First report of Pectobacterium carotovorum subsp. brasiliensis causing blackleg and stem rot in commercial and seed potato fields in Minnesota and North Dakota

Blackleg and stem rot are common diseases affecting potato (Solanum tuberosum) production in the Northern Great Plains of the United States. Changes in the taxonomy of the causal agents of blackleg and stem rot diseases and concerns in the potato industry about newly emerging pathogens, such as Dickeya spp., prompted a study to identify the soft rot pathogens present in Minnesota and North Dakota (Jiang et al. 2016). Surveys conducted in 2015–16 of commercial and seed potato fields produced multiple samples exhibiting soft rot and blackleg symptoms. Bacteria were isolated from symptomatic tissues by suspending about 1-cm sections from the margins of lesions in 0.02 M potassium phosphate buffer, pH 7.2, for 30 min, followed by serial dilutions on crystal violet pectate (CVP) agar. Bacterial colonies producing pitting on CVP were reisolated on CVP and further purified on nutrient broth yeast extract medium (NBY). Characterization of four isolates provided evidence that a previously unreported soft rot pathogen was present in the region. SR36 and SR72, from Sherburne County in Minnesota, and SR124 and SR162 from Pembina and Walsh counties in North Dakota, produced symptoms associated with blackleg and stem rot of potato. Symptoms appeared near the stem base as black-brown necrotic lesions exhibiting variable levels of tissue cracking and moisture. Phenotypic analyses revealed that all four isolates were gram-negative, nonfluorescent on King’s B (KB) agar medium, facultatively anaerobic in Hugh-Leifson medium, and able to grow at 37°C on nutrient agar (NA). All isolates produced typical soft rot symptoms in potato tubers inoculated with 10⁷ cfu/ml of the pathogen and incubated at 25°C for 48 h. DNA was extracted from pure cultures of each isolate with DNeasy Blood & Tissue Kits (Qiagen, Valencia, CA) and used as template for genetic identification. 16S rDNA was amplified using universal primer set F27/R1492 (Monciardini et al. 2002). Resultant amplicons were sequenced and submitted for GenBank nucleotide BLAST. All isolates shared ∼99% sequence identify with Pectobacterium carotovorum subsp. brasiliensis type strain BC1 (NC16_21220). To further validate this identification, PCR was conducted using P. carotovorum subsp. brasiliensis-specific primers BR1f/L1r (Duarte et al. 2004). All isolates produced the predicted 322-bp amplicons expected for P. carotovorum subsp. brasiliensis. Additional loci representing housekeeping genes dnaA (NC16_00005), dnaJ (NC16_18375), dnaX (NC16_05325), gyrB (NC16_00015), and recN (NC16_03605) were amplified and sequenced. Primer sets used were as reported by Marrero et al. (2013). Per isolate, housekeeping gene sequences were concatenated creating a 3,545-bp fragment. Pairwise percent similarity was determined via CLC Main Workbench (Qiagen, Valencia, CA). All isolates displayed 97 to 98% similarity with type strain P. carotovorum subsp. brasiliensis BC1. P. carotovorum subsp. brasiliensis is present in other potato-producing states in the U.S. Here we provide the first report of the occurrence of P. carotovorum subsp. brasiliensis in commercial and seed potato fields in Minnesota and North Dakota.