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

Abstract

Abstract Persian walnut (Juglans regia) has a considerable economic importance worldwide. However, the vigor and vitality of walnut trees have been heavily affected by bark canker during the last few years. Irregular longitudinal cankers in the outer bark, stem tissue necrosis, bleeding with black-colored exudates and dark lesions in the inner bark of walnut trees have been observed in Kermanshah, Hamedan, Markazi, Alborz, Isfahan, Qom, Semnan, and Razavi Khorasan provinces in western, central and eastern Iran during 2018 and 2019. Therefore in order to identification of bacteria associated with walnut decline, a total of 150 symptomatic samples were collected from affected walnut trees. Two-hundred sixty strains with a metallic green sheen were isolated on EMB-agar medium. The pathogenicity of 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 branches 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 (601 bp) and infB (615 bp), indicated that strains including I1, Q6, and S6 were grouped in a cluster with G. quercinecans FBR97T as well as strains I2, I5, and KE6 were clustered with Rahnella victoriana FRB 225T. Moreover, strains MR1, MR3, and MR5 were grouped with the E. hormaechei subsp. hoffmannii DSM 14563T. Phylogenetic analyses based on partial sequencing of housekeeping genes including fusA (633 bp), pyrG (305), and leuS (640 bp) revealed that KH1, KH3, and KH7 strains belong to C. 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.