Molecular characterization of a novel non-segmented double-stranded RNA mycovirus isolated from the phytopathogenic fungus Lasiodiplodia pseudotheobromae

Molecular characterization of a novel non-segmented double-stranded RNA mycovirus isolated from the phytopathogenic fungus Lasiodiplodia pseudotheobromae | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Molecular characterization of a novel non-segmented double-stranded RNA mycovirus isolated from the phytopathogenic fungus Lasiodiplodia pseudotheobromae Zhengzhe Guan, Mengjiao Wang, Jiayi Ma, Mengyuan Mu, Xinyu Li, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5757021/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 08 Mar, 2025 Read the published version in Archives of Virology → Version 1 posted 5 You are reading this latest preprint version Abstract In this study, we isolated a novel mycovirus from Lasiodiplodia pseudotheobromae strain YY-1, which we named “Lasiodiplodia pseudotheobromae Mycovirus 1” (LpMyV1). The complete genome of LpMyV1 is 2877 bp in length and contains two non-overlapping open reading frames (ORF1 and ORF2). ORF1 encodes a putative protein of 316 amino acids with a molecular weight of 34.1 kDa, which shares similarity with the coat proteins of several mycoviruses. ORF2 encodes a protein of 561 amino acids with a molecular weight of 63.2 kDa, which contains a conserved RNA-dependent RNA polymerase (RdRp) domain. A BLASTp results show that the RdRp of LpMyV1 shares the highest similarity with non-segmented dsRNA viruses. Multiple sequence alignment and phylogenetic analysis indicate that LpMyV1 is a new member of the proposed genus “ Unirnavirus ”. It is the first report of a mycovirus genome sequence from L. pseudotheobromae . Figures Figure 1 Figure 2 Introduction Mycoviruses are widely distributed among various eukaryotic organisms, including mushrooms, yeasts, filamentous fungi, and notably, plant-pathogenic fungi [ 1 , 2 ]. According to the International Committee on Taxonomy of Viruses (ICTV, https://ictv.global/ ), the genomes of mycoviruses are classified into four types: single-stranded DNA (ssDNA), negative-sense single-stranded RNA (-ssRNA), positive-sense single-stranded RNA (+ ssRNA), and double-stranded RNA (dsRNA) [ 3 – 5 ]. The dsRNA viruses are further grouped into eleven families ( Amalgaviridae , Chrysoviridae , Curvulaviridae , Endornaviridae , Megabirnaviridae , Partitiviridae , Picobirnaviridae , Polymycoviridae , Quadriviridae , Spinareoviridae , and Totiviridae ) and one unassigned genus ( Botybirnavirus ) [ 6 – 8 ]. As high-throughput sequencing technologies, including virome analysis, advance, an increasing number of mycoviruses have been identified. Consequently, numerous novel viral taxa are anticipated to be established. Most mycovirus infections do not elicit conspicuous phenotypic alterations in their fungal hosts. Nonetheless, a select few mycoviruses can modify host characteristics such as growth rate, sporulation efficiency, and virulence, resulting in hypovirulence or hypervirulence [ 4 , 9 , 10 ]. The application of hypovirulent mycoviruses as biocontrol agents for plant diseases has garnered increasing interest. For example, Cryphonectria hypovirus 1 (CHV1) has been effectively utilized to combat chestnut blight [ 11 , 12 ]. Similarly, Sclerotinia sclerotiorum hypovirulence-associated DNA virus 1 (SsHADV-1) demonstrates the capacity to mitigate diseases caused by S. sclerotiorum . Notably, the SsHADV-1-infected strain DT-8 not only suppresses the pathogen but also endophytically colonizes rapeseed, fostering plant growth and bolstering host immunity [ 13 – 15 ]. These findings underscore the substantial potential of mycoviruses as biocontrol tools for managing crop diseases. Lasiodiplodia pseudotheobromae belongs to the order Botryosphaeriales and the family Botryosphaeriaceae. This fungus infects a wide range of plants, including papaya, citrus, and peanut, causing rot diseases [ 16 – 18 ]. The use of mycoviruses to control diseases caused by L. pseudotheobromae is a topic currently under discussion. To the best of our knowledge, no mycoviruses that infect L. pseudotheobromae have been reported to date. Here, we isolated a novel non-segmented double-stranded RNA (dsRNA) mycovirus from the L. pseudotheobromae strain YY-1, which we have designated as "Lasiodiplodia pseudotheobromae Mycovirus 1" (LpMyV1), and its genome was subsequently characterized. Provenance of the virus material The L. pseudotheobromae