (each) of
primers PEa1-A and PEa1-B (Norgen Biotek, St. Catharines, Canada), 1 PCR
buffer, 0.2 mM (each) deoxynucleoside triphosphates, 1.5 mM MgCl2, and 1.5 U
of Taq polymerase (MBI Fermentas). One l of a 108-PFU/ml phage suspension
in phosphate buffer was used as a template. Reactions were run in a GeneAmp
9600 thermocycler (Perkin-Elmer, Norwalk, Conn.) under the following conditions: 95C, 2 min; 95C, 30 s, 53C, 30 s, and 72C, 30 s, 30 cycles.
RFLPs. Bacteriophage DNA was digested with EcoRI, BglII, BamHI (all from
Promega), or ThaI (Life Technologies, Rockville, Md.) according to the suppliers instructions and using 0.5 to 1 g of DNA, 3 U of enzyme, and 0.1-mg/ml
acetylated bovine serum albumin per 50-l reaction mixture. Samples were
digested with EcoRI, BglII, or BamHI overnight at 37C or with ThaI for 1 to 2 h
at 60C. Fragments were separated on a 1% agarose gel in Tris-acetate-EDTA
and stained in 1 g of ethidium bromide/ml.
Transmission electron microscopy. High-titer phage liquid cultures were prepared and centrifuged at 8,000 g to separate phage from the host cells. The
supernatant containing the phages was centrifuged at 16,000 g for 1 h at 5C.
The phage pellet was resuspended in sterile distilled water. One drop of the
suspension was placed onto a 200-by-200-mesh nickel grid coated with formvar
and allowed to sit for 2 min. The phages on the grid were negatively stained with
2% uranyl acetate, 3% sodium phosphotungstate, or 1% ammonium molybdate.
To each stain solution, 4 mM MgCl2 was added in a 1:1 (vol/vol) ratio. This was
allowed to sit for 3 min before the solution was wicked off and the grid was air
dried. The electron microscope was calibrated with catalase crystals at the same
magnification used for viewing the phages (5). Each phage isolate was enriched
a minimum of three times on its original host, and each replicate was examined
under the electron microscope. Specimens were viewed using a Philips CM10
transmission electron microscope with an accelerating voltage of 80 kV.

RESULTS
Phage isolation. A total of 50 bacteriophage isolates were
collected from the Botanical Gardens and southern Ontario
apple and pear orchards. Eight bacteriophages isolates failed
to flourish under the laboratory storage and enrichment regimes. The majority of the 42 phages were collected from soil
samples adjacent to infected trees, while five phages were
isolated from diseased aerial tissue (Table 2).
Plaque morphology. The bacteriophage isolates in the collection produced one of four general plaque sizes on lawns of
host E. amylovora (Table 2). Those phages, identified as related to phage PEa1 by PCR and RFLP analysis (groups 3A,
3B, and 3C), produced distinctive 4- to 6-mm plaques with a
distinct translucent halo which continued to expand after the
plaque itself stopped growing. The remaining groups of phages
produced plaques of variable diameters ranging from 0.5 to 3
mm (Table 2).
Host range. The phages in groups 1, 2, 4, and 5 were all able
to form plaques on 11 or more of the 13 E. amylovora stains
tested (Table 3). Phages in Groups 3A, 5, and 6 exhibited
narrower host range patterns (Table 3). The phages in these
groups could not infect the British Columbia Erwinia isolates
and some of the Ontario isolates. Phages belonging to groups
3C (phage PEa1) and 3B showed little or no visible lytic activity
against E. amylovora strains Ea29-7, BC29, Ea34A, BC34A,
and BC1280 or against bacterial strains EaG-5 and Ea6-4,
isolated from Harrow, Ontario (data not shown). Host resistance was evident in the bacterial strains EaG-5, Ea6-4, and

BACTERIOPHAGES OF ERWINIA AMYLOVORA

VOL. 69, 2003

2135

TABLE 2. Phage grouping by RFLP pattern, bacterial host, origin, and morphotypes
Groupa

