High-Quality Genome Assembly of <i>Neopestalotiopsis rosae</i> ML1664, the Pathogen Causing Strawberry Leaf Blight and Crown Rot

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HomeMolecular Plant-Microbe Interactions®Vol. 35, No. 10High-Quality Genome Assembly of Neopestalotiopsis rosae ML1664, the Pathogen Causing Strawberry Leaf Blight and Crown Rot PreviousNext RESOURCE ANNOUNCEMENT OPENOpen Access licenseHigh-Quality Genome Assembly of Neopestalotiopsis rosae ML1664, the Pathogen Causing Strawberry Leaf Blight and Crown RotSheng-Yu Hsu, Yu-Chen Lin, Yuan-Cheng Xu, Hao-Xun Chang, Pei-Che Chung, and Hiran A. AriyawansaSheng-Yu Hsuhttp://orcid.org/0000-0001-6227-0936Department of Plant pathology and Microbiology, National Taiwan University, No. 1, Sec. 4, Roosevelt Rd., Taipei City 10617, Taiwan, Yu-Chen Linhttp://orcid.org/0000-0002-2319-0869Department of Plant pathology and Microbiology, National Taiwan University, No. 1, Sec. 4, Roosevelt Rd., Taipei City 10617, Taiwan, Yuan-Cheng Xuhttp://orcid.org/0000-0002-3885-498XDepartment of Plant pathology and Microbiology, National Taiwan University, No. 1, Sec. 4, Roosevelt Rd., Taipei City 10617, Taiwan, Hao-Xun Changhttp://orcid.org/0000-0002-4667-7741Department of Plant pathology and Microbiology, National Taiwan University, No. 1, Sec. 4, Roosevelt Rd., Taipei City 10617, Taiwan, Pei-Che ChungMiaoli District Agricultural Research and Extension Station, Council of Agriculture, Executive Yuan, Miaoli County, 36346, Taiwan, and Hiran A. Ariyawansa†Corresponding author: H. A. Ariyawansa; E-mail Address: [email protected]://orcid.org/0000-0001-8526-7721Department of Plant pathology and Microbiology, National Taiwan University, No. 1, Sec. 4, Roosevelt Rd., Taipei City 10617, TaiwanAffiliationsAuthors and Affiliations Sheng-Yu Hsu1 Yu-Chen Lin1 Yuan-Cheng Xu1 Hao-Xun Chang1 Pei-Che Chung2 Hiran A. Ariyawansa1 † 1Department of Plant pathology and Microbiology, National Taiwan University, No. 1, Sec. 4, Roosevelt Rd., Taipei City 10617, Taiwan 2Miaoli District Agricultural Research and Extension Station, Council of Agriculture, Executive Yuan, Miaoli County, 36346, Taiwan Published Online:26 Sep 2022https://doi.org/10.1094/MPMI-04-22-0077-AAboutSectionsView articlePDFSupplemental ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InRedditEmailWechat View articleGenome AnnouncementThe genus Neopestalotiopsis was introduced by Maharachchikumbura et al. (2014) to accommodate Pestalotiopsis-like taxa (conidia usually five-celled, with three brown median cells and hyaline end cells and with two or more apical appendages arising from the apical cell) with versicolorous median cells, based on morphology coupled with phylogenetic analysis of combined genes. Species in genus Neopestalotiopsis are cosmopolitan, with either multiple species occurring in a single host or a single species found in multiple hosts (Darapanit et al. 2021; Diogo et al. 2021; Tsai et al. 2021). Currently over 70 species of Neopestalotiopsis are listed in Index Fungorum (2022). Diseases caused by Neopestalotiopsis species in Taiwan as well as globally are an emerging issue (Ayoubi and Pari 2016; Ayoubi and Soleimani 2016; Baggio et al. 2021; Chamorro et al. 2016; Hsu et al. 2020; Jayawardena et al. 2015; Liu et al. 2017; Machín et al. 2019; Obregón et al. 2018; Sun et al. 2021; Tsai et al. 2021). Recent studies done by our group and other researchers found that Neopestalotiopsis species are associated with numerous diseases in various hosts such as tea (Tsai et al. 2021) and strawberry (Wu et al. 2021) in Taiwan.Strawberry (Fragaria spp.) is one of the most economically important crops worldwide (Simpson 2018). In Taiwan, strawberries are grown in various counties and annually generate over NT$1.5 to 1.8 billion (Chung et al. 2021). Strawberry plants are, however, prone to many fungal diseases. The first report of Neopestalotiopsis rosae causing leaf blight and crown rot on strawberry was recently published