strain YY-1 was isolated from a peanut plant exhibiting symptoms of stem rot of peanut in Yuanyang County, Xinxiang City, Henan Province, China. The initial culture was grown on PDA plates at 28°C in darkness for 2–3 days. Subsequently, fresh mycelium was transferred to PDA medium overlaid with sterile cellophane and further incubated for an additional 2–3 days. Extraction and purification of double-stranded RNA (dsRNA) were performed as previously described [ 19 ], using CF11 cellulose powder (Sigma-Aldrich, China) for selective dsRNA adsorption. The purified dsRNA was treated with RNase-free DNase I and S1 nuclease (Takara, Dalian, China) to eliminate contaminating nucleic acids. Finally, the processed dsRNA was analyzed by electrophoresis on a 1.5% agarose gel at 120 V for 50 minutes. Following the methodology outlined in [ 19 ], initial sequence cloning was executed employing random primers through RACE-RT. Subsequently, specific primers were crafted based on these initially obtained sequences to amplify and bridge gaps between fragments via RT-PCR. To access the untranslated regions (UTRs) at the 5' and 3' termini, an anchor primer (pC3-T7loop: 5'-p) was ligated to the ends of double-stranded RNA using T4 RNA ligase. This step facilitated the completion of the UTRs. RT-PCR was then carried out using the pC2 primer alongside terminal-specific primers. All resulting PCR products were cloned into the pMD19-T vector (Takara, Dalian, China) and transformed into Escherichia coli DH5α cells (Takara, Dalian, China) for sequencing purposes. A comprehensive list of primers utilized for cDNA cloning and sequencing is provided in Supplementary Table S1 . All sequencing results were assembled using SnapGene 6.0.2 software. Open reading frames (ORFs) and conserved domains of the dsRNA were predicted through NCBI (ncbi.nlm.nih.gov). Sequence alignment of the mycovirus under study with other mycoviruses members was conducted using the CLUSTALX (2.0) program [ 20 ]. Phylogenetic analysis was performed with MEGA11 software, employing the maximum likelihood (ML) method with 1000 bootstrap replicates [ 21 ]. The resulting phylogenetic tree was further refined and visualized using ITOL (iTOL: Interactive Tree of Life, embl.de) [ 22 ]. Sequence properties From L. pseudotheobromae strain YY-1 (Fig. 1 A), we isolated two double-stranded RNAs (dsRNA1 and dsRNA2) (Fig. 1 B). dsRNA2 was identified as LpMyV1. The complete genome of LpMyV1 is 2877 nucleotides (nt) in length, with a GC content of 58%. The GenBank accession number for LpMyV1 is PQ790082. The 5’-untranslated region (UTR) and 3’-UTR of LpMyV1 are 17 nt and 45 nt, respectively. LpMyV1 contains two non-overlapping open reading frames (ORF1 and ORF2), separated by a 179 nt non-coding region. The genomic structure is shown in Fig. 1 C. The nucleotide sequence of LpMyV1-ORF1 spans positions 17 to 967, encoding a putative protein of 316 amino acids (aa) with an estimated molecular weight of 34.1 kDa. A BLASTp search indicates that the protein encoded by LpMyV1-ORF1 shows significant similarity to the coat proteins of several mycoviruses, including Colletotrichum higginsianum non-segmented dsRNA virus 1 (accession number YP_009177216.1; identity, 52.41%; coverage, 98%; E-value, 2.00E-83) and Fusarium culmorum virus 1 (accession number QIC51518.1; identity, 51.85%; coverage, 85%; E-value, 3.00E-83). The nucleotide sequence of LpMyV1-ORF2 spans positions 1147 to 2832, encoding a protein of 561 amino acids with a predicted molecular weight of 63.2 kDa. This protein contains a conserved RNA-dependent RNA polymerase (RdRp) domain (accession number cl40470) spanning amino acids 181 to 411. BLASTp results further reveal that LpMyV1-ORF2 shares high similarity with the RdRp of Aspergillus lentulus non-segmented dsRNA virus 1 (accession number BCH36647.1; identity, 67.28%; coverage, 96%; E-value, 0) and Colletotrichum higginsianum non-segmented dsRNA virus 1 (accession number YP_009177217.1; identity, 63.14%; coverage, 97%; E-value, 0). The analysis of RdRp amino acid sequences encoded by LpMyV1-ORF2 and other unassigned dsRNA viruses revealed three conserved motifs (Ⅳ-Ⅵ) characteristic of viral RNA-dependent polymerases, as determined through multiple sequence alignment (Fig. 2 A). To further investigate the relationship between LpMyV1 and other dsRNA mycoviruses, a phylogenetic analysis based on the RdRp sequence was conducted (Fig. 2 B). The results indicated that LpMyV1 clusters with the proposed genus " Unirnavirus " and shares a distant relationship with members of the family Curvulaviridae . However, it is important to note that mycoviruses in the Curvulaviridae family possess two dsRNA segments, whereas viruses in the " Unirnavirus " genus have a single dsRNA segment. Based on the above results, we propose that LpMyV1 is a novel non-segmented dsRNA mycovirus belonging to the proposed genus " Unirnavirus .". The LpMyV1 is the first unassigned dsRNA virus reported to infect L. pseudotheobromae , and it also the first report of a mycovirus genome sequence from L. pseudotheobromae . Declarations Acknowledgements We express our deep gratitude to Lin zhou (Henan Agricultural University) for providing mate-rials and advices. Funding This work was financially supported by the National Natural Science Foundation of China (32472642; 32302331); Natural Science Foundation of Henan Province (232300420013); Science and Technology Planning Project of Henan Province of China (242102110190); Henan Provincial Science and Technology Major Project (221100110100). Authors' contributions YiZ designed the research. ZG, MW, JM, MM, XL, PS, MZ and YuZ performed the experimental work. ZG, MW, LS, CW and YiZ analyzed the data and wrote the manuscript. Conflict of interest All authors declare no competing interests. Ethical approval This article does not contain any studies involving human participants or animals. References Xie J, Jiang D (2014) New insights into mycoviruses and exploration for the biological control of crop fungal diseases. Annu Rev Phytopathol 52(1):45–68 Ghabrial SA, Caston JR, Jiang D, Nibert ML, Suzuki N (2015) 50-plus years of fungal viruses. Virology 479–480:356–368 Xie J, Jiang D (2024) Understanding the diversity, evolution, ecology, and applications of mycoviruses. Annu Rev Microbiol 78(1):595–620 Kotta-Loizou I (2021) Mycoviruses and their role in fungal pathogenesis. 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J Fungi 10(3):179 Shahi S, Chiba S, Kondo H, Suzuki N (2021) Cryphonectria nitschkei chrysovirus 1 with unique molecular features and a very narrow host range. Virology 554:55–65 Kondo H, Botella L, Suzuki N (2022) Mycovirus diversity and evolution revealed/inferred from recent. Annu Rev Phytopathol 60(1):307–336 Anagnostakis SL (1982) Biological control of chestnut blight. Science 215(4532):466–471 Milgroom MG, Cortesi PJARP (2004) Biological control of chestnut blight with hypovirulence: a critical analysis. Annu Rev Phytopathol 42(1):311–338 Yu X, Li B, Fu Y, Xie J, Cheng J, Ghabrial SA, Li G, Yi X, Jiang D (2013) Extracellular transmission of a DNA mycovirus and its use as a natural fungicide. Proc Natl Acad Sci U S A 110(4):1452–1457 Zhang H, Xie J, Fu Y, Cheng J, Qu Z, Zhao Z, Cheng S, Chen T, Li B, Wang Q, Liu X, Tian B, Collinge DB, Jiang D (2020) A 2-kb mycovirus converts a pathogenic fungus into a beneficial endophyte for Brassica protection and yield enhancement. Mol Plant 13(10):1420–1433 Qu Z, Fu Y, Lin Y, Zhao Z, Zhang X, Cheng J, Xie J, Chen T, Li B, Jiang D (2021) Transcriptional responses of Sclerotinia sclerotiorum to the infection by SsHADV-1. J Fungi 7(7):493 Gomes ACA, da Costa Lima M, de Oliveira KÁR, Dos Santos Lima M, Magnani M, Câmara MPS, de Souza EL (2020) Coatings with chitosan and phenolic-rich extract from acerola ( Malpighia emarginata D.C.) or jabuticaba ( Plinia jaboticaba (Vell.) Berg) processing by-product to control rot caused by Lasiodiplodia spp. in papaya ( Carica papaya L.) fruit. Int J Food Microbiol 331:108694 Chen J, Zhu Z, Fu Y, Cheng J, Xie J, Lin Y (2021) Identification of Lasiodiplodia pseudotheobromae causing fruit rot of citrus in China. Plants 10(2):202 Zhang X, Li Y, Xu M, Guo Z, Yu J, Song X, He K, Zhang Z, Chi Y (2022) First report of Lasiodiplodia pseudotheobromae causing collar rot of peanut in Shandong Province, China. Plant Dis 106(7):1982 Zhao Y, Zhang Y, Wan X, She Y, Li M, Xi H, Xie J, Wen C (2020) A novel Ourmia-Like mycovirus confers hypovirulence-associated traits on Fusarium oxysporum . Front Microbiol 11:569869 Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG (1997) The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 25(24):4876–4882 Tamura K, Stecher G, Kumar S (2021) MEGA11: Molecular Evolutionary Genetics Analysis Version 11. Mol Biol Evol 38(7):3022–3027 Letunic I, Bork P (2021) Interactive Tree of Life (iTOL) v5: an online tool for phylogenetic tree display and annotation. Nucleic Acids Res (W1): W293–W296 Supplementary Files