RESOURCE ANNOUNCEMENTOpen Access icon OPENOpen Access license

Genome Sequence Resource of ‘Candidatus Liberibacter asiaticus’ from Diaphorina citri Kuwayama (Hemiptera: Liviidae) from La Réunion

    Authors and Affiliations
    • Jinming Lu1
    • Hélène Delatte2
    • Bernard Reynaud2
    • George A. C. Beattie3
    • Paul Holford3
    • Yijing Cen4
    • Yanjing Wang5
    1. 1College of Forestry and Biotechnology, Zhejiang Agriculture & Forestry University, Lin’an, Zhejiang 311300, China
    2. 2CIRAD, UMR PVBMT CIRAD-Universitéde La Réunion, Pôle de Protection des Plantes, France
    3. 3School of Science, Western Sydney University, Penrith, NSW 2751, Australia
    4. 4Key Laboratory of Bio-Pesticide Innovation and Application/Citrus Huanglongbing Research Laboratory, College of Plant Protection, South China Agricultural University, Guangzhou 510642, China
    5. 5College of Agriculture and Food Sciences, Zhejiang A&F University, Lin’an, Zhejiang 311300, China


    Candidatus Liberibacter asiaticus’ is an insect-transmitted, phloem-restricted α-proteobacterium associated with huanglongbing. Here, we provide the whole genome sequence of ‘Ca. L. asiaticus’ strain ReuSP1 from its insect vector Diaphorina citri (Hemiptera: Liviidae) collected in La Réunion. The genome is composed of 1,230,064 bp and has a 36.5% G+C content. This study reports the first ‘Ca. L. asiaticus’ genome sequence from La Réunion, which will add to ‘Ca. L. asiaticus’ genome resources and help elucidate our understanding of the introduction pathway into La Réunion.

    Huanglongbing (HLB) or citrus greening is the most severe citrus disease. It is well established that HLB is associated with ‘Candidatus Liberibacter asiaticus’, ‘Ca. L. americanus’, and ‘Ca. L. africanus’ (Bové 2006; Lin 1956). ‘Ca. L. asiaticus’ occurs in many citrus-growing regions around the world, whereas ‘Ca. L. africanus’ is associated with commercial citrus orchards in Africa (Ajene et al. 2020; Bové 2006; Pietersen et al. 2010; Saponari et al. 2010), Saudi Arabia, Yemen, the Comoros Islands, Madagascar, La Réunion, and Mauritius in the Indian Ocean (Bové 2006; EPPO 2014) and the island of St. Helena in the Atlantic Ocean (EPPO 2014; van den Berg and Greenland 2000).

    HLB was recorded in La Réunion (France: Indian Ocean) in 1967 (Bové and Cassin 1968; Moreria 1967). Both ‘Ca. L. asiaticus’ and ‘Ca. L. africanus’, as well as their associated insect vectors Diaphorina citri Kuwayama (Hemiptera: Sternorrhyncha: Liviidae) and Trioza erytreae Del Guercio (Sternorrhyncha: Triozidae), are present in La Réunion (Aubert 1977; Bové and Cassin 1968; Catling 1970, 1972; Garnier et al. 1996). Sequencing the genome of the bacterium may advance our understanding of the genetic diversity of ‘Ca. L. asiaticus’ and the nature of host–pathogen interactions, thereby assisting in the development of more effective disease control measures. In this study, we report the first whole-genome sequence for a strain of ‘Ca. L. asiaticus’ from La Réunion.

    D. citri was collected from Citrus (Rutaceae: Aurantioideae: Aurantieae) in Pierrefond, Saint Pierre, La Réunion (21°17′S, 55°26′E) in October 2017. DNA was extracted from a single adult using the Qiagen DNeasy Blood and Tissue Kit (Qiagen, Valencia, CA, U.S.A.). Real-time PCR targeting the 16S ribosomal RNA gene was used to confirm the presence of ‘Ca. L. asiaticus’ (Li et al. 2006). A double-index Illumina sequencing library was prepared from DNA extracts with an average insert size of 300 bp using an Illumina TruSeq, version 2 paired-end library preparation kit (300-bp insert size) (Illumina, San Diego, CA, U.S.A.). Genome sequencing was performed using a HiSeq PE150 Illumina platform (Illumina Inc.).

