Alfalfa (Medicago sativa)is the most important forage crop in Hamedan province, located in west of Iran. Under field condition, plants showed growth stunting, chlorosis and wilting symptoms not previously reported. To survey the causal agent of the disease, symptomatic plants were collected from the main alfalfa growing area in this province. Plants samples were disinfected, grinded in phosphate buffer, and a loop full of the prepared suspension was stroked on nutrient agar medium containing 5% yeast extract and 1% glycerol. Isolation and characterization of the causal agent, especially from plants in primary growth stage of the disease, is difficult. Isolated bacteria caused wilting and chlorosis after 40 days on alfalfa plants, under green house condition. Phenotypic feature determination of the isolated bacteria, together with two defined strains, by standard bacteriological methods indicated that they belong to P. viridiflava. PCR assay confirmed results of identification of the causal bacterial agent, determined by classical methods. Tested representative bacterial isolates showed an 860 pb PCR band, using primers designed for 16S rDNA sequence of P. viridiflava LMG 2352T (type strain). This new reported bacterium is the second pathogenic bacterium agent, which causes alfalfa yield losses in Hamedan province of Iran.