Submission2906990.pdf supplementarymaterial.pdf Cite Share Download PDF Status: Published Journal Publication published 08 Mar, 2025 Read the published version in Archives of Virology → Version 1 posted Editorial decision: Major Revision 03 Feb, 2025 Reviewers agreed at journal 09 Jan, 2025 Reviewers invited by journal 09 Jan, 2025 Editor assigned by journal 08 Jan, 2025 First submitted to journal 02 Jan, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5757021","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":400018974,"identity":"e0f43b43-1179-4281-9292-723ec1ec3032","order_by":0,"name":"Zhengzhe Guan","email":"","orcid":"","institution":"Henan Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Zhengzhe","middleName":"","lastName":"Guan","suffix":""},{"id":400018975,"identity":"56d50f6c-a439-4f58-9537-60d9ee6c6894","order_by":1,"name":"Mengjiao Wang","email":"","orcid":"","institution":"Henan Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Mengjiao","middleName":"","lastName":"Wang","suffix":""},{"id":400018976,"identity":"0fe8139a-dca8-45c5-a355-847d4f6af2c4","order_by":2,"name":"Jiayi Ma","email":"","orcid":"","institution":"Henan Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Jiayi","middleName":"","lastName":"Ma","suffix":""},{"id":400018977,"identity":"f76420cc-7c19-430f-86b7-37b2f28caf32","order_by":3,"name":"Mengyuan Mu","email":"","orcid":"","institution":"Henan Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Mengyuan","middleName":"","lastName":"Mu","suffix":""},{"id":400018978,"identity":"7d17ddee-75d5-448d-b7e0-04610b386d69","order_by":4,"name":"Xinyu Li","email":"","orcid":"","institution":"Henan Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Xinyu","middleName":"","lastName":"Li","suffix":""},{"id":400018979,"identity":"96dcdd8b-e559-41c0-ade3-c1c00aac2ac7","order_by":5,"name":"Peimeng Sun","email":"","orcid":"","institution":"Henan Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Peimeng","middleName":"","lastName":"Sun","suffix":""},{"id":400018980,"identity":"03864798-0ffe-4526-9833-8abbf6d665f3","order_by":6,"name":"Mengyuan Zhang","email":"","orcid":"","institution":"Henan Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Mengyuan","middleName":"","lastName":"Zhang","suffix":""},{"id":400018981,"identity":"0e7b78b2-d722-4366-985d-2a2d41eb9946","order_by":7,"name":"Yuanyuan Zhang","email":"","orcid":"","institution":"Henan Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Yuanyuan","middleName":"","lastName":"Zhang","suffix":""},{"id":400018982,"identity":"33d2b21a-f596-43e8-8e4e-755dfc80d251","order_by":8,"name":"Luyang Song","email":"","orcid":"","institution":"Henan Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Luyang","middleName":"","lastName":"Song","suffix":""},{"id":400018983,"identity":"5bf175cc-ea3f-481d-901f-81aa727c453b","order_by":9,"name":"Caiyi Wen","email":"","orcid":"","institution":"Henan Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Caiyi","middleName":"","lastName":"Wen","suffix":""},{"id":400018984,"identity":"ea5dde70-c8b9-4def-8552-f3cfb0aee157","order_by":10,"name":"Ying Zhao","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA70lEQVRIiWNgGAWjYDACCRBhwMDALwETOUCsFskZpGkB6bpBrBb52c0PH/MU3LHbfLv52IOfbQxyfDcSGD8X4NHCOOeYsTGPwbPkbXeOpRv2tjEYS95IYJaegUcLs0SCmTSPweFksxs5ZhK8bQyJG24ksDHz4NHCJpH+DazFeEb+N8m/bQz1BLXwSOSAbbEzkMhhkwbakmBASIuERE6x4RyDwwkSN9LMpGXOSRjOPPOwWRqfFvkZ6RsfvPlz2J5/RvIzyTdlNvJ8x5MPfsanBQSYgAoSG6C2AjFjAwENQCU/GBjsCaoaBaNgFIyCkQsA+3ZIgz2rkfUAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0002-3948-5442","institution":"Henan Agricultural University","correspondingAuthor":true,"prefix":"","firstName":"Ying","middleName":"","lastName":"Zhao","suffix":""}],"badges":[],"createdAt":"2025-01-03 09:18:25","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5757021/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5757021/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s00705-025-06263-z","type":"published","date":"2025-03-08T15:58:37+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":73651953,"identity":"6372966f-023a-49bb-8220-2a8442b565f3","added_by":"auto","created_at":"2025-01-13 09:48:49","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":616758,"visible":true,"origin":"","legend":"\u003cp\u003eThe colony morphology of strain YY-1; electrophoresis of LpMyV1 and its genome structure diagram. \u003cstrong\u003e(A)\u003c/strong\u003e Colony morphology of \u003cem\u003eL. pseudotheobromae\u003c/em\u003e strain YY-1. \u003cstrong\u003e(B)\u003c/strong\u003e Agarose gel electrophoresis of dsRNA treated with DNase I and S1 Nuclease. \u003cstrong\u003e(C)\u003c/strong\u003e Genome structure diagram of LpMyV1.