INTRODUCTION

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Fire blight, caused by the bacterium Erwinia amylovora, is a devastating disease of apple and pear trees in North America, Europe, the Mediterranean region, and New Zealand. Most pear and apple cultivars currently in commercial production are moderately to highly susceptible to fire blight. The pathogen is also able to infect a few other members of the Rosaceae, including pyracantha, hawthorn, and cotoneaster. A distinct subtype of E. amylovora is able to cause fire blight on Rubus spp., especially raspberry and blackberry, but not on apple and pear shoots or seedlings (14, 19). Except for differences in genetic fingerprints (11, 12), strains isolated from Rubus spp. are indistinguishable from tree fruit E. amylovora strains.

Preventing the buildup of epiphytic populations of E. amylovora on nutrient-rich stigmatic surfaces of blossoms in the spring is the main strategy for controlling fire blight (6, 22). Streptomycin is no longer effective for controlling epiphytic E. amylovora on blossoms in many fruit-growing regions because of the emergence of streptomycin-resistant strains of the pathogen (7). A new strategy for hindering the establishment of epiphytic E. amylovora on stigmas is the use of bacteria such as Pseudomonas fluorescens strain A506 (9), Pantoea agglomerans strain C9-1 (13, 20), and a few other species (8, 13), but so far the level and consistency of control with microbial agents are lower than those with antibiotics (6). Phages of E. amylovora have been proposed as possible control agents for fire blight. The control potential of an E. amylovora phage was first demonstrated by Erskine in 1973 (3). Subsequently, symptoms of fire blight were attenuated in apple seedlings inoculated with Ea1 in conjunction with E. amylovora (15) and in pear fruit inoculated with E. amylovora in the presence of a Ea1-encoded polysaccharide depolymerase (5). Phages of E. amylovora have commonly been found on aerial parts and in soils associated with fire blight-infected apple trees (3, 15, 16), but the association of phage with fire blight-infected Rubus spp. has not been investigated. Except for cloning a polysaccharide depolymerase gene from Ea1 (5), genetic methods have not been used to study E. amylovora-specific phages.

In this study the diversity of E. amylovora phages recovered from soils and shoots of fire blight-infected plants was evaluated. Phages were characterized using PCR, restriction fragment length polymorphism analysis, and pulsed-field gel electrophoresis (PFGE). The phage sensitivity of a panel of E. amylovora strains and the ability of the phage to lyse broth cultures of E. amylovora were examined. The ability to definitively identify phages is prerequisite to future epidemiological and control studies of fire blight using phage.

	MATERIALS AND METHODS

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Bacterial strains, phages, and media. E. amylovora strain Ea110, isolated in 1975 as strain MSU110 from a canker on Jonathan apple in an experimental orchard near East Lansing, Mich. (15), and phage Ea1, isolated in 1975 as phage PEa1 from blighted Jonathan apple shoots collected near Paw Paw, Mich. (15), were obtained from John S. Hartung, Agricultural Research Service, U.S. Department of Agriculture, Beltsville, Md. (5, 16). Phage Ea7, isolated in 1976 as phage PEa7 from blighted apple shoots collected near Berrien Springs, Mich. (15), was obtained from the American Type Culture Collection (ATCC 29780-B2). Phages Ea100, Ea104, Ea125, and Ea116C were isolated in this study and deposited at the American Type Culture Collection as ATCC 29780-B4, 29780-B5, 29780-B3, and 29780-B6, respectively. Forty strains of E. amylovora were collected from 14 apple orchards located in six counties of Michigan during 1997 and 1998. Their identity was confirmed based on their colony color and morphology on MM2Cu and Luria-Bertani (LB) media (2) and by PCR assay with the AJ75-AJ76 primer pair (10). E. amylovora strains MR1, -2, -3, and -4; RBA4, -8, -10, and -E; and RKK2, -3, -4, and -5 were collected from three raspberry farms in Michigan (11). Other bacteria included seven nonfluorescent and nine fluorescent strains of Pseudomonas spp. and 12 strains of Pantoea agglomerans previously isolated from Michigan apple orchards (18). Bacteria were routinely cultured on LB agar and in LB broth. The double-layer agar technique (1) was used to produce phage plaques on bacterial hosts, using a top agar consisting of 11.5 g of nutrient agar, 5 g of glucose, and 5 g of yeast extract per liter on a bottom agar of 23 g of nutrient agar and 5 g of glucose per liter.