3A

3B
3C
4
5
6

Isolateb

Bacterial hostc

Crop

Sourced

Plaque type
(diameter, mm)

Head
(Length,e nm)

Morphotypesf

PEa10-2
PEa10-3
PEa10-4
PEa21-1
PEa21-2
PEa21-3
PEa21-4
PEa31-1
PEa35-2
PEa10-6
PEa31-2
PEa31-4
PEa35-7
PEa10-7
PEa10-8
PEa10-9
PEa10-10
PEa10-11
PEa10-13
PEa10-14
PEa10-15
PEa31-3
PEa46-2
PEa1
PEa10-12
PEa10-16
PEa9-2
PEa9-4
PEa9-5
PEa51-1
PEa51-2
PEa51-4
PEa51-6
PEa51-7
PEa51-8

Ea6-4
EaD-7
EaG-5
110R
EaG-5
EaD-7
Ea6-4
110R
Ea17-1-1
Ea29-7
EaD-7
Ea29-7
Ea29-7
Ea29-7
Ea29-7
110R
110R
Ea17-1-1
Ea6-4
Ea110
110R
Ea29-7
EaD-7
110R
EaG-5
Ea17-1-1
Ea17-1-1
EaG-5
Ea6-4
Ea17-1-1
110R
Ea29-7
Ea6-4
Ea29-7
Ea17-1-1

Apple
Apple
Apple
Pear
Pear
Pear
Pear
Apple
Pear
Apple
Apple
Apple
Pear
Apple
Apple
Apple
Apple
Apple
Apple
Apple
Apple
Apple
Crabapple
Apple
Apple
Apple
Pear
Pear
Pear
RBGh
RBG
RBG
RBG
RBG
RBG

Aerial
Aerial
Aerial
Soil
Soil
Soil
Soil
Soil
Soil
Aerial
Soil
Soil
Soil
Soil
Soil
Soil
Soil
Soil
Soil
Soil
Soil
Soil
Aerial
Aerial
Soil
Soil
Soil
Soil
Soil
Soil
Soil
Soil
Soil
Soil
Soil

13
13
13
13
13
13
13
13
13
0.5
0.5
0.5
0.5
46
46
46
46
46
46
46
46
46
46
46
0.5
0.5
13
13
13
12
12
12
12
12
12

NTg
NT
5769
6071
NT
5371
NT
86110
6372
NT
84114
NT
NT
4654
NT
NT
NT
NT
NT
NT
NT
NT
NT
5363
6071
NT
5565
NT
NT
NT
6677
NT
NT
NT
NT

A1
A1
A1
A1
A1
A1
A1
A1
NT
NT
A1
NT
NT
C1
C1
C1
C1
NT
NT
NT
NT
C1
C1
C1
C1
C1
C1
NT
C1
NT
C1
C1
NT
NT
NT

a
Group designation based on PCR and RFLP analyses. PEa9-3, PEa10-1, PEa35-3, PEa35-4, PEa35-5, PEa35-6, PEa45-1, and PEa45-3 were left ungrouped. The
first six phages listed could not be digested with the four restriction enzymes used in this study, and the remaining two phages were not tested by RFLP.
b
Phage isolates were collected from seven different sites in southern Ontario, Canada. Designation for each phage is PEa, followed by site number-isolate number.
c
Bacterial host E. amylovora isolate designations are described in Table 1.
d
Source of phages in the orchard.
e
Length measurement represents a range of values obtained by measuring a minimum of five phages/isolate.
f
Phage morphology for phage families as described by Ackermann, Fig. 1 (3). Tailed phages belonging to morphotype A1 to A3 are Myoviridae with contractile tails;
morphotype B1 to B3 are Siphoviridae with noncontractile tails; C1 to C3 are Podoviridae with short tails.
g
NT, not tested.
h
RBG, Royal Botanical Gardens, Hamilton, Canada. Collected from trees, Sorbus spp., demonstrating fire blight symptoms.