\u003c/p\u003e","description":"","filename":"Figrue1.png","url":"https://assets-eu.researchsquare.com/files/rs-5757021/v1/57cad6260ee3bd25eb434ae1.png"},{"id":73653725,"identity":"4bcc0a91-40ef-4d76-b719-dce0b1f9a6e1","added_by":"auto","created_at":"2025-01-13 09:56:49","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1221654,"visible":true,"origin":"","legend":"\u003cp\u003eMultiple sequence alignment and phylogenetic analysis of LpMyV1 \u003cstrong\u003e(A)\u003c/strong\u003e Amino acid multisequence alignment of LpMyV1 with RdRp of other nonsegmented dsRNA mycoviruses. Similar amino acid residues are shaded in gray. \u003cstrong\u003e(B)\u003c/strong\u003e Phylogenetic analysis of the RdRp of LpMyV1 and other mycoviruses via the maximum likelihood (ML) method in MEGA 11 with 1000 bootstrap replicates. LpMyV1 was shown in red.\u003c/p\u003e","description":"","filename":"Figrue2.png","url":"https://assets-eu.researchsquare.com/files/rs-5757021/v1/c3ba7874833e1746d829719c.png"},{"id":78190688,"identity":"fea50a75-35bc-479e-9bdc-477bff4cae88","added_by":"auto","created_at":"2025-03-10 19:50:33","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2160987,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5757021/v1/375bcf61-0959-44f7-adce-814a77cb51d0.pdf"},{"id":73651957,"identity":"631963a9-2b0c-4f87-b0cf-5eefe94f1b96","added_by":"auto","created_at":"2025-01-13 09:48:49","extension":"pdf","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":16589,"visible":true,"origin":"","legend":"","description":"","filename":"Submission2906990.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5757021/v1/640be7f809ca9205ea51ff24.pdf"},{"id":73653726,"identity":"46eb7f5c-ecbb-4847-b66c-fd2116cfa07b","added_by":"auto","created_at":"2025-01-13 09:56:49","extension":"pdf","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":102354,"visible":true,"origin":"","legend":"","description":"","filename":"supplementarymaterial.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5757021/v1/d24e1c5a15b19b40eb115e35.pdf"}],"financialInterests":"","formattedTitle":"Molecular characterization of a novel non-segmented double-stranded RNA mycovirus isolated from the phytopathogenic fungus Lasiodiplodia pseudotheobromae","fulltext":[{"header":"Introduction","content":"\u003cp\u003eMycoviruses are widely distributed among various eukaryotic organisms, including mushrooms, yeasts, filamentous fungi, and notably, plant-pathogenic fungi [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. According to the International Committee on Taxonomy of Viruses (ICTV, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://ictv.global/\u003c/span\u003e\u003cspan address=\"https://ictv.global/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), the genomes of mycoviruses are classified into four types: single-stranded DNA (ssDNA), negative-sense single-stranded RNA (-ssRNA), positive-sense single-stranded RNA (+ ssRNA), and double-stranded RNA (dsRNA) [\u003cspan additionalcitationids=\"CR4\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e–\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. The dsRNA viruses are further grouped into eleven families (\u003cem\u003eAmalgaviridae\u003c/em\u003e, \u003cem\u003eChrysoviridae\u003c/em\u003e, \u003cem\u003eCurvulaviridae\u003c/em\u003e, \u003cem\u003eEndornaviridae\u003c/em\u003e, \u003cem\u003eMegabirnaviridae\u003c/em\u003e, \u003cem\u003ePartitiviridae\u003c/em\u003e, \u003cem\u003ePicobirnaviridae\u003c/em\u003e, \u003cem\u003ePolymycoviridae\u003c/em\u003e, \u003cem\u003eQuadriviridae\u003c/em\u003e, \u003cem\u003eSpinareoviridae\u003c/em\u003e, and \u003cem\u003eTotiviridae\u003c/em\u003e) and one unassigned genus (\u003cem\u003eBotybirnavirus\u003c/em\u003e) [\u003cspan additionalcitationids=\"CR7\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e–\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. As high-throughput sequencing technologies, including virome analysis, advance, an increasing number of mycoviruses have been identified. Consequently, numerous novel viral taxa are anticipated to be established.