by Wu et al. (2021) from Miaoli County in Taiwan. The infection became predominant and more severe during 2019 to 2020, causing up to 30% plant loss after transplanting (Wu et al. 2021). Subsequently, the same species was recently reported in Florida by Baggio et al. (2021), devastating strawberry fields in the United States and thus becoming a serious issue for the strawberry industry.The development of whole-genome sequencing technology and successive application of the Nanopore and Illumina approaches can produce genome assemblies for various important pathogenic fungi (Grigoriev et al. 2013). To date however, no genome data has been available for N. rosae. Here, we provide the first draft genome assembly of N. rosae, which will facilitate future studies of its genomic features and the pathogenic mechanism of this fungus.The Neopestalotiopsis rosae ML1664 strain (Supplementary Fig. S1) used in the present study was initially isolated from crown tissue of diseased strawberry in Hsinchu County, Taiwan (Wu et al. 2021). This strain was identified as N. rosae based on both morphological data and multilocus phylogenetic analyses (internal transcribed spacer, β-tubulin, translation elongation factor 1-α) by Wu et al. (2021). The isolate was cultured on potato dextrose agar for 7 days at 25°C. The mycelium was harvested, and mycelial DNA was extracted using the Qiagen Genomic-tip 20/G kit following manufacturer protocol. Genome sequencing of N. rosae ML1664 was performed using a combination of Nanopore and Illumina sequencing platforms at Tri-I Biotech Inc. (Taipei, Taiwan).N. rosae ML1664 long-read sequences were generated with Nanopore GridION model Mk1 (software MinKNOW v21.05.20), and Guppy v5.0.13 was used for base calling (super-accurate basecalling model). Demultiplexing and adapter trimmed analysis were done using Nanoplot version 1.28.1 (de Coster et al. 2018). NECAT was used to perform de novo assembly of N. rosae ML1664 with Nanopore long reads (Chen et al. 2021). Illumina short-read sequences were generated by Illumina NovaSeq 6000 (150 base, paired-end reads) and the Qiagen CLC Genomic Workbench version 10 was used to remove low quality sequences (Q20) and adapter sequences. Trimmed Illumina sequences were used to polish the Nanopore de novo assembly contigs, using Qiagen CLC Genomic Workbench v10 (map reads to reference). Assembly statistics and quality assessment of the genome were estimated in QUAST (Gurevich et al. 2013). The completeness of the genome was assessed using BUSCO (benchmarking universal single-copy orthologs) v5.3.0 (Seppey et al. 2019) in genome and protein modes with Fungi (n = 758), Ascomycota (n = 1,706), and Sordariomycetes (n = 3,817) lineage datasets from OrthoDB v10 (Kriventseva et al. 2019). Putative protein-coding genes were predicted by AUGUSTUS v3.3.3 (Hoff and Stanke 2013), using Fusarium graminearum as the model organism, and the putative secreted proteins were identified via SignalP version 6.0 in slow model mode (Teufel et al. 2022). Putative biosynthesis gene clusters were predicted by antiSMASH version 6.1.0 (Blin et al. 2021).The total assembly length of the genome was 53.78 Mbp. The genome consisted of 18 contigs (N50 = 6.34 Mbp) with an average length of 2.98 Mbp and GC content of 49.88%. Table 1 gives the major statistical data of gene assembly results, and Table 2 gives the statistical data of each contig. The numbers of putative protein-coding genes and candidate secreted peptides in the N. rosae ML1664 genome were 15,966 and 1,822, respectively.Table 1. Assembly statistics of the Neopestalotiopsis rosae ML1664 genomeGenome featuresValuesNanopore-generated raw reads6,155,951Genome size (bp)53,779,277Coverage (×)155.01GC content (%)49.88Contig number18Minimum length of contigs (bp)8,128Maximum length of contigs (bp)8,327,510Average length of contigs (bp)2,987,737.61Contig N50 (bp)6,346,464BUSCO v5.3.0 completeness (%)98.4AUGUSTUS v3.3.3 protein-coding sequencing15,966SignalP