First report of Erwinia aphidicola from Phaseolus vulgaris and Pisum sativum in Spain

During 2003 and 2004, leaf spot disease of common bean (Phaseolus vulgaris) was observed in southeastern Spain (Almeria, Granada and Malaga) (Gonzalez et al. 2005) and symptoms of generalised chlorosis as well as necrosis in leaves and tendrils were observed in Pisum sativum cv. Tirabeque (Gonzalez et al.2007).In 2006 and 2007, samples of common bean (cv. Donna) with chlorotic and necrotic leaf spots were collected from Almeria to determine the pathogen. Bacteria isolated from leaves with spots exhibited the biochemical characteristics of the family Enterobacteriaceae. They were Gram-negative, oxidase negative, catalase positive, fermentative, rod shaped, motile, facultatively anaerobic.

Two isolates were selected for pathogenicity tests. Bacterial suspensions (10⁸ cfu/ml) were spray inoculated on bean seedlings (Phaseolus vulgaris) cv. Donna (2-3 true leaves). Beans were covered with transparent plastic bags for two days and held in an incubation chamber at 22 ºC and 80% relative humidity with a 12 h photoperiod. Assays were conducted twice. Symptoms that developed were similar to those originally observed in the field. No symptoms were observed on control plants (inoculated with distilled water).

Preliminary identification of the pathogenic isolates based on 16S rDNA sequencing was as either Erwinia persicina or E. aphidicola (99–100% homology). Primers for PCR amplification of partial sequences of dnaJ, recA and gapDH were manually designed from sequences of Erwinia persicina from GenBank (Accession Nos. AB272647, DQ859883 and AF165028, respectively). The amplified sequences were compared with available DNA sequences by using BLAST giving 100 % homology with recA and gapDH, and 99% with dnaJ of E. aphidicola. The same results were obtained for isolates from Phaseolus vulgaris (LPPA 373) and Pisum sativum (LPPA 408) obtained by González et al. (2005, 2007). Additional biochemical and pathogenicity tests and molecular analysis were performed using E. persicina ATCC 49742, E. persicina ATCC 35998 and E. aphidicola GTC 1688 as controls in which 91%, 85% and 88% homology with recA, gapDH, and dnaJ of E. persicina was found.

Previously, E. aphidicola was isolated from the pea aphid, Acyrthosiphon pisum (Harada et al. 1997), so to our knowledge, this is the first report of E. aphidicola as a plant pathogen of Phaseolus vulgaris and Pisum sativum.Our results confirm that sequence analysis of 16S rDNA may not provide sufficient resolving power in discriminating closely related species.