\u003c/p\u003e \u003cp\u003eMost mycovirus infections do not elicit conspicuous phenotypic alterations in their fungal hosts. Nonetheless, a select few mycoviruses can modify host characteristics such as growth rate, sporulation efficiency, and virulence, resulting in hypovirulence or hypervirulence [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. The application of hypovirulent mycoviruses as biocontrol agents for plant diseases has garnered increasing interest. For example, Cryphonectria hypovirus 1 (CHV1) has been effectively utilized to combat chestnut blight [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Similarly, Sclerotinia sclerotiorum hypovirulence-associated DNA virus 1 (SsHADV-1) demonstrates the capacity to mitigate diseases caused by \u003cem\u003eS. sclerotiorum\u003c/em\u003e. Notably, the SsHADV-1-infected strain DT-8 not only suppresses the pathogen but also endophytically colonizes rapeseed, fostering plant growth and bolstering host immunity [\u003cspan additionalcitationids=\"CR14\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e–\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. These findings underscore the substantial potential of mycoviruses as biocontrol tools for managing crop diseases.\u003c/p\u003e \u003cp\u003e \u003cem\u003eLasiodiplodia pseudotheobromae\u003c/em\u003e belongs to the order Botryosphaeriales and the family Botryosphaeriaceae. This fungus infects a wide range of plants, including papaya, citrus, and peanut, causing rot diseases [\u003cspan additionalcitationids=\"CR17\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e–\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. The use of mycoviruses to control diseases caused by \u003cem\u003eL. pseudotheobromae\u003c/em\u003e is a topic currently under discussion. To the best of our knowledge, no mycoviruses that infect \u003cem\u003eL. pseudotheobromae\u003c/em\u003e have been reported to date. Here, we isolated a novel non-segmented double-stranded RNA (dsRNA) mycovirus from the \u003cem\u003eL. pseudotheobromae\u003c/em\u003e strain YY-1, which we have designated as \"Lasiodiplodia pseudotheobromae Mycovirus 1\" (LpMyV1), and its genome was subsequently characterized.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Provenance of the virus material","content":"\u003cp\u003eThe \u003cem\u003eL. pseudotheobromae\u003c/em\u003e strain YY-1 was isolated from a peanut plant exhibiting symptoms of stem rot of peanut in Yuanyang County, Xinxiang City, Henan Province, China. The initial culture was grown on PDA plates at 28°C in darkness for 2–3 days. Subsequently, fresh mycelium was transferred to PDA medium overlaid with sterile cellophane and further incubated for an additional 2–3 days. Extraction and purification of double-stranded RNA (dsRNA) were performed as previously described [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], using CF11 cellulose powder (Sigma-Aldrich, China) for selective dsRNA adsorption. The purified dsRNA was treated with RNase-free DNase I and S1 nuclease (Takara, Dalian, China) to eliminate contaminating nucleic acids. Finally, the processed dsRNA was analyzed by electrophoresis on a 1.5% agarose gel at 120 V for 50 minutes.\u003c/p\u003e\u003cp\u003eFollowing the methodology outlined in [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], initial sequence cloning was executed employing random primers through RACE-RT. Subsequently, specific primers were crafted based on these initially obtained sequences to amplify and bridge gaps between fragments via RT-PCR. To access the untranslated regions (UTRs) at the 5' and 3' termini, an anchor primer (pC3-T7loop: 5'-p) was ligated to the ends of double-stranded RNA using T4 RNA ligase. This step facilitated the completion of the UTRs. RT-PCR was then carried out using the pC2 primer alongside terminal-specific primers. All resulting PCR products were cloned into the pMD19-T vector (Takara, Dalian, China) and transformed into \u003cem\u003eEscherichia coli\u003c/em\u003e DH5α cells (Takara, Dalian, China) for sequencing purposes. A comprehensive list of primers utilized for cDNA cloning and sequencing is provided in Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e.