A survey on sugarcane mosaic symptoms leads to the first report of sugarcane chlorotic streak virus in Cameroon

A survey on sugarcane mosaic symptoms leads to the first report of sugarcane chlorotic streak virus in Cameroon | 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 A survey on sugarcane mosaic symptoms leads to the first report of sugarcane chlorotic streak virus in Cameroon Jean-Heinrich Daugrois, Benoît Ahondopké, Serge Galzi, Charlotte Julian, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4604985/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Four sugarcane leaf samples from Cameroon showing mosaic like symptoms tested negative by RT-PCR for potyviruses and a poacevirus causing mosaic in sugarcane. Virus sequences sharing 98–99% nucleotide identity with Sugarcane chlorotic streak virus from Nigeria were found in these four samples by virion-associated nucleic acid metagenomics. Five complete genome sequences were obtained after cloning five isolates from Cameroon. These genome sequences shared less than 75–76% identity with the closest Mastrevirus species, thus confirming that Sugarcane chlorotic streak virus is a new Mastrevirus species. To our knowledge, this is the first report of Sugarcane chlorotic streak virus in Cameroon. Figures Figure 1 Figure 2 Figure 3 Introduction Mosaic caused by the potyviruses sugarcane mosaic virus (SCMV) and sorghum mosaic virus (SrMV) is a well-known disease of sugarcane in Africa [ 1 ]. However, mosaic-like symptoms including chlorotic streaks can also be caused in sugarcane by other viruses belonging to different species. Sugarcane streak mosaic virus (SCSMV) is a poacevirus of the Potyviridae family that was recently discovered in Africa and leaves infected by this virus exhibit a mix of mosaic patterns and streaks [ 2 ]. Maize streak virus (MSV) is a mastrevirus from Africa that was initially discovered on maize but that also infects sugarcane in South Africa and in Nigeria [ 3 , 4 ]. Besides MSV, six other sugarcane-infecting mastreviruses have been reported in African locations or on the adjacent islands of the South West Indian Ocean [ 5 , 6 ], namely saccharum streak virus (SacSV) in South Africa [ 7 ], sugarcane chlorotic streak virus (SCSV) in Nigeria [ 4 ], sugarcane streak virus (SSV) in South Africa [ 8 ], sugarcane streak Egypt virus (SSEV) in Egypt [ 9 ], sugarcane white streak virus (SWSV) in Egypt, Sudan, Reunion Island and germplasm collections in Barbados and Guadeloupe [ 10 – 12 ], and sugarcane streak Reunion virus (SSRV) in Nigeria, Mauritius, and Reunion [ 9 ]. SCMV has been present in Cameroon for several decades. Following the analysis of 26 symptomatic leaf samples collected from 1979–2000, this virus was considered the unique causal agent of mosaic symptoms in this country [ 13 ]. Similarly, mosaic symptoms were only attributed to SCMV in Côte d’Ivoire for numerous years until the emergence and spread of SCSMV in the late 2010s [ 6 ]. Consequently, to update the identity of viruses causing sugarcane mosaic symptoms in Cameroon, the Société Sucrière du Cameroun (SOSUCAM) conducted a survey of symptomatic plants in 2022. Plants found with mosaic-like symptoms were subsequently subjected to molecular identification of the causal agent(s). Materials and methods First fully developed leaves were collected from 23 sugarcane varieties from SOSUCAM’s breeding program and commercial fields (Supplementary Table 1). After sampling, the green leaves were shipped to Montpellier, France where they were stored at -20°C until further processing. Samples from plants infected by SCMV or SCSMV and grown in a greenhouse in Montpellier were used as control. Total RNA was extracted from 100 mg of green leaf tissue with the Qiagen RNAeasy plant mini kit. For the SCMV/SrMV and SCSMV assays, RT-PCR was performed with the Qiagen one step RT-PCR kit using 1 µL of RNA and 0.4µM of oligo1/2 primers for detection of SCMV/SrMV [ 14 ] or 0.4µM of ST2/ST5 primers for detection of SCSMV [ 15 ]. Amplification conditions for all viruses were 50°C for 30 min; 95°C for 15 min; 30 cycles at 94°C for 1min, 55°C (SCMV/SrMV) or 50°C (SCSMV) for 1 min, 72°C for 30 sec; and 72°C for 10 min. Amplification products were visualized by 1% agarose gel electrophoresis. Expected amplicons had a size of 327 and 400 bp for SCMV/SrMV and SCSMV, respectively. The symptomatic samples that tested negative for SCMV/SrMV and SCSMV were processed using the virion-associated nucleic acid (VANA)-based metagenomics approach [ 16 ]. Bioinformatics analyses were performed as described previously [ 17 ]. Diamond (v2.1.6) was used to taxonomically assign the contigs against the nr GenBank protein database [ 18 ]. For cloning, total DNA extracted with the VANA protocol was used. Circular DNA molecules were amplified by rolling circle amplification (RCA) using the Phi29 DNA polymerase (TempliPhi™, GE Healthcare, USA) as