HomePlant DiseaseAhead of PrintGenome Sequence Resource of Curvularia clavata Causing Leaf Spot Disease on Tobacco by Oxford Nanopore PromethION PreviousNext RESOURCE ANNOUNCEMENT OPENOpen Access licenseGenome Sequence Resource of Curvularia clavata Causing Leaf Spot Disease on Tobacco by Oxford Nanopore PromethIONYi Cao, Qiuyan Cai, Changquan Li, Guanglong Song, Ning Lu, and Zhixiao YangYi Cao†Corresponding authors: Y. Cao; E-mail Address: [email protected], and Z. Yang; E-mail Address: [email protected]https://orcid.org/0000-0003-4154-3258Guizhou Provincial Academician Workstation of Microbiology and Health, Guizhou Academy of Tobacco Science, Guiyang 550081, ChinaSearch for more papers by this author, Qiuyan CaiGuizhou Agricultural Science and Technology Information Institute, Guiyang 550006, ChinaSearch for more papers by this author, Changquan LiLiupanshui Branch of Guizhou Tobacco Company, Liupanshui 553001, ChinaSearch for more papers by this author, Guanglong SongTongren Branch of Guizhou Tobacco Company, Tongren 564300, ChinaSearch for more papers by this author, Ning Luhttps://orcid.org/0000-0002-6418-9717Guizhou Provincial Academician Workstation of Microbiology and Health, Guizhou Academy of Tobacco Science, Guiyang 550081, ChinaSearch for more papers by this author, and Zhixiao Yang†Corresponding authors: Y. Cao; E-mail Address: [email protected], and Z. Yang; E-mail Address: [email protected]Guizhou Provincial Academician Workstation of Microbiology and Health, Guizhou Academy of Tobacco Science, Guiyang 550081, ChinaSearch for more papers by this authorAffiliationsAuthors and Affiliations Yi Cao1 † Qiuyan Cai2 Changquan Li3 Guanglong Song4 Ning Lu1 Zhixiao Yang1 † 1Guizhou Provincial Academician Workstation of Microbiology and Health, Guizhou Academy of Tobacco Science, Guiyang 550081, China 2Guizhou Agricultural Science and Technology Information Institute, Guiyang 550006, China 3Liupanshui Branch of Guizhou Tobacco Company, Liupanshui 553001, China 4Tongren Branch of Guizhou Tobacco Company, Tongren 564300, China Published Online:22 May 2023https://doi.org/10.1094/PDIS-09-22-2283-AAboutSectionsView articlePDFSupplemental ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InRedditEmailWechat View articleGenome AnnouncementPlant pathogens cause destructive crop diseases leading to significant yield and quality decline. Curvularia is an important plant pathogen (Tovar-Pedraza et al. 2023; Zhang and Zhang 2007). It can harm many gramineous plants, such as corn, oats, rice, sugarcane, and wheat (AbdElfatah et al. 2021; Bengyella et al. 2019; Zhang et al. 2020), causing leaf spots, seed discoloration, and seedling withering (Marin-Felix et al. 2020). Curvularia clavata belongs to Ascomycota, Dothideomycetes, Pleosporales, Pleosporaceae, and Curvularia. It has been reported that the fungus has a wide host range and can infect a variety of crops, such as Curcuma, Jatropha, pineapple, and corn (Chen et al. 2013; Zhong et al. 2016). In addition to causing plant diseases, the fungus of this genus can also cause cutaneous phaeohyphomycosis (Fan et al. 2009). In recent years, the diseases caused by the fungi of this genus have been found continuously (Z. Liu et al. 2019; Ul Haq et al. 2021a, b), which has a serious impact on agricultural production.In previous studies, our team collected leaves with leaf spot diseases in Fenggang County of Guizhou Province, China (27.43°N, 107.43°E), and determined the pathogenicity by tissue isolation and in vitro leaf inoculation. The pathogen was classified and identified as C. clavata YC1106 by morphological characteristics and molecular biology methods based on ITS gene sequences (He et al. 2021). The strain was isolated and preserved in the Microbial Library of the Southwest China Wild Germplasm Bank (accession number: YMF1.07864).Genome sequencing technology can comprehensively analyze the molecular biological characteristics of pathogenic fungi at the gene level and play an important role in clarifying the infection of related fungal pathogens (Dong et al. 2021; He et al. 2021; B. Liu et al. 2019). Hence, it is important to deeply explore the secretory enzymes, protein composition, stimulating metabolites, and pathways of C. clavata based on genome sequencing to clarify its pathogenesis and subsequent prevention. This study sequenced the genome of a previously isolated C. clavata YC1106.In this project, genomic DNA was extracted using the CTAB method. The quality and integrity of genomic DNA were assessed using 1% agarose gel electrophoresis and densitometry compared with the appropriate size standards. Meanwhile, DNA yield and purity were measured using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA) and TBS-380 fluorometer (Turner BioSystems Inc., Sunnyvale, CA). High-quality DNA (OD260/280 = 1.8–2.0, >1 μg) was sequenced by Shanghai Winnerbio Technology Co., Ltd. (Shanghai, China) using Illumina NovaSeq 6000 (Illumina, San Diego, CA) and Oxford Nanopore PromethION (Oxford Nanopore Technologies, Oxford, U.K.). For Illumina sequencing, 1 μg of genomic DNA of the strain was used to construct the library using the NEB Next Ultra DNA Library Prep Kit for Illumina (New England Biolabs, Ipswich, MA) according to the manufacturer’s protocol. DNA samples were sheared into 400- to 500-bp fragments using a Covaris M220 Focused Acoustic Shearer (Covaris, Woburn, MA) following the manufacturer’s protocol. Illumina sequencing libraries were prepared from the sheared fragments. The prepared libraries were then used for paired-end Illumina sequencing (2 × 150 bp) on an Illumina NovaSeq 6000 machine. For ONT sequencing, DNA fragments larger than 20 kb were screened using beads (Agencourt AMPure XP, Beckman Coulter, Inc., Pasadena, CA). The ends of the DNA