First Report of Dickeya dianthicola Causing Potato Blackleg in Oregon

In May 2023, one potato seed lot (variety 'Russet Norkotah') submitted to Oregon State University's Hermiston Agricultural Research and Extension Center seed lot trials (Umatilla County, OR) had stunted plants expressing wilt and stem rot at the base of the stem. Disease incidence was 24% (43/182 plants). Four symptomatic plants were sampled and shipped overnight on ice to the USDA Emerging Pests and Pathogens Research Unit in Ithaca, NY. To isolate pathogens associated with disease, we incubated minced stem tissue from diseased plants in double distilled water for 20 minutes and streaked the resulting suspension on crystal violet pectate (CVP) media and incubated overnight at 28°C. White colonies causing depressions on CVP were re-streaked to isolate pure cultures. Pectinolytic colonies were isolated from two out of eight samples tested. One colony from each of the two samples was named KF23-6 and KF23-8, respectively and stored at -80°C in 16% glycerol. We extracted genomic DNA from each isolate using ZymoBIOMICS DNA miniprep kit (Zymo Research D4300) and submitted them for 2X150 paired-end Illumina sequencing at the Cornell University Biotechnology Resource Center. Bacterial genomes were assembled and annotated as previously described (Ma et al., 2024). The genome sequences were deposited in the NCBI GenBank database (Accession number: JBKQBI000000000, JBKQBH000000000). We used genomic DNA sequences to construct phylogenetic trees (Fig. S1) to examine the relationship between our isolates and all known Dickeya species. The two query strains isolated in this study formed a monophyletic clade with D. dianthicola. Genome-wide average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values between the query strains and D. dianthicola ME23 were above the thresholds for species assignment (Table S1). The ANI between the two isolates from Oregon is 99.9936%, suggesting they are clonal (Rodriguez-R et al. 2024). Importantly, these two isolates were not clonal to the ME23 (Table S1), which is the representative isolate from widespread outbreaks between 2014-2019 in the Eastern US and Canada (Ge et al., 2021; Ma et al., 2024; Charkowski, 2018). To complete Koch's postulates, we inoculated yellow flesh potato tubers by adding 10 ul overnight culture (1 x 108 CFU/ml) of each isolate individually to a 0.5 mm deep wound formed by a 2 mm diameter wooden applicator. After a 24-hour incubation at 28°C, macerated regions formed around the inoculation sites. Tissue in those regions was resuspended in distilled water and placed on CVP. Colonies that formed pits on CVP were confirmed to be the same bacteria used in the inoculum by comparing the partial dnaX gene sequences, amplified by colony DNA using (Sławiak,et al., 2009). Stems of four-week-old potato plants (cv. Upstate Abundance) propagated from cuttings were inoculated using sterile toothpicks lightly smeared with a bacterial colony. Similarly, pectinolytic bacteria were isolated on CVP three days after stem inoculation and confirmed using dnaX PCR and sequence. All experiments were conducted in duplicate. Dickeya dianthicola is one of the main causes of soft rot disease in the northeastern and mid-Atlantic states, Florida, two provinces in Canada, and is linked to the soft rot and blackleg outbreaks in the Northeast US between 2014-2019 (Ge et al., 2021; Ma et al., 2024; Charkowski, 2018). This study marks the first time D. dianthicola has been reported in Oregon. The emergence of D. dianthicola in the Northwest US production region means that farmers should remain vigilant.