previously described [ 19 ]. Two partially overlapping PCR primer pairs (SCSV_RepA_F 5’-CATAGTTCCAAACCTACAGCCT-3’, SCSV_RepA_R 5’-ACTGCAACTTAGAGCCAGAGGC-3’ and SCSV_IR_F 5’-GGCGCCAAGGACTATAAGATG-3’, SCSV_IR_R 5’-TGCGATCCCACATACAAGCATC-3’) were used to amplify the complete genome of SCSV isolates from Cameroon as reported by Yahaya et al. [ 4 ]. Amplicons of approximatively 2700 bp obtained with both primer pairs were extracted from the electrophoresis agarose gel and purified (Monarch DNA Gel Extraction T1020L). Purified amplicons were ligated into pGEM-T easy vector following the supplier’s protocol (PROMEGA pGEM-T Easy vector system ref. A1360), cloned into Escherichia coli DH5α. and sequenced by standard Sanger sequencing using a primer walking approach by Azenta (Leipzig, Germany). Genetic distance and phylogenetic analyses were conducted using the entire genome sequences of five SCSV isolates obtained in this study, 12 isolates of SCSV from Nigeria, and 44 isolates representing the overall diversity of Mastrevirus species currently validated by the International Committee on Taxonomy of Viruses (ICTV) [ 20 ]. Full genome nucleotide sequences of selected mastreviruses were rearranged before alignment to fit the TAATATT motif at the 3’ end of the genome. Genome alignment was performed with the MUSCLE program [ 21 ] and the identity matrix of the 61 mastrevirus genomes was determined with the Sequence Demarcation Tool (SDT 1.3) using pairwise genetic identity calculations [ 22 ]. A phylogenetic tree based on these sequences was constructed with Geneious 8.1.9 (Biomatters New Zealand) using the genetic distance model HKY and the Neighbor-Joining method. Protein sequences of single genes were compared between the 17 SCSV isolates from Cameroon and Nigeria and the 44 Mastrevirus species currently validated by ICTV [ 20 ]. Comparisons were performed for the movement protein (MP), the coat protein (CP), and the replication-associated protein (Rep). Protein sequences were aligned using ClustalW [ 23 ]. Each protein tree was constructed with the Geneious Tree builder program and the Neighbor Joining method. Finally, recombination events between SCSV and the other 44 Mastrevirus species were investigated with the RDP5 program [ 24 ]. Results Eleven sugarcane leaf samples exhibited mosaic-like symptoms in a field disease survey conducted in 2022 in Cameroon (Fig. 1 ). Seven of those tested positive in RT-PCR assays for SCMV/SrMV, whereas all were negative for SCSMV (Supplementary Table 1). The 14 asymptomatic leaf samples tested all negative for these three viruses. The four remaining symptomatic samples (RB92579, RCMR180357, KN07-0032, and FR81258) that were RT-PCR negative for SCSMV, SrMV and SCSMV, respectively yielded 1 (342 reads), 2 (4 reads), 12 (135 reads), and 2 (2 reads) VANA metagenomics-based contigs that were assign to SCSV. Five complete genome sequences (2755–2756 nt) were obtained after cloning and sequencing of five SCSV isolates recovered from 3 of the 4 infected plants (Supplementary Table 1), including two genomes named RB92579-I (GenBank accession number PP840300) and RB92579-R (PP840301) from sugarcane variety RB92579, two genomes KN07-0032-I (PP840302) and KN07-032-R (PP840303) from variety KN07-0032, and one genome RCMR180357-R (PP840304) from variety RCMR180357. These five sequences were compared to the 12 full SCSV genome sequences (2757 nt) from Nigeria available in GenBank. Genome identities among isolates from Cameroon ranged from 98.2–99.8% whereas identities among Nigerian isolates varied from 99–100%. Identities between isolates from the two African geographic locations ranged from 97.8–98.7%. In the phylogenetic tree constructed with the entire nucleotide genome sequences of 45 mastreviruses species, the 17 SCSV isolates from Cameroon and Nigeria formed a unique group (Fig. 2 ). SDT analysis showed that this group of SCSV isolates was closest to eragrostis streak virus (ESV, EU244915) and urochloa streak virus (USV, EU245699) with 75-75.7% and 75.2–75.9% identity, respectively. Additionally, the SCSV isolates from Nigeria and Cameroon were also distributed in different subgroups (Fig. 2 ). The distribution of the SCSV isolates into subgroups was also found in phylogenetic trees based on single gene amino-acid sequences such as the MP and the Rep proteins (Fig. 3). The separation into subgroups was not observed in the phylogenetic tree constructed with the CP protein sequences as the CP protein sequence was identical for 13 isolates from the two locations. The diversity observed for the remaining four isolates (two from Cameroun and two from Nigeria) was caused by one or two single non-synonymous nucleotide mutations per site. When compared to the 44 other mastreviruses, the 17 SCSV isolates formed a separate group regardless of the protein analyzed (Fig. 3). The closest protein sequences for the SCSV