# Draft genome sequences of 284 Xanthomonas citri pv. citri strains causing Asiatic citrus canker

Damien Richard, Adrien Rieux, Pierre Lefeuvre, Abdou Azali Hamza, Kanta

Kumar Lobin, Marc Naiken, Randy Stravens, Claudine Boyer, Karine Boyer,

Stéphanie Javegny, et al.

► To cite this version:

Damien Richard, Adrien Rieux, Pierre Lefeuvre, Abdou Azali Hamza, Kanta Kumar Lobin, et al. Draft genome sequences of 284 Xanthomonas citri pv. citri strains causing Asiatic citrus canker. Microbiology Resource Announcements, 2021, 10 (1), pp.e01024-20. 10.1128/MRA.01024-20. hal-05177330

HAL Id: hal-05177330

https://hal.science/hal-05177330v1

Submitted on 22 Jul 2025

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_1.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
<figure><img src="image_2.png" /></figure>

# Draft Genome Sequences of 284 *Xanthomonas citri* pv. citri Strains Causing Asiatic Citrus Canker

Damien Richard,ª,ᵇ,ᶜ Adrien Rieux,ª Pierre Lefeuvre,ª Azali Hamza,ᵈ Kanta Kumar Lobin,ᵉ Mark Naiken,>f Randy Stravens,>f
Claudine Boyer,ª Karine Boyer,ª Stéphanie Javegny,ª Olivier Pruvostª

**ABSTRACT** High-quality Illumina assemblies were produced from 284 *Xanthomonas citri* pv. citri pathotype A strains mostly originating from the Southwest Indian Ocean region, a subset of which was also sequenced using MinION technology. Some strains hosted chromosomally encoded transcription activator-like effector (TALE) genes, an atypical feature for this bacterium.

Most citrus-producing areas currently face Asiatic citrus canker due to the dissemi-
nation of its causative agent, *Xanthomonas citri* pv. citri pathotype A (*XccA*), from
its native area (Asia) (1). This pathotype infects most cultivated citrus species. Based on
minisatellite data, a single genetic lineage was found to be mostly responsible for the
worldwide expansion of *XccA* (2). To better reconstruct the expansion of *XccA* and
understand the genetic basis of its evolution, we sequenced the genome of 284 strains.
Our sampling had a special focus on the Southwest Indian Ocean (SWIO) area (210
strains out of 284). The SWIO region comprises several geographically distant and
administratively distinct islands, representing a good setting to apprehend *XccA*
genetic structure and accessory gene content. In this region, Asiatic citrus canker man-
agement is attempted through integrated strategies, including using copper-based
pesticides. Copper resistance involving a conjugative plasmid hosting the *copLAB sys-
tem* recently emerged in Réunion, an outermost EU territory in the SWIO region (3).
The sequenced strains were isolated from diseased citrus tissue using standard proce-
dures (4) or were obtained from international culture collections, indicated by CFBP
(Collection Française de Bactéries Associées aux Plantes, Beaucouzé, France) or NCPPB
(National Collection of Plant Pathogenic Bacteria, York, UK) numbers. Copper-resistant
strains (phenotypes were assessed as in reference 5) originating from all three regions of
the world where this phenotype was reported were included. A further 60 *XccA* strains
were selected from 16 countries to capture the global diversity of *XccA*. Genomic DNA
(gDNA) was extracted from bacterial colonies grown on yeast extract-peptone-glucose
agar (YPGA) medium using the Promega Wizard kit. Illumina sequencing was performed
by GATC Biotech on a HiSeq 4000 instrument (2 × 150-bp format) using the genomic
Nextera XT protocol. Long-read Oxford Nanopore MinION sequencing was additionally
performed on 13 selected strains from gDNA purified using the SQK-RBK004 library prepa-
ration kit on a single R9.4.1 flow cell using the manufacturer's instructions. Default parame-
ters were used for all software unless otherwise specified. The Illumina reads were trimmed
based on the quality score using Trimmomatic v. 0.36 with the options LEADING:15,
TRAILING:15, SLIDINGWINDOW:4:20, and MINLEN:100 (6). *De novo* genome assemblies

**Citation** Richard D, Rieux A, Lefeuvre P, Hamza A, Lobin KK, Naiken M, Stravens R, Boyer C, Boyer K, Javegny S, Pruvost O. 2021. Draft genome sequences of 284 *Xanthomonas citri* pv. citri strains causing Asiatic citrus canker. Microbiol Resour Announc 10:e01024-20. https://doi.org/10.1128/MRA.01024-20.

