Effect of two double-strand RNA viruses on the virulence of the phytopathogenic fungus Fusarium oxysporum

Effect of two double-strand RNA viruses on the virulence of the phytopathogenic fungus Fusarium oxysporum | 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 Effect of two double-strand RNA viruses on the virulence of the phytopathogenic fungus Fusarium oxysporum Jing wang, Yunxia Ni, xintao Liu, hui Zhao, xinbei Zhao, rui Qiu, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8194396/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 5 You are reading this latest preprint version Abstract Fusarium root rot, a persistent soil-borne disease, pose a serious threat to crop production, quality, and ultimately to food security. We identified two double-stranded RNA viruses co-infecting the phytopathogenic fungus Fusarium oxysporum strain 3S-18: Fusarium oxysporum partitivirus 1 isolate 3S-18 (FoPV1/3S18) and Fusarium oxysporum virus 1 (FoV1). The genome of FoPV1/3S18 consists of two segments. dsRNA1 is 1,761 nt in length with a large open reading frame (ORF) encoding an RNA-dependent RNA polymerase (RdRp) of 539 amino acids (aa). dsRNA2 is 1,556 nt in length with an ORF encoding a putative coat protein (CP) of 430 aa. Phylogenetic analysis based on both RdRp and CP amino sequences indicated that FoPV1/3S18 clusters with the members of the genus G ammapartitivirus within the family Partitiviridae . FoV1 was identified as a new monopartite dsRNA virus with 2,944 nt, containing two ORFs which encode a encoding a protein of 590 aa RdRp and 134 aa nucleocapsid protein, respectively. Its belonging to the genus Unirnavirus. Furthermore, we demonstrated that both FoPV1/3S18 and FoV1 can be successfully transmitted via hyphal anastomosis to a virus-free strain. Co-infection with FoV1 and FoPV1/3S18 reduced conidial production but did not attenuate fungal virulence. In contrast, Infection by FoV1 alone not only reduced conidial production but also induced hypovirulence. Fusarium root rot Partitiviridae Gammapartitivirus Hypovirulence Biological control Figures Figure 1 Figure 2 Figure 3 Figure 4 Intruction Mycoviruses, which infect fungi, are ubiquitous across all major fungi taxa, typically manifest as latent infection and seldom cause apparent symptoms in their hosts [ 1 – 3 ]. Advances in high-throughput sequencing and metaviromic have greatly expanded our understanding of mycoviruses diversity, revealing that a single fungal strain can be co-infected by multiple viruses. Interestingly, some mycoviruses enhance the virulence of their hosts, others attenuate pathogenic traits, thereby modulating the host biology. These alterations may include irregular colony morphology, reduced growth rate, abnormal pigmentation, impaired sporulation, and changes in sexual reproduction [ 4 , 5 ]. The most concerning effect is mycovirus induced hypovirulence, which infection attenuates the pathogenicity of plant pathogenic fungi. This phenomenon presents mycoviruses as promising biocontrol agents for plant diseases [ 6 , 7 ]. Mycoviruses with linear double-stranded RNA (dsRNA) genomes are currently classified into eight families: Amalgaviridae , Chrysoviridae , Megabirnaviridae , Partitiviridae , Quadriviridae , Reoviridae , Totiviridae , and Polymycoviridae (ICTV, https://talk.ictvonline.org/ictv-reports/ictv_online_report/ ). The family Paritiviridae currently comprises of five established genera: Alphapartitivirus , Betapartitivirus , Cryspovirus , Deltapartitivirus , and Gammapartitivirus , along with two proposed genera, Epsilonpartitivirus and Zetapartitivirus [ 8 , 9 ]. With the except of Cryspovirus and Deltapartitivirus , members of the other genera have been reported to infect fungus. Partitiviruses are small, isometric, non-enveloped viruses with bisegmented dsRNA genomes ranging from 3.0 to 4.8 kbp in size [ 8 ]. The two essential genome segments, dsRNA1 and dsRNA2, typically encode the RNA-dependent RNA polymerase (RdRp) and coat protein (CP), respectively [ 10 ]. Occasionally, partitiviruses harbor defective interfering RNA (DI-RNA) that can modulate viral infection symptom in its hosts. For instance, Rosellinia necatrix partitivirus 2 (RnPV2) carries a DI-RNA that alters viral induction in Cryphonectria parasitica [ 11 ]. Similarly, an additional dsRNA segment has been identified in also found in certain isolates of Aspergillus flavus partitivirus 2 [ 12 , 13 ]. The fungus infected by partitiviruses are generally asymptomatic, However, some members, such as Sclerotinia sclerotiorum partitivirus 1 (SsPV1/WF-1), Botrytis cinerea partitivirus 2 (BcPV2), and Colletotrichum liriopes partitivirus 1 (ClPV1), have been reported to reduce host virulence, mycelial growth, or conidial production [ 14 – 16 ]. beyond fungi, partitiviruses like the Osugoroshi viruses (OGVs) are associated with male-specific mortality after hatching in oriental tea tortrix, Homona magnanima [ 17 ]. Additionally, partitiviruses can exhibit broad host adaptability. A notable example is Penicillium aurantiogriseum partiti-like virus 1 (PaOLV1), which can stably replicate in a new fungal host, Cryphonectria parasitica , and confers enhanced resistance to salinity stress [ 18 ]. Fusarium oxysporum is an important plant pathogenic fungus that causes Fusarium wilt and Fusarium root rot in many crops worldwide [ 19 – 21 ]. Compared to other pathogenic fungi, relatively few mycoviruses have been reported in F. oxysporum . The identified dsRNA viruses belong to the family Chrysoviridae , Alternaviridae , Polymycoviridae , and Partitiviridae , as well as the proposed genus Unirnavirus [ 22 – 26 ]. Reported positive-sense single-stranded RNA [(+)ssRNA] viruses fall within the family Hypoviridae , Botourmiaviridae , and Mitoviridae [ 27 – 30 ]. One negative-sense single-standed RNA [(-)ssRNA] virus from family Mymonaviridae has been described [ 31 ]. Co-infection of a single fungal strain by multiple viruses is common in species such as Sclerotinia sclerotiorum , Magnaporthe oryzae , Rosellinia necatrix , and Macrophomina phaseolina [ 32 , 33 ]. However, the co-infection of F. oxysporum by two distinct viruses has not been previously reported. In our previous studies, we found two dsRNA viruses, designated Fusarium oxysporum virus 1 (FoV1) and Fusarium oxysporum partitivirus 1 isolate 3S-18 (FoPV1/3S18) co-infect a single strain 3S-18 of F. oxysporum . We only reported the genomic characteristics and phylogenetic relationships of FoV1, but its effects on the host have not yet been investigated [ 25 ]. In this study, we analyzed the molecular characteristics and phylogenetic relationships of the other virus FoPV1/3S18. Furthermore, we demonstrated that FoV1 is the core virus responsible for hypovirulence in the host, and further evaluated the potential of FoV1 as biocontrol agents against Fusarium root rot. Materials and Methods Fungal Strains and Culture Conditions The Fusarium oxysporum strain 3S-18 was originally obtained by single-spore isolation from tobacco root rot sample collected in the city of Sanmenxia, Henan Province, China, 2020. The F. oxysproum strain B9 Hyg R harbored no virus and isolated from diseased tobacco root (Xuchang, Henan Province, China, 2020), which has normal colony morphology, high virulence in its host. In this study, strain B9 was used as a recipient strain in a horizontal transmission test. Methods for pathogen isolation, purification and identification are referred to the previous description [ 31 ]. For identification the Fusarium species, the primers of translation elongation factor 1-alpha (EF-1a), RNA polymerase II subunit I gene (RPB1), and RNA polymerase II subunit II gene (RPB2) [ 34 , 35 ] were used, and the corresponding amplicons were analyzed by Nucleotide BLAST in the NCBI database ( https://www.ncbi.nlm.nih.gov ). All strains were cultured on potato dextrose agar (PDA) in darkness at 25 ℃, for long-term storage with 25% glycerin at -80 ℃. dsRNA, Total RNA extraction and RNA sequencing DsRNA from F. oxysporum strain 3S-18 was extracted and purified according to the procedures described by Wu et al. [ 36 ], and further confirmed based on resistance to DNase 1 and S1 nuclease (TaKaRa, Dalian, China). Finally, the extracted dsRNA was fractionated by agarose gel (1%, w/v) electrophoresis and visualized by staining with GelGreen (0.1%) and viewing on a UV transilluminator. Total RNA was extracted from 1.0 g of mycelia of each isolates using an RNAiso Plus Kit (TaKaRa, China) following the manufacturer’s instructions and further purified by RNAClean XP Kit (Cat A63987, Bechman Coulter, Inc, Kraemer Boulevard brea, CA, USA) and RNase-Free DNase set (Cat79254, QIAGEN, GmBH, Germany), and rRNA was depleted by a Ribo-Zero Tm rRNA Removal Kit (Illumina, CA, USA). Then, the qualified samples were used for high-throughput sequencing on an Illumina HiSeq 2500 platform at Shanghai Bohao Biotechnology Co., Ltd. The detailed parameters in this bioinformatics pipeline were performed according to the procedures described by Wang et al. [ 31 ]. The clean reads was 6.6×10 7 bp after data processing, and the final UniGene counts for 62,690 processed by CLC Genomics Workbench (version:6.0.4) and CAP3 EST based on standard parameter. These contigs were blasted by the non-redundant protein sequences (NR) database in NCBI, the contigs which represented partial genome sequences of “virus” or “viral” were collected and subjected to further analysis. RT-PCR detection and RACE To determine whether the “viral” sequences identified in the transcriptome data are present in the tested strains, the specific primers were designed based on each contig to conduct Reverse Transcription-PCR (RT-PCR) detection on individual strains. The cDNA of each F. oxysporum strains were synthesized by PrimerScriptTM 1st Strand cDNA synthesis Kit (TaKaRa, Dalian, China) following the manufacturer’s instructions. The RT-PCR investigation suggested that strain 3S-18 harbored two different viruses, FoPV1 (contig350, contig360) and FoV1 (contig375). The 5’- and 3’-terminal sequences of these contigs were completed using a SMARTer RACE Amplification Kit (Clontech, Mountain View, CA, United States) following the manufacturer’s instructions using gene-specific primers (GSPs). Four pairs GSP primers were used as the inner and out primers for 5’-RACE and 3’-RACE, respectively (Supplementary Table S1 ). All these amplicons of expected size were purified and cloned into the Escherichia coli Trelief 5α (TSINGKE Biotech, Zhengzhou, China). At least three recombinant clones were sequenced at the TSINGKE Biotech to obtain the full-length cDNA sequences of FoPV1. Sequence analysis of FoPV1/3S18 and FoV1 The full-length cDNA sequences of dsRNA viruses were used as queries to BLASTX search in NCBI database ( https://www.ncbi.nlm.nih.gov ). The putative open reading frames (ORFs) of FoPV1 was deduced using the ORF Finder program in NCBI ( http://www.ncbi.nlm.nih.gov/orffinder/ ). A search for the conserved domains of FoPV1 and FoV1 were deduced using CDD database ( http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi ). Multiple sequence alignments of the RNA-dependent RNA polymerase (RdRp) were conducted using DNAMAN program (Version 9) and Clustal X program (Version 2.0). The phylogenetic tree was constructed using the maximum-likelihood (ML) method and tested with 1,000 bootstrap replicates to determined the reliability of a given branch pattern in MEGA-X (Version 10.1.8). Virus Transmission Assay To investigate the influences of viruses FoPV1 and FoV1 on its host, the pairing-culture technique [ 36 ] was used to test the viral transmission from F. oxysporum strain 3S-18 to strain B9. Strain 3S-18 and strain B9 served as the donor and recipient, respectively. Strain B9 was resistant to hygromycin, which could be used to preclude the contamination by strain 3S-18 as a recipient. The derivative strains of B9 were transplanted to a new PDA plate containing hygromycin B (50 µg ml -1 ) for three times. The presence of FoPV1 and FoV1 in all derivative strains and its biological properties were determined as described above. Finally, two derivative stains were obtained. The isogenic strain B9-VI-1 harbored one virus FoV1, and the other isogenic strain B9-VI-2 harbored two viruses FoV1 and FoPV1. Biological Characterization and Virulence Assay To assess the effects of FoPV1 and FoV1 on its host biological property and pathogenicity, three isogenic strains B9 (virus-free), B9-VI-1 (FoV1+), and B9-VI-2 (FoV1+; FoPV1+) were used. The radial mycelial growth rate and conidial production of the isogenic strains were determined according the procedures described by Wu et al [ 36 ] and Wang et al [ 31 ], respectively. The pathogenicity of isogenic strains on Tobacco Nicotiana benthamiana were conducted using the procedures described in our previous studies [ 31 ]. Furthermore, the pathogens was re-isolated from plant inoculated with B9-VI-1 and B9-VI-2, and also detected to carry corresponding viruses, respectively. The assay treatments were repeated three times with three tobacco seedlings each. Statistical analysis The fungal radial mycelial growth rate, conidial production, phenotypic values and disease index of isogenic strains of F. oxysporum were analyzed by using analysis of variance in open source software R-4.1.0. Results Biological properties of F. oxysporum strain 3S-18 The F. oxysproum strain 3S-18 was confirmed by PCR detection using primers of EF-1a, RPB1, and RPB2 (Supplementary Table S2). Strain 3S-18 was cultured at 25℃ on PDA for 10 days to observe its morphology (Fig. 1A). The average radial mycelial growth of 3S-18 was 10.5 mm/day, which was significantly difference with the virus-free strain AJ3-8 (15.2 mm/day) (Fig. 1B). The average conidial production of 3S-18 was 1.7×10 7 ml − 1 , which was significantly ( p < 0.05) lower than that of strain AJ3−8 (21.5×10 7 ml − 1 ) (Fig. 1C). Overall, strain 3S−18 infected with FoPV1 and FoV1, exhibited a reduced growth rate and decreased conidial production. Genome Analysis of FoPV1/3S18 Based on sequencing results of the RT-PCR products, strain 3S-18 contained two dsRNA viruses, FoPV1 and FoV1 (Fig. 2A). The full-length genome of FoPV1 comprises two segments, designated dsRNA1 (Accession No. PX306286) and dsRNA2 (Accession No. PX306285). The dsRNA1 segment is 1,761 nt in length, while dsRNA2 segment is 1,556 nt long (Fig. 2B). The GC content of two segments are 45.9% and 50%, respectively. The dsRNA1 contains a single largeORF(nt 111−1,403) of 1,293 nt that encodes a 430-aa protein (46.8 kDa) on the plus strand, with a predicted isoelectric point of 7.68. CD-Search showed that large ORF (nt 475−1,311) encoded a conserved polymerase domain RdRp_1 (pfam00680). Multiple alignment of RdRp amino acid sequences encoded by dsRNA1 and other partitiviruses revealed six conserved motifs (Ⅲ-Ⅷ) (Fig. 2C), which are characteristic and unique to viral RNA-dependent RNA polymerases [37]. were identified. The dsRNA2 contains a single ORF (nt 52−1,671) of 1,620 nt that encodes a 539-aa protein (62.7 kDa), with a predicted isoelectric point of 8.79. However, the CD-Search revealed that this ORF contains no putative conserved domains. The untranslated regions (UTRs) of dsRNA1 at the 5’- and 3’- ends are 51 nt and 90 nt long, respectively. The UTRs of dsRNA2 are 110 nt and 153 nt long, respectively. Moreover, semi-conserved CU-rich sequences are found in the 5’-UTRs of both dsRNA1 and dsRNA2. Similarly, the semi-conserved AU-rich sequences are found in the 3’-UTRs of both dsRNA1 and dsRNA2 (Fig. 2D). The 5’- and 3’-terminal sequences of dsRNA1 and dsRNA2 are predicted to fold into stable stem-loop structures with ΔG values of−4.20 kcal mol − 1 ,−22.00 kcal mol − 1 ,−19.8kcal mol − 1 , and−42.30 kcal mol − 1 , respectively (Figure S1). A BLASTX search of NCBI database indicated that the nucleotide sequences of dsRNA1 and dsRNA2 are closely related to those of other partitiviruses with 44-94% identity (Supplementary table S3). The protein encoded by dsRNA1 showed 94% identity to the RdRp of Fusarium mangiferae partitivirus 2 (GenBank Acc No. UBZ25878.1). The protein encoded by dsRNA2 showed 99% identity to the coat protein of Fusarium oxysporum partitivirus 1 isolate FCR51 (GenBank Acc No. OQ418725). In summary, the genome structure of FoPV1 conforms to the typical features of the family Partitivirdae , and