The drippy nut disease of oak was first described in California in 1967 and, since then, the causal agent has not been reported in any other area. This study describes for the first time in Europe the isolation of Brenneria (Erwinia) quercina from bark canker in addition to drippy bud and drippy nut in Quercus ilex and Q. pyrenaica. The bark canker and drippy bud symptoms were not previously described as caused by this bacterium. No fungal pathogens were associated with any of the symptoms. Physiological and biochemical characterization identified the pathogenic isolates from Spain as belonging to B. quercina, similar to the reference strain CFBP 1266. Fatty acid profiles of the Spanish isolates also were similar to the strain of B. quercina from California. Serological analysis by indirect immunofluorescence and enzyme-linked immunosorbent assay using polyclonal antisera against the reference strain of B. quercina and one Spanish oak isolate revealed some antigenic heterogeneity between isolates of different origins. Pathogenicity tests demonstrated that the Spanish isolates were able to reproduce internal symptoms of necrosis and acorn exudation in Q. ilex and Q. pyrenaica and suggest that B. quercina may be associated, among other causes, with the oak decline syndrome affecting Spanish oak forests.

Brenneria quercina has been reported as one of the causal agents of oak decline in Spain. To investigate the bacterial variability of this pathogen from different Spanish oak forests, a collection of 38 bacterial isolates from seven geographic locations and from different oak species was analysed by sequencing 16S rDNA and rep-PCR fingerprinting. All Spanish isolates of B. quercina were grouped by rep-PCR into a homogenous cluster that differed significantly from B. quercina reference strains from California. 16S rDNA analysis revealed that 34 out of 38 isolates were Brenneria . However, four isolates belonged to the genus Serratia , suggesting that this bacterium could cause cankers in oak trees. The information obtained by rep-PCR fingerprint analysis was used to develop PCR primers for the sensitive and specific detection of B. quercina from infected plant tissues. Pathogenicity tests performed with Brenneria and Serratia isolates showed that both were able to grow and cause cankers in oak trees.

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.