ABSTRACT
Here, we report the complete genome sequences of Xylella fastidiosa subsp. fastidiosa strain AlmaReb2 and X. fastidiosa subsp. multiplex strain AlmaRebR6, causing blueberry bacterial leaf scorch disease in Georgia, USA. The X. fastidiosa subsp. fastidiosa AlmaRebR2 chromosome is 2,549,422 bp, and the X. fastidiosa subsp. multiplex AlmaReb6 chromosome is 2,530,348 bp.
ANNOUNCEMENT
Xylella fastidiosa is a Gram-negative, xylem-limited bacterium that causes blueberry bacterial leaf scorch (BBLS) disease (1). BBLS was initially reported to be caused by a strain of X. fastidiosa subsp. multiplex (2). However, greenhouse experiments have shown that isolates of X. fastidiosa subsp. fastidiosa can also cause BBLS (3), and blueberry isolates of X. fastidiosa subsp. fastidiosa and X. fastidiosa subsp. multiplex have been identified from the same field in southern Georgia through single-locus and multilocus sequence analyses (4). This study reports the complete genome sequences of an X. fastidiosa subsp. fastidiosa strain and an X. fastidiosa subsp. multiplex strain from BBLS plants in Georgia.
X. fastidiosa subsp. fastidiosa strain AlmaReb2 and X. fastidiosa subsp. multiplex strain AlmaReb6 were originally isolated in fall 2017 and 2018, respectively, from the same field block (31.5350 N, 82.4185 W) of Southern highbush (SHB) (Vaccinium corymbosum interspecific hybrids) cv. Rebel in Bacon County (GA, USA) (4). Both strains were originally isolated on periwinkle wilt (PW) medium (5) according to the methods described by Di Genova et al. (4). For DNA extraction, strains AlmaReb2 and AlmaReb6 were both grown for 10 days at 28°C on PW medium, and cells were harvested using the DNeasy blood and tissue kit (Qiagen, Germantown, MD) following the manufacturer’s instructions for Gram-negative bacteria. DNA was checked for quality and quantity using a Qubit 4 fluorometer (Invitrogen, Waltham, MA).
The two strains were sequenced using Oxford Nanopore long read-based technology. Libraries were prepared using the ligation sequencing kit (SQK-LSK109) and native barcoding expansion kit (EXP-NBD104) and sequenced on the ONT MinION device (Oxford Nanopore Technologies, UK). A barcoded library was prepared for each strain, and the barcoded libraries were sequenced in multiplex on the same flow cell. Fast base calling was performed using Guppy v2.3.5. Related metrics are listed in Table 1.
TABLE 1.
Features of the complete whole-genome sequences of Xylella fastidiosa subsp. fastidiosa strain AlmaReb2 and X. fastidiosa subsp. multiplex strain AlmaReb6
| Feature | Data for strain: |
|
|---|---|---|
| X. fastidiosa subsp. fastidiosa AlmaReb2 | X. fastidiosa subsp. multiplex AlmaReb6 | |
| Sequence reads | ||
| No. of reads (≥0 bp) | 117,818 | 127,101 |
| Total length (bp) | 1,215,285,643 | 1,284,168,600 |
| Longest read (bp) | 123,372 | 102,678 |
| N50 (bp) | 17,525 | 18,455 |
| L50 | 23,317 | 23,272 |
| Assembly | ||
| Genome size (bp) | 2,549,422 | 2,530,348 |
| Coverage (×) | 477 | 508 |
| G+C content (%) | 51.37 | 51.43 |
| No. of protein-coding genes | 2,731 | 2,714 |
| No. of rRNA genes | 6 | 6 |
| No. of tRNA genes | 49 | 48 |
The genome sequences were assembled de novo using Canu software v2.2 (6) with default parameters. The genomes of X. fastidiosa subsp. fastidiosa AlmaReb2 and X. fastidiosa subsp. multiplex AlmaReb6 each consist of a single circular chromosome contig (Table 1). While Nanopore long reads allowed for completion of a circular genome assembly, Nanopore reads alone can be error prone and are challenged by homopolymer regions. Accordingly, proovframe v0.9.7 software (7) was used to correct possible frameshift errors referencing the X. fastidiosa subsp. fastidiosa M23 (GenBank accession number CP001011) and X. fastidiosa subsp. multiplex M12 (CP000941) genomes for the AlmaReb2 and AlmaReb6 contigs, respectively. Both genome sequences were annotated using Prokka v1.14.5 (8) and rotated manually based on annotations, so that the first base corresponded to the start of dnaA.
The assembly completeness was evaluated using Benchmarking Universal Single-Copy Orthologs (BUSCO) v5.2.2 based on the generic domain bacteria_odb10 (9), with the following results for both the AlmaReb2 and AlmaReb6 genomes: C: 95.2% [S: 95.2%, D: 0.0%], F: 2.4%, M: 2.4%, n: 124. This study reports the whole-genome sequences of X. fastidiosa subsp. fastidiosa (AlmaReb2) and X. fastidiosa subsp. multiplex (AlmaReb6) strains causing BBLS in the same field block in Georgia. This genome pair (AlmaReb2 and AlmaReb6) can benefit research into X. fastidiosa horizontal gene transfer and genome structure variation analysis.
Data availability.
The whole-genome sequences have been deposited at GenBank under accession numbers CP117467 for X. fastidiosa subsp. multiplex strain AlmaReb6 and CP117468 for X. fastidiosa subsp. fastidiosa strain AlmaReb2. All sequence information is available under BioProject accession number PRJNA930171, BioSample accession numbers SAMN32989530 and SAMN32989531, and Sequence Read Archive (SRA) accession numbers SRR24459456 and SRR24459457. The versions described here are the first versions.
ACKNOWLEDGMENTS
We thank R. Holland and D. Di Genova for assistance with the original isolation and identification of the X. fastidiosa strains utilized in this study. We also thank Y. Andrade for technical assistance.
This research was partially supported by the USDA-NIFA (HATCH project number 1016575), Georgia Farm Bureau, Georgia Agricultural Commodity Commission for Blueberries, and a grant jointly funded by UGA-CAES, Office of Research, and Department of Plant Pathology.
The mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture.
Contributor Information
Jonathan E. Oliver, Email: jonathanoliver@uga.edu.
David A. Baltrus, University of Arizona
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Associated Data
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Data Availability Statement
The whole-genome sequences have been deposited at GenBank under accession numbers CP117467 for X. fastidiosa subsp. multiplex strain AlmaReb6 and CP117468 for X. fastidiosa subsp. fastidiosa strain AlmaReb2. All sequence information is available under BioProject accession number PRJNA930171, BioSample accession numbers SAMN32989530 and SAMN32989531, and Sequence Read Archive (SRA) accession numbers SRR24459456 and SRR24459457. The versions described here are the first versions.