belongs to the same species as FmPV2 and FoPV1 isolate FCR51 [38, 26]. Figure 2. The genome organization of a partitivirus from F. oxysporum strain 3S-18. (A) Agar gel electrophoresis detecting the dsRNA segments in the strain 3S-18. (B) Schematic diagram of the genome organization of Fusarium oxysporum partitivirus 1 isolate 3S-18. (C) Multiple alignments of the amino acid sequences of RdRp encoded by FoPV1 isolate 3S-18 and other partitiviruses .. (D) Alignment of 5’- and 3’-UTR sequences between the dsRNA1 and dsRNA2 of FoPV1. Phylogenetic Analysis of FoPV1/3S18 and FoV1 To examine the relationship between FoPV1 and other partitiviruses, phylogenetic analyses were performed using protein alignments of the conserved RdRp domain and coat protein of FoPV1 and 29 other dsRNA viruses, respectively(Supplementary table S4). It including representative members of seven genera in family Partitiviridae ( Alphapartitivirus , Betapartitivirus , Deltapartitivirus , Gammapartitivirus , Epsilonpartitivirus , Zetapartitivirus , and Cryspovirus ). The result of phylogenetic analyses based on RdRp domain showed that FoPV1/3S-18 clustered with FoPV1/FCR51 to form a distinct clade with a bootstrap support value of 98%, indicating a close evolutionary relationship. These two viruses clustered with 10 other gammapartitivirus forming a large independent clade with a bootstrap support value of 99%. The remaining 19 partitiviruses also formed an independent clade corresponding to the viral genus of the family Partitiviridae (Fig. 3A). Similarity, the phylogenetic tree based on coat protein also indicated that FoPV1/3S-18 belongs to the genus Gammapartitivirus in the family Partitiviridae (Fig. 3B). These results confirm that FoPV1 is a new isolate of the genus Gammapartitivirus , family Partitiviridae. Preliminary study indicates that RdRp domain of FoV1 shares 60.00% to 84.28% sequence identity with non-segmented dsRNA viruses, and phylogenetic analysis further suggested it is a new member of the genus Unirnaviru s, which currently consists of unclassified monopartite dsRNA viruses [25]. Horizontal Transmission of FoPV1/3S18 and FoV1 between Fusarium oxysporum strains The F. oxysporum strain B9 was used as a recipient for horizontal transmission of FoPV1 and FoV1. Finally, two derivative isolates, B9-VI-1 and B9-VI-2, were obtained from the B9 recipient colony in the contact cultures between 3S-18 and B9 (Fig. 4A). The isolate B9-VI-1 contains one virus, FoV1, whereas the isolate B9-VI-2 contains two viruses, FoV1 and FoVP1/3S18. There is no significant difference in the growth rate between B9-VI-1 and B9-VI-2 (Fig. 4B). The conidial production of B9-VI-1 and B9-VI-2 was 25.67×10 7 ml − 1 and 24.67×10 7 ml − 1 , respectively, which was significantly ( p < 0.05) lower than that of B9 (53.33×10 7 ml − 1 ; p < 0.05; Fig. 4C). Moreover, RT-PCR detection revealed that FoV1 was successfully transmitted from 3S-18 to the virus-free strain B9, and both FoV1 and FoPV1/3S18 were simultaneously transmitted to B9 (Figure S2). In summary, FoV1 and FoPV1/3S18 significantly reduced the conidial production of the derivative strain but had no significant effect on colony morphology or growth rate. Effect of FoV1 and FoPV1/3S18 on Host Virulence To examine the impact of FoV1 and FoPV1/3S18 on fungal virulence, we evaluated the pathogenicity of three isogenic strains, B9 (virus-free), B9-VI-2 (FoV1 + FoPV1), and B9-VI-1 (FoV1), on Nicotiana benthamiana . The disease index of B9, B9-VI-2, and B9-VI-1 were 70.37, 62.96, and 11.11, respectively (Supplementary Table S5). There was no significant difference ( p < 0.05) in fresh weight, root length, or plant height between the viurs-free strain B9 and the FoV1 + FoPV1-containing strain B9-VI-2 (Fig. 4D). By contrast, all three parameters were significantly lower in the FoV1-only strain B9-VI-1 than in either B9 or B9-VI-2 ( p < 0.05; Fig. 4D). Meanwhile, we re-isolated the pathogens from the roots of plants infected with B9-VI-1 and B9-VI-2 and detected for FoV1 and FoPV1. Each re-isolated strains retained its original viruses (Figure S3), indicating that FoPV1/3S18 does not alter host pathogenicity, whereas FoV1 does. Discussion In this study, we identified and characterized two doubel-stranded RNA (dsRNA) mycovirus co-infecting Fusarium oxysporum strain 3S-18. Genomic and phylogenetic analyses indicate that one virus, FoPV1 isolate 3S-18 (FoPV1/3S18), belongs to the same species as FoPV1 isolate FCR51 [26] within the genus Gammapartitivirus , family Partitiviridae . The other virus, FoV1, we previous identified as a new member of the genus Unirnavirus , which comprise monopartite dsRNA viruses [25, 39]. Advances in high-throughput sequencing and metaviromics have led to the discovery of an increasing number of fungal viruses, revealing their extensive diversity. Co-infection of a single fungal strain by multiple viruses is a common phenomenon, as reported in species such as Sclerotinia sclerotiorum , Rosellinia necatrix , Botrytis cinerea , Rhizoctonia solani , Macrophomina phaseolina [40–43, 33]. These strains have been found to harbor 2 to 17 distinct viruses, often exhibiting hypovirulence. Similarly, strain 3S−18 of Fusarium oxysporum is co-infected by two different dsRNA viruses: FoV1 and FoPV1/3S18. Furthermore, FoPV1/3S18 and the previously reported FoPV1/FCR51 belong to the same species as Fusarium mangiferae partitivirus 2 [38]. The widespread occurrence of such co-infections may be attributed to the ubiquity of fungi in nature and their diverse dissemination mechanisms. It is generally accepted that fungal viruses are transmitted through the production of asexual or sexual spores, as well as via hyphal fusion [1]. Liu et al. [44] demonstrated that SsHADV-1 can be transmitted by the mycophagous insect Lycoriella ingenua . This is because S. sclerotiorum infected with SsHADV-1 attracts L. ingenua for feeding, and the virus can replicate and persist throughout the insect's life stages, including larvae, pupae, adults, and eggs. Furthermore, recent studies have documented instances of mycoviruses being transmitted across species, and even between different kingdoms [45]. For example, the fungal virus Leptosphaeria biglobosa botybirnavirus 1 was successfully transmitted to the distantly related phytopathogenic fungus Botrytis cinerea through mixed spore inoculation on both culture medium and rapeseed stems, with transmission frequencies of 4.6% and 18.8%, respectively[46]. Similarly, Valsa mali negative-strand RNA virus 1 (VmNSRV1), which infects the apple Valsa canker fungus ( Valsa mali ), can be bidirectionally transmitted between V. mali and its apple tree host [47]. Fusarium root rot as a soil born disease, that hyphal spread is influenced by the diverse microbial community in the soil. This interaction may consequently facilitate the transmission of the virus. Such phenomena have been increasingly reported, indicating that the cross-kingdom transmission of fungal viruses may be common in agricultural ecosystems [48, 49]. This mode of transmission also helps explain the extensive diversity observed among fungal viruses. Fungal viruses often impair host fitness by reducing growth, sporulation, pigmentation, and most notably, pathogenicity [3, 5]. Owing to this ability to confer hypovirulence, they are regarded as promising biocontrol agents against plant diseases. For instance, SsHADV−1, which infect S. sclerotiorum strain DT−8, suppresses the expression of key pathogenesis-related genes-including those encoding oxalate synthase, cell wall-degrading enzymes, and effector proteins, thereby abolishing fungal pathogenicity [50]. Moreover, SsHADV−1 can endophytes grow in plant and help it mitigating the risks associated with the field release of pathogens [51]. Other viruses, such as VmNSRV1 and Diaporthe sojae circular DNA virus (DsCDV1), also induce strong hypovirulence in their hosts, show potential for population-wide prevalence, and hold promise for managing fruit tree diseases [47]. In this study, while infection by FoV1 did not affect the host’s growth rate, it significantly reduced conidial production and also attenuated its pathogenicity. Conidia serve as the primary