First report of Bacillus pumilus on Phaseolus vulgaris in Spain

Since 2003, new symptoms of interveinal purple-brown leaf spots have been observed sporadically in greenhouse-grown Phaseolus vulgaris cv. Donna in Almería, southeastern Spain (Fig.1). In February 2008, this new symptomatology was observed in approximately 30% of plants in affected greenhouses. Bacteria isolated from leaves with symptoms were Gram positive, rod shaped with peritrichous flagellae, motile, oxidase negative, catalase positive, mannitol positive, nitrate reduction positive and facultatively anaerobic, which coincided with the general characteristics of the genus Bacillus. The gene encoding the 16S rRNA from two isolates (50/08-C1 and 71/08-C2) was sequenced after PCR amplification (Edwards et al., 1989). The two sequences were almost identical (GenBank Accession Nos. FJ648765 and FJ648766, respectively) and showed 99% nucleotide identity with Bacillus pumilus strains(AY373359, DQ275671, EU869252 and AM237360). These two isolates were also tested for pathogenicity on beans. Bacterial suspensions (10⁸ cfu/ml) were spray-inoculated on 10 seedlings (with three true leaves), which were covered with transparent plastic bags for three days and kept in a greenhouse (20±5°C) for seven weeks. Assays were repeated at least twice. Five weeks later, symptoms that developed (Fig.2) were similar to those originally observed in the greenhouses (Fig.1), while symptoms did not occur on control seedlings sprayed with sterile distilled water. PCR amplification of the gene encoding the 16S rRNA (Edwards et al., 1989) from the two cultures re-isolated from inoculated plants (P5-50/08-C1 and P1-71/08-C2) was sequenced (FJ817343 and FJ883626, respectively) and showed 100 % nucleotide identity with the original bacterial isolates.

B. pumilus has been previously isolated from peach in Egypt causing bacterial blotch (Saleh et al., 1997). To our knowledge, this is the first report of B. pumilus on common bean in Spain and in Europe. B. pumilus has been proposed as a potential biological control agent against a variety of pathogens, and research into epidemiology of this cross-domain pathogen merits further study.