Ea4-96 of E. amylovora. The phages in groups 1, 4, and 6 were

unable to lyse these host bacterial isolates.
A limited number of isolates were selected for evaluation of
their ability to form plaques on lawns composed of bacterial
species other than E. amylovora (data not shown). Of the 14
phage isolates used in these experiments, none was able to lyse
lawns composed of E. coli DH5, Pseudomonas syringae MB-4,
or Pseudomonas fluorescens A506. Two phage isolates from
group 1 were able to produce plaques on both P. agglomerans
strains evaluated. Of the six members of group 6, five were
tested and all were able to lyse P. agglomerans 49018.
PCR. Using the primers specific for bacteriophage PEa1,
phages PEa10-7, PEa10-8, PEa10-9, PEa10-10, PEa10-11,
PEa10-13, PEa10-14, PEa10-15, PEa31-3, and PEa46-2 produced a ca. 300-bp PCR product, indicating relatedness to
phage PEa1 (data not shown). Phage PEa1(h) obtained from

the American Type Culture Collection also produced this fragment.

RFLPs. DNA was extracted from 40 bacteriophage isolates
collected from the field and phage PEa1(h). The arrangement
of phage isolates into groups based on RFLP data is shown in
Table 2. All of the phages which were identified as related to
PEa1 using PCR produced similar restriction patterns; these
phages were placed into Group 3. Isolates PEa10-7, PEa10-8,
PEa10-9, PEa10-10, PEa10-11, PEa10-13, PEa10-14, PEa1015, and PEa31 produced identical restriction patterns and were
designated group 3A. Phage PEa46-2, which produced two
more fragments than the phages in group 3A when digested
with BglII, was undigested by EcoRI and produced two different fragments when digested with ThaI. Based on this information, PEa46-2 was placed into group 3B. Phage PEa1(h) was
also undigested by EcoRI and produced two fragments which

2136

GILL ET AL.

APPL. ENVIRON. MICROBIOL.

TABLE 3. Phage host range according to RFLP groups and Erwinia host
No. of phage isolates in RFLP grouph

Erwinia host

EaG5a
EaD7
Ea6-4
Ea17-1-1
Ea29-7
110Rb
BC20Ac
BC29
BC34A
BC1280
Ea4-96d
Ea6-96b
Ea1-97

Group 1

Group 2

Group 3A

Group 4

Group 5

Group 6

Ungrouped

()f

()

()

()

()

()

()

6
9
9
9
4
9
8
9
4
4
5
8
9

1
0
0
0
4
0
0
0
3
4
1
0
0

3
0
0
0
0
0
0
0
0
0
1
0
0

2
4
4
4
4
4
4
4
4
4
4
4
4

0
0
0
0
0
0
0
0
0
0
0
0
0

2
0
0
0
0
0
0
0
0
0
0
0
0

2
9
2
9
9
9
9
2
2
2
2
9
9

7
0
4
0
0
0
0
0
0
1
2
0
0

0
0
3
0
0
0
0
7
7
6
0
0
0

2
2
2
2
1
2
2
2
1
0
0
2
2

0
0
0
0
1
0
0
0
1
2
0
0
0

0
0
0
0
0
0
0
0
0
0
1
0
0

3
3
3
3
3
3
3
2
0
0
0
3
3

0
0
0
0
0
0
0
0
2
1
2
0
0

0
0
0
0
0
0
0
1
1
2
0
0
0

2
5
2
6
5
6
6
2
0
0
0
6
6

4
1
0
0
0
0
0
0
2
3
0
0
0

0
0
4
0
1
0
0
4
4
3
6
0
0

7
8
8
8
8
8
8
8
5
4
8
8
8

0
0
0
0
0
0
0
0
2
2
0
0
0

1
0
0
0
0
0
0
0
0
2
0
0
0

Bacteria isolated from pear and apple orchards in southern Ontario.