source of infection for Fusarium root rot, which directly influences the severity of disease development. In this study, a transmission assay confirmed a significantly reduction in both conidial production and pathogenicity in the host infected with the derivative isolate B9-VI-1 (FoV1+), compared to the virus-free strain B9. Furthermore, the other derivative isolate B9-VI-2 (FoV1 + FoPV1) also showed reduced conidial production, but its pathogenicity did not differ significantly from that of the virus-free strain B9. Those results indicated that FoV1 was the core virus that significantly attenuated the pathogenicity of the host, suggesting its potential as a biocontrol agent worthy of further investigation for managing the Fusarium root rot. Declarations Data Availability Statement: The sequence file of FoV1 and FoPV1/3S18 are available from the NCBI, GeneBank Accession No. OR372790, PX306286, PX306285. All the supplementary materials can be download in this article. Author Contributions: JW designed the research. YXN, HZH, XTL, XBZ, SYF, and XYT collected the materials. JW, YXN and RQ perform the experiments. JW and YXN wrote the first draft of the manuscript, HYL, SJL, and SYS reviewed the manuscript. All authors critically reviewed the manuscript and approved the final submission. Funding: This research work was financially supported by National Natural Science Foundation of China (32302445), the Science and technology innovation team of Henan Academy of Agricultural Sciences (2022TD26), Joint Fund of Science and Technology R&D Program of Henan Province (232301420117), China Agriculture Research System of MOF and MARA (CARS-14), The Key Project of Science and Technology of Henan Province (251100110100), and Major Science and Technology Project of China National Tobacco Corporation (110202101051). The funder had no role in the study design, data collection and analysis, decision to publish or preparation of the manuscript. Conflicts of Interest: The authors declare no conflict of interest. Ethical approval: This article does not contain any studies with human participants or animals performed by any of the authors. References Ghabrial SA, Castón JR, Jiang D, Nibert ML, Suzuki N (2015) 50-plus years of fungal viruses. 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ISME J 14:3120–3135 Supplementary Files SupplementaryFigure2.tif SupplementaryFigure3.tif SupplementaryTable1.docx SupplementaryTable2.docx SupplementaryTable3.docx SupplementaryTable4.docx SupplementaryTable5.docx renamedbec54.jpg Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Major Revision - English Corrections 05 Jan, 2026 Reviewers agreed at journal 03 Dec, 2025 Reviewers invited by journal 01 Dec, 2025 Editor assigned by journal 26 Nov, 2025 First submitted to journal 24 Nov, 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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(C).\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8194396/v1/735d536aa175ffdf67587d26.jpeg"},{"id":97544942,"identity":"bd4265c3-4de9-4275-8eb1-d95531bf478a","added_by":"auto","created_at":"2025-12-05 15:50:59","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":769920,"visible":true,"origin":"","legend":"\u003cp\u003eThe genome organization of a \u003cem\u003epartitivirus\u003c/em\u003efrom \u003cem\u003eF. oxysporum\u003c/em\u003e strain 3S-18. (A) Agar gel electrophoresis detecting the dsRNA segments in the strain 3S-18. (B) Schematic diagram of the genome organization of Fusarium oxysporum partitivirus 1 isolate 3S-18. (C) Multiple alignments of the amino acid sequences of RdRp encoded by FoPV1 isolate 3S-18 and other \u003cem\u003epartitiviruses\u003c/em\u003e.. (D) Alignment of 5’- and 3’-UTR sequences between the dsRNA1 and dsRNA2 of FoPV1.\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8194396/v1/40545636087b44e21850a2d6.jpeg"},{"id":97544940,"identity":"fd4ade28-7b34-4d4f-ad5f-89651d3da08f","added_by":"auto","created_at":"2025-12-05 15:50:59","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":168866,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenetic analysis of FoPV1 isolate 3S-18 (marked with a red dot) and other \u003cem\u003epartitivirus\u003c/em\u003ein the family \u003cem\u003ePartitiviridae\u003c/em\u003e. The phylogenetic tree was generated by the Maximum-likelihood method (1,000 bootstrap replicates) based on the amino acid sequences of the RdRp domains and coat protein using MEGA-X.\u003c/p\u003e","description":"","filename":"Onlinefloatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-8194396/v1/3ab8ed4703bcc968b5a6adf9.png"},{"id":97544944,"identity":"d0446db3-01bd-42e8-a875-670b10d5dd7e","added_by":"auto","created_at":"2025-12-05 15:50:59","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":286674,"visible":true,"origin":"","legend":"\u003cp\u003eThe comparison of different biological characteristic of recipient strain B9, derivative strain B9-VI-2 and B9-VI-1. (A) Culture morphology (25℃, 4 days) of three tested strains. (B, C) Comparison of radical mycelial growth rate (B) on PDA and conidial production (C) in CMC (28℃, 180 rpm) of three strains. (D) Average plant height, root length, and fresh weight of tobacco cultivar inoculated with strain B9 (virus-free), B9-VI-2 (FoV1+FoPV1/3S18), B9-VI-1 (FoV1). (E) Comparison of growth status of plants inoculated with strain B9, B9-VI-2, and B9-VI-1. The different capital letters indicated a significantly difference (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05) among the tested strains, and the “ns” indicated no significantly difference (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05) among the three strains.\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8194396/v1/c24a72691d82b9ad80d3086b.jpeg"},{"id":97678467,"identity":"db6080d2-0f7a-4f3d-9b95-43e095d54d05","added_by":"auto","created_at":"2025-12-08 09:55:31","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2079090,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8194396/v1/bf267799-e324-4e78-8d5e-8c0c8d11759a.pdf"},{"id":97544959,"identity":"e8a25d11-2e4b-4e55-9c07-8356637b4742","added_by":"auto","created_at":"2025-12-05 15:50:59","extension":"tif","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":3070000,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigure2.tif","url":"https://assets-eu.researchsquare.com/files/rs-8194396/v1/20fd3f9648ca40e0a9e24cf6.tif"},{"id":97544958,"identity":"06883f15-b21f-4d96-970c-ed6479158881","added_by":"auto","created_at":"2025-12-05 15:50:59","extension":"tif","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":4162860,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigure3.tif","url":"https://assets-eu.researchsquare.com/files/rs-8194396/v1/9ad484b9ca438420b273d9f2.tif"},{"id":97544945,"identity":"3a36c828-a46f-46d6-9271-7f86cac0bd5b","added_by":"auto","created_at":"2025-12-05 15:50:59","extension":"docx","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":13950,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTable1.docx","url":"https://assets-eu.researchsquare.com/files/rs-8194396/v1/428547f894ebfcf3601fb226.docx"},{"id":97671965,"identity":"1af80250-6451-493b-83bd-d6544ade93ec","added_by":"auto","created_at":"2025-12-08 09:33:33","extension":"docx","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":17335,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTable2.docx","url":"https://assets-eu.researchsquare.com/files/rs-8194396/v1/46fc828f1689b6aea9f5a30c.docx"},{"id":97544954,"identity":"77f754e4-c795-49f2-a889-e59dc0686c04","added_by":"auto","created_at":"2025-12-05 15:50:59","extension":"docx","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":15543,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTable3.docx","url":"https://assets-eu.researchsquare.com/files/rs-8194396/v1/43cf323b53810521af1cc5a3.docx"},{"id":97544949,"identity":"2c4a2c2c-b8cf-4fc8-8fdb-c7911e4e98fb","added_by":"auto","created_at":"2025-12-05 15:50:59","extension":"docx","order_by":9,"title":"","display":"","copyAsset":false,"role":"supplement","size":17218,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTable4.docx","url":"https://assets-eu.researchsquare.com/files/rs-8194396/v1/bfada7f97b64c794a2e79f57.docx"},{"id":97672054,"identity":"dd37d409-cd50-41d9-8e8b-c0b69f86e1e7","added_by":"auto","created_at":"2025-12-08 09:33:57","extension":"docx","order_by":10,"title":"","display":"","copyAsset":false,"role":"supplement","size":12146,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTable5.docx","url":"https://assets-eu.researchsquare.com/files/rs-8194396/v1/e6c9c4d89ebaedddfe7df7ed.docx"},{"id":97544952,"identity":"c7c04b3f-6629-4f45-afe2-56eacfa5a110","added_by":"auto","created_at":"2025-12-05 15:50:59","extension":"jpg","order_by":11,"title":"","display":"","copyAsset":false,"role":"supplement","size":797938,"visible":true,"origin":"","legend":"","description":"","filename":"renamedbec54.