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Genetic Analysis of Streptomycin-Resistant (Sm^R) Strains of Erwinia amylovora Suggests that Dissemination of Two Genotypes Is Responsible for the Current Distribution of Sm^R E. amylovora in Michigan

First, second, third, fourth, sixth, and seventh authors: Department of Plant Pathology, Michigan State University, East Lansing 48824; fifth author: Michigan State University, Southwest Michigan Research and Extension Center, Benton Harbor 49022; and sixth author: Michigan State University, Clarksville Horticulture Experiment Station, Clarksville 48815.

Streptomycin-resistant (Sm^R) strains of the fire blight pathogen Erwinia amylovora were first isolated in southwest Michigan in 1991. Since that time, resistant strains have progressed northward to other apple-producing regions in the state. A total of 98.7% of Sm^R strains isolated between 2003 and 2009 in Michigan harbored the strA-strB genes on transposon Tn5393. strA and strB encode phosphotransferase enzymes that modify streptomycin to a nonbactericidal form. Mutational resistance to streptomycin, caused by a point mutation-mediated target-site alteration of the ribosomal S12 protein, occurred in 1.3% of E. amylovora strains from Michigan. Tn5393 was originally introduced to E. amylovora on the plasmid pEa34; thus, the first Sm^R strains isolated contained both pEa34 and the ubiquitous nonconjugative plasmid pEA29. More recently, we have observed Sm^R strains in which Tn5393 is present on pEA29, suggesting that the transposon has moved via transposition from pEa34 to pEA29. Almost all of the strains containing Tn5393 on pEA29 had lost pEa34. Of 210 pEA29::Tn5393 plasmids examined, the transposon was inserted at either nucleotide position 1,515 or 17,527. Both of these positions were in noncoding regions of pEA29. Comparative sequencing of the housekeeping genes groEL and potentially variable sequences on pEA29 was done in an attempt to genetically distinguish Sm^R strains from streptomycin-sensitive (Sm^S) strains isolated in Michigan. Only 1 nucleotide difference within the total 2,660 bp sequenced from each strain was observed in 2 of 29 strains; multiple sequence differences were observed between the Michigan strains and E. amylovora control strains isolated in the western United States or from Rubus spp. Alterations in virulence observable using an immature pear fruit assay were detected in three of eight Sm^R strains examined. Our current genetic data indicate that only two Sm^R strain genotypes (strains containing pEA29::Tn5393 with Tn5393 inserted at either nucleotide position 1,515 or 17,527 on the plasmid) are responsible for the dissemination of Tn5393-encoded streptomycin resistance in Michigan, and that the Sm^R and Sm^S strains in Michigan compose a homogenous group.