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Bacteriophages for Plant

Disease Control
J.B. Jones,1 L.E. Jackson,2 B. Balogh,1
A. Obradovic,3 F.B. Iriarte,4 and M.T. Momol5
1

Department of Plant Pathology, University of Florida, Gainesville, Florida 32611;

email: jbjones@u.edu

Layton, Utah 84040

University of Belgrade, Faculty of Agriculture, Plant Pathology Department,

11080 Belgrade-Zemun, Serbia

USDA ARS, Ft Pierce, Florida 34945

North Florida Research and Education Center, University of Florida, Quincy,

Florida 32351

Annu. Rev. Phytopathol. 2007. 45:24562

Key Words

First published online as a Review in Advance on

March 26, 2007

biocontrol, phage therapy, phage ecology

The Annual Review of Phytopathology is online at

phyto.annualreviews.org
This articles doi:
10.1146/annurev.phyto.45.062806.094411
c 2007 by Annual Reviews.
Copyright 
All rights reserved
0066-4286/07/0908-0245$20.00

The U.S. Government has the right to retain a

nonexclusive, royalty-free license in and to any
copyright covering this paper.

Abstract
The use of phages for disease control is a fast expanding area of plant
protection with great potential to replace the chemical control measures now prevalent. Phages can be used effectively as part of integrated disease management strategies. The relative ease of preparing
phage treatments and low cost of production of these agents make
them good candidates for widespread use in developing countries
as well. However, the efcacy of phages, as is true of many biological control agents, depends greatly on prevailing environmental
factors as well as on susceptibility of the target organism. Great
care is necessary during development, production and application of
phage treatments. In addition, constant monitoring for the emergence of resistant bacterial strains is essential. Phage-based disease
control management is a dynamic process with a need for continuous adjustment of the phage preparation in order to effectively ght
potentially adapting pathogenic bacteria.

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SAR: systemic
acquired resistance

246

10:33

CHALLENGES FOR
CONTROLLING BACTERIAL
DISEASES ON PLANTS
Plant diseases caused by bacteria are a major
economic liability to agricultural production.
Disease control has been a major challenge
for many bacterial diseases (21). This challenge is a direct result of pathogen variability,
high probability for mutation or gene transfer
in the pathogen when confronted with resistance genes or bactericides, high pathogen
multiplication rate during optimal conditions
for disease development, and lack of adequate
chemical-based approaches for control.
Disease control is best achieved using an integrated management approach by combining
proper cultural practices, chemicals such as
bactericides or plant activators where applicable, introgression of plant resistance genes,
and biological control strategies (75, 76).
Copper-based bactericides introduced in
the 1880s are still used extensively to combat many bacterial plant diseases, although
copper resistance has been reported in many
bacterial pathogens, including plasmid-borne
resistance and chromosomal resistance (7, 13
15, 55, 86). Bacterial diseases of plants have
been difcult to contain because few effective,
economical bactericides have been developed.
Streptomycin, an aminoglycoside antibiotic,
has been utilized in agriculture since the 1950s
to control phytopathogenic bacteria (95). Extensive use of this antibiotic for control of
various bacterial diseases resulted in increased
prevalence of streptomycin-resistant strains in
bacterial populations and reduced efcacy of
control of bacterial spot of tomato and pepper (95), re blight of apple and pear (60), as
well as many other bacterial plant pathogens
(25). Resistance develops as a result of both
selection pressure and the widespread distribution of plasmid-encoded streptomycin resistance genes (20, 68, 74, 90).
Systemic acquired resistance (SAR) plant
inducers have shown activity against bacterial
diseases of tomato and pepper (58, 75, 81),
Xanthomonas leaf blight on onion (32), and re

Jones et al.

blight on apple (63). Although SAR inducers

may reduce disease, they may also have deleterious effects on certain plant species and/or
affect yield (32, 81). Plant inducers have also
been ineffective for disease control in some
pathosystems such as for control of citrus
canker (38).
Deployment of resistance genes has been
a goal of many research programs. Although
plant resistance genes have been deployed
in some crop systems, effectiveness has been
compromised due to development of strains
that overcome the resistance (31, 52, 79). In
other situations, it has been difcult to introgress plant resistance genes into commercially acceptable cultivars.
Given the difculties in controlling bacterial diseases using conventional strategies,
considerable efforts have been made to use
biologically based strategies. Biological control of bacterial diseases on tomato has been
attempted using nonpathogenic strains of the
pathogen (30, 57a), saprophytic bacteria (47),
and plant growth-promoting inducing plant
rhizobacteria (47). In all cases disease control
has been variable. Recently, there has been
resurgence in interest in use of bacteriophages
for control of bacterial plant diseases (28,
33, 34).