Isolate obtained from Michigan.
Bacteria isolated from apple orchards in British Columbia.
d
Bacteria isolated from raspberry plantings in Nova Scotia.
e
, Visible plaque formation
f
(), weak plaque formation.
g
, No plaque formation.
h
Numbers in the columns represent the number of phage isolates that produced a plaque, weak plaque, and/or no plaque on the designated Erwinia host.
b
c

were of differing lengths from those found in group 3A when

digested with BglII and one extra fragment when digested with
ThaI. Phage PEa1 (h) was placed into group 3C.
Transmission electron microscopy. E. amylovora bacteriophages were placed into morphotype groups according to the
method of Ackermann (3). The phage morphology within each
RFLP grouping is described in Table 2. The phages placed into
RFLP group 1 consisted of an icosahedral head (variable
lengths from 53 to 110 nm), contractile tail, and A1 morphotype. Two distinct phage shapes and sizes were evident in this
group: the smaller phages, as seen in (Fig. 1A and C), or the
larger phages (Fig. 1B and D). Phages PEa31-2 and -4 belonged to RFLP group 2 and morphotype A1. The contracted
stage was never observed for these two phage isolates. The
morphology of the distinct rigid tail places the phages in the
Myoviridae family (Hans Ackermann, personal communication). All the phages placed in RFLP groups 3A, 3B, 3C, 4, 5,
and 6 belonged to morphotype C1 (Fig. 1E).
DISCUSSION
Of the 42 bacteriophages collected from the pear and apple
orchards and the Royal Botanical Gardens, 37 phages were
isolated from the soil surrounding the infected trees. Five
phages were recovered from aerial portions of infected
branches that exhibited fire blight symptoms. This finding is in
agreement with the work of Erskine (11), who isolated phages
from the soil surrounding infected trees and not from aerial
samples. Phages have been shown to be stabilized in soil environments due to adsorption to charged colloids, such as clays
(15, 22). Additionally, soil provides protection against desiccation and UV light, two factors which will inactivate a wide
variety of phages, including those of E. amylovora (7, 11, 12,
16). In this study, E. amylovora phages could be recovered from
soil samples after 12 months storage at 4C (data not shown).

It is unlikely that populations of E. amylovora phages are able

to reside permanently in aerial tissues, since Ritchie (16, 17)
was unable to isolate E. amylovora phages from aerial tissue in
the winter months.
The independent methods used to characterize the phages,
molecular identification, plaque morphology, and electron microscopy, proved to be remarkably congruent in organizing
phages in this collection. Phages were placed into six groups
based on restriction patterns, with one group, group 3, subdivided into three subgroups. Transmission electron microscopy
of phages in each of the restriction groupings revealed that the
morphology was consistent within the same group. Based on
the system devised by Bradley (9) and refined by Ackermann
and DuBow (1, 5), the morphology will place these phages into
the virus order Caudovirales. In our study all phages belonging
to groups 1 and 2 had contractile tails and belonged to the
family Myoviridae (Fig. 1A to E). Within the Myoviridae two
distinct morphologies were evident, a smaller contractile phage
and a larger contractile phage. Both morphotypes, however,
are consistent with the A1 type description of Ackerman and
Dubow (1, 5), and both produced the same plaque morphology. The phages seen in Fig. 1A and D are similar in appearance to Pseudomonas spp. phages, while those in Fig. 1B and D
have not been previously observed in the Enterobacteriaceae
(H.-W. Ackermann, personal communication). The phages belonging to groups 3A, 3B, 3C, 4, 5, and 6 were short-tailed
phages in the family Podoviridae. In this group the morphology
varied from short tails to barely discernible openings with or
without decorations. Group 3 phages were all identifiable as
PEa1 type both by the PCR protocol and by their production
of distinctive, large plaques with expanding haloes. The majority of these phages, placed into group 3A, produced identical
restriction patterns which differed slightly from the type phage,
PEa1, and from phage PEa46-2. Group 6 phages had a unique
restriction pattern and plaque morphology but shared phage

VOL. 69, 2003

FIG. 1. Electron micrographs of representative phages belonging

to restriction groups 1 and 3. (A to D) Erwinia bacteriophages belonging to the Myoviridae. Small phage in uncontracted state (A), larger
phage in uncontracted state (B) (note the decorations at the bottom of
the tail [arrow]), small phage in the contracted state (C), and the larger
phage in the contracted state (D) are shown. Panel E shows group 3A
phages belonging to the Podoviridae. The arrow points to the tail
region. Micron marker, 100 nm.

