Association of Rahnella victoriana, Enterobacter hormaechei subsp. hoffmannii and Citrobacter braakii with walnut decline

Persian walnut (Juglans regia) has a considerable economic importance worldwide. However, the vigor and vitality of walnut trees were heavily affected by bark canker during the last few years. Irregular longitudinal cankers in the outer bark, stem tissue necrosis, and bleeding with black-colored exudates walnut trees were observed in Kermanshah, Hamedan, Markazi, Alborz, Isfahan, Qom, Semnan, and Razavi Khorasan provinces in western, central and eastern Iran during 2018 and 2019. A total of 150 symptomatic samples were collected from affected walnut trees in order to identify bacteria associated with walnut decline. Two-hundred sixty strains with a metallic green sheen were isolated on EMB-agar medium. The pathogenicity of all strains was proved by inoculating a suspension of the bacterial strains under the bark of immature walnut fruits cv. ‘Hartley’. Ninety-five strains caused necrosis and a dark-colored region in the mesocarp around the inoculation site 14 days post-inoculation. Moreover, 12 representative strains induced necrotic and black-colored tissues in the bark of young green twigs of two-year old walnut seedling cv. ‘Chandler’. The strains were classified into four categories based on conventional phenotypic characters confirmed with the 16S rRNA gene sequences. A phylogenetic tree based on the concatenated sequences of two housekeeping gene fragments, gyrB and infB, indicated that strains including I1, Q6, and S6 were grouped in a cluster with Gibbsiella quercinecans FBR97(T) as well as strains I2, I5, and KE6 were clustered with Rahnella victoriana FRB 225(T). Moreover, strains MR1, MR3, and MR5 were grouped with the Enterobacter hormaechei subsp. hoffmannii DSM 14563(T). The phylogenetic analyses based on the partial sequencing of housekeeping genes including fusA, pyrG, and leuS revealed that strains KH1, KH3, and KH7 belong to Citrobacter braakii species. To the best of our knowledge, this is the first report of C. braakii and E. hormaechei as plant pathogens and R. victoriana associated with walnut decline.