First report of bleeding canker caused by Rahnella sp. on Populus nigra in Iran

In 2019, a bleeding canker with a sour-smelling and brownish liquid exudate was observed on mature (>10 years old) black poplar (Populus nigra) trees in eight poplar plantations in the Hamadan, Kermanshah, Isfahan, and Kohgiluyeh and Boyer-Ahmad provinces of Iran. Up to 85% of trees were affected in some stands and the trees showed a general decline and yellowing of leaves. The exudates and cankers were found on the trunk in spring and summer (Fig. 1). The symptoms were similar to bark canker of Populus × euramericana in Spain (Biosca et al., 2006).

Samples were collected from symptomatic trees. Fifteen isolations of bacteria were made on nutrient agar (NA; Merck, Germany) from the interface between diseased and healthy tissue based on Moradi-Amirabad et al. (2019). In all cases the predominant bacteria were cream, round, convex and smooth with entire margins on NA. Phenotypic tests were performed based on standard methods (Schaad et al., 2001). All isolates were Gram-negative, and facultatively anaerobic, positive for catalase, nitrate reduction, and hypersensitive reaction on geranium (Pelargonium × hortorum), but negative for oxidase, arginine dihydrolase, urease, lysine and ornithine decarboxylase, hydrogen sulphide and indole production, citrate utilization, and fluorescent pigment production on King's medium B. All isolates produced acid from D-fructose, D-galactose, D-glucose, D-lactose, D-maltose, D-mannitol, D-mannose, D-melibiose, D-ribose, D-sorbitol, D-trehalose, D-xylose, L-arabinose, and salicin. The isolates were phenotypically identical to members of the Yersiniaceae, specifically Rahnella spp. (Brady et al., 2014).

DNA was extracted from a representative isolate (P1) (Moradi‐Amirabad et al., 2019). House-keeping genes gyrB and infB were partially sequenced (Brady et al., 2008) (Genbank Accession Nos. MT107177 and MT107178) and had 100 and 99.67% identity with Rahnella sp. strain L31-1-12, respectively. Using a neighbour-joining clustering algorithm based on the concatenated gyrB, and infB sequences, the P1 isolate clustered with Rahnella sp. strain L31-1-12 on a separate branch from other Rahnella spp. (Fig. 2). This suggests that the isolates from poplar trees in Iran belong to the genus Rahnella but are distinct from other named species.

Pathogenicity tests were performed as described by Li et al. (2014). Four strains (P1, P3, P7, and P12) were cultured on NA for 48 hr at 28°C. Bacteria were suspended in sterile distilled water (c. 10⁷ CFU/ml) and 100 µl was pipetted into cross-shaped wounds (5 mm long) made in the bark of three excised stems with a sterile knife. Control stems were inoculated with sterile distilled water. Inoculated stems were enclosed with polyethylene bags and kept in darkness at 28°C. Canker symptoms with copious brownish exudate appeared seven days after inoculation (Fig. 3). The inoculated isolates were re-isolated and their identity confirmed based on phenotypic characteristics. Control stems did not produce any symptoms and no bacteria were isolated from them.

Rahnella spp. have been associated with acute oak decline symptoms (e.g. bleeding stem) on oak and hornbeam (Brady et al., 2014; Moradi‐Amirabad et al., 2019). This is the first report of a Rahnella sp. causing a bleeding canker on poplar trees in Iran and worldwide.