**Editor** David A. Baltrus, University of Arizona

**Copyright** © 2021 Richard et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.

Address correspondence to Olivier Pruvost,
olivier.pruvost@cirad.fr.

**Received** 29 September 2020

**Accepted** 8 December 2020

**Published** 7 January 2021

<footnote>ªCentre de Coopération Internationale en Recherche Agronomique pour le Développement (CIRAD), UMR PVBMT, St. Pierre, Réunion, France</footnote>

<footnote>ᵇAgence Nationale de Sécurité Sanitaire de l'Alimentation, de l'Environnement et du Travail (ANSES), Plant Health Laboratory, St. Pierre, Réunion, France</footnote>

<footnote>ᶜUniversité de la Réunion, UMR PVBMT, St. Denis, Réunion, France</footnote>

<footnote>ᵈInstitut National de Recherche pour l'Agriculture, la Pêche et l'Environnement (INRAPE), Moroni, Union of the Comoros</footnote>

<footnote>ᵉFood and Agricultural Research & Extension Institute (FAREI), Le Réduit, Mauritius</footnote>

<footnote>fNational Biosecurity Agency (NBA), Mahé, Seychelles</footnote>
<figure><img src="image_3.png" /></figure>

were generated using SPAdes v. 3.6.2 (7) for strains solely sequenced using short-read technology and Canu v. 1.8 (8), specifying a genome size of 5.3 Mbp, followed by Pilon v. 1.22 (9) for strains additionally sequenced using long-read technology. The genome size, number of contigs, GC content, and other metrics are detailed in Dataverse (10). Automatic annotation was performed using the NCBI Prokaryotic Genome Annotation Pipeline (11).

All the strains sequenced herein contained contigs similar to plasmids previously described in XccA. Accordingly, plasmids were identified in all strains submitted to long-read sequencing, most of them (ca. 60%) suggested as being circular by Canu. BLASTN searches identified the previously described copper resistance plasmid only in strains from Réunion but not in those collected from other SWIO islands.

In XccA, transcription activator-like effector (TALE) genes, including *pthA4*, which is necessary for disease development, and other so-called minor TALE genes are primarily plasmid borne (12). The sole exception was reported recently for pathotype A\* strains (with a host range restricted to Mexican lime and alemow) from Texas (13). Herein, 7 out of 13 long-read-based *de novo* assemblies contained minor chromosomal TALE genes, identified using Prodigal (14) and BLASTN. Whether these reads represent stable chromosomal integrations is presently being investigated further.

**Data availability.** The raw data are available under SRA accession numbers SRR11234551 to SRR11234624 and SRR11234626 to SRR11234855 and the corresponding BioProject information under accession number PRJNA599566. Details for each strain and the associated data accession numbers are available on the Dataverse platform (https://dataverse.cirad.fr/dataset.xhtml?persistentId=doi:10.18167/DVN1/TOVWWL) (10).

## ACKNOWLEDGMENTS

We thank the Plant Protection Platform (3P, IBISA). INRAPE (Union of the Comoros), FAREI (Mauritius), and NBA (Seychelles) provided biological material from their respective countries.

The European Regional Development Fund (ERDF; contract number GURDT I2016-1731-0006632), the Réunion Region, the French Agropolis Foundation (LabEx Agro-Montpellier; E-SPACE project number 1504-004), ANSES, and CIRAD provided financial support.

## REFERENCES

1. Graham JH, Gottwald TR, Cubero J, Achor DS. 2004. *Xanthomonas axonopodis* pv. *citri*: factors affecting successful eradication of citrus canker. Mol Plant Pathol 5:1–15. https://doi.org/10.1046/j.1364-3703.2004.00197.x.