MP, CP and Rep proteins belonged to ESV (EU244915) with 53–54% identity, USV (EU445697) with 90% identity, and ESV (EU244915) with 77.5–79% identity, respectively. No recombination event was identified for SCSV with the 61 mastrevirus dataset of our study. Discussion The field survey and the laboratory analyses performed in this study revealed that mosaic-like symptoms observed on sugarcane leaves in Cameroon can no longer be associated with a single virus species (SCMV). At least one other virus species, namely SCSV, infects sugarcane in this geographic location of Africa. If the presence of SCMV (one of the causal agents of mosaic disease with SrMV) was well known for several decades [ 13 ], this is to our knowledge the first report of SCSV in Cameroon. The only other sugarcane growing location where this virus has been found so far is Nigeria, a border country of Cameroon [ 4 ]. The prevalence of SCSV in sugarcane commercial fields and the impact of this virus on crop yields are currently unknown. Nevertheless, SCSV will have an impact on the local breeding program with the need to screen for resistance to SCMV and to SCSV. Aphids are the insect vector of SCMV but the vector of SCSV remains to be identified. Other sugarcane-infecting mastreviruses, such as SSV and MSV, are usually vectored by leafhopper species of the genus Cicadulina [ 25 ]. As described for mastreviruses [ 26 ], the genome structure of SCSV comprises four coding sequences (CDS), a long intergenic region (LIR), and a short intergenic region (SIR). The CDS are expected to encode four proteins, namely the MP (114 aa), the CP (248 aa), the Rep (363 aa), and the RepA (287 aa). The MP and the CP are expressed in virion sense transcripts whereas the Rep and RepA are expressed in complementary sense transcripts with a splicing of 92 nt for the Rep. The SCSV isolates from Cameroon and Nigeria differed by one-two nucleotide insertions/deletions in the SIR region. Overall, diversity based on percent nucleotide identity was slightly greater for the SCSV isolates from Cameroon than for the isolates from Nigeria. Furthermore, the diversity of SCSV in Cameroon appeared to be related to the host variety. ESV and USV are the closest mastreviruses to SCSV with 75–76% full genome identity, which is below the 78% demarcation threshold for Mastrevirus species. For undetermined reasons, these values for ESV and USV identity with SCSV are higher in our study as compared to the ones reported previously [ 4 ]. Furthermore, no significant recombination event was found in the SCSV genome of 17 isolates using RPD5 program, which differs also with the previous report on SCSV in Nigeria that described occurrence of one recombination in the CP gene [ 4 ]. We believe that the previously reported lower levels of nucleotide identity between SCSV and ESV or USV, as well as the previously described recombination event, are artefacts related to an incorrect alignment of the virus genome sequences. This statement is supported by the lack of standard setting to the same nucleotide pattern (e.g. the conserved nonanucleotide sequence TAATATT/AC) of the nucleotide sequences of the complete genomes of the 45 Mastrevirus species currently validated by ICTV [ 20 ] and stored in GenBank. This lack of uniform sequence setting leads to misalignments that produce inconsistent analyses of recombination or calculations of pairwise identity scores. Sequence homologies of SCSV proteins with other mastreviruses varied according to the viral protein. However, the CP sequences of SCSV isolates from Cameroon and Nigeria were identical. This conserved CP structure suggests that the same insect vector spreads SCSV in both countries. Mastreviruses are persistently transmitted viruses [ 27 , 28 ], and their CP is involved in the vectorization [ 29 ]. Identification of this vector should greatly improve the management of SCSV infections in sugarcane fields. Declarations Funding The authors declare that no funds, grants, or other support were received during the preparation of this manuscript Author Contributions All authors contributed to the study conception and design. Material preparation, data collection, vana and cloning were performed by Benoît Ahondopké, Serge Galzi1and Charlotte Julian and analysis were performed by Jean H. Daugrois, Denis Filloux, Philippe Rott and Philippe Roumagnac. The first draft of the manuscript was written by Jean H. Daugrois and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript. Data Availability The datasets generated during and/or analysed during the current study are available in the Genbank repository, NCBI” References Grisham MP (2000) Mosaic. 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Virology 479–480:278–289. https://doi.org/10.1016/j.virol.2015.03.026 Supplementary Files Supplementary.docx Cite Share Download PDF Status: Posted Version 1 posted 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. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-4604985","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":318250993,"identity":"2e4bcc75-fdbb-4637-b04e-2e731968b0d9","order_by":0,"name":"Jean-Heinrich Daugrois","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABNklEQVRIie3PMUvDQBQH8HccJMtR10CEfIULhbbS2n6VC4F0cRAK4hgJXJfQOU5+AiEu4hgIxCWYNVCQZrFroVA6RPSaalEuiKNg/nDvuHf3u+MAmjT5g0HuvpBdxQB432YwAEXMmph/TZwP4vzwFFTn8WcvrmodwVPvZYEeBsddPb5an0PfoNltsViUmdHS4yIH9iw94CddilKHnMwsTw9gbIb5sk0tPjd5y2n3gE0kErCO/spjQlPEMYEYhXmiaJY7ZwqBjoa2TCI3y42G+NuBjCrCyidB1I0GTCYBEVfx6ECsMOOCKJEgYquO+GcXgtg74umEju3rXMHiL7bJCZn0mEzM6eO9IMMRTdViTS77p7MsQcW2HBqGr97lqxrifltSMbSvhyQAYMito0juNWnSpMm/zjsbJWbEGLRqTgAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0002-7282-5859","institution":"CIRAD","correspondingAuthor":true,"prefix":"","firstName":"Jean-Heinrich","middleName":"","lastName":"Daugrois","suffix":""},{"id":318250994,"identity":"ca065920-578a-4c09-b001-81772cb708f5","order_by":1,"name":"Benoît Ahondopké","email":"","orcid":"","institution":"Societe Sucriere du Cameroun","correspondingAuthor":false,"prefix":"","firstName":"Benoît","middleName":"","lastName":"Ahondopké","suffix":""},{"id":318250995,"identity":"aa16a52f-f8ad-4ffc-8f6b-b6048763bb68","order_by":2,"name":"Serge Galzi","email":"","orcid":"","institution":"CIRAD","correspondingAuthor":false,"prefix":"","firstName":"Serge","middleName":"","lastName":"Galzi","suffix":""},{"id":318250996,"identity":"6147b885-b490-4c29-a632-9322ccb5d8bc","order_by":3,"name":"Charlotte Julian","email":"","orcid":"","institution":"CIRAD","correspondingAuthor":false,"prefix":"","firstName":"Charlotte","middleName":"","lastName":"Julian","suffix":""},{"id":318250997,"identity":"d59e979d-1ccc-4617-9ffc-50fd58423f07","order_by":4,"name":"Denis Filloux","email":"","orcid":"","institution":"CIRAD","correspondingAuthor":false,"prefix":"","firstName":"Denis","middleName":"","lastName":"Filloux","suffix":""},{"id":318250998,"identity":"43bfb525-cfda-450c-9474-d8aaad225be6","order_by":5,"name":"Philippe C. Rott","email":"","orcid":"","institution":"CIRAD","correspondingAuthor":false,"prefix":"","firstName":"Philippe","middleName":"C.","lastName":"Rott","suffix":""},{"id":318250999,"identity":"0c04341a-c5d3-4e55-9351-35e72f68ef98","order_by":6,"name":"Philippe Roumagnac","email":"","orcid":"","institution":"CIRAD","correspondingAuthor":false,"prefix":"","firstName":"Philippe","middleName":"","lastName":"Roumagnac","suffix":""}],"badges":[],"createdAt":"2024-06-19 09:55:35","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4604985/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4604985/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":60346670,"identity":"3b69f0c3-9bd4-4ac8-bb93-ce41bd4a28db","added_by":"auto","created_at":"2024-07-15 20:09:28","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":51636,"visible":true,"origin":"","legend":"\u003cp\u003eMosaic-like symptoms observed on sugarcane leaves in Cameroon. A. Symptoms caused by sugarcane mosaic virus (SCMV); B. Symptoms caused by sugarcane chlorotic streak virus (SCSV).\u003c/p\u003e","description":"","filename":"F1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4604985/v1/501b22002b16c904cdf3333d.jpg"},{"id":60346378,"identity":"ce2a80b2-f8c7-4e97-b6b4-feac9d4c3719","added_by":"auto","created_at":"2024-07-15 20:01:28","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":191376,"visible":true,"origin":"","legend":"\u003cp\u003eNeighbor-joining phylogenetic tree constructed with the complete nucleotide genome sequence of five isolates of sugarcane chlorotic streak virus (SCSV) from Cameroon, 12 SCSV isolates from Nigeria, and 44 isolates of \u003cem\u003eMastrevirus\u003c/em\u003e species. Isolates from Cameroon obtained in this study are colored in blue. Bootstrap values of 1000 replications are given at the nodes. Scale bar unit is in number of substitutions per nucleotide.\u003c/p\u003e","description":"","filename":"F2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4604985/v1/0ead301eae5dbc82de73988e.jpg"},{"id":60346381,"identity":"3172fd0b-e6fa-4dd6-8ae2-1a22179aeb0a","added_by":"auto","created_at":"2024-07-15 20:01:28","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":133694,"visible":true,"origin":"","legend":"\u003cp\u003eNeighbor-joining phylogenetic trees constructed with the movement protein (MP), the coat protein (CP) and the replication-associated protein (REP) sequences of five isolates of sugarcane chlorotic streak virus (SCSV) from Cameroon (in blue), 12 SCSV isolates from Nigeria (NGA), and 44 isolates of \u003cem\u003eMastrevirus\u003c/em\u003e species.