Open access
Genomics and Proteomics
Announcement
15 September 2023

Complete genome sequence of “Candidatus Phytoplasma cynodontis” GY2015, a plant pathogen associated with Bermuda grass white leaf disease in Taiwan

ABSTRACT

The complete genome sequence of “Candidatus Phytoplasma cynodontis” strain GY2015, which consists of one 498,922-bp circular chromosome, is presented in this work. This uncultivated plant-pathogenic bacterium is associated with Bermuda grass white leaf disease in Taoyuan, Taiwan.

ANNOUNCEMENT

An uncultivated bacterium, “Candidatus Phytoplasma cynodontis,” has been identified as the causal pathogen of Bermuda grass white leaf disease (1). This disease was first reported in Taiwan in 1972 (2) and later found in other Asian countries, Australia, Africa, and Europe (1). In addition to extensive chlorosis, the infection leads to stunting and death, causing economic damages in turf maintenance and worsening soil erosion. To facilitate the study of this pathogen, we conducted shotgun sequencing of a symptomatic plant collected in Guanyin District (Taoyuan City, Taiwan) in 2015 and assembled the complete genome sequence of strain GY2015 associated with this sample. All kits were used according to the manufacturer’s protocols, and all bioinformatics tools were used with the default settings unless stated otherwise.
Approximately 0.5 g of leaves was collected for DNA extraction using Wizard Genomic DNA Purification Kit (Promega), then used for both sequencing platforms. For Illumina sequencing, the library was prepared using KAPA LTP Library Preparation Kit (Roche) with a targeted insert size of 550 bp and sequenced on a MiSeq using Reagent Kit v3 (600 cycles) to generate 15.3 Gb of paired-end reads, then trimmed with a Q20 cutoff and filtered using a 200-bp cutoff. For Oxford Nanopore Technologies (ONTs) sequencing, the library was prepared using ONT Ligation Sequencing Kit (SQK-LSK109) without shearing and without size selection, then sequenced on a MinION (R9.4.1; FLO-MIN106). Basecalling was performed using Guppy v3.6.1 with the high-accuracy mode, which produced 546,782 reads (N50: 32.2 kb; combined: 11.7 Gb).
The first de novo assembly used only the Illumina reads for Velvet v1.2.10 (3). Putative phytoplasma contigs were identified by BLASTX (4) searches against phytoplasma proteins available in the NCBI nr database (5). All raw reads were mapped to these contigs using BWA v0.7.17 (6) and Minimap2 v2.15 (7). The Illumina reads with an alignment score >30 and the ONT reads with a score >500 were extracted for hybrid assembly using Unicycler v0.4.9b (8), which produced one circular chromosomal contig. This contig was polished iteratively using the Illumina reads based on BWA mapping and SAMtools v1.9 (9) checks. The procedure of gene prediction and annotation was based on that described in our previous work (10, 11). For gene prediction, RNAmmer v1.2 (12), tRNAscan-SE v1.3.1 (13), and Prodigal v2.6.3 (14) were used. The annotation was based on the homologs in other phytoplasma genomes available from GenBank (5) as identified by OrthoMCL v1.3 (15), followed by manual curation based on BlastKOALA v.2.2 (16). Putative-secreted proteins were identified using SignalP v5.0 (17) and filtered by TMHMM v2.0 (18).
The assembly consists of one 498,922 bp circular chromosome with 21.1% G + C content, which was rotated to have dnaA as the first gene. There were 1.0 million Illumina reads mapped to the phytoplasma genome, providing 597.5-fold coverage. For ONT reads, 2,256 reads totaling 56.4 Mb (N50: 33.9 kb) provided 113.1-fold coverage. The annotation contains 6 rRNA genes, 23 tRNA genes, 413 protein-coding genes, and 4 pseudogenes. No plasmid was found.

ACKNOWLEDGMENTS

The library preparation service was provided by the Genomic Technology Core (Institute of Plant and Microbial Biology, Academia Sinica, Taiwan). The Illumina sequencing service was provided by the Genomics Core (Institute of Molecular Biology, Academia Sinica, Taiwan). The funding was provided by Academia Sinica and the Ministry of Science and Technology of Taiwan (106-2311-B-001-028-MY3) to C.-H.K. The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

