First Report of Stem Rot on Potato Caused by Dickeya chrysanthemi in Minnesota

Abstract

Bacterial soft rot and black leg diseases of potato occur sporadically in the Upper Midwestern United States. Multiple members of the soft rot Enterobacteriaceae (SRE) cause diseases in potato. The taxonomy of SREs has undergone extensive revision, resulting in the establishment of new genera and species to better reflect the phylogeny and diversity of strains formerly classified as Erwinia carotovora or E. chrysanthemi (Marrero et al. 2013). One such species, Dickeya dianthicola, has been associated with recent severe black leg outbreaks in the eastern United States. To identify the SREs in the Upper Midwest, a 2016 survey of commercial and seed potato fields in Minnesota and North Dakota was conducted. SREs (123) were isolated from 78 tissue samples exhibiting symptoms of black leg, stem rot, and soft rot. Small tissue samples from the margin of symptomatic and healthy tissues were suspended in 0.02 M potassium phosphate buffer, pH 7.2, for 30 min. Cell suspensions were serially diluted and spread onto crystal violet pectate (CVP) agar. Bacterial colonies producing pitting on CVP were subsequently reisolated on CVP and purified on nutrient broth yeast extract medium. Three isolates from one sample were obtained from a stem sample collected in Sherburne County, Minnesota. One isolate, SR64, was retained for phenotypic characterization and genetic identification. Symptoms associated with SR64 included dry, black-brown necrotic stem lesions with cracking and exposed vasculature. SR64 was determined to be gram negative based on reaction in 3% KOH. Growth on King’s B agar medium failed to produce fluorescence characteristic of Pseudomonas. SR64 also exhibited facultative anaerobic respiration in Hugh-Leifson medium and the ability to grow at 37°C on nutrient agar. Reinoculation of potato tuber slices with SR64 and incubation at 25°C for 2 days produced tissue maceration and dark ooze typical of SRE. DNA extracted from a pure culture of SR64 using DNeasy blood and tissue kits (Qiagen, Valencia, CA, U.S.A.) was used as template for genetic analysis. PCR was conducted using two primer sets: ADE1/ADE2 (Nassar et al. 1996) and Df/Dr (Laurila et al. 2010). SR64 template DNA produced 420 and 119 bp amplicons via ADE1/ADE2 and Df/Dr primers, respectively, as expected for Dickeya. The 16S rDNA was amplified using primer set F27/R1492 (Monciardini et al. 2002), and the ∼1.2 kb amplicon was sequenced and submitted for GenBank nucleotide BLAST analysis. SR64 shared 98% identity with that of the 16S rDNA sequence of type strain Dickeya chrysanthemi LMG 2804 (NZ_CM001974) (Samson et al. 2005). To confirm the species identification, additional housekeeping genes, dnaJ (DCH402_RS02710), dnaX (DCH402_RS15110), and gyrB (DCH402_RS00180) were amplified using primer sets reported by Marrero et al. (2013). Housekeeping gene sequences were concatenated creating a 1,955 bp fragment for analysis with CLC Main Workbench software (Qiagen). The pairwise percent identity between the concatenated sequences of SR64 and D. chrysanthemi LMG 2804ᵀ, D. zeae LMG 2505ᵀ, D. dianthicola LMG 2485ᵀ, D. dadantii subsp. dadantii LMG 25991ᵀ, and D. solani LMG 25993ᵀ was 99.13, 89.42, 90.85, 89.88, and 85.89%, respectively. The low incidence of Dickeya observed in this survey could be owing to limitations of the protocols used to isolate SREs. Alternatively, it could indicate that Dickeya is not currently prevalent in the region. This is the first report of D. chrysanthemi on potato in Minnesota.