\u003c/p\u003e","description":"","filename":"F3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4604985/v1/6c7f499ebd6ee2df821daa80.jpg"},{"id":60350365,"identity":"3ec4219b-9b9c-49a2-beac-d3048c7a5eb1","added_by":"auto","created_at":"2024-07-15 22:01:52","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":647362,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4604985/v1/91213921-d312-47b3-8b88-830c39537be4.pdf"},{"id":60346379,"identity":"c312036f-3910-4a80-bd26-6bc572568189","added_by":"auto","created_at":"2024-07-15 20:01:28","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":164713,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementary.docx","url":"https://assets-eu.researchsquare.com/files/rs-4604985/v1/2188c75d4492a9c972fcf5a1.docx"}],"financialInterests":"","formattedTitle":"A survey on sugarcane mosaic symptoms leads to the first report of sugarcane chlorotic streak virus in Cameroon","fulltext":[{"header":"Introduction","content":"\u003cp\u003eMosaic caused by the potyviruses sugarcane mosaic virus (SCMV) and sorghum mosaic virus (SrMV) is a well-known disease of sugarcane in Africa [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. However, mosaic-like symptoms including chlorotic streaks can also be caused in sugarcane by other viruses belonging to different species. Sugarcane streak mosaic virus (SCSMV) is a poacevirus of the \u003cem\u003ePotyviridae\u003c/em\u003e family that was recently discovered in Africa and leaves infected by this virus exhibit a mix of mosaic patterns and streaks [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Maize streak virus (MSV) is a mastrevirus from Africa that was initially discovered on maize but that also infects sugarcane in South Africa and in Nigeria [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Besides MSV, six other sugarcane-infecting mastreviruses have been reported in African locations or on the adjacent islands of the South West Indian Ocean [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], namely saccharum streak virus (SacSV) in South Africa [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], sugarcane chlorotic streak virus (SCSV) in Nigeria [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], sugarcane streak virus (SSV) in South Africa [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], sugarcane streak Egypt virus (SSEV) in Egypt [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], sugarcane white streak virus (SWSV) in Egypt, Sudan, Reunion Island and germplasm collections in Barbados and Guadeloupe [\u003cspan additionalcitationids=\"CR11\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], and sugarcane streak Reunion virus (SSRV) in Nigeria, Mauritius, and Reunion [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eSCMV has been present in Cameroon for several decades. Following the analysis of 26 symptomatic leaf samples collected from 1979\u0026ndash;2000, this virus was considered the unique causal agent of mosaic symptoms in this country [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Similarly, mosaic symptoms were only attributed to SCMV in C\u0026ocirc;te d\u0026rsquo;Ivoire for numerous years until the emergence and spread of SCSMV in the late 2010s [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Consequently, to update the identity of viruses causing sugarcane mosaic symptoms in Cameroon, the Soci\u0026eacute;t\u0026eacute; Sucri\u0026egrave;re du Cameroun (SOSUCAM) conducted a survey of symptomatic plants in 2022. Plants found with mosaic-like symptoms were subsequently subjected to molecular identification of the causal agent(s).\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003eFirst fully developed leaves were collected from 23 sugarcane varieties from SOSUCAM\u0026rsquo;s breeding program and commercial fields (Supplementary Table\u0026nbsp;1). After sampling, the green leaves were shipped to Montpellier, France where they were stored at -20\u0026deg;C until further processing. Samples from plants infected by SCMV or SCSMV and grown in a greenhouse in Montpellier were used as control.\u003c/p\u003e \u003cp\u003eTotal RNA was extracted from 100 mg of green leaf tissue with the Qiagen RNAeasy plant mini kit. For the SCMV/SrMV and SCSMV assays, RT-PCR was performed with the Qiagen one step RT-PCR kit using 1 \u0026micro;L of RNA and 0.4\u0026micro;M of oligo1/2 primers for detection of SCMV/SrMV [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e] or 0.4\u0026micro;M of ST2/ST5 primers for detection of SCSMV [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Amplification conditions for all viruses were 50\u0026deg;C for 30 min; 95\u0026deg;C for 15 min; 30 cycles at 94\u0026deg;C for 1min, 55\u0026deg;C (SCMV/SrMV) or 50\u0026deg;C (SCSMV) for 1 min, 72\u0026deg;C for 30 sec; and 72\u0026deg;C for 10 min. Amplification products were visualized by 1% agarose gel electrophoresis. Expected amplicons had a size of 327 and 400 bp for SCMV/SrMV and SCSMV, respectively.