Characterization of Pectobacterium carotovorum subsp. brasiliense as a Causal Agent of Sugarbeet Soft Rot
View/ Open
Abstract
A soft rot decay of sugarbeet was observed in commercial fields in North Dakota and Minnesota from 2012 to 2016. Symptoms reported are similar to those for bacterial vascular necrosis and rot caused by Pectobacterium betavasculorum including soft decay of internal root tissues, reddening of affected tissue after cutting, blackening of petiole vascular bundles, half-leaf yellowing, and root frothing. The disease can cause serious yield losses in the field, and additional economic losses in storage and during processing due to accumulation of invert sugars that reduce sugarbeet quality. Sap from the margin of diseased root tissue was streaked on pectate agar medium and incubated. Single pectolytic colonies were selected and transferred to nutrient broth for bacterial identification and completion of Koch’s postulates. Pathogenicity of isolates was assessed by inoculating greenhouse-grown sugarbeet roots. Symptoms characteristic of the disease were observed at 30 days after inoculation included all of the aforementioned, previously stated symptomology. Bacterial DNA was extracted from 46 pathogenic isolates and analyzed by restriction-associated DNA genotype-by-sequencing (RAD-GBS). Ion-torrent sequencing reads (n = 8.54 million) were assembled de novo producing ∼6,000 sequence tags representing approximately 21% of each bacterial genome analyzed. Partial sequences of five of the seven genes previously used in Pectobacterium subspecies phylogenetic analysis were represented in the RAD-GBS isolate sequences. Gene sequences were aligned using Workbench 8.0.3 software to the corresponding reference gene sequences of P. carotovorum subsp. carotovorum, P. atrosepticum, P. betavasculorum, P. carotovorum subsp. odoriferum, and P. wasabiae. The alignments showed 99.76% nucleotide sequence identity on average across all five genes to the P. carotovorum subsp. brasiliense reference sequences. The alignments to P. cartovoroum subsp. carotovorum, P. atrosepticum, P. betavasculorum, P. carotovorum subsp. odoriferum and P. wasabiae reference sequences showed 96, 95.4, 94.3, 97 and 94.4% identity, respectively, on average across the five genes. The nearly 100% identity across all five genes previously utilized in multi-locus sequencing and divergence from the closely related subspecies strongly suggests that the isolates are P. carotovorum subsp. brasiliense. To our knowledge, this is the first report of this pathogen causing field decay of sugarbeet in North America.