EARLY STUDIES WITH PHAGES

Phages were discovered in the early part of
the twentieth century by Twort in 1915 and
by dHerelle in 1917 (89), who independently
reported about lterable and transmissible
agents of bacterial lysis. They did not agree,
though, on the origin of this lytic principle. Whereas Twort proposed that a bacterial enzyme with ability to grow caused
the lysis, dHerelle speculated that a virus
was responsible for the phenomenon. Even
though dHerelles bacterial virus theory was
not proven conclusively for over 30 more
years, the idea of using phages as control
agents of bacterial diseases was a direct consequence of his concept.

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There was considerable enthusiasm in the

medical eld regarding possible use of phages
as tools for ghting bacterial diseases that did
not have any effective control measures. Four
years after dHerelles discovery, Brunoghe &
Maisin (18) reported control of Bacillus anthracis and staphylococcus infections with bacteriophage treatment; a year later phages were
evaluated for control of typhoid fever and
bacillary dysentery (9, 27). Phages were also
used extensively to control cholera outbreaks
in India (71). Over 800 papers were published
over the next 40 years concerning disease control or prevention with phages (54).
A few years after dHerelles discovery,
phages were proposed as plant disease control agents (70) and were evaluated for controlling bacterial plant diseases. In 1924,
Mallman & Hemstreet (59) isolated Xanthomonas campestris pv. campestris from diseased cabbage tissue and observed that ltrate
of liquid collected from decomposed cabbage
inhibited in vitro growth of the pathogen. The
following year, Coons & Kotila (26) inoculated moist carrot disks with Erwinia carotovora subsp. carotovora alone or in combination with its homologous phage. Rotting
occurred only where bacteria were applied
alone. They believed that as a result of the
widespread presence of this lytic principle
(phages), phage could play an important role
in limiting bacterial organisms in the soil in
nature. Kotila & Coons (51) also isolated bacteriophages from soil samples active against
the causal agent of blackleg disease of potato.
They demonstrated in growth chamber experiments that coinoculation of Erwinia carotovora subsp. atroseptica with phage successfully inhibited the pathogen and prevented
rotting of tubers.
In 1934, Massey (62) suggested that phage
was a prime factor in limiting severity of bacterial blight disease in eld-grown cotton. The
incidence of bacterial blight disease was reduced in elds that had been ooded by the
Nile River. Massey attributed this reduction
to destruction of Xanthomonas malvacearum by
phage carried in Nile River water. A year ear-

lier he had obtained phage for X. malvacearum

from previously ooded soil, but not from soil
that had not come in contact with river water.
Thomas (96) reported effective control of
Stewarts wilt disease of corn. He treated corn
seeds that were infected with Pantoea stewartii,
the causal agent of Stewarts wilt of corn, with
phage isolated from diseased plant material.
The seed treatment reduced disease incidence
from 18% to 1.4%.
Despite the promising early work, phage
therapy did not prove to be a reliable and
effective means of controlling phytobacteria. Several workers questioned whether positive results were possible. In 1963, Okabe
stated, in general, the phage seems to be ineffective for [controlling] the disease development (77). Three decades later, Goto concluded, practical use of phages for control of
bacterial plant disease in the eld has not been
successful. (36). Furthermore, given the narrow spectrum of activity of phages and broad
spectrum activity of antibiotics, it was a common belief in the medical eld that phages
were much more likely to fail if the causal
agent was not accurately identied (89). Interest in phages waned because of this limitation
as well as the abundance of effective antibiotics. Chemical control with antibiotics and
copper compounds became the standard for
controlling bacterial plant diseases (61, 95).

REVIVED INTEREST IN USE

OF BACTERIOPHAGE FOR
CONTROL OF BACTERIA
Recent events such as the appearance of bacterial strains that are resistant to all known antibiotics as well as a slowdown in introducing
effective new antibiotics renewed interest in
phage therapy in the eld of medicine. The
greater knowledge and advanced research
techniques led to successful treatment of several bacterial diseases in humans in Poland
(84) and to the development and commercial
application of a wide range of phage-based
medical products in the former Soviet republics of Georgia and Russia, such as sprays,

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Xcp: Xanthomonas
campestris pv. pruni

10:33

ampoules, and pills for treatment of various

skin and enteric diseases (54). In these systems, there was a great emphasis on building a bank of pathogenic bacterial strains and
phages that were capable of lysing them.
Initially, the host range, lytic spectrum, and
cross-resistance properties of phage strains
were evaluated. Only lytic phages were used
for therapy, thereby avoiding problems caused
by lysogeny. To prevent the problem of resistance, a phage cocktail was prepared from
strains with different receptor specicity for
the pathogenic bacterium, sometimes for several different species of pathogenic bacteria
(54). For example, a formulation called IntestiPhage, developed by the Eliava Institute
in Georgia, contained 23 different phages active against a wide range of enteric bacteria (54). Phage formulations were successfully
used not only for disease treatment but also
for prevention (85) as well as for sanitation
purposes (54).
There are several potential advantages for
using phages in disease control:
1. Phages are self-replicating and selflimiting; they replicate only as long as
the host bacterium is present in the environment, but are quickly degraded in
its absence (54).
2. Phages are natural components of the
biosphere; they can readily be isolated
from wherever bacteria are present, including soil, water, plants, animals (1,
37, 100), and the human body (78).
3. Phages can be targeted against bacterial
receptors that are essential for pathogenesis, so resistant mutants are attenuated in virulence (54).
4. Phages are nontoxic to the eukaryotic
cell (39). Thus, they can be used in situations where chemical control is not allowed owing to legal regulations, such
as for treatment of peach fruit before
harvest (101) or for control of human
pathogens in fresh-cut produce (56, 57).
5. Phages are specic or highly discriminatory, eliminating only target bacteria

