Open access
Genomics and Proteomics
Announcement
25 May 2023

Complete Genome Sequence of “Candidatus Phytoplasma asteris” QS2022, a Plant Pathogen Associated with Lettuce Chlorotic Leaf Rot Disease in China

ABSTRACT

The complete genome sequence of “Candidatus Phytoplasma asteris” QS2022, which consists of one 834,303-bp circular chromosome, is presented in this work. This bacterium is associated with lettuce chlorotic leaf rot disease in Fujian Province, China.

ANNOUNCEMENT

Lettuce chlorotic leaf rot disease (LCLRD) is an emerging disease in China. It was first reported in Yong’an County, Fujian Province, in 2005. Since then, it has posed a persistent threat to the local lettuce production industry, causing significant economic losses every year. The causative agent, a 16SrI-B subgroup phytoplasma, is transmitted by the leafhopper Macrosteles striifrons and specifically invades the plant phloem (1). After infection, apical leaves become elongated and pale, growing buds rot, and plants eventually die. Because phytoplasmas are uncultivated, we conducted shotgun sequencing of an infected plant to study the genome of this bacterium. All kits were used according to the manufacturer’s protocols, and all bioinformatics tools were used with the default settings unless stated otherwise.
A symptomatic lettuce plant was collected in Qingshui Village of Yong’an County in October 2022. Pale apical leaves and buds were used for DNA extraction with the Hi-DNAsecure plant kit (DP350; Tiangen Biotech). For Illumina sequencing, the library was prepared using the VAHTS Universal Plus DNA library prep kit (ND617-C3-02; Vazyme), followed by sequencing on the Illumina NovaSeq 6000 platform to generate 9.1 Gb of 150-bp paired-end reads. For Oxford Nanopore Technologies (ONT) sequencing, DNA fragments of >10 kb were collected using the BluePippin system (Sage Science), followed by processing with NEBNext formalin-fixed, paraffin-embedded (FFPE) DNA repair mix and the NEBNext Ultra II end repair/deoxyribosyladenine (dA)-tailing module (New England Biolabs). The library was prepared using the ONT ligation sequencing kit (SQK-LSK109) without shearing and sequenced using PromethION flow cells (FLO-PRO002) on PromethION48. Guppy v3.2.6 was used for base calling and adapter trimming, which produced 618,488 reads totaling 9.9 Gb and with an N50 of 25.4 kb.
All Illumina and ONT reads were mapped to complete genome assemblies of 16SrI-B phytoplasmas (accession numbers GCF_000009845, GCF_001712875, and GCF_004214875) to identify bacterial reads using BWA v0.7.17 (2) and Minimap2 v2.15 (3), respectively. “Candidatus Phytoplasma asteris” OY-M (4) was found to have ~99% genome-wide average nucleotide identity with this new strain, QS2022, and was used to extract bacterial reads. The Illumina reads with an alignment score above 30 were extracted, trimmed using a Q20 cutoff, and combined with the ONT reads with an alignment score above 1,000 for assembly based on Trycycler v0.5.3 (5). The procedure of gene prediction and annotation was based on that described in our previous work (6, 7). For gene prediction, RNAmmer v1.2 (8), tRNAscan-SE v1.3.1 (9), and Prodigal v2.6.3 (10) were used. The annotation was based on the homologs in other phytoplasmas as identified by OrthoMCL v1.3 (11), followed by manual curation based on BlastKOALA v.12 (12) and GenBank (13). Putative secreted proteins were identified using SignalP v5.0 (14) and filtered by TMHMM v2.0 (15).
The assembly produced a single 834,303-bp circular contig with 27.6% G+C content, which represents the chromosome; no plasmid was found. The Illumina and ONT reads provided 547- and 136-fold coverage, respectively. The contig was rotated to have dnaA as the first gene. The annotation contains 6 rRNA genes, 32 tRNA genes, 819 protein-coding genes, and 36 pseudogenes.

Data availability.

This whole-genome shotgun project has been deposited in NCBI under the accession number PRJNA937408 (https://www.ncbi.nlm.nih.gov/bioproject/PRJNA937408). The raw reads have been deposited at the NCBI Sequence Read Archive under the accession numbers SRX19478431 (https://www.ncbi.nlm.nih.gov/sra/SRX19478431) and SRX19478432 (https://www.ncbi.nlm.nih.gov/sra/SRX19478432). The genome sequence has been deposited at GenBank under the accession number CP120448 (https://www.ncbi.nlm.nih.gov/nuccore/CP120448).