\u003c/p\u003e\u003cp\u003eAll sequencing results were assembled using SnapGene 6.0.2 software. Open reading frames (ORFs) and conserved domains of the dsRNA were predicted through NCBI (ncbi.nlm.nih.gov). Sequence alignment of the mycovirus under study with other mycoviruses members was conducted using the CLUSTALX (2.0) program [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Phylogenetic analysis was performed with MEGA11 software, employing the maximum likelihood (ML) method with 1000 bootstrap replicates [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. The resulting phylogenetic tree was further refined and visualized using ITOL (iTOL: Interactive Tree of Life, embl.de) [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e"},{"header":"Sequence properties","content":"\u003cp\u003eFrom \u003cem\u003eL. pseudotheobromae\u003c/em\u003e strain YY-1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA), we isolated two double-stranded RNAs (dsRNA1 and dsRNA2) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). dsRNA2 was identified as LpMyV1. The complete genome of LpMyV1 is 2877 nucleotides (nt) in length, with a GC content of 58%. The GenBank accession number for LpMyV1 is PQ790082. The 5’-untranslated region (UTR) and 3’-UTR of LpMyV1 are 17 nt and 45 nt, respectively. LpMyV1 contains two non-overlapping open reading frames (ORF1 and ORF2), separated by a 179 nt non-coding region. The genomic structure is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC.\u003c/p\u003e\u003cp\u003e \u003c/p\u003e\u003cp\u003eThe nucleotide sequence of LpMyV1-ORF1 spans positions 17 to 967, encoding a putative protein of 316 amino acids (aa) with an estimated molecular weight of 34.1 kDa. A BLASTp search indicates that the protein encoded by LpMyV1-ORF1 shows significant similarity to the coat proteins of several mycoviruses, including Colletotrichum higginsianum non-segmented dsRNA virus 1 (accession number YP_009177216.1; identity, 52.41%; coverage, 98%; E-value, 2.00E-83) and Fusarium culmorum virus 1 (accession number QIC51518.1; identity, 51.85%; coverage, 85%; E-value, 3.00E-83). The nucleotide sequence of LpMyV1-ORF2 spans positions 1147 to 2832, encoding a protein of 561 amino acids with a predicted molecular weight of 63.2 kDa. This protein contains a conserved RNA-dependent RNA polymerase (RdRp) domain (accession number cl40470) spanning amino acids 181 to 411. BLASTp results further reveal that LpMyV1-ORF2 shares high similarity with the RdRp of Aspergillus lentulus non-segmented dsRNA virus 1 (accession number BCH36647.1; identity, 67.28%; coverage, 96%; E-value, 0) and Colletotrichum higginsianum non-segmented dsRNA virus 1 (accession number YP_009177217.1; identity, 63.14%; coverage, 97%; E-value, 0).\u003c/p\u003e\u003cp\u003eThe analysis of RdRp amino acid sequences encoded by LpMyV1-ORF2 and other unassigned dsRNA viruses revealed three conserved motifs (Ⅳ-Ⅵ) characteristic of viral RNA-dependent polymerases, as determined through multiple sequence alignment (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). To further investigate the relationship between LpMyV1 and other dsRNA mycoviruses, a phylogenetic analysis based on the RdRp sequence was conducted (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). The results indicated that LpMyV1 clusters with the proposed genus \"\u003cem\u003eUnirnavirus\u003c/em\u003e\" and shares a distant relationship with members of the family \u003cem\u003eCurvulaviridae\u003c/em\u003e. However, it is important to note that mycoviruses in the \u003cem\u003eCurvulaviridae\u003c/em\u003e family possess two dsRNA segments, whereas viruses in the \"\u003cem\u003eUnirnavirus\u003c/em\u003e\" genus have a single dsRNA segment. Based on the above results, we propose that LpMyV1 is a novel non-segmented dsRNA mycovirus belonging to the proposed genus \"\u003cem\u003eUnirnavirus\u003c/em\u003e.\". The LpMyV1 is the first unassigned dsRNA virus reported to infect \u003cem\u003eL. pseudotheobromae\u003c/em\u003e, and it also the first report of a mycovirus genome sequence from \u003cem\u003eL. pseudotheobromae\u003c/em\u003e.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u0026nbsp;\u003c/strong\u003eWe express our deep gratitude to Lin zhou (Henan Agricultural University) for providing mate-rials and advices.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003eThis work was financially supported by the National Natural Science Foundation of China (32472642; 32302331); Natural Science Foundation of Henan Province (232300420013); Science and Technology Planning Project of Henan Province of China (242102110190); Henan Provincial Science and Technology Major Project (221100110100).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u0026nbsp;\u003c/strong\u003eYiZ designed the research. ZG, MW, JM, MM, XL, PS, MZ and YuZ performed the experimental work. ZG, MW, LS, CW and YiZ analyzed the data and wrote the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e All authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval\u0026nbsp;\u003c/strong\u003eThis article does not contain any studies involving human participants or animals.