morphological characteristics with groups 3, 4, and 5. Based

upon these observations, it should be possible to categorize
with a fair degree of accuracy any new E. amylovora bacteriophage isolates.
The one characteristic which did not show any consistent
correlation with any of the above phenotypes was host range.
This characteristic could not be used to place individual phage
into any a specific RFLP group. This lack of association of host
range with any other characteristic could very likely be the
reason for the great diversity of phages isolated in this study.
Historically, studies involved in the isolation of E. amylovora
bacteriophages have consistently used a single host system (16,
17, 18). Schnabel and Jones (18) isolated 48 bacteriophages on
a single host, E. amylovora strain 110R. They recovered five
distinct phage types based on differences in genome size and
restriction patterns; however, the diversity of phages was not
extensive in that 41 of the 48 isolates were of the PEa1 type, 4
of the remaining 7 were PEa7-like, and 3 were novel (18). In
the present study, six bacterial hosts, including strain 110R,
were used to isolate bacteriophages. The use of the six hosts

BACTERIOPHAGES OF ERWINIA AMYLOVORA

2137

had a profound influence on the variety of bacteriophages

recovered. Analysis of host ranges revealed that phages were
able to infect a wide range of host bacteria. We noted that
phages in groups 3, 5, and 6, with few exceptions, were unable
to infect the E. amylovora bacteria from British Columbia.
Therefore, the diversity of our collection may in part be attributed to the use of the six-host system in the initial isolation and
purification protocol. The bacterial host, E. amylovora 110R,
was used both in our study and by Schnabel and Jones (18).
This particular host bacterium was lysed by all the phages in
our collection.
Individual phage isolates grouped together on restriction
patterns were isolated from the same site. The eight phages in
group 1, for instance, were collected from three sites; the six
inhabitants of group 6 were all isolated from the same location.
In these cases, it is not unreasonable to make the assumption
that phage isolates exhibiting identical restriction patterns and
isolated from the same sample are the same phages, isolated
multiple times on different bacterial host strains. Phages with
identical restriction patterns can be said to be highly related to
each other regardless of site of isolation. The groups based on
molecular markers will aid in streamlining future research.
Based on the above observation, the authors would suggest
that in fact phage populations are quite homogeneous in an
orchard. In our collection only the phages designated with the
location/phage number PEa10 were placed into four out of the
six possible restriction groups.
In this study we examined a collection of phages based on
restriction endonuclease patterns, phage particle morphology,
plaque morphology, and host range. Future research will explore the potential of using phages as biological control agents
under field conditions.
ACKNOWLEDGMENTS
This work was supported by a Matching Investment Initiatives grant
from Agriculture and Agri-Food Canada. Financial support for graduate student J. J. Gill was provided by Horizons Canada and the
Ontario Apple Marketing Commission.
We thank Alison Myers and Charlene Green for their patience and
hard work during the collection and isolation of the bacteriophages
from the orchards. Ed Barszcz and Brent Wiens, Agriculture and
Agri-Food Canada, Vineland Station, provided commendable computer and photography skills in the layout of the electron micrograph
plates. A. L. Jones, Department of Plant Pathology, Michigan State
University, provided invaluable assistance and provision of PCR primers for PEa1 phage. Hans-Wolfgang Ackermann, Department of Medical Biology, Laval University, Quebec, Canada, provided invaluable
advice and guidance on the interpretation of the bacteriophage morphology and general comments and advice on the manuscript.
REFERENCES
1. Ackermann, H.-W. 1987. Bacteriophage taxonomy in 1987. Microbiol. Sci.
4:214218.
2. Ackermann, H.-W. 1996. Frequency of morphological phage descriptions in
1995. Arch. Virol. 141:209218.
3. Ackermann, H.-W. 2001. Frequency of morphological phage descriptions in
the year 2000. Arch. Virol. 146:843857.
4. Ackermann, H. W., A. Audurier, L. Berthiaume, L. A. Jones, J. A. Mayo, and
A. K. Vidaver. 1978. Guidelines for bacteriophage characterization. Adv.
Virus Res. 23:124.
5. Ackermann, H.-W., and M. S. Dubow (ed.). 1987. General properties of
tailed phages, p. 1113. In Viruses of prokaryotes. CRC Press, Boca Raton,
Fla.
6. Ackermann, H.-W., M. S. DuBow, A. W. Jarvis, A. L. Jones, V. N. Krylov, J.
Maniloff, J. Rocourt, R. S. Safferman, J. Schneider, L. Seldin, T. Sozzi, P. R.
Stewart, M. Werquin, and L. Wu
nsche. 1992. The species concept and its
application to tailed phages. Arch. Virol. 124:6982.