Isolation of phage. Samples were collected from an asymptomatic apple orchard at Michigan State University, several fire blight-infected commercial apple and pear orchards in southern Michigan, a fire blight-infected apple orchard in northern California, and a raspberry farm in northern Michigan in 1996 and 1997. Aerial tissues (i.e., branches, leaves, and fruit) and/or soil from within the tree dripline were mixed with sterile water. The collected washes were treated with chloroform at a final concentration of 5%. Aliquots (100 µl) were mixed with 100 µl of Ea110 culture at an optical density at 600 nm of 1.0 to 3.0, plated, and assessed for the presence of plaques following 18 h of incubation. Phage were recovered from individual plaques by soaking isolated agar plugs in 1 ml of 100 mM NaCl-50 mM Tris-HCl(pH 7.5)-8 mM MgSO₄ for at least 1 h. Phage were purified using successive rounds of single-plaque isolation.

Isolation of phage DNA. Ea110 was grown overnight in LB broth with agitation at 28°C and diluted to an optical density at 600 nm of 0.14 (approximately 2 × 10⁸ CFU/ml). Bacteriophage (2 × 10⁷ PFU) were mixed with 1 ml of diluted bacteria. Following a 10-min incubation, 9 ml of LB broth was added and the cultures were grown overnight at 28°C with agitation. Chloroform (30 µl) was added, and bacterial debris was removed by centrifugation at 4,000 × g for 15 min. DNA was purified either by using the Wizard Lambda DNA Preps DNA Purification System (Promega, Madison, Wis.) or by phenol extraction of concentrated phage. In the latter case, the cleared bacterial lysate was incubated for 30 min at 37°C following addition of 40 µl of nuclease mix (0.25 mg each of DNase and RNase per ml in 150 mM NaCl-50% [wt/vol] glycerol). Four milliliters of 33% polyethylene glycol 8000-3.3 M NaCl was then added, and the mixture was incubated on ice for at least 30 min followed by centrifugation in 15-ml Corex tubes (Corning, Corning, N.Y.) at 10,000 × g for 10 min. The pellet was resuspended in 400 µl of 150 mM NaCl-40 mM Tris-HCl (pH 7.4)-10 mM MgSO₄ and clarified by centrifugation in a microcentrifuge for 2 min. The supernatant fluid was extracted twice with chloroform. Phage DNA was then released by gentle mixing in an equal volume of Tris-buffered phenol (pH 7.9) for 5 min. After centrifugation for 5 min, the upper layer was extracted again with phenol and then with chloroform. DNA was precipitated by the addition of 1 ml of 95% ethanol and 50 µl of 3 M sodium acetate (pH 5.2), collected by centrifugation for 15 min, rinsed with 300 µl of 70% ethanol, allowed to dry, and gently resuspended in 50 µl of 10 mM Tris-HCl (pH 8.0)-0.1 mM EDTA.

Ea1 PCR assay. BglII restriction fragments of Ea1 were ligated into BamHI-digested, dephosphorylated pGEM3zf(+); ligation products were introduced into Escherichia coli strain JM109 by electroporation, and selected transformants were analyzed. A clone containing a 1.8-kb BglII fragment was partially sequenced (GenBank accession no. AF222715). Based on this sequence, PCR primers were designed to amplify a 304-bp fragment from Ea1 (PEa1A 5'-AATGGGCACCGTAAGCAGT and PEa1B 5'-TAATGGGTATGATAGAAGGCAGAC). PCR reaction mixtures (20 µl) consisted of 20 mM Tris-HCl (pH 8.4), 50 mM KCl, 1.5 mM MgCl₂, 0.5 µM (each) primer, 0.16 mM deoxynucleoside triphosphates (Gibco BRL, Grand Island, N.Y.), 0.5 U of Taq polymerase (Gibco BRL) and 1 µl of phage lysate containing 10₄ to 10₇ PFU of phage. Reactions were performed in a PTC-150 minicycler (MJ Research, Watertown, Mass.) with cycling parameters of 95°C for 2 min followed by 35 cycles of 95°C for 30 s, 53°C for 30 s, and 72°C for 30 s. Reaction products were analyzed on 1.5% (wt/vol) agarose gels in 0.5× Tris-borate-EDTA (TBE) buffer run at 10 V/cm, followed by ethidium bromide staining.