fragments of interest were repaired, and barcode tags were added using the EXP-NBD104/114 kit after bead purification. The product of DNA was purified by beads and ligated using the sequencing adapter in the SQK-LSK109 kit. The constructed library was added to the flow cell and transferred to the Oxford Nanopore PromethION platform for sequencing.The Illumina and ONT data were quality controlled to obtain clean data for downstream analysis. Specifically, for Illumina data, sequences containing adapter and N bases were removed, and reads with sequencing quality values less than Q20 were removed. Finally, 4.3 Gb of clean data with 28,703,866 clean reads was obtained after filtering. ONT data were used to remove sequences with an average quality value ≤7, and 5.4 Gb of data with 504,813 reads were obtained after filtering. Then, ONT sequences were assembled using the Canu assembler (version 2.2, https://github.com/marbl/canu), and Pilon software (version 1.24, https://github.com/broadinstitute/pilon) was used for assembly polishing with Illumina short reads to improve genome quality with 2,416 bases corrected. BUSCO (Benchmarking Universal Single-Copy Orthologs: http://busco.ezlab.org/) evaluation uses a single-copy orthologous gene library combined to evaluate the integrity of the assembled genomes.The rRNA genes were found by using Barrnap (version 0.9), and tRNA genes were found by using tRNAscan-SE (version 2.0.8) with default settings. MAKER (version 3.01.03) was used to predict the open reading frame (ORF) with default parameters. The predicted gene sequences were translated and searched against the National Center for Biotechnology Information (NCBI) nonredundant database. The UniProt/Swiss-Prot, protein families (Pfam), Clusters of Orthologous Group (COG), and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases were used for annotation by blastP with E-values of ≤1e−5. Additional annotation was carried out using the following databases: Pathogen Host Interactions (PHI), Antibiotic Resistance Genes Database (ARDB), and Carbohydrate Active enZYmes (CAZy) by blastP with E-values of ≤1e−5.The results showed that the genome size was 32,360,171 bp, average gene length (bP) was 1,855.58 bp, GC content in the gene region was 50.65%, KEGG gene number was 3,722, COG gene number was 9,557, GO gene number was 7,006, total number of tRNA was 110, and total number of rRNA was 18 (Fig. 1; Table 1). BUSCO assessed that the completeness of assembly result was 98.3% (Supplementary Table S1).Fig. 1. Circos-plot of genomic features of Curvularia clavata YC1106. From outside to inside, it represents chromosome, positive-stranded gene, negative-stranded gene, ncRNA, GC content, and GC skew.Download as PowerPointTable 1. Genome features of Curvularia clavata YC1106 strainFeaturesNumbersGenome size (bp)32,360,171Scaffold no.8GC Content (%)50.65CDS no.10,838Genes of KEGG3,722Genes of COG9,557Genes of GO7,006tRNA no.110rRNA no.18Scaf N50 (bp)4,749,760Scaf N90 (bp)2,896,455Gene no.10,838 Download table Table 1. Genome features of Curvularia clavata YC1106 strainView as image HTML Furthermore, the CAZy database was used to perform functional annotation of the genes of C. clavata. CAZy is a professional data annotation database related to carbohydrate enzymes (Zhao et al. 2013). In this study, the CAZy database was used to annotate C. clavata. The number of genes annotated as CAZymes was 526, and a total of five database classification types were annotated. The number of genes from high to low were glycoside hydrolases (GHs, 227), auxiliary activities (AAs, 128), carbohydrate esterases (CEs, 87), glycosyl transfers (GTs, 65), and polysaccharide lyases (PLs, 16). The CARD database is constructed in the form of antibiotic resistance ontology (ARO), which is used to correlate antibiotic modules and their targets, resistance mechanisms, gene variations, and other information. In this study, the information on drug resistance genes contained in each genome was obtained through the annotation of the CARD database. It was found that the drug resistance genes of C. clavata mainly targeted the antibiotics tetracycline, fluoroquinolone, macrolide, rifamycin, and cephalosporin. Based on the PHI database, the pathogen-host interaction was analyzed, and the phenotype was classified according to the number of annotated genes. Among them, the number of interacting phenotypes of key pathogenic genes, including increased virus (hypervirulence), effector (plant availability determinant), and enhanced antigen genes, was 124, 17, and one, respectively. 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Cai contributed equally to this work.Funding: This research was supported by the Major Science and Technology Program of China Tobacco Corporation (110202101055 [LS-15]), Science and Technology Program of Guizhou Tobacco Company [2021XM12], National Natural Science Foundation of China (No. 32160522), and Guizhou Provincial Academician Workstation of Microbiology and Health project ([2020] 4004).The author(s) declare no conflict of interest.DetailsFiguresLiterature CitedRelated Just PublishedSubscribeISSN:0191-2917e-ISSN:1943-7692 Metrics Article History Published: 22 May 2023Accepted: 22 Nov 2022 Information© 2023 The American Phytopathological SocietyFundingMajor Science and Technology Program of China Tobacco CorporationGrant/Award Number: 110202101055 [LS-15]Science and Technology Program of Guizhou Tobacco CompanyGrant/Award Number: 2021XM12National Natural Science Foundation of ChinaGrant/Award Number: 32160522Guizhou Provincial Academician Workstation of Microbiology and HealthGrant/Award Number: [2020] 4004KeywordsCurvularia clavatagenome sequencingOxford nanopore technologyThe author(s) declare no conflict of interest.PDF download