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8194396/v1/38e3bcf41efd95b3730b2bec.jpg"}],"financialInterests":"","formattedTitle":"Effect of two double-strand RNA viruses on the virulence of the phytopathogenic fungus Fusarium oxysporum","fulltext":[{"header":"Intruction","content":"\u003cp\u003eMycoviruses, which infect fungi, are ubiquitous across all major fungi taxa, typically manifest as latent infection and seldom cause apparent symptoms in their hosts [\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Advances in high-throughput sequencing and metaviromic have greatly expanded our understanding of mycoviruses diversity, revealing that a single fungal strain can be co-infected by multiple viruses. Interestingly, some mycoviruses enhance the virulence of their hosts, others attenuate pathogenic traits, thereby modulating the host biology. These alterations may include irregular colony morphology, reduced growth rate, abnormal pigmentation, impaired sporulation, and changes in sexual reproduction [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. The most concerning effect is mycovirus induced hypovirulence, which infection attenuates the pathogenicity of plant pathogenic fungi. This phenomenon presents mycoviruses as promising biocontrol agents for plant diseases [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eMycoviruses with linear double-stranded RNA (dsRNA) genomes are currently classified into eight families: \u003cem\u003eAmalgaviridae\u003c/em\u003e, \u003cem\u003eChrysoviridae\u003c/em\u003e, \u003cem\u003eMegabirnaviridae\u003c/em\u003e, \u003cem\u003ePartitiviridae\u003c/em\u003e, \u003cem\u003eQuadriviridae\u003c/em\u003e, \u003cem\u003eReoviridae\u003c/em\u003e, \u003cem\u003eTotiviridae\u003c/em\u003e, and \u003cem\u003ePolymycoviridae\u003c/em\u003e (ICTV, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://talk.ictvonline.org/ictv-reports/ictv_online_report/\u003c/span\u003e\u003cspan address=\"https://talk.ictvonline.org/ictv-reports/ictv_online_report/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The family \u003cem\u003eParitiviridae\u003c/em\u003e currently comprises of five established genera: \u003cem\u003eAlphapartitivirus\u003c/em\u003e, \u003cem\u003eBetapartitivirus\u003c/em\u003e, \u003cem\u003eCryspovirus\u003c/em\u003e, \u003cem\u003eDeltapartitivirus\u003c/em\u003e, and \u003cem\u003eGammapartitivirus\u003c/em\u003e, along with two proposed genera, \u003cem\u003eEpsilonpartitivirus\u003c/em\u003e and \u003cem\u003eZetapartitivirus\u003c/em\u003e [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. With the except of \u003cem\u003eCryspovirus\u003c/em\u003e and \u003cem\u003eDeltapartitivirus\u003c/em\u003e, members of the other genera have been reported to infect fungus. \u003cem\u003ePartitiviruses\u003c/em\u003e are small, isometric, non-enveloped viruses with bisegmented dsRNA genomes ranging from 3.0 to 4.8 kbp in size [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. The two essential genome segments, dsRNA1 and dsRNA2, typically encode the RNA-dependent RNA polymerase (RdRp) and coat protein (CP), respectively [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Occasionally, partitiviruses harbor defective interfering RNA (DI-RNA) that can modulate viral infection symptom in its hosts. For instance, Rosellinia necatrix partitivirus 2 (RnPV2) carries a DI-RNA that alters viral induction in \u003cem\u003eCryphonectria parasitica\u003c/em\u003e [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Similarly, an additional dsRNA segment has been identified in also found in certain isolates of Aspergillus flavus partitivirus 2 [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. The fungus infected by \u003cem\u003epartitiviruses\u003c/em\u003e are generally asymptomatic, However, some members, such as Sclerotinia sclerotiorum partitivirus 1 (SsPV1/WF-1), Botrytis cinerea partitivirus 2 (BcPV2), and Colletotrichum liriopes partitivirus 1 (ClPV1), have been reported to reduce host virulence, mycelial growth, or conidial production [\u003cspan additionalcitationids=\"CR15\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. beyond fungi, \u003cem\u003epartitiviruses\u003c/em\u003e like the Osugoroshi viruses (OGVs) are associated with male-specific mortality after hatching in oriental tea tortrix, \u003cem\u003eHomona magnanima\u003c/em\u003e [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Additionally, partitiviruses can exhibit broad host adaptability. A notable example is Penicillium aurantiogriseum partiti-like virus 1 (PaOLV1), which can stably replicate in a new fungal host, \u003cem\u003eCryphonectria parasitica\u003c/em\u003e, and confers enhanced resistance to salinity stress [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003cem\u003eFusarium oxysporum\u003c/em\u003e is an important plant pathogenic fungus that causes Fusarium wilt and Fusarium root rot in many crops worldwide [\u003cspan additionalcitationids=\"CR20\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Compared to other pathogenic fungi, relatively few mycoviruses have been reported in \u003cem\u003eF. oxysporum\u003c/em\u003e. The identified dsRNA viruses belong to the family \u003cem\u003eChrysoviridae\u003c/em\u003e, \u003cem\u003eAlternaviridae\u003c/em\u003e, \u003cem\u003ePolymycoviridae\u003c/em\u003e, and \u003cem\u003ePartitiviridae\u003c/em\u003e, as well as the proposed genus \u003cem\u003eUnirnavirus\u003c/em\u003e [\u003cspan additionalcitationids=\"CR23 CR24 CR25\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Reported positive-sense single-stranded RNA [(+)ssRNA] viruses fall within the family \u003cem\u003eHypoviridae\u003c/em\u003e, \u003cem\u003eBotourmiaviridae\u003c/em\u003e, and \u003cem\u003eMitoviridae\u003c/em\u003e [\u003cspan additionalcitationids=\"CR28 CR29\" citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. One negative-sense single-standed RNA [(-)ssRNA] virus from family \u003cem\u003eMymonaviridae\u003c/em\u003ehas been described [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Co-infection of a single fungal strain by multiple viruses is common in species such as \u003cem\u003eSclerotinia sclerotiorum\u003c/em\u003e, \u003cem\u003eMagnaporthe oryzae\u003c/em\u003e, \u003cem\u003eRosellinia necatrix\u003c/em\u003e, and \u003cem\u003eMacrophomina phaseolina\u003c/em\u003e [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. However, the co-infection of \u003cem\u003eF. oxysporum\u003c/em\u003e by two distinct viruses has not been previously reported.\u003c/p\u003e\u003cp\u003eIn our previous studies, we found two dsRNA viruses, designated Fusarium oxysporum virus 1 (FoV1) and Fusarium oxysporum partitivirus 1 isolate 3S-18 (FoPV1/3S18) co-infect a single strain 3S-18 of \u003cem\u003eF. oxysporum\u003c/em\u003e. We only reported the genomic characteristics and phylogenetic relationships of FoV1, but its effects on the host have not yet been investigated [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. In this study, we analyzed the molecular characteristics and phylogenetic relationships of the other virus FoPV1/3S18. Furthermore, we demonstrated that FoV1 is the core virus responsible for hypovirulence in the host, and further evaluated the potential of FoV1 as biocontrol agents against Fusarium root rot.