248

Jones et al.

without damaging other, possibly benecial, members of the indigenous ora.

Thus their use can also be coupled with
the application of antagonistic bacteria
for increased pressure on the pathogen
(94); or they can be used to promote a
desired strain against other members of
the indigenous ora (8).
6. Phage preparations are fairly easy
and inexpensive to produce and can
be stored at 4 C for months without
signicant reduction in titer (39). Application can be carried out with standard
farm equipment. Since phages are not
inhibited by the majority of agrochemicals (4, 101), they can be tank-mixed
with many agrochemicals without
signicant loss in titer.

RECENT USE OF PHAGES

IN PLANT PATHOLOGY
More than 30 years after the earliest work on
using phages for control of bacterial plant diseases, Stanier et al. (87) reported that fewer
than ten phage particles present at the beginning of a 21-h induction period completely
inhibited tumor induction by a highly virulent
strain of Agrobacterim tumefaciens. Civerolo &
Keil (24) found that by treating peach foliage
with Xanthomonas campestris pv. pruni (Xcp)
phage 1 h before inoculation of the bacterium,
the percentage of infected leaves was 22%
compared to 58% for control plants. Foliage
treated 24 h prior to inoculation with Xcp resulted in 29% infected leaves.
Boyd et al. (17) demonstrated that tomato
plants absorbed virus within three hours after
root immersion in a phage lysate of A. tumefaciens. Phage was detected two weeks later in
roots and stems. A signicant gall size reduction (43%72%) was obtained when wounded
plants absorbed phage lysates three hours before bacterial inoculation.
A survey by Kuo et al. (53) revealed that
phage of X. oryzae were widely distributed in
water of paddy elds. When puried phage
was applied 1, 3, and 7 days before X. oryzae

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inoculation, there were 100%, 96%, and 86%

reductions of bacterial leaf blight, respectively. They found phage to be stable for
seven days at the site of infection in the absence of host bacteria, and for at least six
months in lysates or on dry lter paper at room
temperature.
Civerolo (22) reported that preinoculation
treatment of foliage of peach seedlings with
crude lysates of a phage mixture gave 6%8%
fewer infected leaves and a 17%31% reduction of disease compared to water-treated controls. Civerolo reasoned that slight to moderate leaf infection under natural conditions
may not result in severe defoliation. He observed that phages conceivably have a role in
regulating populations of Xcp. Civerolo was
able to recover pruniphage from apparently
healthy apricot leaves in the eld to support
this suggestion.
Saccardi et al. (82, 101) reduced fruit spot
incidence on peaches with biweekly spray
applications of phage suspension effective
against Xcp. The process involved isolating eight phages active against the pathogen,
screening them for host range and lytic ability, and selecting a lytic phage strain with the
broadest host range for disease control.
Tanaka et al. (94) reduced tobacco bacterial wilt incited by Ralstonia solanacearum
by coapplication of an avirulent strain of R.
solanacearum and its phage that was active
against both the virulent and avirulent strains.
They reduced the ratio of wilted plants from
the 95.8% of control plants, to 39.5% and
17.6% with application of avirulent strain and
avirulent strain plus phage, respectively.
Control of Erwinia amylovora, the re
blight pathogen of apple, pear, and raspberry,
with bacteriophages is currently under investigation in Canada and the United States.
Schnabel et al. (83) used a mixture of three
phages to control re blight on apple blossoms and achieved signicant (37%) disease
reduction. Gill et al. (34) isolated 47 phages
capable of lysing E. amylovora and categorized
them based on plaque morphology and host
range. Later phages were evaluated for disease