2138

GILL ET AL.

7. Adams, M. H. 1959. Bacteriophages. Interscience Publishers, New York,

N.Y.
8. Baldwin, C. H., Jr., and R. N. Goodman. 1963. Prevalence of Erwinia amylovora in apple buds as detected by phage typing. Phytopathology 53:1299
1303.
9. Bradley, D. E. 1965. The morphology and physiology of bacteriophages as
revealed by the electron microscope. J. R. Microsc. Soc. 84:257316.
10. Crosse, J. E., and M. K. Hingorani. 1958. A method for isolating Pseudomonas mors-prunorum phages from soil. Nature 181:6061.
11. Erskine, J. M. 1973. Characteristics of Erwinia amylovora bacteriophage and
its possible role in the epidemiology of fire blight. Can. J. Microbiol. 19:837
845.
12. Hendry, A. T., J. A. Carpenter, and E. H. Gerrard. 1967. Bacteriophage
studies of isolates from fire blight sources. Can. J. Microbiol. 13:13571364.
13. Manfioletti, G., and C. A. Schneider. 1988. A new and fast method for
preparing high quality lambda DNA suitable for sequencing. Nucleic Acids
Res. 16:28732885.
14. Maniloff, J., and H. W. Ackerman. 1998. Taxonomy of bacterial viruses:
establishment of tailed virus genera and the order Caudovirales. Arch. Virol.
143:20512063.
15. Reanney, D. C., P. C. Gowland, and J. H. Slater. 1983. Genetic interactions
among microbial communities, p. 379422. In J. H. Slater (ed.), Microbes in

APPL. ENVIRON. MICROBIOL.

16.

17.
18.
19.

20.
21.
22.

their natural environments. Cambridge University Press, Cambridge, United

Kingdom.
Ritchie, D. F. 1978. Bacteriophages of Erwinia amyovora: their isolation,
distribution, characterization, and possible involvement in the etiology and
epidemiology of fire blight. Ph.D. thesis. Michigan State University, Ann
Arbor, Mich.
Ritchie, D. F., and E. J. Klos. 1979. Some properties of Erwinia amylovora
bacteriophages. Phytopathology 69:10781083.
Schnabel, E. L., and A. L. Jones. 2001. Isolation and characterization of five
Erwinia amylovora bacteriophages and assessment of phage resistance in
strains of Erwinia amylovora. Appl. Env. Microbiol. 67:5964.
van der Zwet, T., and S. V. Beer. 1991. Fire blight: its nature, prevention and
controla practical guide to integrated disease management. Agriculture
information bulletin no. 631. U.S. Department of Agriculture, Washington,
D.C.
Van Der Zwet, T., and H. Keil. 1979. Fire blight: a bacterial disease of
rosaceous plants. U.S. Department of Agriculture, Washington, D.C.
Vanneste, J. L. 2000. Fire blight: the disease and its causative agent, Erwinia
amylovora. CAB International, New York, N.Y.
Williams, S. T., A. M. Mortimer, and L. Manchester. 1987. Ecology of soil
bacteriophages, p. 320. In S. M. Goyal, C. P. Gerba, and G. Bitton (ed.),
Phage ecology. John Wiley & Sons, New York, N.Y.