ACKNOWLEDGMENTS

The sequencing service was provided by Biomarker Technologies (Beijing, China).
The funding for this work was provided by the Natural Science Foundation of Shanghai (grant number 23ZR1470300) and the CAS Center for Excellence in Molecular Plant Sciences to W.H. and Academia Sinica 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.
Lin J, Yang C, Liu J, Yu S, Xing J, Huang P, Chen W, Bao Y, Hu Q, Chen C, Zhang M. 2020. Identification and characterization of the phytoplasma associated with lettuce chlorotic leaf rot disease together with its natural reservoirs and leafhopper vectors in China. Crop Prot 138:105318.
2.
Li H, Durbin R. 2009. Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics 25:1754–1760.
3.
Li H. 2018. Minimap2: pairwise alignment for nucleotide sequences. Bioinformatics 34:3094–3100.
4.
Oshima K, Kakizawa S, Nishigawa H, Jung H-Y, Wei W, Suzuki S, Arashida R, Nakata D, Miyata S, Ugaki M, Namba S. 2004. Reductive evolution suggested from the complete genome sequence of a plant-pathogenic phytoplasma. Nat Genet 36:27–29.
5.
Wick RR, Judd LM, Cerdeira LT, Hawkey J, Méric G, Vezina B, Wyres KL, Holt KE. 2021. Trycycler: consensus long-read assemblies for bacterial genomes. Genome Biol 22:266.
6.
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.
7.
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.
8.
Lagesen K, Hallin P, Rodland EA, Staerfeldt H-H, Rognes T, Ussery DW. 2007. RNAmmer: consistent and rapid annotation of ribosomal RNA genes. Nucleic Acids Res 35:3100–3108.
9.
Lowe T, Eddy S. 1997. tRNAscan-SE: a program for improved detection of transfer RNA genes in genomic sequence. Nucleic Acids Res 25:955–964.
10.
Hyatt D, Chen G-L, LoCascio P, Land M, Larimer F, Hauser L. 2010. Prodigal: prokaryotic gene recognition and translation initiation site identification. BMC Bioinformatics 11:119.
11.
Li L, Stoeckert CJ, Roos DS. 2003. OrthoMCL: identification of ortholog groups for eukaryotic genomes. Genome Res 13:2178–2189.
12.
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.
13.
Benson DA, Cavanaugh M, Clark K, Karsch-Mizrachi I, Ostell J, Pruitt KD, Sayers EW. 2018. GenBank. Nucleic Acids Res 46:D41–D47.
14.
Armenteros JJA, 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.
15.
Krogh A, Larsson B, von Heijne G, Sonnhammer ELL. 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 620 June 2023
eLocator: e00306-23
Editor: Vanja Klepac-Ceraj, Wellesley College
PubMed: 37227269

History

Received: 12 April 2023
Accepted: 11 May 2023
Published online: 25 May 2023

Contributors

Authors

Xiao-Hua Yan
Institute of Plant and Microbial Biology, Academia Sinica, Taipei, Taiwan
Jixiu Lin
Yongan Crop Protection and Quarantine Station, Yongan, Fujian, China
Yanzhi Liu
Shanghai Center for Plant Stress Biology, CAS Center for Excellence in Molecular Plant Sciences, Chinese Academy of Sciences, Shanghai, China
Peizhi Huang
Yongan Crop Protection and Quarantine Station, Yongan, Fujian, China
Jianmi Liu
Sanming Crop Protection and Quarantine Station, Sanming, Fujian, China
Qibin Hu
Sanming Crop Protection and Quarantine Station, Sanming, Fujian, China
Yalu Li
Shanghai Center for Plant Stress Biology, CAS Center for Excellence in Molecular Plant Sciences, Chinese Academy of Sciences, Shanghai, China
Shen-Chian Pei
Institute of Plant and Microbial Biology, Academia Sinica, Taipei, Taiwan
Weijie Huang [email protected]
Shanghai Center for Plant Stress Biology, CAS Center for Excellence in Molecular Plant Sciences, Chinese Academy of Sciences, Shanghai, China
Institute of Plant and Microbial Biology, Academia Sinica, Taipei, Taiwan

Editor

Vanja Klepac-Ceraj
Editor
Wellesley College

Notes

Xiao-Hua Yan, Jixiu Lin, Yanzhi Liu, and Yalu Li made equal contributions. The order of names was decided by an agreement among all authors.
The authors declare no conflict of interest.

Metrics & Citations

Metrics

Note:

  • For recently published articles, the TOTAL download count will appear as zero until a new month starts.
  • There is a 3- to 4-day delay in article usage, so article usage will not appear immediately after publication.
  • Citation counts come from the Crossref Cited by service.

Citations

If you have the appropriate software installed, you can download article citation data to the citation manager of your choice. For an editable text file, please select Medlars format which will download as a .txt file. Simply select your manager software from the list below and click Download.

View Options

Figures and Media

Figures

Media

Tables

Share

Share

Share the article link

Share with email

Email a colleague

Share on social media

American Society for Microbiology ("ASM") is committed to maintaining your confidence and trust with respect to the information we collect from you on websites owned and operated by ASM ("ASM Web Sites") and other sources. This Privacy Policy sets forth the information we collect about you, how we use this information and the choices you have about how we use such information.
FIND OUT MORE about the privacy policy