Open access
Genomics and Proteomics
Announcement
11 January 2024

Complete genome sequence of Candidatus Phytoplasma australasiaticum WF_GM2021, a plant pathogen associated with soybean witches’ broom disease in Taiwan

ABSTRACT

The complete genome sequence of Candidatus Phytoplasma australasiaticum strain WF_GM2021, which consists of one 633,005-bp circular chromosome, is presented in this work. This uncultivated plant-pathogenic bacterium is associated with soybean (Glycine max) witches’ broom disease in Wufeng District, Taichung City, Taiwan.

ANNOUNCEMENT

Phytoplasmas are plant-pathogenic bacteria that induce developmental abnormalities in their hosts, causing agricultural losses worldwide (1). Among the described Candidatus Phytoplasma species (2), Ca. P. australasiaticum is prevalent in Australia and Asia (3) and has been developed as a major representative for phytoplasma genomics research (4). To improve the sampling of these bacteria, a strain WF_GM2021 associated with soybean witches’ broom disease was collected for whole-genome sequencing. Unless stated otherwise, the methods were based on the cited references; the kits were used according to the manufacturer’s instructions; and the bioinformatics tools were used with the default settings.
A soybean plant with witches’ broom symptom was collected in Wufeng District (Taichung City, Taiwan; 24.01495 N 120.69428 E) in August 2021. The uncultivated phytoplasma was transmitted via dodder (Cuscuta australis) to periwinkle (Catharanthus roseus) for maintenance (5). After propagation via side grafting for one passage, the leaves of a periwinkle plant showing yellowing and witches’ broom symptoms were collected for DNA extraction without cultivation using a cetyltrimethylammonium bromide method (6). For Illumina sequencing, the DNA sample was processed using KAPA LTP Library Preparation Kit (Roche, Switzerland) and sequenced on a NovaSeq 6000 using S4 Reagent Kit v1.5 (Illumina, USA). A total of 87,826,828 pairs of 151-bp paired-end reads were generated. After quality trimming using Trimmomatic v.0.39 (7) with the “TrimmomaticPE” mode, 83,518,753 read pairs with a combined length of 24,467,418,087 bp were obtained.
By mapping reads to representative phytoplasma genomes that originated from Asian countries (4) using BWA v.0.7.17 (8), WF_GM2021 was found to share 100% 16S rRNA gene sequence identity with strain NCHU2014 (National Center for Biotechnology Information assembly accession number GCA_001307505.2, from a symptomatic Echinacea purpurea plant collected in Wufeng District in June 2014) (9, 10). Therefore, a resequencing strategy was used with NCHU2014 as the reference without re-assembly. The BWA mapping result was programmatically checked using SAMtools v.1.9 (11) to identify the polymorphisms. Polymorphic sites that have a QUAL score above 100 in the variant call format file produced by SAMtools were manually inspected using IGV v.2.11.1 (12) both before and after correcting the polymorphisms. The chromosomal contig was determined to be complete and circular based on the mapping of read pairs that span both ends of the contig. The procedure of gene prediction and annotation was based on that described in our previous work (5, 13). For gene prediction, RNAmmer v.1.2 (14), tRNAscan-SE v.1.3.1 (15), and Prodigal v.2.6.3 (16) were used. The annotation was based on the homologs in NCHU2014 (9, 10) as identified by OrthoMCL v.1.3 (17). Putative secreted proteins were identified using SignalP v.5.0 (14) and filtered by TMHMM v.2.0 (15).
The assembly produced a single 633,005-bp circular chromosomal contig with 24.5% G + C content. Among the quality-trimmed reads, 8,709,260 reads (5.2% of total) can be mapped to this uncultivated bacterium, providing 1,455.7-fold coverage. The same as the reference genome, which shares 99.96% average nucleotide identity (18), this new assembly has dnaA as the first gene. The annotation contains 6 rRNA genes, 27 tRNA genes, 507 protein-coding genes, and 12 pseudogenes.

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 Genomics BioSci and Tech Co, Ltd (New Taipei, Taiwan).
The funding was provided by Academia Sinica to C.-H.K. The funder had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

