In November 2017, vertical cankers were observed in the trunk of 120 trees growing in a plantation of hybrid poplar (Populus × euramericana) of 12 ha in the district of Santarem (Portugal), suggesting a bacterial disease. The trees had been imported from France and planted in 2011. The vertically cracked bark presented a whitish creamy mass delimited by dark brown exudates. Under the microscope, the creamy mass was found to be composed by a large number of individuals of the free-living, bacte-riophagous nematode Panagrellus redivivus, as also reported by Toth et al. (2013), who suggested that large numbers of bacteria are present in the bark canker. Samples of cankers and exudates were macerated and extracts plated on King’s B medium. Cultures yielded bacterial isolates showing colonies white-ivory colored, bright, round, slightly convex, and not fluorescent under ultraviolet light. Nine isolates were selected and further characterized by phenotypic (biochemical data, fatty acid profiles, and carbon source utilization by Biolog GEN III and API 20E test plates) and phylogenetic analysis. A Lonsdalea populi strain was also included for comparison. Colonies were gram-negative, facultative anaerobic, levan and esculin hydrolysis positive, but did not have cytochrome c oxidase or urease activity and did not induce a hypersensitivity response on tobacco plants. The symptomatology in the affected poplars and the results of the phenotypic analyses of the isolates were similar to those reported for L. populi (Berruete et al. 2016; Li et al. 2014, 2017; Toth et al. 2013). Partial sequences of the 16S rDNA gene and housekeeping genes gapA, atpD, gyrB, and infB showed 99 to 100% identity with isolates of L. populi available at the GenBank database. The neighbor-joining phylogenetic tree, inferred by the CIPRES Science Gateway after online alignment by the MAFFT program version 7 of 16S rDNA and housekeeping gene sequences, further identified the isolates as L. populi. Sequences from two representative isolates (EFA-L5 and EFA-L6) from this study were submitted to GenBank (accession nos. MH469711 and MH469712 for 16S rRNA gene, MH496625 and MH496626 for gapA, MH559348 and MH559349 for atpD, MH824634 and MH824635 for gyrB, and MH899112 and MH899113 for infB). Pathogenicity of EFA-L5 and EFA-L6 isolates was tested on nursery plants of P. × euramericana ‘I-214’. For each bacterial isolate, a suspension in water was prepared as inoculum (10⁷ CFU/ml). Five plants were inoculated with EFA-L5 and another five with EFA-L6. In each inoculated plant, two incisions were made in the trunk, and 100 µl of the corresponding bacterial suspension applied in each incision. Sterile distilled water was applied in incisions similarly made in the trunk of five control plants. All plants were placed in a growth chamber at 85 to 90% humidity and 22 ± 3°C. A watery necrosis area was observed in all inoculated plants 7 days after inoculation, but not in control plants. After 3 months, decay and death of most inoculated plants were observed. L. populi was reisolated from symptomatic plants, thus fulfilling Koch’s postulates. L. populi has been reported causing bark canker disease in poplar in Hungary, China, and Spain (Berruete et al. 2016; Li et al. 2014; Toth et al. 2013). To our knowledge this is the first report of L. populi on poplar in Portugal. More research is needed to know the distribution of bark canker disease in this country, because it could have a significant economic impact on poplar plantations.
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