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Announcement
27 September 2018

Near-Complete Genome Sequence of Ralstonia solanacearum T523, a Phylotype I Tomato Phytopathogen Isolated from the Philippines

ABSTRACT

Ralstonia solanacearum strain T523 is the major phytopathogen causing tomato bacterial wilt in the Philippines. Here, we report the complete chromosome and draft megaplasmid genomes with predicted gene inventories supporting rhizosphere processes, extensive plant virulence effectors, and the production of bioactive signaling metabolites, such as ralstonin, micacocidin, and homoserine lactone.

ANNOUNCEMENT

Ralstonia solanacearum causes bacterial wilt, one of the most important plant diseases worldwide (1). Bacterial wilt affects 200 species in 50 different families, including tobacco, banana, and solanaceous crops, such as potato and tomato (2). Widespread outbreaks in the Philippines have affected various economically important crops, with severe effects on tomato production (3). Here, we report the genome of R. solanacearum strain T523, isolated from wilting tomatoes in the Philippines (3).
Genomic DNA was extracted from R. solanacearum strain T523 cells grown in Kelman’s tetrazolium chloride medium (24 h, 28°C) using an MG genomic DNA purification kit (MGmed-Doctor Protein, Republic of Korea), according to the manufacturer’s protocol. The whole genome was sequenced at Macrogen, Inc. (Republic of Korea), from 10 µg of genomic DNA using a PacBio P6 DNA polymerase binding kit and a PacBio version 4.0 sequencing kit with eight single-molecule real-time (SMRT) cells (C4 chemistry) on the PacBio RS II platform. This generated 139,215 reads from a 20-kb SMRT library (mean subread length, 6,474 bp; N50, 9,102 bp). The 9.01-Mb reads were de novo assembled into contigs using the Hierarchical Genome Assembly Process (HGAP version 2.3) (4) to generate a final genome of 5,722,229 bp. One contig is a complete, closed, circular chromosome with a size of 3,652,934 bp, a G+C content of 67%, and a coverage of 98×. A second contig is the megaplasmid, with a size of 2,069,295 bp, a G+C content of 67%, and a coverage of 112×. Gene prediction was performed independently using the NCBI Prokaryotic Genome Annotation Pipeline (PGAP) (5) and the Joint Genome Institute-Integrated Microbial Genomes and Microbiomes (JGI-IMG/M) pipeline (6). Species identity was determined from the genome-wide average nucleotide identity (gANI) and alignment fraction (AF) using the Microbial Species Identifier (MiSI) calculator employed in IMG/M (7). Strain identity was ascertained by the digital DNA-DNA hybridization score using the Genome-to-Genome Distance Calculator (GGDC) version 2.1 (8). Bioactive secondary metabolites and virulence-associated genes were predicted using the antiSMASH version 4 (9) and Ralsto T3E (10) servers, respectively. All programs were run with default parameters unless otherwise noted.
The T523 genome has an ANI of >99% (AF, 0.9) and a dDDH (formula 2) of <70% with R. solanacearum GMI1000 and other phylotype I strains, thereby supporting the nomenclature. The genome revealed an extensive repertoire of biosynthetic gene clusters and type III virulence effectors supporting rhizosphere processes and plant symbiotic associations. The chromosome encodes a complete gene cluster for micacocidin biosynthesis, a siderophore utilizing a hybrid pathway of nonribosomal peptide synthetase, and a type I iterative polyketide synthase (11). The megaplasmid encodes genes involved in the production of the antibiotic lipopeptide ralstonin/ralsolamycin, with established phytotoxic (12, 13) and antifungal (12, 14) activities, and a putative bacteriocin. Biosynthetic gene clusters for exopolysaccharide, terpene, and homoserine lactone production were detected. Virulence-associated enzyme loci were identified, including pectinase, cellulase, and phospholipase C. Finally, the Ralsto T3E server predicted 37 and 45 rip 77 (Ralstonia-injected proteins) genes (10) located in the chromosome and megaplasmid, respectively.

Data availability.

The sequences were deposited in DDBJ/ENA/GenBank under accession numbers CP022702 and CP022703 for the chromosome and megaplasmid, respectively. The sequencing reads were deposited in the SRA under the accession number SRP159038.

ACKNOWLEDGMENTS

This work, including the efforts of A.D.M., was funded by the University of the Philippines Office of the Vice President for Academic Affairs through an Enhanced Creative Work and Research Grant (ECWRG 2015-01-25) and by a research fellowship to A.K.R. from the National Academy of Science and Technology Philippines. G.M.B.A. was supported by the Core Project for “University-Wide Capacity Building in Bioinformatics: Introduction to Principles and Techniques for Agriculture, Forestry, and Fisheries Research” of the University of the Philippines Los Baños (UPLB) Office of the Vice Chancellor for Research and Extension. I.A.P. was supported by the National Institute of Molecular Biology and Biotechnology, UPLB, Philippines. A.R.R.R. was funded by the Vanier Canada Graduate Scholarship, Alberta Innovates–Technology Futures and President’s Doctoral Prize of Distinction.