REFERENCES

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Bertaccini A. 2007. Phytoplasmas: diversity, taxonomy, and epidemiology. Front Biosci 12:673–689.
2.
Bertaccini A, Arocha-Rosete Y, Contaldo N, Duduk B, Fiore N, Montano HG, Kube M, Kuo C-H, Martini M, Oshima K, Quaglino F, Schneider B, Wei W, Zamorano A. 2022. Revision of the ‘Candidatus Phytoplasma’ species description guidelines. Int J Syst Evol Microbiol 72:005353.
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Rodrigues Jardim B, Tran-Nguyen LTT, Gambley C, Al-Sadi AM, Al-Subhi AM, Foissac X, Salar P, Cai H, Yang J-Y, Davis R, Jones L, Rodoni B, Constable FE. 2023. The observation of taxonomic boundaries for the 16SrII and 16SrXXV phytoplasmas using genome-based delimitation. Int J Syst Evol Microbiol 73:005977.
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Huang C-T, Pei S-C, Kuo C-H. 2023. Genomic studies on Asian phytoplasmas, p 67–83. In Tiwari AK, Oshima K, Yadav A, Esmaeilzadeh-Hosseini SA, Hanboonsong Y, Lakhanpaul S (ed), Characterization, epidemiology, and management. Academic Press.
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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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Fulton TM, Chunwongse J, Tanksley SD. 1995. Microprep protocol for extraction of DNA from tomato and other herbaceous plants. Plant Mol Biol Rep 13:207–209.
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Bolger AM, Lohse M, Usadel B. 2014. Trimmomatic: a flexible trimmer for illumina sequence data. Bioinformatics 30:2114–2120.
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Li H, Durbin R. 2009. Fast and accurate short read alignment with burrows–wheeler transform. Bioinformatics 25:1754–1760.
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Tan CM, Lin Y-C, Li J-R, Chien Y-Y, Wang C-J, Chou L, Wang C-W, Chiu Y-C, Kuo C-H, Yang J-Y. 2021. Accelerating complete phytoplasma genome assembly by immunoprecipitation-based enrichment and MiniON-based DNA sequencing for comparative analyses. Front Microbiol 12:766221.
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Chang S-H, Cho S-T, Chen C-L, Yang J-Y, Kuo C-H. 2015. Draft genome sequence of a 16SrII-A subgroup phytoplasma associated with purple coneflower (Echinacea purpurea) witches’ broom disease in Taiwan. Genome Announc 3:e01398-15.
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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.
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Robinson JT, Thorvaldsdóttir H, Winckler W, Guttman M, Lander ES, Getz G, Mesirov JP. 2011. Integrative genomics viewer. Nat Biotechnol 29:24–26.
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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.
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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.
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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.
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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.
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Jain C, Rodriguez-R LM, Phillippy AM, Konstantinidis KT, Aluru S. 2018. High throughput ANI analysis of 90K prokaryotic genomes reveals clear species boundaries. Nat Commun 9:5114.

Information & Contributors

Information

Published In

cover image Microbiology Resource Announcements
Microbiology Resource Announcements
Volume 13Number 215 February 2024
eLocator: e00993-23
Editor: David A. Baltrus, The University of Arizona, Tucson, Arizona, USA
PubMed: 38206024

History

Received: 18 October 2023
Accepted: 21 December 2023
Published online: 11 January 2024

Keywords

  1. genome
  2. phytoplasma
  3. pathogens
  4. phytopathogens

Data Availability

This genome project has been deposited in National Center for Biotechnology Information (NCBI) under the accession number PRJNA1008853. The raw reads have been deposited at the NCBI Sequence Read Archive under the accession number SRX21482278. The genome sequence has been deposited at GenBank under the accession number CP133702.

Contributors

Authors

Nian-Pu Li
Institute of Plant and Microbial Biology, Academia Sinica, Taipei, Taiwan
Department of Plant Pathology, National Chung Hsing University, Taichung, Taiwan
Author Contributions: Data curation, Formal analysis, Validation, and Writing – original draft.
Xiao-Hua Yan
Institute of Plant and Microbial Biology, Academia Sinica, Taipei, Taiwan
Author Contributions: Data curation, Formal analysis, and Validation.
Shen-Chian Pei
Institute of Plant and Microbial Biology, Academia Sinica, Taipei, Taiwan
Department of Plant Pathology and Microbiology, National Taiwan University, Taipei, Taiwan
Author Contributions: Data curation and Methodology.
Ting-Hsuan Hung
Department of Plant Pathology and Microbiology, National Taiwan University, Taipei, Taiwan
Author Contributions: Resources and Supervision.
Tsu-Cheng Chang
Agricultural Chemicals Research Institute, Ministry of Agriculture, Taichung, Taiwan
Author Contribution: Investigation.
Qiong-Zhuan Bai
Agricultural Chemicals Research Institute, Ministry of Agriculture, Taichung, Taiwan
Author Contribution: Investigation.
Agricultural Chemicals Research Institute, Ministry of Agriculture, Taichung, Taiwan
Author Contributions: Conceptualization, Funding acquisition, Project administration, Resources, and Writing – review and editing.
Institute of Plant and Microbial Biology, Academia Sinica, Taipei, Taiwan
Biotechnology Center, National Chung Hsing University, Taichung, Taiwan
Author Contributions: Conceptualization, Funding acquisition, Methodology, Project administration, Supervision, Writing – original draft, and Writing – review and editing.

Editor

David A. Baltrus
Editor
The University of Arizona, Tucson, Arizona, USA

Notes

The authors declare no conflict of interest.

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