Isolation and characterization of five Erwinia amylovora bacteriophages and assessment of phage resistance in strains of Erwinia amylovora

Abstract

Phages able to infect the fire blight pathogen Erwinia amylovora were isolated from apple, pear, and raspberry tissues and from soil samples collected at sites displaying fire blight symptoms. Among a collection of 50 phage isolates, 5 distinct phages, including relatives of the previously described phages phiEa1 and phiEa7 and 3 novel phages named phiEa100, phiEa125, and phiEa116C, were identified based on differences in genome size and restriction fragment pattern. phiEa1, the phage distributed most widely, had an approximately 46-kb genome which exhibited some restriction site variability between isolates. Phages phiEa100, phiEa7, and phiEa125 each had genomes of approximately 35 kb and could be distinguished by their EcoRI restriction fragment patterns. phiEa116C contained an approximately 75-kb genome. phiEa1, phiEa7, phiEa100, phiEa125, and phiEa116C were able to infect 39, 36, 16, 20, and 40, respectively, of 40 E. amylovora strains isolated from apple orchards in Michigan and 8, 12, 10, 10, and 12, respectively, of 12 E. amylovora strains isolated from raspberry fields (Rubus spp.) in Michigan. Only 22 of 52 strains were sensitive to all five phages, and 23 strains exhibited resistance to more than one phage. phiEa116C was more effective than the other phages at lysing E. amylovora strain Ea110 in liquid culture, reducing the final titer of Ea110 by >95% when added at a ratio of 1 PFU per 10 CFU and by 58 to 90% at 1 PFU per 10(5) CFU.

FIG. 1

Restriction analysis and pulsed-field gel analysis of DNAs from E. amylovora phages φEa1, φEa7, φEa100, φEa125, and φEa116C. (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.).

FIG. 2

BglII restriction analysis of DNAs from φEa1 and putative φEa1-type phages isolated from Michigan (φEa101, φEa104, and φEa109) and California (φEa123). Lane M, 1-kb Ladder Plus (Gibco BRL). An additional 14 phages, each yielding a 0.3-kb PCR product typical for φEa1, had restriction patterns similar to these.

FIG. 3

Restriction maps of E. amylovora phage isolates φEa1 and φEa104. Fragments a to j represent the longest to shortest BglII fragments of φEa1. Corresponding BglII fragments of φEa104 are indicated with the same letter designation; fragments a¹, c¹, and c² are unique to φEa104. Other restriction sites in common between φEa1 and φEa104 are indicated between the two maps, and those unique to each phage are indicated above or below the φEa1 and φEa104 maps, respectively. B, BglII; K, KpnI; Nc, NcoI; N, NdeI; Pv, PvuII; X, XbaI.

FIG. 4

Control of growth of E. amylovora strain Ea110 in the presence of phage(s). Single phage types (φEa1, φEa7, φEa100, φEa125, or φEa116C) or combinations of equal proportions of two or three phages (φEa1 plus φEa7 or φEa1 plus φEa7 plus φEa116C) 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.