\u003c/p\u003e \u003cp\u003eThe symptomatic samples that tested negative for SCMV/SrMV and SCSMV were processed using the virion-associated nucleic acid (VANA)-based metagenomics approach [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Bioinformatics analyses were performed as described previously [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Diamond (v2.1.6) was used to taxonomically assign the contigs against the nr GenBank protein database [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eFor cloning, total DNA extracted with the VANA protocol was used. Circular DNA molecules were amplified by rolling circle amplification (RCA) using the Phi29 DNA polymerase (TempliPhi\u0026trade;, GE Healthcare, USA) as previously described [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Two partially overlapping PCR primer pairs (SCSV_RepA_F 5\u0026rsquo;-CATAGTTCCAAACCTACAGCCT-3\u0026rsquo;, SCSV_RepA_R 5\u0026rsquo;-ACTGCAACTTAGAGCCAGAGGC-3\u0026rsquo; and SCSV_IR_F 5\u0026rsquo;-GGCGCCAAGGACTATAAGATG-3\u0026rsquo;, SCSV_IR_R 5\u0026rsquo;-TGCGATCCCACATACAAGCATC-3\u0026rsquo;) were used to amplify the complete genome of SCSV isolates from Cameroon as reported by Yahaya et al. [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Amplicons of approximatively 2700 bp obtained with both primer pairs were extracted from the electrophoresis agarose gel and purified (Monarch DNA Gel Extraction T1020L). Purified amplicons were ligated into pGEM-T easy vector following the supplier\u0026rsquo;s protocol (PROMEGA pGEM-T Easy vector system ref. A1360), cloned into \u003cem\u003eEscherichia coli\u003c/em\u003e DH5α. and sequenced by standard Sanger sequencing using a primer walking approach by Azenta (Leipzig, Germany).\u003c/p\u003e \u003cp\u003eGenetic distance and phylogenetic analyses were conducted using the entire genome sequences of five SCSV isolates obtained in this study, 12 isolates of SCSV from Nigeria, and 44 isolates representing the overall diversity of \u003cem\u003eMastrevirus\u003c/em\u003e species currently validated by the International Committee on Taxonomy of Viruses (ICTV) [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Full genome nucleotide sequences of selected mastreviruses were rearranged before alignment to fit the TAATATT motif at the 3\u0026rsquo; end of the genome. Genome alignment was performed with the MUSCLE program [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e] and the identity matrix of the 61 mastrevirus genomes was determined with the Sequence Demarcation Tool (SDT 1.3) using pairwise genetic identity calculations [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. A phylogenetic tree based on these sequences was constructed with Geneious 8.1.9 (Biomatters New Zealand) using the genetic distance model HKY and the Neighbor-Joining method. Protein sequences of single genes were compared between the 17 SCSV isolates from Cameroon and Nigeria and the 44 \u003cem\u003eMastrevirus\u003c/em\u003e species currently validated by ICTV [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Comparisons were performed for the movement protein (MP), the coat protein (CP), and the replication-associated protein (Rep). Protein sequences were aligned using ClustalW [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Each protein tree was constructed with the Geneious Tree builder program and the Neighbor Joining method. Finally, recombination events between SCSV and the other 44 \u003cem\u003eMastrevirus\u003c/em\u003e species were investigated with the RDP5 program [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e].\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eEleven sugarcane leaf samples exhibited mosaic-like symptoms in a field disease survey conducted in 2022 in Cameroon (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Seven of those tested positive in RT-PCR assays for SCMV/SrMV, whereas all were negative for SCSMV (Supplementary Table\u0026nbsp;1). The 14 asymptomatic leaf samples tested all negative for these three viruses. The four remaining symptomatic samples (RB92579, RCMR180357, KN07-0032, and FR81258) that were RT-PCR negative for SCSMV, SrMV and SCSMV, respectively yielded 1 (342 reads), 2 (4 reads), 12 (135 reads), and 2 (2 reads) VANA metagenomics-based contigs that were assign to SCSV.