First Report of Dickeya dianthicola Causing Blackleg on Potato (Solanum tuberosum) in Bulgaria.

Potato (Solanum tuberosum) is an important and widespread crop in Bulgaria. A new disease was observed on a single potato plot (Plovdiv region) without a history of potato cultivation in the spring of 2011. Initially, single lower leaves wilted on recently emerged plants (approx. 15% incidence) with subsequent desiccation of the leaf margins. The wilting progressed over time and eventually the whole stem became desiccated. A blackleg-like necrosis was noticed at the stem base when symptomatic plants were uprooted. Most diseased stems remained green above ground but pith tissue was heavily macerated and some of the stems became hollow as the pith dried out. Mother tubers were partially or entirely macerated. In most cases, the decay was initiated from the stolon end. Bacterial strains were obtained from symptomatic stems and tubers by dilution plating on King's B medium. The strains produced indigoidin pigment and induced a hypersensitive response 24 h after infiltration into tobacco and Sedum hybridum leaves (2). The strains were identified as Dickeya spp. by the production of the PCR amplicon of the pectate lyase ADE gene cluster (3) and of the pectate lyase I gene (4). The partial sequence of the fliC PCR amplicon (1) of strain SB2589 (GenBank Accession No. KF442436) displayed 100% homology with four whole genome shotgun sequences of Dickeya dianthicola in GenBank. Pectinolytic activity was demonstrated by inoculation of surface disinfested potato tubers of cv. Kondor. Conical core tissue was removed at the apical end and 100 μl bacterial suspension (10⁷ CFU in sterile 10 mM phosphate buffer) was deposited in the cavity. The cap was reattached to the tuber and immobilized by Parafilm. Positive control tubers were inoculated with D. dianthicola reference strain GBBC 2039 (LMG 25864) and negative control tubers were inoculated with sterile 10 mM phosphate buffer. All tubers were incubated for 48 h at 28°C under micro-aerobic conditions reducing the air pressure to 90 mb in a vacuum incubator. The D. dianthicola reference strain and Bulgarian strains produced maceration of tuber tissue. Maceration was not observed in the negative control tubers. Potato plants cv. Kondor were grown from minitubers in sandy soil in plastic nursery containers. The plants were inoculated by root drenching (one application of cell suspension at 10⁹ CFU/liter) when the stems were 15 to 20 cm high (tuber initiation stage). Plants were incubated at 25 to 28°C with regular watering. Wilting symptoms developed within 10 days of inoculation, followed by necrosis of the pith. Strains obtained from the inoculated stems were confirmed as D. dianthicola as described above. Based on the disease symptoms, the cultural, molecular, and pathological features of the strains, we conclude that the disease was caused by D. dianthicola and to our knowledge this is the first report of the pathogen on potato in Bulgaria. Furthermore, this incident warrants further surveys of pectinolytic bacteria causing blackleg-like symptoms in potato crops in Bulgaria. References: (1) S. Diallo et al. Eur. J. Plant Pathol. 125:349, 2009. (2) Y-.A. Lee and C-.P. Yu. J. Microbiol. Methods 64:200, 2006. (3) A. Nassar et al. Appl. Environ. Microbiol. 62:2228, 1996. (4) J. Van Vaerenbergh et al. PLoS ONE 7(5):e35738, 2012.