control ability in pear blossom bioassays, and

the ones with broad host ranges and best disease control ability were selected for orchard
trials (91). P. agglomerans, a bacterial antagonist that was also sensitive to the phages, was
used to deliver and propagate them on the leaf
surface. Disease control comparable to streptomycin was achieved (91).
There has been extensive research on suppression of tomato bacterial spot with phage.
Flaherty et al. (29) effectively controlled the
disease in greenhouse and eld experiments
with a mixture of four phages active against
two predominant races of the pathogen X.
campestris pv. vesicatoria. Balogh et al. (5) enhanced the efcacy of phage treatment with
protective formulations that increased phage
persistence on tomato foliage. Obradovic et al.
(75, 76) used formulated phages in combination with other biological control agents
and plant inducers, as a part of an integrated
disease management approach. Phage-based
integrated management of tomato bacterial
spot is now ofcially recommended to tomato
growers in Florida (69), and bacteriophage
mixtures against the pathogen are commercially available (Agriphage from OmniLytics
Inc., Salt Lake City, UT, EPA Registration
# 67986-1).
Other important work includes reduction
of incidence of bacterial blight of geraniums with foliar applications of a mixture
of phages (28), disinfection of Streptomyces
scabies-infected seed potatoes using a wide
host range phage (66), control of Xanthomonas
blight on onion (54a), citrus bacterial canker
and citrus bacterial spot (2, 6), and reduction
in the loss of cultivated mushrooms caused
by bacterial blotch with phage applications
(72, 73).

ADDRESSING HOST
RESISTANCE TO PHAGE
A major limiting factor in using phages for
control of plant diseases was the probability
of developing bacterial strains resistant to the
phage. This risk was addressed early on by

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4
X

1
E1

E1r

2
6
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5
h-mutant

Figure 1
Development of phage-resistant bacterial strains and host-range mutant phages (h-mutant) in nature.
1, wild-type phage; 2, wild-type, specic bacterial host (E1); 3, 1 per 106 mutate to phage-resistance
(E1r); 4, wild-type phage cannot kill phage-resistant mutant (E1r); 5, 1 per 107 to 109 spontaneously
mutate to h-mutant; h = host-range (extended host range); 6, h-mutant kills both wild-type parent
bacteria and phage-resistant mutants.

h-mutant: host
range mutant phage
Xcpl: Xanthomonas
campestris pv.
pelargonii

250

Katznelson (50) and later in two reviews on

phages (77, 97).
In 1989, a patented process (46) was developed to prevent occurrence of phage-resistant
mutants. The process involved preparing
a mixture of host range mutant phages
(h-mutants) for disease control. H-mutants
possess the ability to lyse bacterial strains
that are resistant to the parent phage (1),
while still being capable of lysing the wildtype bacterium. Thus, they have an extended
host range compared to the parent phage.
(Figure 1).
Using the strategy of phage application
proposed by Jackson (46), a mixture of four
phages including wild-type and h-mutant
phages were applied twice weekly in the
early morning prior to sunrise to determine control efcacy of bacterial spot of
tomato. The phage applications provided signicantly better disease control than the standard copper-mancozeb treatment (29). Additionally, the yield of extralarge fruits was
signicantly higher on phage-treated plants
than on copper-mancozeb treated ones. The
phage mixture reduced disease severity of bacJones et al.

terial spot by 17%, whereas copper-mancozeb

application caused 11% reduction.
A similar strategy was used to control Xanthomonas campestris pv. pelargonii (Xcpl), the
causal agent of bacterial blight of geranium
(28). Sixteen phages were evaluated for ability
to lyse Xcpl strains isolated from around the
world, and then h-mutants were developed
from ve phages that exhibited the broadest
host range and included in the mixture that
was used for disease control. Foliar applications of the phage mixture applied daily signicantly reduced spread of disease. The disease incidence was reduced by 50% or more in
phage-treated plots compared to the control,
and was signicantly less than in plots treated
with the recommended bactericide.

CHALLENGES IN USING
PHAGES FOR DISEASE
CONTROL
The success of any particular biocontrol treatment is inuenced by agent and target densities (48). In the case of phage therapy, it is critical that the phage comes in contact with its

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host before it is destroyed (35) and the probability of phage-bacterium contact depends
on several factors: initial phage concentration,
rates of virion decay, phage replication ability in the target environment, concentration
and location of target bacteria, and presence
of adequate water as a medium for phage diffusion (33). Additionally, disease control efcacy may be inuenced by timing of phage
application, relative tness of phage-resistant
bacterial mutants (33), and the surrounding
environment.
Phages are utilized for controlling plant
pathogens either in the rhizosphere or phyllosphere. Gill & Abedon (33) identied several factors that can hinder success of disease
control in the rhizosphere. The rate of diffusion through the heterogeneous soil matrix is
low and changes as a function of available free
water. Phages can become trapped in biolms
(88), reversibly adsorb to particles of soil, such
as clay (98), and be inactivated by low soil
pH (92). Physical refuges can protect bacteria
from coming into contact with phages. Because of low rates of phage diffusion and high
rates of phage inactivation, only a low number
of viable phages is available to lyse target bac-

teria (33). An additional problem is the need

for high populations of both phage and bacterium, in order to start the chain reaction of
bacterial lysis (33).
The phyllosphere is a harsh environment
and phages applied to aerial tissues degrade
extremely rapidly (3, 5, 24, 67). This transient survival of bacteriophages on plant leaf
surfaces is a major limiting factor of phage
treatment. Field and laboratory studies have
demonstrated that viruses are inactivated by
exposure to high temperature, high and low
pH and sunlight, and dislodged by rain leaching (42, 44). The most destructive environmental factor was determined to be the UV-A
and UV-B spectra (280400 nm) of sunlight
(41). In trials when phage was applied in
the mid-morning, control of bacterial spot of
tomato was not achieved (Jones, unpublished
results). We speculated that short residual activity of the control agents hindered efcacy
of phage treatment when applied during daytime. In an experiment, phages on tomato
foliage exposed to high intensities of sunlight during daytime were eliminated from
the phyllosphere within hours after application under eld conditions (Figures 2, 3) (45).
Nonformulated