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eXie J, Jiang D (2014) New insights into mycoviruses and exploration for the biological control of crop fungal diseases. Annu Rev Phytopathol 52(1):45\u0026ndash;68\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGhabrial SA, Caston JR, Jiang D, Nibert ML, Suzuki N (2015) 50-plus years of fungal viruses. 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Nucleic Acids Res 25(24):4876\u0026ndash;4882\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTamura K, Stecher G, Kumar S (2021) MEGA11: Molecular Evolutionary Genetics Analysis Version 11. Mol Biol Evol 38(7):3022\u0026ndash;3027\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLetunic I, Bork P (2021) Interactive Tree of Life (iTOL) v5: an online tool for phylogenetic tree display and annotation. Nucleic Acids Res (W1): W293\u0026ndash;W296\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"archives-of-virology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"arvi","sideBox":"Learn more about [Archives of Virology](https://www.springer.com/journal/705)","snPcode":"705","submissionUrl":"https://submission.nature.com/new-submission/705/3","title":"Archives of Virology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-5757021/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5757021/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eIn this study, we isolated a novel mycovirus from \u003cem\u003eLasiodiplodia pseudotheobromae\u003c/em\u003e strain YY-1, which we named \u0026ldquo;Lasiodiplodia pseudotheobromae Mycovirus 1\u0026rdquo; (LpMyV1). The complete genome of LpMyV1 is 2877 bp in length and contains two non-overlapping open reading frames (ORF1 and ORF2). ORF1 encodes a putative protein of 316 amino acids with a molecular weight of 34.1 kDa, which shares similarity with the coat proteins of several mycoviruses. ORF2 encodes a protein of 561 amino acids with a molecular weight of 63.2 kDa, which contains a conserved RNA-dependent RNA polymerase (RdRp) domain. A BLASTp results show that the RdRp of LpMyV1 shares the highest similarity with non-segmented dsRNA viruses. Multiple sequence alignment and phylogenetic analysis indicate that LpMyV1 is a new member of the proposed genus \u0026ldquo;\u003cem\u003eUnirnavirus\u003c/em\u003e\u0026rdquo;. It is the first report of a mycovirus genome sequence from \u003cem\u003eL. pseudotheobromae\u003c/em\u003e.\u003c/p\u003e","manuscriptTitle":"Molecular characterization of a novel non-segmented double-stranded RNA mycovirus isolated from the phytopathogenic fungus Lasiodiplodia pseudotheobromae","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-01-13 09:48:44","doi":"10.21203/rs.3.rs-5757021/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major Revision","date":"2025-02-03T14:58:05+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2025-01-09T14:41:12+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-01-09T14:38:36+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-01-08T14:48:03+00:00","index":"","fulltext":""},{"type":"submitted","content":"Archives of Virology","date":"2025-01-03T04:18:00+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"archives-of-virology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"arvi","sideBox":"Learn more about [Archives of Virology](https://www.springer.com/journal/705)","snPcode":"705","submissionUrl":"https://submission.nature.com/new-submission/705/3","title":"Archives of Virology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"6935480a-7a96-4c35-9bd9-deb224d24264","owner":[],"postedDate":"January 13th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-03-10T19:47:39+00:00","versionOfRecord":{"articleIdentity":"rs-5757021","link":"https://doi.org/10.1007/s00705-025-06263-z","journal":{"identity":"archives-of-virology","isVorOnly":false,"title":"Archives of Virology"},"publishedOn":"2025-03-08 15:58:37","publishedOnDateReadable":"March 8th, 2025"},"versionCreatedAt":"2025-01-13 09:48:44","video":"","vorDoi":"10.1007/s00705-025-06263-z","vorDoiUrl":"https://doi.org/10.1007/s00705-025-06263-z","workflowStages":[]},"version":"v1","identity":"rs-5757021","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5757021","identity":"rs-5757021","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}