REFERENCES

1.
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2.
Vailleau F, Sartorel E, Jardinaud MF, Chardon F, Genin S, Huguet T, Gentzbittel L, Petitprez M. 2007. Characterization of the interaction between the bacterial wilt pathogen Ralstonia solanacearum and the model legume plant Medicago truncatula. Mol Plant Microbe Interact 20:159–167.
3.
Orlina-Villareal ME, Opina NL, Raymundo AK. 2008. A hypervirulent isolate identified from a race 1 Ralstonia solanacearum strain from tomato (Lycopersicon esculentum mill. cv. L-180). Phil Agric Scientist 91:94–98.
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Chin C-S, Alexander DH, Marks P, Klammer AA, Drake J, Heiner C, Clum A, Copeland A, Huddleston J, Eichler EE, Turner SW, Korlach J. 2013. Nonhybrid, finished microbial genome assemblies from long-read SMRT sequencing data. Nat Methods 10:563–569.
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Markowitz VM, Chen I-MA, Palaniappan K, Chu K, Szeto E, Pillay M, Ratner A, Huang J, Woyke T, Huntemann M, Anderson I, Billis K, Varghese N, Mavromatis K, Pati A, Ivanova NN, Kyrpides NC. 2014. IMG 4 version of the integrated microbial genomes comparative analysis system. Nucleic Acids Res 42:D560–D567.
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Varghese NJ, Mukherjee S, Ivanova N, Konstantinidis KT, Mavrommatis K, Kyrpides NC, Pati A. 2015. Microbial species delineation using whole genome sequences. Nucleic Acids Res 43:6761–6771.
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Meier-Kolthoff JP, Auch AF, Klenk H-P, Göker M. 2013. Genome sequence-based species delimitation with confidence intervals and improved distance functions. BMC Bioinformatics 14:60.
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Blin K, Wolf T, Chevrette MG, Lu X, Schwalen CJ, Kautsar SA, Suarez Duran HG, de los Santos ELC, Kim HU, Nave M, Dickschat JS, Mitchell DA, Shelest E, Breitling R, Takano E, Lee SY, Weber T, Medema MH. 2017. antiSMASH 4.0—improvements in chemistry prediction and gene cluster boundary identification. Nucleic Acids Res 45:W36–W41.
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Peeters N, Carrère S, Anisimova M, Plener L, Cazalé A-C, Genin S. 2013. Repertoire, unified nomenclature and evolution of the type III effector gene set in the Ralstonia solanacearum species complex. BMC Genomics 14:859.
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Kreutzer MF, Kage H, Gebhardt P, Wackler B, Saluz HP, Hoffmeister D, Nett M. 2011. Biosynthesis of a complex yersiniabactin-like natural product via the mic locus in phytopathogen Ralstonia solanacearum. Appl Environ Microbiol 77:6117–6124.
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Murai Y, Mori S, Konno H, Hikichi Y, Kai K. 2017. Ralstonins A and B, lipopeptides with chlamydospore-inducing and phytotoxic activities from the plant pathogen Ralstonia solanacearum. Org Lett 19:4175–4178.
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Baldeweg F, Kage H, Schieferdecker S, Allen C, Hoffmeister D, Nett M. 2017. Structure of ralsolamycin, the interkingdom morphogen from the crop plant pathogen Ralstonia solanacearum. Org Lett 19:4868–4871.
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Spraker JE, Sanchez LM, Lowe TM, Dorrestein PC, Keller NP. 2016. Ralstonia solanacearum lipopeptide induces chlamydospore development in fungi and facilitates bacterial entry into fungal tissues. ISME J 10:2317–2330.

Information & Contributors

Information

Published In

cover image Microbiology Resource Announcements
Microbiology Resource Announcements
Volume 7Number 1227 September 2018
eLocator: 10.1128/mra.01048-18
Editor: Irene L. G. Newton, Indiana University Bloomington

History

Received: 24 July 2018
Accepted: 30 August 2018
Published online: 27 September 2018

Contributors

Authors

Andrew D. Montecillo
Microbiology Division, Institute of Biological Sciences, University of the Philippines Los Baños, Los Baños, Laguna, Philippines
Departament de Produccio Vegetal I Ciencia Forestal, Universitat de Lleida, Lleida, Spain
Asuncion K. Raymundo
Microbiology Division, Institute of Biological Sciences, University of the Philippines Los Baños, Los Baños, Laguna, Philippines
National Academy of Science and Technology Philippines, Bicutan, Taguig, Philippines
Irene A. Papa
National Institute of Molecular Biology and Biotechnology, University of the Philippines Los Baños, Los Baños, Laguna, Philippines
Genevieve Mae B. Aquino
Philippine Genome Center, Program for Agriculture, Livestock, Fisheries and Forestry, Office of the Vice-Chancellor for Research and Extension University of the Philippines Los Baños, Los Baños, Laguna, Philippines
Arian J. Jacildo
Institute of Computer Science, University of the Philippines Los Baños, Los Baños, Laguna, Philippines
Paul Stothard
Department of Agricultural Food and Nutritional Science, University of Alberta, Edmonton, Alberta, Canada
Department of Chemistry, University of Alberta, Edmonton, Alberta, Canada

Editor

Irene L. G. Newton
Editor
Indiana University Bloomington

Notes

Address correspondence to Asuncion K. Raymundo, [email protected], or Albert Remus R. Rosana, [email protected].
A.D.M. and A.R.R.R. contributed equally to this work.

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