Restriction analysis of phage DNA. Purified phage DNA was subjected to restriction analysis according to the manufacturers' protocols (Gibco BRL and Roche Molecular Biochemicals, Indianapolis, Ind.). The samples were electrophoresed either through 0.8% agarose in 0.5× TBE buffer at 6 V/cm for 1 to 2 h or through 0.4% agarose in 0.5× TBE buffer at 1 V/cm for 18 to 24 h. The sizes of the fragments were estimated by comparison to HindIII-digested  DNA, high-molecular-weight DNA standards, or the 1-kb DNA Plus ladder (Gibco BRL). Restriction maps were constructed by compiling the data from single- and double-enzyme digestions of intact and cloned BglII fragments of phage DNA.

Contour-clamped homogeneous electric field gel analysis. Pulsed-field gels were run using a CHEF-DR II PFGE system (Bio-Rad Laboratories, Hercules, Calif.). The 1% gels were made with Seakem (Rockland, Maine) Gold agarose in 0.5× TBE buffer. The pulsed-field parameters were as follows: 0.5× TBE running buffer, 0.1-s initial switch time, 10-s final switch time, 6.0 V/cm, 15-h run time, and 14°C buffer temperature. The gels were stained with ethidium bromide and visualized using a Foto/Eclipse system (Fotodyne, Inc., Harland, Ws.).

Infection and lysis experiments. Aliquots (50 µl) of overnight cultures of various strains were mixed with 10³ PFU of phage (as determined by plaque formation using strain Ea110 as a standard), incubated for 10 min, mixed with 3 ml of top agar, and plated onto bottom agar. Following incubation at 22°C for 18 to 42 h, the plates were evaluated for the presence of plaques. Strain and phage combinations which yielded no plaques were retested at least twice.

	RESULTS

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Identification of phage types. The collection of phages that formed plaques on the E. amylovora Ea110 host consisted of reference phages Ea1 and Ea7 and 48 new isolates collected in 1996 and 1997. Forty-four of the new isolates were from Michigan, including 41 from soil and plant material of fire blight-infected apple orchards, 2 from blighted raspberry canes, and 1 from a blighted pear shoot. Four additional isolates came from soil collected in a fire blight-infected apple orchard in California.

View larger version (56K): [in this window] [in a new window]   FIG. 1.   Restriction analysis and pulsed-field gel analysis of DNAs from E. amylovora phages Ea1, Ea7, Ea100, Ea125, and Ea116C. (A) EcoRI restriction digestion analysis of phage DNA. Lane M, 1-kb Ladder Plus (Gibco BRL). (B) PFGE of phage DNA. Lane M, MidRange I PFG Marker (New England BioLabs, Inc.).

Restriction analysis of Ea1-type isolates. To verify that the presence of the 0.3-kb PCR product was accurately identifying Ea1-type phage, BglII restriction digests of DNAs from four PCR-positive isolates and Ea1 were compared (Fig. 2). The digest of Ea1 DNA yielded fragments of approximately 9, 7, 6.5, 6.2, 5.2, 3.5, 3, 2.1, 1.6, and 1.4 kb. Isolate Ea123 yielded a pattern identical to that of Ea1. The three other isolates exhibited restriction patterns similar to, but distinct from, that of Ea1. These three isolates were distinguished from Ea1 by the absence of the 9-, 6.5-, and 5.2-kb fragments and the presence of fragments of 14 kb; 3.8, 4.3, or 4.8 kb for isolates Ea101, Ea104, and Ea109, respectively; and 1.7 kb.