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFive complete genome sequences (2755\u0026ndash;2756 nt) were obtained after cloning and sequencing of five SCSV isolates recovered from 3 of the 4 infected plants (Supplementary Table\u0026nbsp;1), including two genomes named RB92579-I (GenBank accession number PP840300) and RB92579-R (PP840301) from sugarcane variety RB92579, two genomes KN07-0032-I (PP840302) and KN07-032-R (PP840303) from variety KN07-0032, and one genome RCMR180357-R (PP840304) from variety RCMR180357. These five sequences were compared to the 12 full SCSV genome sequences (2757 nt) from Nigeria available in GenBank. Genome identities among isolates from Cameroon ranged from 98.2\u0026ndash;99.8% whereas identities among Nigerian isolates varied from 99\u0026ndash;100%. Identities between isolates from the two African geographic locations ranged from 97.8\u0026ndash;98.7%. In the phylogenetic tree constructed with the entire nucleotide genome sequences of 45 mastreviruses species, the 17 SCSV isolates from Cameroon and Nigeria formed a unique group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). SDT analysis showed that this group of SCSV isolates was closest to eragrostis streak virus (ESV, EU244915) and urochloa streak virus (USV, EU245699) with 75-75.7% and 75.2\u0026ndash;75.9% identity, respectively. Additionally, the SCSV isolates from Nigeria and Cameroon were also distributed in different subgroups (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe distribution of the SCSV isolates into subgroups was also found in phylogenetic trees based on single gene amino-acid sequences such as the MP and the Rep proteins (Fig.\u0026nbsp;3). The separation into subgroups was not observed in the phylogenetic tree constructed with the CP protein sequences as the CP protein sequence was identical for 13 isolates from the two locations. The diversity observed for the remaining four isolates (two from Cameroun and two from Nigeria) was caused by one or two single non-synonymous nucleotide mutations per site. When compared to the 44 other mastreviruses, the 17 SCSV isolates formed a separate group regardless of the protein analyzed (Fig.\u0026nbsp;3). The closest protein sequences for the SCSV MP, CP and Rep proteins belonged to ESV (EU244915) with 53\u0026ndash;54% identity, USV (EU445697) with 90% identity, and ESV (EU244915) with 77.5\u0026ndash;79% identity, respectively. No recombination event was identified for SCSV with the 61 mastrevirus dataset of our study.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe field survey and the laboratory analyses performed in this study revealed that mosaic-like symptoms observed on sugarcane leaves in Cameroon can no longer be associated with a single virus species (SCMV). At least one other virus species, namely SCSV, infects sugarcane in this geographic location of Africa. If the presence of SCMV (one of the causal agents of mosaic disease with SrMV) was well known for several decades [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], this is to our knowledge the first report of SCSV in Cameroon. The only other sugarcane growing location where this virus has been found so far is Nigeria, a border country of Cameroon [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. The prevalence of SCSV in sugarcane commercial fields and the impact of this virus on crop yields are currently unknown. Nevertheless, SCSV will have an impact on the local breeding program with the need to screen for resistance to SCMV and to SCSV. Aphids are the insect vector of SCMV but the vector of SCSV remains to be identified. Other sugarcane-infecting mastreviruses, such as SSV and MSV, are usually vectored by leafhopper species of the genus \u003cem\u003eCicadulina\u003c/em\u003e [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAs described for mastreviruses [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], the genome structure of SCSV comprises four coding sequences (CDS), a long intergenic region (LIR), and a short intergenic region (SIR). The CDS are expected to encode four proteins, namely the MP (114 aa), the CP (248 aa), the Rep (363 aa), and the RepA (287 aa). The MP and the CP are expressed in virion sense transcripts whereas the Rep and RepA are expressed in complementary sense transcripts with a splicing of 92 nt for the Rep.\u003c/p\u003e \u003cp\u003eThe SCSV isolates from Cameroon and Nigeria differed by one-two nucleotide insertions/deletions in the SIR region. Overall, diversity based on percent nucleotide identity was slightly greater for the SCSV isolates from Cameroon than for the isolates from Nigeria. Furthermore, the diversity of SCSV in Cameroon appeared to be related to the host variety. ESV and USV are the closest mastreviruses to SCSV with 75\u0026ndash;76% full genome identity, which is below the 78% demarcation threshold for \u003cem\u003eMastrevirus\u003c/em\u003e species. For undetermined reasons, these values for ESV and USV identity with SCSV are higher in our study as compared to the ones reported previously [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Furthermore, no significant recombination event was found in the SCSV genome of 17 isolates using RPD5 program, which differs also with the previous report on SCSV in Nigeria that described occurrence of one recombination in the CP gene [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. We believe that the previously reported lower levels of nucleotide identity between SCSV and ESV or USV, as well as the previously described recombination event, are artefacts related to an incorrect alignment of the virus genome sequences. This statement is supported by the lack of standard setting to the same nucleotide pattern (e.g. the conserved nonanucleotide sequence TAATATT/AC) of the nucleotide sequences of the complete genomes of the 45 \u003cem\u003eMastrevirus\u003c/em\u003e species currently validated by ICTV [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e] and stored in GenBank. This lack of uniform sequence setting leads to misalignments that produce inconsistent analyses of recombination or calculations of pairwise identity scores.