In this study, we provide a polyphasic characterization of 18 spp. strains associated with alfalfa leaf spot symptoms in Iran. All of the strains were pathogenic on alfalfa, although the aggressiveness and symptomology varied among the strains. All strains but one were pathogenic on broad bean, cucumber, honeydew, and zucchini, whereas only a fraction of the strains were pathogenic on sugar beet, tomato, and wheat. Syringomycin biosynthesis genes ( and ) were detected using the corresponding PCR primers in all of the strains isolated from alfalfa. Phylogenetic analyses using the sequences of four housekeeping genes (, , , and ) revealed that all of the strains except one (Als34) belong to phylogroup 2b of , whereas strain Als34 placed within phylogroup 1 close to the type strain of pv. . Among the phylogroup 2b strains, nine strains were phylogenetically close to the pv. clade, whereas the remainder were scattered among pv. and pv. strains. Pathogenicity and host range assays of the bacterial strains evaluated in this study on a set of taxonomically diverse plant species did not allow us to assign a "pathovar" status to the alfalfa strains. However, these results provide novel insight into the host range and phylogenetic position of the alfalfa-pathogenic members of , and they reveal that phenotypically and genotypically heterogeneous strains of the pathogen cause bacterial leaf spot of alfalfa.
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