10

Foliar phage populations

(Log PFU/g leaf tissue)

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Skim milk

Skim milk + sucrose

5
4
3
2
1
0

12

16

20

24

Time after application (h)

Figure 2
Effect of protective formulations on the persistence of bacteriophage Xacm 2004-16 in the tomato
canopy under eld conditions. Phages were applied at 6 a.m. on May 25, 2005, in Citra, Florida, in water
(nonformulated) or 0.75% (wt/V) skim milk powder (skim milk) or 0.75% (wt/V) skim milk powder and
0.5% (wt/V) sucrose (skim milk + sucrose). Error bars indicate the standard error.
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6 AM

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10

Effect of
application time on
the persistence of
bacteriophage
Xacm 2004-16 in
the tomato canopy
under eld
conditions. Phages
were applied at
6 a.m., 10 a.m.,
2 p.m. and 7 p.m.
on May 25, 2005,
in Citra, Florida.
Error bars indicate
the standard error.

[Log PFU/g leaf tissue]

Figure 3

Foliar phage populations

10 AM

2 PM

7 PM

4
3
2
1
0

10

20

25

30

Time after application (h)

In glasshouses, where sunlight UV irradiation

cannot penetrate, phages can persist up to a
week (2).
Temperature may affect both phage
longevity on plant foliage (45) and the ability of phage to lyse bacteria (23). Suboptimal
pH can hinder disease control by inactivating
phages. In a study by Leverentz et al. (57),
phages were unstable on apple slices owing to
low pH (surface pH 4.37), but persisted on
melons, which had a relatively high pH (surface pH 5.77). Phage treatment caused a signicant population reduction of Listeria monocytogenes on melons but not on apples (57).
Phages may come into contact with a number
of pesticides on plant foliage. Although most
chemicals do not affect phage persistence (4,
101), copper compounds are detrimental if applied less than 3 days before phage treatment
(4, 45).
Several approaches have been explored for
increasing efcacy of control in the phyllosphere by enhancing phage longevity including using protective formulations (3, 5,
75) (Figure 2), using carrier bacteria for
phage propagation in the target environment
(91), using phages that are better adapted
to the target environment, and applying
treatments in the evening or early morning
(5, 29).
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15

Jones et al.

DEVELOPMENT OF
PROTECTIVE FORMULATIONS
Various materials have been characterized to
determine if they could extend the life of
viruses following exposure to various physical factors. A number of materials were identied that increased residual activity of insect
viruses and other microbial pesticides by providing protection against sunlight UV and/or
rain leaching, including antioxidants and oxidative enzymes (41), aromatic/heterocyclic
amino acids (40), light adsorbing or reecting dyes (43, 93), activated charcoal (43),
starch- and our- (64, 65), alkaline gluten(10), casein- (11), and lignin-based formulations (12).
Several of these materials were evaluated
for extending phage persistence on tomato foliage and three formulations were identied
that had pronounced effects (3). These included (a) 0.5% pregelatinized corn our and
0.5% sucrose (PCF), (b) 0.5% Casecrete NH400 (a water-soluble casein polymer), 0.25%
pregelatinized corn our and 0.5% sucrose
(Casecrete), and (c) 0.75% skim milk powder
and 0.5% sucrose (skim milk) (3). These formulations increased phage persistence from
several hours to one to two days under
eld conditions compared to phage preparations applied without adding any protective

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materials (Figure 2) (3). The effect was signicant under greenhouse conditions as well.
Phages applied in PCF, Casecrete, and skim
milk formulations had 4700-, 38,500- and
100,000-fold higher populations, respectively,
than nonformulated phage two days after application (3). These same formulations were
evaluated for disease control efcacy in greenhouse and eld trials. Under greenhouse conditions they provided only minimal improvement in disease control, as phage treatments
applied in Casecrete and in skim milk formulations reduced disease severity compared
to nonformulated phage only in 1 of 3 experiments, whereas PCF formulation did not
have any signicant effect. Under eld conditions, however, the effect was more evident:
Casecrete-formulation improved disease control in 3 of 3 trials, PCF in 1 of 3 trials, and
the skim milk in the single trial in which it was
evaluated (5).
Although the formulations above enhanced phage persistence on leaf surfaces,
they also enhanced disease development: All
three of these formulations contributed to
signicant increases in tomato bacterial spot
severity when applied in the greenhouse without phages (5). Furthermore, the skim milk
formulation negated disease control of citrus
canker achieved with phage treatment in the
greenhouse (2). Proteins and sugars in the formulations most likely provided a nutritional
source for the pathogen and also may have facilitated ingress by breaking down surface tension and forming a continuous aqueous layer
on the leaf surface. There is clearly a need for
identifying biologically inert formulations to
improve efcacy of phages without enhancing
ingress.

