HomePhytopathology®Vol. 112, No. 9Genome Sequence Resource for Strains of Pseudomonas syringae Phylogroup 2b and P. viridiflava Phylogroup 7a Causing Bacterial Stem Blight of Alfalfa PreviousNext Resource Announcement OPENOpen Access licenseGenome Sequence Resource for Strains of Pseudomonas syringae Phylogroup 2b and P. viridiflava Phylogroup 7a Causing Bacterial Stem Blight of AlfalfaSavana M. Lipps, Deborah A. Samac, and Satoshi IshiiSavana M. Lipps†Corresponding author: S. M. Lipps; E-mail Address: [email protected]https://orcid.org/0000-0002-2028-4333Department of Plant Pathology, University of Minnesota, St. Paul, MN 55108Search for more papers by this author, Deborah A. SamacDepartment of Plant Pathology, University of Minnesota, St. Paul, MN 55108U.S. Department of Agriculture-Agriculture Research Service-Plant Science Research Unit, St. Paul, MN 55108Search for more papers by this author, and Satoshi IshiiDepartment of Soil, Water, and Climate, University of Minnesota, St. Paul, MN 55108Search for more papers by this authorAffiliationsAuthors and Affiliations Savana M. Lipps1 † Deborah A. Samac1 2 Satoshi Ishii3 1Department of Plant Pathology, University of Minnesota, St. Paul, MN 55108 2U.S. Department of Agriculture-Agriculture Research Service-Plant Science Research Unit, St. Paul, MN 55108 3Department of Soil, Water, and Climate, University of Minnesota, St. Paul, MN 55108 Published Online:4 Aug 2022https://doi.org/10.1094/PHYTO-12-21-0511-AAboutSectionsPDF ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InRedditEmailWechat Genome AnnouncementBacterial stem blight (BSB) of alfalfa (Medicago sativa L.) is a disease characterized by necrotic lesions on stems, chlorosis and necrosis of leaves, and wilt resulting in curled shoot tips resembling a shepherd’s crook (Gray and Hollingsworth 2015). BSB of alfalfa was first reported in 1904 and associated with frost damage to alfalfa stands in late spring (Sackett 1910). It is widespread in the central and western United States, including the Pacific Coast, and occasionally occurs in the eastern United States. It has been reported in Australia, England, the former Yugoslavia, Russia, and Iran. Damage from BSB in the United States has been sporadic and localized, but the disease appears to be emerging as a serious problem of alfalfa, particularly in the western United States (Getts et al. 2019). Yield losses of up to 50% from the first harvest can occur with severe disease outbreaks (Gray and Hollingsworth 2015). The disease is caused by the ice-nucleation active bacterium Pseudomonas syringae phylogroup 2b (PG2b) and is often associated with frost damage. Damage from BSB may be under-recognized and attributed to frost damage to the crop. Recently, strains of P. viridiflava phylogroup 7a (PG7a), previously found to cause crown rot in alfalfa (Lukezic et al. 1983), were shown to also cause symptoms of bacterial stem blight (Lipps et al. 2019). Since 2000, P. viridiflava has been responsible for 13 economically relevant disease outbreaks on annual plants (Lamichhane et al. 2015) and has been reported to cause disease on over 50 hosts since its discovery (Lipps and Samac 2021). Only one draft genome sequence with 130 contigs has been published for a P. syringe PG2b strain causing bacterial stem blight (Harrison et al. 2016) and no previous genome sequences for PG7a strains isolated from alfalfa have been published.The 12 P. syringae PG2b and eight P. viridiflava PG7a strains sequenced here were isolated from BSB-diseased alfalfa stems collected from western and midwestern states (Table 1). The diseased stems were sonicated in 0.1 mM K2PO4 buffer, and the sonicate was serially diluted and then cultured on King’s B agar medium. Identification of PG2b and PG7a strains was based on phenotypic characterization, fluorescence of colonies on King’s B agar medium under UV light, a multiplex PCR assay designed for discriminating between P. syringae and P. viridiflava (Lipps et al. 2019), and sequence of the citrate synthase (cts) gene (Berge et al. 2014) (Fig. 1). Each strain was tested for pathogenicity by inoculation of alfalfa stems. To inoculate, stems were wounded with a tuberculin needle at the second or third internode and a bacterial suspension (OD600 = 0.1) was applied with a sponge. Disease severity on leaves and stems was measured on the scale of 1 (least severe) to 5 (most severe) and five or six replicates were performed for each strain. Strains were selected for whole genome sequencing to represent a range of pathogenicity phenotypes including low (<2.5), moderate (2.5 to 3.5), and high (>3.5) disease severity. Strains were also selected to represent the range of locations of original diseased plants. For DNA sequencing, bacteria were grown in Luria-Bertani (LB) liquid medium for 48 h, pelleted by centrifugation, washed in sterile distilled water, and submitted to the Microbial Genome Sequencing Center for DNA extraction, library preparation, and sequencing. DNA extractions were performed with DNeasy Blood & Tissue Kit (Qiagen), and the Illumina DNA Prep Kit (Illumina) and Oxford Nanopore Ligation Sequencing Kit were used for library preparation. Samples were run on NextSeq 550 (Illumina), which provided 2 × 150 bp paired end reads, and MinION (Oxford Nanopore) sequencers to obtain short and long read DNA sequences, respectively. Hybrid assembly of NextSeq 550 and MinION reads was performed with Unicycler (v0.4.8). All genomes were assembled with well over 100× coverage. Genome sequences were deposited in the NCBI assembly database (accession numbers in Table 1) and annotated via the NCBI Prokaryotic Genome Annotation Pipeline.Table 1. Genome data and accession numbers of strains of Pseudomonas syringae phylogroup 2b (PG2b) and P. viridiflava phylogroup 7a (PG7a)aStrainPhylogroupLocationCollection yearAverage disease severity (leaf; stem)Assembly accession numberCoverageTotal genome length (bp)N50GC (%)Number of CDSNumber of tRNAU643PG2bCornish, UT20174.2; 2.4GCA_018388525.1140×6,191,7936,191,79358.95,30964T230PG2bTulelake, CA20163.5; 3.5GCA_018383435.1165×6,117,5993,790,62158.95,21463SV921PG2bScott Valley, CA20172.7; 2.0GCA_018383455.1402×6,115,6843,102,43058.95,21663KF529PG2bKlamath Falls, OR20194.5; 4.3GCA_018388485.1355×6,077,2916,077,29159.15,18864SHV323PG2bShasta Valley, CA20172.8; 2.4GCA_018383485.1340×6,035,6145,933,52559.15,14564SHV1866PG2bShasta Valley, CA20173.6; 3.0GCA_018383395.1478×5,988,6915,921,05559.15,07964StP26PG2bSt. Paul, MN20193.2; 3.0GCA_018604405.1530×5,969,3085,969,30859.15,06264SV1599PG2bScott Valley, CA20174.2; 3.3GCA_018383755.1357×5,961,5424,095,87759.15,02064U888PG2bCornish, UT20192.0; 2.0GCA_018383465.1310×5,959,5233,177,46159.05,02564Susan762PG2bSusanville, CA20172.6; 2.4GCA_018388505.1376×5,887,9925,887,99259.14,98564T1434PG2bTulelake, CA20173.0; 2.6GCA_018385455.1389×5,886,5523,819,63259.15,01664Susan2139PG2bSusanville, CA20174.2; 2.5GCA_018394375.1382×5,868,3735,827,96459.05,11063StP3PG7aSt. Paul, MN20193.8; 2.2GCA_019104085.1244×6,376,7016,376,70159.05,67865KF485PG7aKlamath Falls, OR20192.6; 1.8GCA_019083885.1274×6,221,4256,219,08659.15,44866SV1779PG7aScott Valley, CA20173.2; 2.0GCA_019104065.1301×6,106,5896,106,58959.25,34065U658PG7aCornish, UT20173.0; 1.8GCA_019083835.1350×6,098,4354,289,83059.35,36967T1426PG7aTulelake, CA20171.8; 1.6GCA_019104045.1279×6,075,9606,075,96059.35,30965U625PG7aCornish, UT20173.2; 2.0GCA_018388545.1310×5,997,2205,997,22059.25,21364StP4PG7aSt. Paul, MN20192.8; 1.8GCA_019104005.1323×5,994,7795,994,77959.35,24565T157PG7aTulelake, CA20194.0; 2.0GCA_019104025.1301×5,965,7925,965,79259.35,23165aAll L50 values are 1. CDS represents protein coding sequences.Table 1. Genome data and accession numbers of strains of Pseudomonas syringae phylogroup 2b (PG2b) and P. viridiflava phylogroup 7a (PG7a)aView as image HTML Fig. 1. Bayesian tree of the citrate synthase (cts) gene of Pseudomonas syringae