REFERENCES

1.
Marcone C, Schneider B, Seemüller E. 2004. 'Candidatus Phytoplasma cynodontis', the phytoplasma associated with Bermuda grass white leaf disease. Int J Syst Evol Microbiol 54:1077–1082.
2.
Chen CT, Lee CS, Chen MJ. 1972. Mycoplasma-like organisms in Cynodon dactylon and Brachiaria distachya affected by white leaf disease. Rep Taiwan Sugar Exp Stn 56:49–55.
3.
Zerbino DR, Birney E. 2008. Velvet: algorithms for de novo short read assembly using de Bruijn graphs. Genome Res 18:821–829.
4.
Camacho C, Coulouris G, Avagyan V, Ma N, Papadopoulos J, Bealer K, Madden TL. 2009. BLAST+: architecture and applications. BMC Bioinformatics 10:421.
5.
Benson DA, Cavanaugh M, Clark K, Karsch-Mizrachi I, Ostell J, Pruitt KD, Sayers EW. 2018. GenBank. Nucleic Acids Res 46:D41–D47.
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Li H, Durbin R. 2009. Fast and accurate short read alignment with Burrows–Wheeler transform. Bioinformatics 25:1754–1760.
7.
Li H, . 2018. Minimap2: pairwise alignment for nucleotide sequences. Bioinformatics 34:3094–3100.
8.
Wick RR, Judd LM, Gorrie CL, Holt KE. 2017. Unicycler: resolving bacterial genome assemblies from short and long sequencing reads. PLOS Comput Biol 13:e1005595.
9.
Li H, Handsaker B, Wysoker A, Fennell T, Ruan J, Homer N, Marth G, Abecasis G, Durbin R, 1000 Genome Project Data Processing Subgroup. 2009. The sequence alignment/map format and SAMtools. Bioinformatics 25:2078–2079.
10.
Chung W-C, Chen L-L, Lo W-S, Lin C-P, Kuo C-H. 2013. Comparative analysis of the peanut witches’-broom phytoplasma genome reveals horizontal transfer of potential mobile units and effectors. PLoS ONE 8:e62770.
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Cho S-T, Zwolińska A, Huang W, Wouters RHM, Mugford ST, Hogenhout SA, Kuo C-H. 2020. Complete genome sequence of “Candidatus Phytoplasma asteris” RP166, a plant pathogen associated with rapeseed phyllody disease in Poland. Microbiol Resour Announc 9:e00760-20.
12.
Lagesen K, Hallin P, Rødland EA, Staerfeldt H-H, Rognes T, Ussery DW. 2007. RNAmmer: consistent and rapid annotation of ribosomal RNA genes. Nucleic Acids Res 35:3100–3108.
13.
Lowe TM, Eddy SR. 1997. tRNAscan-SE: a program for improved detection of transfer RNA genes in genomic sequence. Nucleic Acids Res 25:955–964.
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.
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Li L, Stoeckert CJ, Roos DS. 2003. OrthoMCL: identification of ortholog groups for eukaryotic genomes. Genome Res 13:2178–2189.
16.
Kanehisa M, Sato Y, Morishima K. 2016. BlastKOALA and GhostkOALA: KEGG tools for functional characterization of genome and metagenome sequences. J Mol Biol 428:726–731.
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Almagro Armenteros JJ, Tsirigos KD, Sønderby CK, Petersen TN, Winther O, Brunak S, von Heijne G, Nielsen H. 2019. SignalP 5.0 improves signal peptide predictions using deep neural networks. Nat Biotechnol 37:420–423.
18.
Krogh A, Larsson B, von Heijne G, Sonnhammer EL. 2001. Predicting transmembrane protein topology with a hidden Markov model: application to complete Genomes. J Mol Biol 305:567–580.

Information & Contributors

Information

Published In

cover image Microbiology Resource Announcements
Microbiology Resource Announcements
Volume 12Number 1019 October 2023
eLocator: e00457-23
Editor: Julie C. Dunning Hotopp, University of Maryland School of Medicine, Baltimore, Maryland, USA
PubMed: 37855628

History

Received: 30 May 2023
Accepted: 1 August 2023
Published online: 15 September 2023

Keywords

  1. phytoplasma
  2. genome
  3. Mollicutes
  4. pathogen
  5. plant pathogens

Data Availability

This genome project has been deposited in NCBI under the accession number PRJNA318828. The metadata have been provided under the accession number SAMN04868714. The raw reads have been deposited at the NCBI Sequence Read Archive under the accession numbers SRX20434751 and SRX20434752. The genome sequence has been deposited at GenBank under the accession number CP126225.

Contributors

Authors

Shu-Ting Cho
Institute of Plant and Microbial Biology, Academia Sinica, Taipei, Taiwan
Department of Plant Pathology and Microbiology, National Taiwan University, Taipei, Taiwan
Ai-Ping Chen
Institute of Plant and Microbial Biology, Academia Sinica, Taipei, Taiwan
Shu-Jen Chou
Institute of Plant and Microbial Biology, Academia Sinica, Taipei, Taiwan
Xiao-Hua Yan
Institute of Plant and Microbial Biology, Academia Sinica, Taipei, Taiwan
Chan-Pin Lin [email protected]
Department of Plant Pathology and Microbiology, National Taiwan University, Taipei, Taiwan
Institute of Plant and Microbial Biology, Academia Sinica, Taipei, Taiwan

Editor

Julie C. Dunning Hotopp
Editor
University of Maryland School of Medicine, Baltimore, Maryland, USA

Notes

The authors declare no conflict of interest.

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