PHAGE PROPAGATION IN
THE TARGET ENVIRONMENT
Another approach for maintaining high phage
populations is to coapply them with bacteria that are able to persist in the plant environment and that are sensitive to the phages.
Thus if the bacterial populations are main-

tained at fairly high concentrations, they will

serve as hosts for the phage and potentially
maintain high phage populations. A mildly
pathogenic strain of Xanthomonas perforans
that caused less disease than the wild type
but was able to become established in the
tomato canopy supported high populations of
its phage in a greenhouse study (2). In the absence of the host, phage populations disappeared from the tomato foliage by six days
after application, whereas in the presence of
the host phage numbers a week after application were comparable to the original population. Svircev et al. (91) controlled re blight
of pear by utilizing a strain of P. agglomerans for delivering and sustaining a mixture of
four phages, which were able to lyse strains
of both P. agglomerans and E. amylovora, the
causal agent of re blight. A similar strategy
was used for controlling tobacco bacterial wilt,
where bacteriophages were applied together
with a phage-sensitive avirulent strain of the
pathogen Ralstonia solanacearum to control the
disease (94).

EFFECT OF PHAGE
DURABILITY AND IN VIVO
MULTIPLICATION ABILITY
ON DISEASE CONTROL
Two opposing factors determine phage population changes: decrease due to virion decay
and increase as a result of multiplication on
the host bacterium. Phages with lower rates
of virion decay and/or better multiplication
ability will maintain higher populations in the
phyllosphere and will likely be more effective in controlling populations of the target
pathogen. Phages differ in their ability to tolerate sunlight irradiation and persist on leaf
surfaces (2). Ability to survive the dry and wet
periods also can increase phage survival on leaf
surfaces. Although the majority of phages are
unable to survive desiccation, a desiccationtolerant phage has been isolated from the
plant phyllosphere (45).
The ability to increase populations by effectively multiplying on the leaf surfaces also

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leads to prolonged survival of the phage population. Balogh (2) found that two of three
phages that in vitro were able to lyse the host
bacterium Xanthomonas axonopodis pv. citri and
multiply to high populations were not able to
multiply on the same bacterial host in vivo on
grapefruit leaf surfaces and declined rapidly
within hours. These phages were also unable
to control citrus canker. A third phage, however, which was isolated from aerial plant tissue originally, was able to efciently multiply
in vivo and increased in numbers more than
threefold within nine hours. This phage exhibited effective control of the disease. It appears that selection of phages that are better
adapted to targeted environment contributes
to disease control and should be an important
consideration.

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EFFECT OF APPLICATION
TIMING ON DISEASE
CONTROL EFFICACY
Timing of bacteriophage applications relative to arrival of the pathogen inuenced efcacy of disease control in several instances.
Civerolo & Keil (24) achieved a marked reduction of peach bacterial spot only if phage
treatment was applied one hour or one day
before inoculation with the pathogen. There
was a slight disease reduction when phage
was applied one hour after inoculation and
no effect if applied one day later. Civerolo
(23) suggested that bacteria were inaccessible to phage in the intercellular spaces, or
there were not enough phages reaching the
pathogen. Schnabel et al. (83) achieved a signicant reduction of re blight on apple blossoms when the phage mixture was applied at
the same time as the pathogen, E. amylovora.
In contrast, disease reduction was not significant when phages were applied a day before inoculation. Bergamin Filho (16) investigated the effect of timing on efcacy of
phage treatment in greenhouse trials with two
pathosystems: black rot of cabbage, caused by
X. campestris pv. campestris, and bacterial spot
of pepper, caused by X. campestris pv. vesicato254

Jones et al.

ria. Phage treatment was applied once varying

from 7 days before to 4 days after pathogen inoculation. On cabbage signicant disease reduction was achieved if phage treatment was
applied 3 days before to 1 day after inoculation, whereas on pepper, from 3 days before
to the day of inoculation. The greatest disease reduction occurred with application of
phages the same day as inoculation in both
pathosystems.
Timing of eld application of phages to
times of day when sunlight irradiation is minimal signicantly improved disease control efcacy (45). Flaherty et al. (29) achieved significant reduction of tomato bacterial spot when
phage treatments were applied at dawn but
not when applied at late morning. Balogh
et al. (5) achieved signicantly better control of tomato bacterial spot with evening
applications than with morning applications
(5, 29).
The effect of phage concentration on disease control efcacy has also been investigated. Treatment of tomato plants with phage
mixtures at 106 or 108 plaque-forming units
(PFU)/ml signicantly reduced tomato bacterial spot severity, but a mixture had no effect
at 104 PFU/ml (3).