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n \u003ch2\u003eFungal Strains and Culture Conditions\u003c/h2\u003e\n \u003cp\u003eThe \u003cem\u003eFusarium oxysporum\u003c/em\u003e strain 3S-18 was originally obtained by single-spore isolation from tobacco root rot sample collected in the city of Sanmenxia, Henan Province, China, 2020. The \u003cem\u003eF. oxysproum\u003c/em\u003e strain B9 Hyg\u003csup\u003e\u003cem\u003eR\u003c/em\u003e\u003c/sup\u003e harbored no virus and isolated from diseased tobacco root (Xuchang, Henan Province, China, 2020), which has normal colony morphology, high virulence in its host. In this study, strain B9 was used as a recipient strain in a horizontal transmission test. Methods for pathogen isolation, purification and identification are referred to the previous description [\u003cspan class=\"CitationRef\"\u003e31\u003c/span\u003e]. For identification the \u003cem\u003eFusarium\u003c/em\u003e species, the primers of translation elongation factor 1-alpha (EF-1a), RNA polymerase II subunit I gene (RPB1), and RNA polymerase II subunit II gene (RPB2) [\u003cspan class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e35\u003c/span\u003e] were used, and the corresponding amplicons were analyzed by Nucleotide BLAST in the NCBI database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov\u003c/span\u003e\u003c/span\u003e). All strains were cultured on potato dextrose agar (PDA) in darkness at 25 ℃, for long-term storage with 25% glycerin at -80 ℃.\u003c/p\u003e\n\u003c/div\u003e\n\u003ch3\u003edsRNA, Total RNA extraction and RNA sequencing\u003c/h3\u003e\n\u003cp\u003eDsRNA from \u003cem\u003eF. oxysporum\u003c/em\u003e strain 3S-18 was extracted and purified according to the procedures described by Wu et al. [\u003cspan class=\"CitationRef\"\u003e36\u003c/span\u003e], and further confirmed based on resistance to DNase 1 and S1 nuclease (TaKaRa, Dalian, China). Finally, the extracted dsRNA was fractionated by agarose gel (1%, w/v) electrophoresis and visualized by staining with GelGreen (0.1%) and viewing on a UV transilluminator. Total RNA was extracted from 1.0 g of mycelia of each isolates using an RNAiso Plus Kit (TaKaRa, China) following the manufacturer\u0026rsquo;s instructions and further purified by RNAClean XP Kit (Cat A63987, Bechman Coulter, Inc, Kraemer Boulevard brea, CA, USA) and RNase-Free DNase set (Cat79254, QIAGEN, GmBH, Germany), and rRNA was depleted by a Ribo-Zero\u003csup\u003eTm\u003c/sup\u003e rRNA Removal Kit (Illumina, CA, USA). Then, the qualified samples were used for high-throughput sequencing on an Illumina HiSeq 2500 platform at Shanghai Bohao Biotechnology Co., Ltd. The detailed parameters in this bioinformatics pipeline were performed according to the procedures described by Wang et al. [\u003cspan class=\"CitationRef\"\u003e31\u003c/span\u003e]. The clean reads was 6.6\u0026times;10\u003csup\u003e7\u003c/sup\u003e bp after data processing, and the final UniGene counts for 62,690 processed by CLC Genomics Workbench (version:6.0.4) and CAP3 EST based on standard parameter. These contigs were blasted by the non-redundant protein sequences (NR) database in NCBI, the contigs which represented partial genome sequences of \u0026ldquo;virus\u0026rdquo; or \u0026ldquo;viral\u0026rdquo; were collected and subjected to further analysis.\u003c/p\u003e\n\u003ch3\u003eRT-PCR detection and RACE\u003c/h3\u003e\n\u003cp\u003eTo determine whether the \u0026ldquo;viral\u0026rdquo; sequences identified in the transcriptome data are present in the tested strains, the specific primers were designed based on each contig to conduct Reverse Transcription-PCR (RT-PCR) detection on individual strains. The cDNA of each \u003cem\u003eF. oxysporum\u003c/em\u003e strains were synthesized by PrimerScriptTM 1st Strand cDNA synthesis Kit (TaKaRa, Dalian, China) following the manufacturer\u0026rsquo;s instructions. The RT-PCR investigation suggested that strain 3S-18 harbored two different viruses, FoPV1 (contig350, contig360) and FoV1 (contig375). The 5\u0026rsquo;- and 3\u0026rsquo;-terminal sequences of these contigs were completed using a SMARTer RACE Amplification Kit (Clontech, Mountain View, CA, United States) following the manufacturer\u0026rsquo;s instructions using gene-specific primers (GSPs). Four pairs GSP primers were used as the inner and out primers for 5\u0026rsquo;-RACE and 3\u0026rsquo;-RACE, respectively (Supplementary Table \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003e). All these amplicons of expected size were purified and cloned into the \u003cem\u003eEscherichia coli\u003c/em\u003e Trelief 5\u0026alpha; (TSINGKE Biotech, Zhengzhou, China). At least three recombinant clones were sequenced at the TSINGKE Biotech to obtain the full-length cDNA sequences of FoPV1.\u003c/p\u003e\n\u003ch3\u003eSequence analysis of FoPV1/3S18 and FoV1\u003c/h3\u003e\n\u003cp\u003eThe full-length cDNA sequences of dsRNA viruses were used as queries to BLASTX search in NCBI database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov\u003c/span\u003e\u003c/span\u003e). The putative open reading frames (ORFs) of FoPV1 was deduced using the ORF Finder program in NCBI (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.ncbi.nlm.nih.gov/orffinder/\u003c/span\u003e\u003c/span\u003e). A search for the conserved domains of FoPV1 and FoV1 were deduced using CDD database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi\u003c/span\u003e\u003c/span\u003e). Multiple sequence alignments of the RNA-dependent RNA polymerase (RdRp) were conducted using DNAMAN program (Version 9) and Clustal X program (Version 2.0). The phylogenetic tree was constructed using the maximum-likelihood (ML) method and tested with 1,000 bootstrap replicates to determined the reliability of a given branch pattern in MEGA-X (Version 10.1.8).\u003c/p\u003e\n\u003ch3\u003eVirus Transmission Assay\u003c/h3\u003e\n\u003cp\u003eTo investigate the influences of viruses FoPV1 and FoV1 on its host, the pairing-culture technique [\u003cspan class=\"CitationRef\"\u003e36\u003c/span\u003e] was used to test the viral transmission from \u003cem\u003eF. oxysporum\u003c/em\u003e strain 3S-18 to strain B9. Strain 3S-18 and strain B9 served as the donor and recipient, respectively. Strain B9 was resistant to hygromycin, which could be used to preclude the contamination by strain 3S-18 as a recipient. The derivative strains of B9 were transplanted to a new PDA plate containing hygromycin B (50 \u0026micro;g ml\u003csup\u003e-1\u003c/sup\u003e) for three times. The presence of FoPV1 and FoV1 in all derivative strains and its biological properties were determined as described above. Finally, two derivative stains were obtained. The isogenic strain B9-VI-1 harbored one virus FoV1, and the other isogenic strain B9-VI-2 harbored two viruses FoV1 and FoPV1.\u003c/p\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\n \u003ch2\u003eBiological Characterization and Virulence Assay\u003c/h2\u003e\n \u003cp\u003eTo assess the effects of FoPV1 and FoV1 on its host biological property and pathogenicity, three isogenic strains B9 (virus-free), B9-VI-1 (FoV1+), and B9-VI-2 (FoV1+; FoPV1+) were used. The radial mycelial growth rate and conidial production of the isogenic strains were determined according the procedures described by Wu et al [\u003cspan class=\"CitationRef\"\u003e36\u003c/span\u003e] and Wang et al [\u003cspan class=\"CitationRef\"\u003e31\u003c/span\u003e], respectively. The pathogenicity of isogenic strains on Tobacco \u003cem\u003eNicotiana benthamiana\u003c/em\u003e were conducted using the procedures described in our previous studies [\u003cspan class=\"CitationRef\"\u003e31\u003c/span\u003e]. Furthermore, the pathogens was re-isolated from plant inoculated with B9-VI-1 and B9-VI-2, and also detected to carry corresponding viruses, respectively. The assay treatments were repeated three times with three tobacco seedlings each.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\n \u003ch2\u003eStatistical analysis\u003c/h2\u003e\n \u003cp\u003eThe fungal radial mycelial growth rate, conidial production, phenotypic values and disease index of isogenic strains of \u003cem\u003eF. oxysporum\u003c/em\u003e were analyzed by using analysis of variance in open source software R-4.1.0.