First Report of Dickeya dianthicola Causing Blackleg on Potato in Texas

Blackleg of potato (Solanum tuberosum) is caused by the genera Pectobacterium and Dickeya. In 2015, multiple outbreaks of blackleg occurred in northeastern United States. Following the outbreaks, surveys of seed potatoes in Wisconsin were conducted in 2015 and 2016, and multiple lots tested positive for Dickeya dianthicola. Whole plant samples received from a seed potato buyer in Texas were also included in the survey. Bacteria were isolated from the Texas samples by soaking symptomatic stem sections in sterile water for 10 min, vortexing at full speed for 10 s, and streaking on nutrient agar (NA) plates supplemented with 100 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) and 20 mg/ml of 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-gal). Dickeya encodes β-galactosidase and thus forms blue colonies on NA plates containing IPTG and X-gal. A pure culture was obtained, and genomic DNA was extracted with the FastDNA Spin kit (MP Biomedicals, Solon, OH). Polymerase chain reaction (PCR) assays were carried out with the extracted DNA as a template and with the primer sets Df/Dr (Laurila et al. 2010) and ADE1/ADE2 (Nassar et al. 1996), resulting in the expected 133- and 420-bp amplicons, respectively. These results indicated the isolate was Dickeya. To identify the species of the Dickeya isolate, D. dianthicola-specific primers sets DIA-A and DIA-C were used (Pritchard et al. 2012). No DNA amplification was observed with the DIA-A primer set, which targets the 2-dehydropantoate 2-reductase gene. DNA amplification occurred with the DIA-C primer set, which targets the dTDP-4-dehydrorhamnose reductase gene, indicating that the DIA-C target is present in the isolate. The isolate was designated D. dianthicola strain TXG3. In addition, the PCR assays described above were used to test potato tuber cores from the remainders of the seed lot, still located in Wisconsin. PCR assay results were congruent across samples tested, indicating that TXG3 likely came from the seed potatoes in Wisconsin. Whole-genome sequencing of TXG3 was completed at University of Wisconsin–Madison Biotechnology Center, which confirmed the isolate as D. dianthicola (Perna, personal communication; genome unpublished). Genome analysis of TXG3 confirmed the absence of the DIA-A target. The pathogenicity of TXG3 was further characterized by inoculating potato stems (cultivar Mesa Russet). Tuber sprouts were planted in plastic pots containing potting mix in a greenhouse. After 5 weeks, stems of three plants were inoculated by wounding using toothpicks that had been swiped across a bacterial culture of TXG3; the wounds were wrapped with Parafilm. Three stems were mock inoculated in the same manner, except with sterile toothpicks. Plants were covered with Ziploc bags to maintain high humidity. Eleven days post-inoculation, stems inoculated with TXG3 showed necrotic lesions, whereas control plants did not show any symptoms. Bacteria were reisolated from symptomatic stems and purified on crystal violet pectate agar (Hélias et al. 2011). DNA was extracted as described above. PCR assays with primer sets ADE1/ADE2 and DIA-C were performed with the extracted DNA as a template, and the expected size fragments were amplified. To our knowledge, this is the first report of D. dianthicola causing blackleg on potato in Texas and associated with a seed lot from Wisconsin.