\u003c/p\u003e \u003cp\u003eSequence homologies of SCSV proteins with other mastreviruses varied according to the viral protein. However, the CP sequences of SCSV isolates from Cameroon and Nigeria were identical. This conserved CP structure suggests that the same insect vector spreads SCSV in both countries. Mastreviruses are persistently transmitted viruses [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e], and their CP is involved in the vectorization [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Identification of this vector should greatly improve the management of SCSV infections in sugarcane fields.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThe authors declare that no funds, grants, or other support were received during the preparation of this manuscript\u003c/p\u003e\u003ch2\u003eAuthor Contributions\u003c/h2\u003e \u003cp\u003eAll authors contributed to the study conception and design. Material preparation, data collection, vana and cloning were performed by Beno\u0026icirc;t Ahondopk\u0026eacute;, Serge Galzi1and Charlotte Julian and analysis were performed by Jean H. Daugrois, Denis Filloux, Philippe Rott and Philippe Roumagnac. The first draft of the manuscript was written by Jean H. Daugrois and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e \u003cp\u003eThe datasets generated during and/or analysed during the current study are available in the Genbank repository, NCBI\u0026rdquo;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eGrisham MP (2000) Mosaic. A guide to sugarcane diseases (eds Rott P, Comstock JC, Croft BJ and Saumtally AS, CIRAD/ISSCT, Montpellier 249\u0026ndash;254\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDaugrois JH, Roumagnac P, Julian C, Filloux D, Putra L, Mollov D, Rott P (2024) Historical review on sugarcane streak mosaic virus that has recently emerged in Africa. Phytopathology 114:668\u0026ndash;680. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1094/PHYTO-08-23-0291-RVW\u003c/span\u003e\u003cspan address=\"10.1094/PHYTO-08-23-0291-RVW\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVan Antwerpen T, Mcfarlane SA, Govender P, Potier BAM, Way M, Flett B, Ramusi M, Varsani A, Shepherd DN, Stiller M, Martin DP, Webster TM (2011) Report on maize streak virus in the South African sugar industry. 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Virology 479\u0026ndash;480:278\u0026ndash;289. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.virol.2015.03.026\u003c/span\u003e\u003cspan address=\"10.1016/j.virol.2015.03.026\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-4604985/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4604985/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eFour sugarcane leaf samples from Cameroon showing mosaic like symptoms tested negative by RT-PCR for potyviruses and a poacevirus causing mosaic in sugarcane. Virus sequences sharing 98\u0026ndash;99% nucleotide identity with \u003cem\u003eSugarcane chlorotic streak virus\u003c/em\u003e from Nigeria were found in these four samples by virion-associated nucleic acid metagenomics. Five complete genome sequences were obtained after cloning five isolates from Cameroon. These genome sequences shared less than 75\u0026ndash;76% identity with the closest \u003cem\u003eMastrevirus\u003c/em\u003e species, thus confirming that \u003cem\u003eSugarcane chlorotic streak virus\u003c/em\u003e is a new \u003cem\u003eMastrevirus\u003c/em\u003e species. To our knowledge, this is the first report of \u003cem\u003eSugarcane chlorotic streak virus\u003c/em\u003e in Cameroon.\u003c/p\u003e","manuscriptTitle":"A survey on sugarcane mosaic symptoms leads to the first report of sugarcane chlorotic streak virus in Cameroon","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-07-15 20:01:23","doi":"10.21203/rs.3.rs-4604985/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"71621839-44c2-40e2-8767-21191dc0a82c","owner":[],"postedDate":"July 15th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-07-15T21:53:45+00:00","versionOfRecord":[],"versionCreatedAt":"2024-07-15 20:01:23","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4604985","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4604985","identity":"rs-4604985","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}