    In total, 115,572,582 raw reads with an average length of 150 bp were generated. Adapters were trimmed and bases below a quality threshold of 30 were discarded using Trimmomatic v0.36 (Bolger et al. 2014). From all filtered reads, a preliminary metaassembly was created using MEGAHIT v1.2.9 (Li et al. 2015). The resulting contigs were queried against the genome sequences of ‘Ca. L. asiaticus’ strain A4 (GenBank: CP010804) and three types of prophage sequences, represented by SC1 (GenBank: HQ377372), SC2 (GenBank: HQ377373), and P-JXGC-3 (GenBank: KY661963) using BLAST+ 2.9.0 (Camacho et al. 2009). Filtered reads were aligned to the contigs identified as ‘Ca. L. asiaticus’ using the Burrows-Wheeler Aligner (BWA v0.7.15) (Li and Durbin 2009). Only reads matching those contigs were used to create a refined assembly with SPAdes v3.14.1 (Bankevich et al. 2012). Gap closure was performed using methods described by Silva et al. (2021). In total, 21 contigs over 1,000 bp were obtained that ranged from 1,434 to 341,352 bp. MUMmer v4.0 (Kurtz et al. 2004) was used to algin the assembled contigs to the genome of ‘Ca. L. asiaticus’ strain A4. For prophage identification, filtered reads were mapped to the three types of prophage sequences using the Burrows-Wheeler Aligner (BWA v0.7.15) (Li and Durbin 2009). The study strain, ReuSP1, was confirmed as having type 1-like prophage (Table 1). The draft genome sequence of ReuSP1, including one prophage, is 1,230,064 bp in length, with a GC content of 36.5%, an average nucleotide coverage of 8.3×, N50 of 111,047 bp, and L50 of 3. The genome was annotated using the RAST server ( (Aziz et al. 2008) and contains 1,184 open reading frames and 53 RNA genes.

    Table 1. Mapping information of prophage in ‘Candidatus Liberibacter asiaticus’ strain ReuSP1

    In this study, we report the first draft genome sequence of ‘Ca. L. asiaticus’ in La Réunion and France, thereby providing a valuable resource for understanding genetic diversity and for further comparative genomics of the ‘Ca. L. asiaticus’.

    Accession number. This whole-genome shotgun project has been deposited at DNA Data Bank of Japan/European Nucleotide Archive/GenBank under the accession number CP061535.


    We thank M. Vinot, F. Hervy, and S. Nibouche for their help in providing the DNA sample from La Réunion.

    The author(s) declare no conflict of interest.