INTEGRATED STRATEGIES
A number of approaches have been used for
incorporating phage applications into part
of an integrated management strategy of
plant bacterial diseases. Tanaka et al. (94)
reduced tobacco bacterial wilt incited by R.
solanacearum by coapplication of an avirulent
strain of R. solanacearum and its phage that was
active against both the virulent and avirulent
strains. They reduced the ratio of wilted plants
with applications of an avirulent strain and
phage compared to the avirulent strain alone.
Recently, another approach to phage application has been reported (91). Field trials
were conducted with phages selected for their
broad host range on E. amylovora and their
ability to control the pathogen in forced pear
blossom bioassays. P. agglomerans, a biological

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control agent for E. amylovora, was used to deliver and sustain phages on the blossom surface. Using an integrated approach by combining two biological control agents (i.e., lytic
bacteriophages and P. agglomerans) effective
control of re blight was achieved that was
comparable to streptomycin (91).
In the past few years, considerable attention has been directed to development
of more sustainable strategies for reducing bacterial spot of tomato. Various combinations of biocontrol agents, including
strains of plant growth-promoting rhizobacteria (PGPR), bacterial antagonists, phages,
and SAR inducers (harpin, acibenzolar-Smethyl), were compared in greenhouse experiments (76). Tomato has been used as a model
system for developing an integrated management strategy for combining phages and other
alternative control strategies in an effort to
reduce severity of foliar tomato bacterial diseases given the extensive research done on
plant activators (58, 80), PGPR (47), and antagonistic bacteria (19, 99) to control bacterial spot of tomato. The idea was to integrate
the positive aspects of these practices in order to optimize benets in control of tomato
bacterial spot in the greenhouse and in eld
production.
Although a previous study proved effectiveness of phages in control of bacterial spot of tomato (29), greenhouse experiments showed inconsistent performance of
phage treatments (76). A single application
of unformulated phages used in this study
probably contributed to this inconsistency.
However, when phage was applied to plants
previously treated with SAR inducers, disease
control signicantly improved. The combination of harpin and phage signicantly reduced disease severity compared to harpin
alone. The SAR compound acibenzolar-Smethyl (ASM) effectively initiated plant defense mechanisms against X. campestris pv.
vesicatoria and prevented the occurrence of
disease symptoms under greenhouse conditions. However, in this experiment, plants
treated with ASM developed HR-like necrotic

spots when challenged with high concentrations of the pathogen. Application of phage
in combination with ASM resulted in elimination of HR-type lesions. Phage applications
were shown to decrease pathogen populations
on the leaf surface and concomitantly reduced
pathogen ingress. This in turn resulted in reduced intensity of the plant defense reaction
induced by ASM (76). Based on results from
greenhouse experiments (76), several combinations of SAR inducers and host-specic
phages were compared in eld trials. Although
ASM was not as effective in controlling bacterial spot in the eld as it was in greenhouse
experiments, it signicantly reduced disease
severity compared to untreated control. The
combination of ASM and phage provided an
additional reduction in disease pressure and
resulted in more effective foliar disease control than ASM, phage, or copper-macozeb
alone (75). Application of formulated hostspecic phages can be an effective alternative
to chemical bactericides for treatment of bacterial plant diseases. Phage treatment, integrated with other practices, is currently widely
used in greenhouses and production elds in
Florida as a part of a standard integrated management program for tomato bacterial spot
control.

PGPR: plant
growth-promoting
rhizobacteria
ASM: acibenzolarS-methyl

COMMERCIALIZATION
The rst commercial company to produce
phages specically for control of bacterial
plant diseases was AgriPhi, Inc., established by
L. E. Jackson (46). To minimize development
of bacterial strains resistant to the phage, mixtures of wild-type phages and h-mutants were
used. Only lytic phages isolated from plant
parts (leaves, stems, etc.) soil, and water (eld
runoff, river, and stream water and sewage
efuents) were utilized. To insure that temperate phages were not selected, phages were
never obtained from their respective bacterial
hosts. Although an h-mutant will attack and
kill both its specic wild-type host and bacterial phage-resistant mutants selected from this
parent, a secondary bacterial mutant resistant

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to the h-mutant could occur. The secondary

phage-resistant mutant and its progeny will
be resistant to the one h-mutant being used.
Any time a phage-resistant mutant is encountered, new h-mutant phages could be developed against those strains and included in the
phage mixture. However, even if h-mutants
are used, they may not infect all strains depending on the bacterial diversity that may
exist. Therefore, microbial diversity needs to
be determined and carefully monitored. The
mixture of phages needs to reect the microbial diversity.
Recently, as a result of greenhouse and eld
research, OmniLytics, Inc., in Salt Lake City,
UT (formerly AgriPhi, Inc.), received the rst
EPA registration (EPA Registration # 67986
1) to use phages in agriculture. The registration is for using host-specic phages on tomatoes in greenhouses and production elds in
Florida as a part of a standard integrated management program to control tomato bacterial
spot (49).