2. Pruvost O, Magne M, Boyer K, Leduc A, Tourterel C, Drevet C, Ravigne V, Gagnevin L, Guerin F, Chiroleu F, Koebnik R, Verdier V, Verniere C. 2014. A MLVA genotyping scheme for global surveillance of the citrus pathogen *Xanthomonas citri* pv. *citri* suggests a worldwide geographical expansion of a single genetic lineage. PLoS One 9:e98129. https://doi.org/10.1371/journal.pone.0098129.

3. Richard D, Ravigne V, Rieux A, Facon B, Boyer C, Boyer K, Grygiel P, Javegny S, Terville M, Canteros BI, Robene I, Verniere C, Chabirand A, Pruvost O, Lefeuvre P. 2017. Adaptation of genetically monomorphic bacteria: evolution of copper resistance through multiple horizontal gene transfers of complex and versatile mobile genetic elements. Mol Ecol 26:2131–2149. https://doi.org/10.1111/mec.14007.

4. Klement Z, Rudolph K, Sands DC. 1990. Methods in phytobacteriology. Akadémiai Kiadó, Budapest, Hungary.

5. Richard D, Tribot N, Boyer C, Terville M, Boyer K, Javegny S, Roux-Cuvelier M, Pruvost O, Moreau A, Chabirand A, Vernière C. 2017. First report of copper-resistant *Xanthomonas citri* pv. *citri* pathotype A causing Asiatic citrus canker in Réunion, France. Plant Dis 101:503. https://doi.org/10.1094/PDIS-09-16-1387-PDN.

6. Bolger AM, Lohse M, Usadel B. 2014. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics 30:2114–2120. https://doi.org/10.1093/bioinformatics/btu170.

7. Bankevich A, Nurk S, Antipov D, Gurevich AA, Dvorkin M, Kulikov AS, Lesin VM, Nikolenko SI, Pham S, Prjibelski AD, Pyshkin AV, Sirotkin AV, Vyahhi N, Tesler G, Alekseyev MA, Pevzner PA. 2012. SPAdes: a new genome assembly algorithm and its applications to single-cell sequencing. J Comput Biol 19:455–477. https://doi.org/10.1089/cmb.2012.0021.

8. Koren S, Walenz BP, Berlin K, Miller JR, Bergman NH, Phillippy AM. 2017. Canu: scalable and accurate long-read assembly via adaptive k-mer weighting and repeat separation. Genome Res 27:722–736. https://doi.org/10.1101/gr.215087.116.

9. Walker BJ, Abeel T, Shea T, Priest M, Abouelliel A, Sakthikumar S, Cuomo CA, Zeng Q, Wortman J, Young SK, Earl AM. 2014. Pilon: an integrated tool for comprehensive microbial variant detection and genome assembly improvement. PLoS One 9:e112963. https://doi.org/10.1371/journal.pone.0112963.

10. Richard D. 2020. Description of the draft genome sequences of 284 *Xanthomonas citri* pv. *citri* strains causing Asiatic citrus canker. CIRAD Dataverse. https://doi.org/10.18167/DVN1/TOVWWL.

11. Tatusova T, DiCuccio M, Badretdin A, Chetvernin V, Nawrocki EP, Zaslavsky L, Lomsadze A, Pruitt KD, Borodovsky M, Ostell J. 2016. NCBI Prokaryotic Genome Annotation Pipeline. Nucleic Acids Res 44:6614–6624. https://doi.org/10.1093/nar/gkw569.

12. Brunings AM, Gabriel DW. 2003. *Xanthomonas citri*: breaking the surface. Mol Plant Pathol 4:141–157. https://doi.org/10.1046/j.1364-3703.2003.00163.x.

13. Munoz Bodnar A, Santillana G, Mavrodieva V, Liu Z, Nakhla M, Gabriel DW. 2017. Complete genome sequences of three *Xanthomonas citri* strains from Texas. Genome Announc 5:e00609-17. https://doi.org/10.1128/genomeA.00609-17.

14. Hyatt D, Chen G-L, LoCascio PF, Land ML, Larimer FW, Hauser LJ. 2010. Prodigal: prokaryotic gene recognition and translation initiation site identification. BMC Bioinformatics 11:119. https://doi.org/10.1186/1471-2105-11-119.