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec11\"\u003e\n \u003ch2\u003eBiological properties of F. oxysporum strain 3S-18\u003c/h2\u003e\n \u003cp\u003eThe \u003cem\u003eF. oxysproum\u003c/em\u003e strain 3S-18 was confirmed by PCR detection using primers of EF-1a, RPB1, and RPB2 (Supplementary Table S2). Strain 3S-18 was cultured at 25℃ on PDA for 10 days to observe its morphology (Fig.\u0026nbsp;1A). The average radial mycelial growth of 3S-18 was 10.5 mm/day, which was significantly difference with the virus-free strain AJ3-8 (15.2 mm/day) (Fig.\u0026nbsp;1B). The average conidial production of 3S-18 was 1.7\u0026times;10\u003csup\u003e7\u003c/sup\u003e ml\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, which was significantly (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) lower than that of strain AJ3\u0026minus;8 (21.5\u0026times;10\u003csup\u003e7\u003c/sup\u003e ml\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e ) (Fig.\u0026nbsp;1C). Overall, strain 3S\u0026minus;18 infected with FoPV1 and FoV1, exhibited a reduced growth rate and decreased conidial production.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec12\"\u003e\n \u003ch2\u003eGenome Analysis of FoPV1/3S18\u003c/h2\u003e\n \u003cp\u003eBased on sequencing results of the RT-PCR products, strain 3S-18 contained two dsRNA viruses, FoPV1 and FoV1 (Fig.\u0026nbsp;2A). The full-length genome of FoPV1 comprises two segments, designated dsRNA1 (Accession No. PX306286) and dsRNA2 (Accession No. PX306285). The dsRNA1 segment is 1,761 nt in length, while dsRNA2 segment is 1,556 nt long (Fig.\u0026nbsp;2B). The GC content of two segments are 45.9% and 50%, respectively. The dsRNA1 contains a single largeORF(nt 111\u0026minus;1,403) of 1,293 nt that encodes a 430-aa protein (46.8 kDa) on the plus strand, with a predicted isoelectric point of 7.68. CD-Search showed that large ORF (nt 475\u0026minus;1,311) encoded a\u003c/p\u003e\n \u003cp\u003econserved polymerase domain RdRp_1 (pfam00680). Multiple alignment of RdRp amino acid sequences encoded by dsRNA1 and other \u003cem\u003epartitiviruses\u003c/em\u003e revealed six conserved motifs (Ⅲ-Ⅷ) (Fig.\u0026nbsp;2C), which are characteristic and unique to viral RNA-dependent RNA polymerases [37]. were identified. The dsRNA2 contains a single ORF (nt 52\u0026minus;1,671) of 1,620 nt that encodes a 539-aa protein (62.7 kDa), with a predicted isoelectric point of 8.79. However, the CD-Search revealed that this ORF contains no putative conserved domains. The untranslated regions (UTRs) of dsRNA1 at the 5\u0026rsquo;- and 3\u0026rsquo;- ends are 51 nt and 90 nt long, respectively. The UTRs of dsRNA2 are 110 nt and 153 nt long, respectively. Moreover, semi-conserved CU-rich sequences are found in the 5\u0026rsquo;-UTRs of both dsRNA1 and dsRNA2. Similarly, the semi-conserved AU-rich sequences are found in the 3\u0026rsquo;-UTRs of both dsRNA1 and dsRNA2 (Fig.\u0026nbsp;2D). The 5\u0026rsquo;- and 3\u0026rsquo;-terminal sequences of dsRNA1 and dsRNA2 are predicted to fold into stable stem-loop structures with \u0026Delta;G values of\u0026minus;4.20 kcal mol\u0026thinsp;\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e,\u0026minus;22.00 kcal mol\u0026thinsp;\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e,\u0026minus;19.8kcal mol\u0026thinsp;\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, and\u0026minus;42.30 kcal mol\u0026thinsp;\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, respectively (Figure S1).\u003c/p\u003e\n \u003cp\u003eA BLASTX search of NCBI database indicated that the nucleotide sequences of dsRNA1 and dsRNA2 are closely related to those of other \u003cem\u003epartitiviruses\u003c/em\u003e with 44-94% identity (Supplementary table S3). The protein encoded by dsRNA1 showed 94% identity to the RdRp of Fusarium mangiferae partitivirus 2 (GenBank Acc No. UBZ25878.1). The protein encoded by dsRNA2 showed 99% identity to the coat protein of Fusarium oxysporum partitivirus 1 isolate FCR51 (GenBank Acc No. OQ418725). In summary, the genome structure of FoPV1 conforms to the typical features of the family \u003cem\u003ePartitivirdae\u003c/em\u003e, and belongs to the same species as FmPV2 and FoPV1 isolate FCR51 [38, 26].\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eFigure 2.\u003c/strong\u003e The genome organization of a \u003cem\u003epartitivirus\u003c/em\u003e from \u003cem\u003eF. oxysporum\u003c/em\u003e strain 3S-18. (A) Agar gel electrophoresis detecting the dsRNA segments in the strain 3S-18. (B) Schematic diagram of the genome organization of Fusarium oxysporum partitivirus 1 isolate 3S-18. (C) Multiple alignments of the amino acid sequences of RdRp encoded by FoPV1 isolate 3S-18 and other \u003cem\u003epartitiviruses\u003c/em\u003e.. (D) Alignment of 5\u0026rsquo;- and 3\u0026rsquo;-UTR sequences between the dsRNA1 and dsRNA2 of FoPV1.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec13\"\u003e\n \u003ch2\u003ePhylogenetic Analysis of FoPV1/3S18 and FoV1\u003c/h2\u003e\n \u003cp\u003eTo examine the relationship between FoPV1 and other partitiviruses, phylogenetic analyses were performed using protein alignments of the conserved RdRp domain and coat protein of FoPV1 and 29 other dsRNA viruses, respectively(Supplementary table S4). It including representative members of seven genera in family \u003cem\u003ePartitiviridae\u003c/em\u003e (\u003cem\u003eAlphapartitivirus\u003c/em\u003e, \u003cem\u003eBetapartitivirus\u003c/em\u003e, \u003cem\u003eDeltapartitivirus\u003c/em\u003e, \u003cem\u003eGammapartitivirus\u003c/em\u003e, \u003cem\u003eEpsilonpartitivirus\u003c/em\u003e, \u003cem\u003eZetapartitivirus\u003c/em\u003e, and \u003cem\u003eCryspovirus\u003c/em\u003e). The result of phylogenetic analyses based on RdRp domain showed that FoPV1/3S-18 clustered with FoPV1/FCR51 to form a distinct clade with a bootstrap support value of 98%, indicating a close evolutionary relationship. These two viruses clustered with 10 other \u003cem\u003egammapartitivirus\u003c/em\u003e forming a large independent clade with a bootstrap support value of 99%. The remaining 19 \u003cem\u003epartitiviruses\u003c/em\u003e also formed an independent clade corresponding to the viral genus of the family \u003cem\u003ePartitiviridae\u003c/em\u003e (Fig.\u0026nbsp;3A). Similarity, the phylogenetic tree based on coat protein also indicated that FoPV1/3S-18 belongs to the genus \u003cem\u003eGammapartitivirus\u003c/em\u003e in the family \u003cem\u003ePartitiviridae\u003c/em\u003e (Fig.\u0026nbsp;3B). These results confirm that FoPV1 is a new isolate of the genus \u003cem\u003eGammapartitivirus\u003c/em\u003e, family \u003cem\u003ePartitiviridae.\u003c/em\u003e Preliminary study indicates that RdRp domain of FoV1 shares 60.00% to 84.28% sequence identity with non-segmented dsRNA viruses, and phylogenetic analysis further suggested it is a new member of the genus \u003cem\u003eUnirnaviru\u003c/em\u003es, which currently consists of unclassified monopartite dsRNA viruses [25].\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec14\"\u003e\n \u003ch2\u003eHorizontal Transmission of FoPV1/3S18 and FoV1 between Fusarium oxysporum strains\u003c/h2\u003e\n \u003cp\u003eThe \u003cem\u003eF. oxysporum\u003c/em\u003e strain B9 was used as a recipient for horizontal transmission of FoPV1 and FoV1. Finally, two derivative isolates, B9-VI-1 and B9-VI-2, were obtained from the B9 recipient colony in the contact cultures between 3S-18 and B9 (Fig.\u0026nbsp;4A). The isolate B9-VI-1 contains one virus, FoV1, whereas the isolate B9-VI-2 contains two viruses, FoV1 and FoVP1/3S18. There is no significant difference in the growth rate between B9-VI-1 and B9-VI-2 (Fig.\u0026nbsp;4B). The conidial production of B9-VI-1 and B9-VI-2 was 25.67\u0026times;10\u003csup\u003e7\u003c/sup\u003e ml\u0026thinsp;\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 24.67\u0026times;10\u003csup\u003e7\u003c/sup\u003e ml\u0026thinsp;\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, respectively, which was significantly (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) lower than that of B9 (53.33\u0026times;10\u003csup\u003e7\u003c/sup\u003e ml\u0026thinsp;\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e; \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05; Fig.