v6.0 secretory proteins1,822antiSMASH v6.1.0 BGCs76Table 1. Assembly statistics of the Neopestalotiopsis rosae ML1664 genomeView as image HTML Table 2. Summary of the Neopestalotiopsis rosae ML1664 genome assembly and gene prediction statistics for each contigSequenceLength (bp)GC (%)Protein-coding genesContig_18,327,51049.62,470Contig_27,578,86750.22,200Contig_36,446,31650.21,925Contig_46,346,46450.12,016Contig_54,977,43150.31,497Contig_64,811,43950.51,404Contig_73,635,56149.81,108Contig_83,563,78550.21,048Contig_93,271,70448.5889Contig_101,320,79849.7402Contig_111,137,00948.2320Contig_121,067,05150.3335Contig_13754,84549.4217Contig_14424,93649.3127Contig_1550,89128.20Contig_1634,93828.10Contig_1721,60441.77Contig_188,12847.81Table 2. Summary of the Neopestalotiopsis rosae ML1664 genome assembly and gene prediction statistics for each contigView as image HTML Completeness of the N. rosae ML1664 genome assembly and the protein-coding gene prediction were evaluated by BUSCO with Fungi_odb10, Ascomycota_odb10, and Sordariomycetes_odb10 lineages. The genome was determined to have 99.3, 98.1, and 98.4% completed BUSCOs in Fungi, Ascomycota, and Sordariomycetes, respectively. Furthermore, gene predictions showed 99.7, 99.3, and 98.6% complete orthologs in Fungi, Ascomycota, and Sordariomycetes, respectively (Fig. 1A).Fig. 1. A, The complete genome assembly and protein-coding gene prediction results assessed by BUSCO v5.3.0 in genome and protein modes, with Fungi (n = 758), Ascomycota (n = 1,706), and Sordariomycetes (n = 3,817) lineage datasets from OrthoDB v10. B, Secondary metabolite gene clusters (SMGCs) predicted by antiSMASH v6.1.0, which identified 76 SMGCs, including one betalactone, one fungal RiPP (ribosomally synthesized and posttranslationally modified peptide), four indoles, ten terpenes, 23 T1PKSs (type I polyketide synthase), one T3PKS (type III polyketide synthase), 11 NRPSs (non-ribosomal peptide synthetases), 14 NRPS-like, and 11 hybrids.Download as PowerPointDuring prediction of the biosynthesis gene clusters, antiSMASH v6.1.0 totally identified 76 biosynthesis gene clusters, including one beta-lactone, one fungal RiPP (ribosomally synthesized and posttranslationally modified peptide), four indoles, ten terpenes, 23 T1PKSs (type I polyketide synthase), one T3PKS (type III polyketide synthase), 11 nonribosomal peptide synthetases (NRPSs), 14 NRPS-like, and 11 hybrids (Fig. 1B).Data AvailabilityThe genome assembly and gene annotation data were deposited at GenBank under accession number JALGAS000000000.AcknowledgmentsThe authors appreciate the kind assistance and valuable suggestions from Y.-H. 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First report of Neopestalotiopsis rosae causing leaf blight and crown rot on strawberry in Taiwan. Plant Dis. 105:487-487. https://doi.org/10.1094/PDIS-05-20-1045-PDN Link, Google ScholarS.-Y. Hsu and Y.-C. Lin contributed equally to the project.Funding: This work was supported by the Ministry of Science and Technology (MOST), Taiwan (grant number 110-2313-B-002-031) and the Council of Agriculture, Executive Yuan, Taiwan (grant number 111AS-1.3.2-ST-aN).The author(s) declare no conflict of interest. Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.DetailsFiguresLiterature CitedRelated Vol. 35, No. 10 October 2022ISSN:0894-0282e-ISSN:1943-7706 Download Metrics Article History Issue Date: 8 Oct 2022Published: 26 Sep 2022Accepted: 8 Jul 2022 Pages: 949-953 InformationCopyright © 2022 The Author(s).This is an open access article distributed under the CC BY-NC-ND 4.0 International license.Funding Ministry of Science and Technology (MOST), TaiwanGrant/Award Number: 110-2313-B-002-031 Council of Agriculture, Executive Yuan, TaiwanGrant/Award Number: 111AS-1.3.2-ST-aN KeywordsPestalotiopsis-like fungistrawberry crown rotstrawberry leaf blightwhole-genome sequencingThe author(s) declare no conflict of interest.PDF download

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