View larger version (108K): [in this window] [in a new window]   FIG. 2.   BglII restriction analysis of DNAs from Ea1 and putative Ea1-type phages isolated from Michigan (Ea101, Ea104, and Ea109) and California (Ea123). Lane M, 1-kb Ladder Plus (Gibco BRL). An additional 14 phages, each yielding a 0.3-kb PCR product typical for Ea1, had restriction patterns similar to these.

View larger version (14K): [in this window] [in a new window]   FIG. 3.   Restriction maps of E. amylovora phage isolates Ea1 and Ea104. Fragments a to j represent the longest to shortest BglII fragments of Ea1. Corresponding BglII fragments of Ea104 are indicated with the same letter designation; fragments a1, c1, and c2 are unique to Ea104. Other restriction sites in common between Ea1 and Ea104 are indicated between the two maps, and those unique to each phage are indicated above or below the Ea1 and Ea104 maps, respectively. B, BglII; K, KpnI; Nc, NcoI; N, NdeI; Pv, PvuII; X, XbaI.

Host range of E. amylovora phages. To assess the prevalence of phage resistance in natural populations of E. amylovora, the ability of phages Ea1, Ea7, Ea100, Ea125, and Ea116C to infect 40 strains of E. amylovora isolated from apple was tested in a plaque formation assay (Table 1). Resistance to at least one phage was detected in 65% of the strains. Resistance to Ea100 and Ea125 was found in 50 and 60% of the strains, respectively; resistance to both of these phages was found in 45% of the strains. Resistance to the other three phages was less common. Ea116C formed plaques on all 40 strains, a single strain was resistant to Ea1, and four strains were resistant to Ea7. Ninety percent of the strains were sensitive to phages Ea1, Ea7, and Ea116C.

Lysis of E. amylovora strain Ea110 in liquid culture. The ability of the phages to control populations of E. amylovora was tested by infecting liquid cultures of E. amylovora with individual phages or with combinations of phages Ea1, Ea7, and Ea116C (Fig. 4). Data for the seven phage treatments and three phage concentrations were analyzed by two-way analysis of variance. F values for treatments and phage concentrations were highly significant (P = 0.00001), and interaction effects were not significant. When Ea1, Ea7, Ea100, Ea125, and Ea116C were added individually to cultures of E. amylovora strain Ea110 at 1 PFU per 10 CFU or 1 PFU per 10³ CFU, only Ea116C was able to effectively control bacterial populations, reducing optical densities to 96% ± 4% below that of bacteria grown without phage. A mixture of phages Ea1 and Ea7 was not significantly better than either phage alone in controlling bacterial populations. A mixture of phages Ea116C, Ea1, and Ea7 was as effective as Ea116C alone.

View larger version (56K): [in this window] [in a new window]   FIG. 4.   Control of growth of E. amylovora strain Ea110 in the presence of phage(s). Single phage types (Ea1, Ea7, Ea100, Ea125, or Ea116C) or combinations of equal proportions of two or three phages (Ea1 plus Ea7 or Ea1 plus Ea7 plus Ea116C) were added at 106) (open bars), 104 (hatched bars), and 102 (solid bars) total PFU to 1-ml aliquots of LB broth containing 107 CFU of E. amylovora strain Ea110. The optical densities of the cultures following 18 h of growth at 28°C were compared to the optical densities of cultures grown without phage. The data shown are the averages of three replications, with the standard deviation of each mean shown.

	DISCUSSION

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Five distinct E. amylovora phages were identified from a collection of 50 phage isolates. By cloning and sequencing a 1.8-kb BglII fragment of Ea1, a set of PCR primers specific for detection of Ea1-type phages was developed and used to screen the collection; others have also found these primers useful for rapidly differentiating Ea1 from other phages (4). Phages Ea7, Ea100, Ea125, and Ea116C, which were distinct from Ea1, were differentiated based on genome size and restriction fragment pattern. The analysis of these phages at the DNA level provides a basis for the characterization of future phage isolates.

Phages specific for E. amylovora were prevalent in Michigan orchard sites with active fire blight infections, and the detection of phages during the later stages of fire blight epidemics in apple orchards is consistent with the results of an earlier Michigan study (15). Nearly every site surveyed yielded Ea1-type isolates, indicating that this phage is commonly associated with E. amylovora in nature. Two major subtypes of Ea1-type phages were observed among the isolates based on restriction analysis. The first group included the original Ea1 isolate and the Ea1-type isolates from California; all of these isolates had identical EcoRI restriction patterns. The second group included the more recent isolates from Michigan, which produced somewhat variable EcoRI restriction patterns which were quite distinct from that of the original Ea1 isolate.

