First Report of Bacterial Deep Bark Canker of Walnut Caused by Brenneria (Erwinia) rubrifaciens in Europe

Abstract

During the summer of 1995 and subsequent years, bark cankers were observed in walnut trees (cv. Hartley grafted on Juglans hindsii) imported from California in 1978 growing in Badajoz, Spain. Two foci were found in an orchard of 200 ha where 80 walnut trees were affected. Cankers were observed on trunks and branches, and dark exudates staining the bark appeared mainly in summer. Isolations were performed from affected tissue using King's B medium, and Brenneria (Erwinia)-like colonies (1) were purified and characterized. Gram reaction, Kovacs' oxidase, O/F metabolism, aesculin hydrolysis, urease activity, and levan production were assayed for five isolates (1). Biochemical characterization was performed by the miniaturized API 20E, API 20NE, and API 50CH systems (BioMérieux, Marcy-l'Etoile, France) as recommended, except for incubations that were made at 25°C for 48 h. Analyses of the cellular fatty acids of selected isolates were performed as described by Sasser (2). They were also tested in indirect enzyme-linked immunosorbent assay (ELISA) using antisera obtained against the reference strain CFBP 1284 and one Spanish isolate. When compared to the reference strain from California, isolates were identified as Brenneria rubrifaciens (1,3) on the basis of physiological and biochemical characteristics, fatty acid profiles, and ELISA. Pathogenicity of two selected Spanish isolates was confirmed using three 2-year-old walnut trees per bacterial isolate by inoculating 108 CFU of each isolate in deep wounds made in the trunk at 40 and 80 cm from the crown. The reference strain and water were also inoculated as controls. Two months later, removal of the outer bark of walnut revealed typical dark lesions in the inner bark at all the inoculation sites on trees inoculated with the Spanish and reference strains, but no external cankers were observed. Four years later, these plants showed internal lesions (20 to 80 cm), from which B. rubrifaciens was reisolated far away from the inoculation site. To our knowledge this is the first report of this bacterium in Europe. References: (1) L. Hauben et al. Syst. Appl. Microbiol 21:384, 1998. (2) M. Sasser. Pages 199–204 in: Methods in Phytobacteriology. Budapest, Hungary, 1990. (3) E. Wilson et al. Phytopathology 57:618, 1966.