Location: Emerging Pests and Pathogens ResearchTitle: Complete genome sequence resource for potato ring rot pathogen, Clavibacter sepedonicus K496
|MA, XING - Cornell University - New York|
|PERRY, KEITH - Cornell University - New York|
Submitted to: Plant Disease
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/15/2022
Publication Date: N/A
Interpretive Summary: Clavibacter sepedonicus is a very serious potato pathogen. This pathogen is very difficult to eliminate from potato cropping systems and there are strict policies prohibiting the import/export of tubers with this disease. We received tubers which had unusual symptoms consisting of dark pits of the surface and significant decay of internal tissue. We grew plants from the tubers and subsequently isolated bacterial colonies. Using laboratory tests, we determined the bacteria belong to the genus Clavibacter. To determine the species whole genome sequencing was performed. Sequence analyses revealed the isolated bacteria belong to Clavibacter sepedonicus. This report will serve as a resource for conducting research on these bacteria and for diagnostic labs who might also encounter plants with this disease.
Technical Abstract: We sequenced the genome of Clavibacter sepedonicus that was isolated from diseased potato tubers that we received in December 2020. These diseased potato tubers (Solanum tuberosum cv. Lady Liberty) exhibited external zones of pitting with necrosis of the subtending areas. Scattered brown or blackened internal symptoms were also observed, and in some cases, there was discoloration of the vascular cylinder or pith tissues. Spores of Fusarium sp. were observed in the necrotic areas, and overall, the symptoms were more consistent with a Fusarium dry rot than of bacterial ring rot caused by C. sepedonicus. To aid in isolation of pathogenic organisms without the confounding growth of non-pathogenic saprophytes, we grew plants so that we could sample at the margins of any symptomatic tissues. All tubers produced healthy sprouts, but after eight weeks the two plants grown from the symptomatic tubers showed wilting lower leaves. These symptoms progressed and within a week, water-soaking lesions developed on the affected leaflets followed by leaf desiccation. No symptoms were observed on plants grown from asymptomatic tubers. To isolate bacteria, we collected 1 cm of petiole tissue from leaves of symptomatic plants and the symptom-free control. The petioles pieces were surface sterilized, finely minced, and soaked in double-distilled water for 20 min. The solution was placed on yeast extract-dextrose CaCO3 (YDC) medium and incubated at room temperature (~21°C). White-yellow mucoid colonies formed after four to five days from the samples taken from symptomatic plants and these were the only type of colonies recovered. In contrast, no colonies grew from the plant grown from the asymptomatic tuber piece. One colony from each symptomatic plant was purified by sub-culture on YDC agar at room temperature for two passages; these isolates were designated K496 and K497 and stored in 16% glycerol at -80 °C. Partial 16S rRNA gene sequences for both isolates were identical, and a BLAST search indicated the isolates were C. sepedonicus. We then sequenced the genome of C. sepedonicus K496 and produced a draft genome sequence using Nanopore R9 flowcell. The genome was then polished using short reads generated using NextSeq2000 platform with the 2x150 bp mode. The complete genome of C. sepedonicus K496 consists of a single circular chromosome and a large linear plasmid. The chromosome is 3,266,016 bp long with a GC content of 72.6%, and the linear plasmid is 135,489 bp with a GC content of 67.9%. This resource announcement is the first to report the complete sequence of a single 140 kb C. sepedonicus linear plasmid. The genome was annotated using the NCBI Prokaryotic Genome Annotation Pipeline (PGAP) v.5.3. This resource announcement is useful for comparative and diagnostic analysis of C. sepedonicus.