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SUMMARY
Bacteriophages are viruses that infect bacteria.
Since their discovery in the early part of the
twentieth century, they have been evaluated
extensively for control of all kinds of bacterial
incited diseases, including plant diseases. In
the recent past, phages have been under evaluation for controlling re blight on apple and
pear, bacterial wilt on tobacco, citrus bacterial
canker, citrus bacterial spot, bacterial blight
on geranium, bacterial blotch of mushroom,
and Xanthomonas blight of onion.
Bacteriophages have great potential because they are widely present in nature; are

self-replicating; can be targeted against bacterial receptors that are essential for pathogenesis; are nontoxic to eukaryotes; and are
specic to certain bacterial species or strains
without damaging other, possibly benecial
members of the indigenous ora. Phages are
fairly easy and inexpensive to isolate, produce,
and store. They may also have application for
use in technologically less developed regions.
There are several challenges associated
with the use of phages. The bacteria can easily mutate and become resistant to an individual phage. Therefore, using a single phage
for control is risky. The environmental factors including exposure to sunlight radiation
and in particular UV, desiccation, temperature, and leaching may reduce phage populations. UV in particular will cause a dramatic
reduction. To increase longevity phages can
be applied with protective formulations or a
carrier bacterium in which they may multiply.
The reduction can also be countered by applying the phage in the evening or early morning
rather than during periods of high sunlight
irradiation. These approaches contribute to
the longer presence of high phage populations
in the target area increasing the likelihood of
control.
Phages have potential for use in integrated disease management strategies, especially with other biocontrol agents. The
combination of ASM and phage provided
additional reduction in disease and resulted
in more efcient foliar disease control than
ASM, phage, or copper-macozeb alone. In
two other instances, phages were coapplied
with bacteria that served as phage hosts as well
as biological control agents providing two levels of biological control simultaneously.

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Contents

Volume 45, 2007

Tell Me Again What It Is That You Do

R. James Cook p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p1
Noel T. KeenPioneer Leader in Molecular Plant Pathology
Alan Collmer and Scott Gold p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 25
Structure and Function of Resistance Proteins in Solanaceous Plants
Gerben van Ooijen, Harrold A. van den Burg, Ben J.C. Cornelissen,
and Frank L. W. Takken p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 43
Family Flexiviridae: A Case Study in Virion and Genome Plasticity
Giovanni P. Martelli, Michael J. Adams, Jan F. Kreuze, and Valerian V. Dolja p p p p p p 73
Cell Wall-Associated Mechanisms of Disease Resistance
and Susceptibility
Ralph Hckelhoven p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p101
Genomic Insights into the Contribution of Phytopathogenic Bacterial
Plasmids to the Evolutionary History of Their Hosts
George W. Sundin p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p129
Identifying Microorganisms Involved in Specic Pathogen Suppression
in Soil
James Borneman and J. Ole Becker p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p153
Safety of Virus-Resistant Transgenic Plants Two Decades After Their
Introduction: Lessons from Realistic Field Risk Assessment Studies
Marc Fuchs and Dennis Gonsalves p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p173
Disease Cycle Approach to Plant Disease Prediction
Erick D. De Wolf and Scott A. Isard p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p203
Virus-Induced Disease: Altering Host Physiology One Interaction
at a Time
James N. Culver and Meenu S. Padmanabhan p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p221
Bacteriophages for Plant Disease Control
J.B. Jones, L E. Jackson, B. Balogh, A. Obradovic, F. B. Iriate,
and M. T. Momol p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p245
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Reniform in U.S. Cotton: When, Where, Why, and Some Remedies

A. Forest Robinson p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p263
Flax Rust Resistance Gene Specicity is Based on Direct
Resistance-Avirulence Protein Interactions
Jeffrey G. Ellis, Peter N. Dodds, and Gregory J. Lawrence p p p p p p p p p p p p p p p p p p p p p p p p p p p p289
Microarrays for Rapid Identication of Plant Viruses
Neil Boonham, Jenny Tomlinson, and Rick Mumford p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p307

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Transcript Proling in Host-Pathogen Interactions

Roger P. Wise, Matthew J. Moscou, Adam J. Bogdanove,
and Steven A. Whitham p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p329
The Epidemiology and Management of Seedborne Bacterial Diseases
Ronald Gitiatis and Ronald Walcott p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p371
Elicitors, Effectors, and R Genes: The New Paradigm and a Lifetime
Supply of Questions
Andrew F. Bent and David Mackey p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p399
Magnaporthe as a Model for Understanding Host-Pathogen
Interactions
Daniel J. Ebbole p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p437
Challenges in Tropical Plant Nematology
Dirk De Waele and Annemie Elsen p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p457
Errata
An online log of corrections to Annual Review of Phytopathology articles may be found
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