Projects per year
Double-layer crystal violet pectate (DL-CVP) medium (Helias et al., 2012) was used to isolate pectinolytic bacteria from the stems of blackleg affected plants. Eleven isolates derived from ten seed lots, positive in the Pcb-specific PCR (Duarte et al., 2004), were characterised with multilocus sequence analysis (MLSA) using sequences of acnA, gapA, icdA, mdh, mtlD, pgi and proA (Panda et al., 2012) (GenBank Accession Nos. KJ576801-KJ576810, KJ596350-KJ596399, KJ608062-KJ608071). The phylogenetic analysis was done by constructing a maximum likelihood tree using sequences of reference strains P. atrosepticum, P. wasabiae, P.c. subsp. carotovorum, P.c. subsp. odororiferum, P. betavasculorum and Dickeya paradisiaca, derived from GenBank, for comparison (Fig. 1). The MLSA confirmed the identity of the Pcb strains. The Dutch strains clustered together in a homogeneous branch, which also showed that these strains were highly similar. Strains were motile, able to reduce sucrose and to grow at 39°C, resistant to erythromycin, and unable to ferment lactose according to the procedures described by Hayman et al. (1998). Two Pcb isolates were tested for virulence in a field experiment in the Netherlands in 2013. Two months after planting vacuum-infiltrated seed tubers, a high percentage of the plants (more than 30%) showed typical symptoms, whereas water-treated plants remained disease-free (Fig. 2). The pathogen was re-isolated from the diseased plants and identified as Pcb using MLSA. This is the first time Pcb is described as a potato blackleg-causing agent in the Netherlands, although a strain of Pcb isolated from potato in the Netherlands has been found in a culture collection before (De Boer et al., 2012).