    Literature Cited

    • Ajene, I. J., Khamis, F. M., van Asch, B., Pietersen, G., Seid, N., Rwomushana, I., Ombura, F. L. O., Momanyi, G., Finyange, P., Rasowo, B. A. M., Tanga, C. M., Mohammed, S., and Ekesi, S. 2020. Distribution of Candidatus Liberibacter species in Eastern Africa, and the first report of Candidatus Liberibacter asiaticus in Kenya. Sci. Rep. 10:3919. Crossref, ISIGoogle Scholar
    • Aubert, B. 1977. Recherches sur la maladie du “greening” des citrus à l’île de la Réunion. Rapport de DEA Université de Bordeaux II. Google Scholar
    • Aziz, R. K., Bartels, D., Best, A. A., De Jongh, M., Disz, T., Edwards, R. A., Formsma, K., Gerdes, S., Glass, E. M., Kubal, M., Meyer, F., Olsen, G. J., Olson, R., Osterman, A. L., Overbeek, R. A., McNeil, L. K., Paarmann, D., Paczian, T., Parrello, B., Pusch, G. D., Reich, C., Stevens, R., Vassieva, O., Vonstein, V., Wilke, A., and Zagnitko, O. 2008. The RAST server: Rapid annotations using subsystems technology. BMC Genomics 9:75. Crossref, ISIGoogle Scholar
    • Bankevich, A., Nurk, S., Antipov, D., Gurevich, A. A., Dvorkin, M., Kulikov, A. S., Lesin, V. M., Nikolenko, S. I., Pham, S., and Prjibelski, A. D. 2012. SPAdes: A new genome assembly algorithm and its applications to single-cell sequencing. J. Comput. Biol. 19:455-477. Crossref, ISIGoogle Scholar
    • Bolger, A. M., Lohse, M., and Usadel, B. 2014. Trimmomatic: A flexible trimmer for Illumina sequence data. Bioinformatics 30:2114-2120. Crossref, ISIGoogle Scholar
    • Bové, J. M. 2006. Huanglongbing: A destructive, newly-emerging, century-old disease of citrus. J. Plant Pathol. 88:7-37. ISIGoogle Scholar
    • Bové, J. M., and Cassin, J. P. 1968. Problème de l’agrumiculture Réunionnaise. Compt-rendu demission. Document IFRA-CIRAD, Paris, France. Google Scholar
    • Camacho, C., Coulouris, G., Avagyan, V., Ma, N., Papadopoulos, J., Bealer, K., and Madden, T. L. 2009. BLAST+: Architecture and applications. BMC Bioinf. 10:421. Crossref, ISIGoogle Scholar
    • Catling, H. D. 1970. Distribution of psyllid vectors of citrus greening disease, with notes on the biology and bionomics of Diaphorina citri. FAO Plant Prot. Bull. 18:8-15. Google Scholar
    • Catling, H. D. 1972. The distribution of psyllid vectors of citrus greening disease in Reunion. Rapport de mission, document IRFA Réunion. Google Scholar
    • EPPO. 2014. PM 7/121 (1) ‘Candidatus Liberibacter africanus’, ‘Candidatus Liberibacter americanus’ and ‘Candidatus Liberibacter asiaticus’. Bull. OEPP/EPPO Bull. 44:376-389. CrossrefGoogle Scholar
    • Garnier, M., Jaqoueix, S., Toorawwaw, P., Grisoni, M., Mallessard, R., Dookun, A., Saumtally, S., Autrey, J. C., and Bové, J. M. 1996. Both huanglongbing (greening) Liberibacter species are present in Mauritius and Réunion. Pages 392-394 in: Proc. Thirteenth Conf. Int. 16 Organ. Citrus Virol.. J. V. de Graça, R. F. Lee, and R. K. Yokomi, eds. University of California Riverside, Riverside, CA, U.S.A. Google Scholar
    • Kurtz, S., Phillippy, A., Delcher, A. L., Smoot, M., Shumway, M., Antonescu, C., and Salzberg, S. L. 2004. Versatile an open software for comparing large genomes. Genome Biol. 5:R12. Crossref, ISIGoogle Scholar
    • Li, D., Liu, C. M., Luo, R., Sadakane, K., and Lam, T. W. 2015. MEGAHIT: An ultra-fast single-node solution for large and complex metagenomics assembly via succinct de Bruijn graph. Bioinformatics 31:1674-1676. Crossref, ISIGoogle Scholar
    • Li, H., and Durbin, R. 2009. Fast and accurate short read alignment with Burrows-Wheeler Transform. Bioinformatics 25:1754-1760. Crossref, ISIGoogle Scholar
    • Li, W. B., Hartung, J. S., and Levy, L. 2006. Quantitative real-time PCR for detection and identification of Candidatus Liberibacter species associated with citrus huanglongbing. J. Microbiol. Methods 66:104-115. Crossref, ISIGoogle Scholar
    • Lin, K. H. 1956. Observation on yellow shoot of citrus. Acta Phytopathol. Sin. 2:1-11. Google Scholar
    • Moreria, S. 1967. Enquête sur les maladies des agrumes, îles Maurice et de la Réunion. Bull. Phytosanit. FAO 15:59-60. Google Scholar
    • Pietersen, G., Arrebola, E., Breytenbach, J. H. J., Korsten, L., Le Roux, H. F., la Grange, H., Lopes, S. A., Meyer, J. B., Pretorius, M. C., Schwerdtfeger, M., van Vuuren, S. P., and Yamamoto, P. 2010. A survey for ‘Candidatus Liberibacter’ species in South Africa confirms the presence of only ‘Ca. L, africanus’ in commercial citrus. Plant Dis. 94:244-249. Link, ISIGoogle Scholar
    • Saponari, M., De Bac, G., Breithaupt, J., Loconsole, G., Yokomi, R. K., and Catalano, L. 2010. First report of ‘Candidatus Liberibacter asiaticus’ associated with huanglongbing in sweet orange in Ethiopia. Plant Dis. 94:482. Link, ISIGoogle Scholar
    • Silva, P. A., Huang, J., Wulff, N. A., Zheng, Z., Krugner, R., and Chen, J. 2021. Genome sequence resource of ‘Candidatus Liberibacter asiaticus’ strain 9PA from Brazil. Plant Dis. 105:199-201. Link, ISIGoogle Scholar
    • van den Berg, M. A., and Greenland, J. 2000. Tamarixia dryi, parasitoid of the citrus psylla, Trioza erytreae: A review. Afr. Plant Prot. 6:25-28. Google Scholar

    The author(s) declare no conflict of interest.

    Funding: This study was supported by Zhejiang A&F University impulse project (2019FR029), the National Key Research and Development Program of China (2018YFD0201502) and the Science and Technology Major Project of Guangxi (Gui Ke AA18118046).