First report of Pectobacterium versatile causing potato soft rot in Oregon and Washington

Potato (Solanum tuberosum cv. Norkotah) tubers with symptoms of soft rot were submitted to Oregon State University, Hermiston Agricultural Research and Extension Center Plant Clinic in 2019. One submission in May, originated from a field with poor emergence and seed piece decay (~20% affected) in Umatilla County, Oregon. The second submission, in September, originated from a field in Washington. From each submission, ~100 mg tissue at the margin of infection was washed with distilled water, excised, macerated in 500 L sterile distilled water for 5 minutes. The resulting solution was streaked on crystal violet pectate (CVP) medium and incubated at 28°C for 24 hours. One colony, representative of the many white colonies that formed depressions on CVP plates, was isolated from each submission. Bacterial isolates from Oregon and Washington were named JB56A and JB133A, respectively, and preserved in Luria-Bertani (LB) broth with 15% glycerol at -80°C for long-term storage. Genomic DNA was extracted from JB56A and JB133A cultures grown in LB broth overnight at 30°C using the Wizard SV Genomic DNA kit. The partial dnaX gene (537 bp) was amplified from genomic DNA of each isolate using dnaXf/dnaXr primers (Slawiak et al. 2009) and sequenced. These sequences were deposited to the NCBI GenBank Database, accession numbers MW930747 (JB56A) and MW930748 (JB133A). BLAST analyses (Altschul et al. 1990) using default parameters indicated that the dnaX sequences of JB56A and JB133A were 99.2% (533/537) and 98.7% (530/537) identical to that of P. versatile SCC1 (CP021894). A condensed maximum likelihood tree was built using the partial dnaX sequence of the two query strains, twelve Pectobacterium reference strains to include all known species of Pectobacterium, and four Dickeya species as an outgroup (Fig. S1). JB56A and JB133A formed a monophyletic clade with P. versatile SCC1. Potato (cv. Upstate Abundance) tuber and stem bioassays (Ma et al. 2018) were conducted twice to assess the pathogenicity of these isolates. Tubers were wounded with a sterile 2 mm wide wooden applicator stick and 5 μl culture grown in LB broth overnight (~109 CFU) was pipetted into the wound. Tubers were incubated at 29°C for 24 hours and cut through puncture sites to observe symptoms. Stems of four- or five-week-old plants were wounded with a sterile toothpick about 10 cm above the soil line and a smear of JB56A or JB133A grown on LB agar was inserted into the wound using a toothpick and incubated in a greenhouse for 72 hours. Positive controls (D. dianthicola ME23) and negative controls (no bacteria) were included in both assays. Tubers and stems exhibited disease symptoms after 24 and 72 hours, respectively, following inoculation with JB56A, JB133A, and D. dianthicola ME23. No symptoms were observed for negative controls. The identity of bacteria re-isolated from the margin of stem lesions was confirmed by partial dnaX sequence analyses. P. versatile was recently described as a distinct species based on whole genome sequence comparisons (Portier et al. 2019). In 2018, we isolated P. versatile from potato stems with blackleg disease in New York, and a recent study found that it was isolated in the US from an iris in 1946 (Ma et al. 2021; Portier et al. 2019). However, the geographic distribution and importance of this pathogen in the US remains largely unknown. To our knowledge, this is the first report of potato soft rot caused by P. versatile in Oregon and Washington, two important potato producing states.