phylogroup 2b (PG2b) and P. viridiflava phylogroup 7a (PG7a) strains causing bacterial stem blight (BSB). A Bayesian tree using PG2b and PG7a strains causing BSB and a reference strain was constructed with reference sequences from P. syringae phylogroups defined by Berge et al. (2014).Download as PowerPointGenomes were comprised of a one to nine contigs, with eight of the assemblies based on one contig. The sizes of the PG2b and PG7a genomes ranged from 5,827,964 to 6,376,701 bp (Table 1). The number of protein coding sequences in each genome ranged from 4,985 to 5,678. The G+C content ranged from 58.9 to 59.3%. The number of tRNAs ranged from 63 to 65, and each genome contained 16 rRNAs and four non-coding RNAs. A plasmid sequence was detected within one strain, PG2b Susan2139, by the presence of the conserved major replication gene of P. syringae plasmids, repA (Gutiérrez-Barranquero et al. 2017). There were no other circularized replicons detected within the other strains. The availability of these P. syringae PG2b and P. viridiflava PG7a genome sequences will enable further research on key virulence genes, an improved understanding of the contribution of each bacterium to the epidemiology of BSB and the disease process, and development of resistant alfalfa cultivars.Data AvailabilityAll sequences in this study have been deposited in GenBank (Table 1 provides accession numbers).AcknowledgmentsThis paper is a joint contribution from the Plant Science Research Unit, U.S. Department of Agriculture (USDA)-Agriculture Research Service, and the Minnesota Agricultural Experiment Station. Mention of any trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the USDA. USDA is an equal opportunity provider and employer.The author(s) declare no conflict of interest.Literature CitedBerge, O., Monteil, C. L., Bartoli, C., Chandeysson, C., Guilbaud, C., Sands, D. C., and Morris, C. E. 2014. A user’s guide to a data base of the diversity of Pseudomonas syringae and its application to classifying strains in this phylogenetic complex. PLoS One 9:e105547. https://doi.org/10.1371/journal.pone.0105547 Crossref, Medline, ISI, Google ScholarGetts, T., Wilson, R., Galdi, G., Loveland, C., Samac, D. A., and Creech, E. 2019. Roundup Ready Alfalfa Injury. In: Proceedings of the 2019 Western Alfalfa and Forage Symposium. https://alfalfa.ucdavis.edu/+symposium/proceedings/2019/Articles/GlyphosateInjury_Getts_Article.pdf Google ScholarGray, F. A., and Hollingsworth, C. R. 2015. 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Google ScholarFunding: Support was provided by the U.S. Department of Agriculture-National Institute of Food and Agriculture (NIFA)-Alfalfa and Forage Research Program award number 2017-70005-27088 and U.S. Department of Agriculture-Agricultural Research Service award number 5026-12210-004-00D.The author(s) declare no conflict of interest.DetailsFiguresLiterature CitedRelated Vol. 112, No. 9 September 2022SubscribeISSN:0031-949Xe-ISSN:1943-7684 DownloadCaptionA grape cluster (‘Merlot’) with natural infection of ripe rot, caused by Colletotrichum spp., in a commercial vineyard in Maryland. The cluster was covered with a pest exclusion paper bag from bloom to bunch closure, as part of field trials to expose phenological cluster stages to naturally occurring Colletotrichum inoculum and environmental conditions, with a goal of identifying phenological stages susceptible to ripe rot (Cosseboom and Hu). Photo credit: Scott Cosseboom Metrics Article History Issue Date: 25 Aug 2022Published: 4 Aug 2022Accepted: 13 Apr 2022 Pages: 2028-2031 Information© 2022 The American Phytopathological SocietyFundingU.S. Department of Agriculture-National Institute of Food and AgricultureGrant/Award Number: 2017-70005-27088U.S. Department of Agriculture-Agricultural Research ServiceGrant/Award Number: 5026-12210-004-00DKeywordsbacterial pathogensgenomicsmicrobe-genome sequencingThe author(s) declare no conflict of interest.PDF download