Isolation and characterization of Pseudomonas syringae pv. syringae which induce leaf spot on walnut

Abstract

In 2012, a leaf spot disease on walnut seedlings was observed in Hamedan province of Iran. The spots were necrotic with yellow halos. Symptomatic samples were collected and suspected bacterial agent was isolated on nutrient agar medium. Phenotypic characteristics such as production of fluorescent pigment on KB medium, LOPAT test and utilization of various carbon sources, revealed that the strains were Pseudomonas syringae pv. syringae. All tested strains were pathogenic on peach, plum and walnut seedling. To investigate the genetic diversity among the strains, rep-PCR using BOX and ERIC primers was performed. Results showed high similarity among the strains from the same region, while variation was found among those from different areas and they were divided into two groups based on geographic regions. The phylogenetic analysis based on 16S rRNA, rpoD and gyrB sequences showed that representative isolates were closely related to P. syringae pv. syringae. To the best of our knowledge, this is the first report of the presence of P. syringae pv. syringae as a causal agent of walnut seedlings leaf spot in Iran and worldwide.