For the other four phages, our collection contained either single isolates (Ea100, Ea125, and Ea116C) or five isolates (Ea7). The five isolates of Ea7 displayed no obvious differences in restriction pattern even though two of the isolates were associated with E. amylovora from raspberry canes, which represent a genetically distinct and often spatially isolated host. It is not clear if the paucity of these phages in our collection was due to their scarcity in the collected samples or to difficulties in identifying the small plaques formed by these phages. Because our discovery of phages distinct from Ea1 was somewhat fortuitous, it may be reasonable to suppose that additional E. amylovora phage types exist.

One explanation for the insensitivity of some strains of E. amylovora to phages may be that they harbor temperate phages that render them resistant to other lytic phages. However, this appears unlikely, because Ritchie (15) tested strains of E. amylovora resistant to Ea1 or Ea7 for lysogeny using UV light and mitomycin C as induction agents; all tests were negative for temperate phage. Another indication of the presence of temperate phages is the formation of hazy plaques; none of the phages were observed to form plaques that became hazy in the center with age. As reported previously (15, 16), Ea1-type phages produced plaques with a distinct halo after about 18 h. This halo was shown to be the result of the production of a polysaccharide depolymerase, capable of hydrolyzing the capsular polysaccharide and not of lysogeny (5, 16).

Ritchie and Klos (15, 16) reported that Ea1 and Ea7 infected each of 20 E. amylovora isolates; however, our data from additional strains indicate that some strains differ in their phage sensitivity. All 52 strains of E. amylovora from Malus and Rubus hosts were infected by phage Ea116C. Although a more exhaustive screen might yield strains with resistance to Ea116C, it appears that resistance to this phage is rare. Among Malus strains of E. amylovora, 40 to 98% were infected by the other four phages. Apparently, despite the high degree of homogeneity among tree fruit strains of E. amylovora, some strains possess differences that prevent adherence, uptake, or replication by certain phages. The specific nature of the mechanisms responsible for phage resistance in the E. amylovora strains is unknown. The majority of the E. amylovora strains from Rubus were sensitive to all five phages, although resistance to Ea1, Ea100, and Ea125 was detected. This is the first study to show that Rubus and Malus strains of E. amylovora were infected by the same phages.

Despite the ability of each of the phages to infect E. amylovora strain Ea110, only Ea116C was able to drastically reduce the final density of the bacteria grown in liquid culture. In the case of Ea1, it has been shown that as phage titers increase, a phage-encoded polysaccharide depolymerase, which is able to degrade the capsule of E. amylovora, accumulates in the medium. When levels of the enzyme are high enough, Ea1 can no longer infect the bacteria, presumably due to destruction of Ea1 binding sites (5, 15). For Ea7, Ea100, and Ea125, the reasons for ineffective growth control are unknown.

The phages isolated in this study may be useful for biocontrol of E. amylovora, in particular Ea116C and, to a lesser extent, Ea1 and Ea7, because of the infrequent occurrence of resistant strains in E. amylovora populations. They might be effective either in orchards or for eliminating E. amylovora from the surface of contaminated budwood and fresh fruit. Control of the blossom-infection stage of fire blight is critical to the overall control of fire blight (6). E. amylovora multiplies on the stigmatic surfaces of blossoms prior to blossom infection (22). If E. amylovora-specific phages were present on the blossoms, they might suppress the growth of E. amylovora on the stigmatic surfaces. However, natural phage populations are below detectable levels during the bloom period (reference 16 and this study). Therefore, any control strategy based on phage would require that blossoms be treated with phage in much the same way as blossoms are treated with antagonistic bacteria. Prior colonization of the blossom by a suitable host, such as an avirulent strain of E. amylovora (21), has been shown to be important for the establishment and maintenance of phage populations (17). Without such a host the population of phage rapidly declined, presumably due to UV light or desiccation effects.