THE ANALYSIS OF PLASMID-MEDIATED STREPTOMYCIN RESISTANCE IN ERWINIA-AMYLOVORA

Streptomycin-resistant mutants of Erwinia amylovora were isolated from an apple orchard in Michigan and from crabapple trees adjacent to the same orchard in 1990. Isolates that grew on King's medium B amended with 100-mu-g/ml of streptomycin sulfate were considered to be resistant strains, whereas isolates that failed to grow on this medium were considered to be sensitive strains. Growth of the resistant strains was not inhibited in a filter-paper disk assay (0.06-5-mu-g of streptomycin sulfate), but growth of sensitive strains was inhibited at concentrations as low as 0.06-mu-g of streptomycin sulfate. Only sensitive strains were detected in an additional 19 apple orchards sampled for resistant strains. In colony blot hybridizations, an internal portion of the streptomycin-resistance gene (probe SMP3) from strain Psp36 of Pseudomonas syringae pv. papulans hybridized with all streptomycin-resistant strains of E. amylovora, but not with streptomycin-sensitive strains. Probe SMP3 hybridized to a 2.7-kb restriction fragment from AvaI-digested total genomic and plasmid DNA of two resistant strains of E. amylovora and to a 1.5-kb fragment in DNA from strain Psp36 of P. s. papulans. The probe did not hybridize with digested DNA from sensitive strains. A 33-kb plasmid was present in all streptomycin-resistant field strains but not in streptomycin-sensitive strains. Streptomycin resistance was transferred by matings to four streptomycin-sensitive recipient strains of E. amylovora from each of two streptomycin-resistant donor strains. Transconjugants also contained the 33-kb plasmid. DNA from resistant strain Ea88-90 from Washington did not hybridize with the probe, indicating that this strain contains a resistance system unrelated to that in streptomycin-resistant strains from Michigan.