Abstract
Three bacteriophages, f20-Xaj, f29-Xaj, and f30-Xaj, with lytic activity against Xanthomonas arboricola pv. juglandis were isolated from walnut trees (VIII Bío Bío Region, Chile). These lytic bacteriophages have double-stranded DNA (dsDNA) genomes of 43,851 bp, 41,865 bp, and 44,262 bp, respectively. These are the first described bacteriophages with lytic activity against X. arboricola pv. juglandis that can be utilized as biocontrol agents.
GENOME ANNOUNCEMENT
Current control of the Gram-negative bacteria Xanthomonas arboricola pv. juglandis, the causative agent of walnut blight disease, is mediated by the utilization of copper-based agrochemicals (1). Walnut blight is the main disease affecting walnut production (2), and losses during wet spring can exceed 80% if not controlled (3, 4).
Lytic bacteriophages with potential biocontrol activity against X. arboricola pv. juglandis have not been described. Here, we sequenced three lytic bacteriophages of X. arboricola pv. juglandis. DNA was purified using routine protocols (5). Sequencing was performed using the next-generation sequencer (NGS) Illumina MiSeq at Universidad Mayor, Center for Genomics and Bioinformatics (Huechuraba, Chile). The sequences were assembled using CLC Genomics Workbench 8.5.1 (Qiagen), resulting in a single contig with unknown ends. The genomic analysis was performed using the PHAST server (6). The assembled sequences were annotated by the National Center for Biotechnology Information (NCBI) Prokaryotic Genome Annotation Pipeline (PGAP; http://www.ncbi.nlm.nih.gov/genome/annotation_prok/). The summary report for the three sequenced bacteriophage genomes is shown in Table 1. Based on the predictions, these phage genomes contain genes for replication, structure, and lysis. Open reading frames (ORFs) were found for putative homing endonuclease, helicase, DNA ligase, and DNA polymerase. Bacteriophages f20-Xaj and f30-Xaj possess their own RNA polymerase, suggesting that they are T7 phage related. The ORFs for terminase, head morphogenesis protein, collar protein, putative tail tubular proteins, and tail fiber protein were found. Lysogenization genes, such as site-specific integrases and repressors, were not found. The ORFs for holin, lysozyme, and endolysin were also found. Alignment and molecular phylogenetic analysis by the maximum likelihood method (7, 8) showed that phages f20-Xaj and f30-Xaj are closely related to each other, having the same clade as f29-Xaj, Xanthomonas campestris pv. citri bacteriophages CP2 (GenBank accession no. NC_020205) and Cf1c (GenBank accession no. NC_001396.1) and Xanthomonas phage Prado (GenBank accession no. NC_022987).
TABLE 1 .
Summary report of the de novo assembly of the three Chilean Xanthomonas arboricola pv. juglandis bacteriophages from this study
| Strain | BioSample no. | GenBank accession no. | G+C content (%) | Genome size (bp) | No. of CDSsa | Avg coverage (×) |
|---|---|---|---|---|---|---|
| f20-Xaj | SAMN04420465 | KU595432 | 59.8 | 43,851 | 53 | 1,418 |
| f29-Xaj | SAMN04420466 | KU595434 | 49.8 | 41,865 | 61 | 370 |
| f30-Xaj | SAMN04420467 | KU595433 | 59.9 | 44,262 | 51 | 395 |
CDSs, coding sequences.
Nucleotide sequence accession numbers.
The genome sequences were deposited in DDBJ/EMBL/GenBank under BioProject PRJNA309087, and the accession numbers and details are listed in Table 1.
ACKNOWLEDGMENTS
This work was supported by the CONICYT/FONDECY Regular competition 1140330 and FIC-BIP 30170275-0, CoPEC-UC 2014.J0.71.
We thank Carolina Sanchez (Center for Genomics and Bioinformatics, Universidad Mayor) for her assistance at the sequencing facility and M. Ignacia Diaz (Microbial Pathogenesis and Vaccinology Laboratory) for her logistic support.
Footnotes
Citation Retamales J, Vasquez I, Santos L, Segovia C, Ayala M, Alvarado R, Nuñez P, Santander J. 2016. Complete genome sequences of lytic bacteriophages of Xanthomonas arboricola pv. juglandis. Genome Announc 4(3):e00336-16. doi:10.1128/genomeA.00336-16.
REFERENCES
- 1.Frampton RA, Pitman AR, Fineran PC. 2012. Advances in bacteriophage-mediated control of plant pathogens. Int J Microbiol 2012:326452. doi: 10.1155/2012/326452. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 2.Loreti S, Gallelli A, Belisario A, Wajnberg E, Corazza L. 2001. Investigation of genomic variability of Xanthomonas arboricola pv. juglandis by AFLP analysis. Eur J Plant Pathol 107:583–591. doi: 10.1023/A:1017951406237. [DOI] [Google Scholar]
- 3.Bandi A, Hevesi M, Szani Z, Toth M. 2015. Assessment of bacterial blight tolerance of Persian walnut based on immature nut test. J Biol 7:253–257. [Google Scholar]
- 4.Miller PW. 1934. Walnut blight and its control in the Pacific Northwest USA, circular no. 331. Department of Agriculture, Washington, DC. [Google Scholar]
- 5.Kaiser K, Murray N, Whittaker P. 1995. Construction of representative genomic DNA libraries using phages lambda replacement vectors, p. 37–83. In Glover D, Hames B (ed), DNA cloning, vol 1: a practical approach. Oxford University Press, New York, NY. [Google Scholar]
- 6.Zhou Y, Liang Y, Lynch KH, Dennis JJ, Wishart DS. 2011. PHAST: a fast phage search tool. Nucleic Acids Res 39:W347–W352. doi: 10.1093/nar/gkr485. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 7.Tamura K, Dudley J, Nei M, Kumar S. 2007. MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Mol Biol Evol 24:1596–1599. doi: 10.1093/molbev/msm092. [DOI] [PubMed] [Google Scholar]
- 8.Saitou N, Nei M. 1987. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4:406–425. [DOI] [PubMed] [Google Scholar]