\u0026nbsp;4C). Moreover, RT-PCR detection revealed that FoV1 was successfully transmitted from 3S-18 to the virus-free strain B9, and both FoV1 and FoPV1/3S18 were simultaneously transmitted to B9 (Figure S2). In summary, FoV1 and FoPV1/3S18 significantly reduced the conidial production of the derivative strain but had no significant effect on colony morphology or growth rate.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec15\"\u003e\n \u003ch2\u003eEffect of FoV1 and FoPV1/3S18 on Host Virulence\u003c/h2\u003e\n \u003cp\u003eTo examine the impact of FoV1 and FoPV1/3S18 on fungal virulence, we evaluated the pathogenicity of three isogenic strains, B9 (virus-free), B9-VI-2 (FoV1\u0026thinsp;+\u0026thinsp;FoPV1), and B9-VI-1 (FoV1), on \u003cem\u003eNicotiana benthamiana\u003c/em\u003e. The disease index of B9, B9-VI-2, and B9-VI-1 were 70.37, 62.96, and 11.11, respectively (Supplementary Table S5). There was no significant difference (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in fresh weight, root length, or plant height between the viurs-free strain B9 and the FoV1\u0026thinsp;+\u0026thinsp;FoPV1-containing strain B9-VI-2 (Fig.\u0026nbsp;4D). By contrast, all three parameters were significantly lower in the FoV1-only strain B9-VI-1 than in either B9 or B9-VI-2 (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05; Fig. 4D). Meanwhile, we re-isolated the pathogens from the roots of plants infected with B9-VI-1 and B9-VI-2 and detected for FoV1 and FoPV1. Each re-isolated strains retained its original viruses (Figure S3), indicating that FoPV1/3S18 does not alter host pathogenicity, whereas FoV1 does.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we identified and characterized two doubel-stranded RNA (dsRNA) mycovirus co-infecting \u003cem\u003eFusarium oxysporum\u003c/em\u003e strain 3S-18. Genomic and phylogenetic analyses indicate that one virus, FoPV1 isolate 3S-18 (FoPV1/3S18), belongs to the same species as FoPV1 isolate FCR51 [26] within the genus \u003cem\u003eGammapartitivirus\u003c/em\u003e, family \u003cem\u003ePartitiviridae\u003c/em\u003e. The other virus, FoV1, we previous identified as a new member of the genus \u003cem\u003eUnirnavirus\u003c/em\u003e, which comprise monopartite dsRNA viruses [25, 39].\u003c/p\u003e\n\u003cp\u003eAdvances in high-throughput sequencing and metaviromics have led to the discovery of an increasing number of fungal viruses, revealing their extensive diversity. Co-infection of a single fungal strain by multiple viruses is a common phenomenon, as reported in species such as \u003cem\u003eSclerotinia sclerotiorum\u003c/em\u003e, \u003cem\u003eRosellinia necatrix\u003c/em\u003e, \u003cem\u003eBotrytis cinerea\u003c/em\u003e, \u003cem\u003eRhizoctonia solani\u003c/em\u003e, \u003cem\u003eMacrophomina phaseolina\u003c/em\u003e [40\u0026ndash;43, 33]. These strains have been found to harbor 2 to 17 distinct viruses, often exhibiting hypovirulence. Similarly, strain 3S\u0026minus;18 of \u003cem\u003eFusarium oxysporum\u003c/em\u003e is co-infected by two different dsRNA viruses: FoV1 and FoPV1/3S18. Furthermore, FoPV1/3S18 and the previously reported FoPV1/FCR51 belong to the same species as Fusarium mangiferae partitivirus 2 [38]. The widespread occurrence of such co-infections may be attributed to the ubiquity of fungi in nature and their diverse dissemination mechanisms.\u003c/p\u003e\n\u003cp\u003eIt is generally accepted that fungal viruses are transmitted through the production of asexual or sexual spores, as well as via hyphal fusion [1]. Liu et al. [44] demonstrated that SsHADV-1 can be transmitted by the mycophagous insect \u003cem\u003eLycoriella ingenua\u003c/em\u003e. This is because \u003cem\u003eS. sclerotiorum\u003c/em\u003e infected with SsHADV-1 attracts \u003cem\u003eL. ingenua\u003c/em\u003e for feeding, and the virus can replicate and persist throughout the insect\u0026apos;s life stages, including larvae, pupae, adults, and eggs. Furthermore, recent studies have documented instances of mycoviruses being transmitted across species, and even between different kingdoms [45]. For example, the fungal virus Leptosphaeria biglobosa botybirnavirus 1 was successfully transmitted to the distantly related phytopathogenic fungus \u003cem\u003eBotrytis cinerea\u003c/em\u003e through mixed spore inoculation on both culture medium and rapeseed stems, with transmission frequencies of 4.6% and 18.8%, respectively[46]. Similarly, Valsa mali negative-strand RNA virus 1 (VmNSRV1), which infects the apple Valsa canker fungus (\u003cem\u003eValsa mali\u003c/em\u003e), can be bidirectionally transmitted between \u003cem\u003eV. mali\u003c/em\u003e and its apple tree host [47]. Fusarium root rot as a soil born disease, that hyphal spread is influenced by the diverse microbial community in the soil. This interaction may consequently facilitate the transmission of the virus. Such phenomena have been increasingly reported, indicating that the cross-kingdom transmission of fungal viruses may be common in agricultural ecosystems [48, 49]. This mode of transmission also helps explain the extensive diversity observed among fungal viruses.\u003c/p\u003e\n\u003cp\u003eFungal viruses often impair host fitness by reducing growth, sporulation, pigmentation, and most notably, pathogenicity [3, 5]. Owing to this ability to confer hypovirulence, they are regarded as promising biocontrol agents against plant diseases. For instance, SsHADV\u0026minus;1, which infect \u003cem\u003eS. sclerotiorum\u003c/em\u003e strain DT\u0026minus;8, suppresses the expression of key pathogenesis-related genes-including those encoding oxalate synthase, cell wall-degrading enzymes, and effector proteins, thereby abolishing fungal pathogenicity [50]. Moreover, SsHADV\u0026minus;1 can endophytes grow in plant and help it mitigating the risks associated with the field release of pathogens [51]. Other viruses, such as VmNSRV1 and Diaporthe sojae circular DNA virus (DsCDV1), also induce strong hypovirulence in their hosts, show potential for population-wide prevalence, and hold promise for managing fruit tree diseases [47]. In this study, while infection by FoV1 did not affect the host\u0026rsquo;s growth rate, it significantly reduced conidial production and also attenuated its pathogenicity. Conidia serve as the primary source of infection for Fusarium root rot, which directly influences the severity of disease development.\u003c/p\u003e\n\u003cp\u003eIn this study, a transmission assay confirmed a significantly reduction in both conidial production and pathogenicity in the host infected with the derivative isolate B9-VI-1 (FoV1+), compared to the virus-free strain B9. Furthermore, the other derivative isolate B9-VI-2 (FoV1\u0026thinsp;+\u0026thinsp;FoPV1) also showed reduced conidial production, but its pathogenicity did not differ significantly from that of the virus-free strain B9. Those results indicated that FoV1 was the core virus that significantly attenuated the pathogenicity of the host, suggesting its potential as a biocontrol agent worthy of further investigation for managing the Fusarium root rot.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData Availability Statement:\u0026nbsp;\u003c/strong\u003eThe sequence file of FoV1 and FoPV1/3S18 are available from the NCBI, GeneBank Accession No. OR372790, PX306286, PX306285. All the supplementary materials can be download in this article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions:\u0026nbsp;\u003c/strong\u003eJW designed the research. YXN, HZH, XTL, XBZ, SYF, and XYT collected the materials. JW, YXN and RQ perform the experiments. JW and YXN wrote the first draft of the manuscript, HYL, SJL, and SYS reviewed the manuscript. All authors critically reviewed the manuscript and approved the final submission.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u0026nbsp;\u003c/strong\u003eThis research work was financially supported by National Natural Science Foundation of China (32302445), the Science and technology innovation team of Henan Academy of Agricultural Sciences (2022TD26), Joint Fund of Science and Technology R\u0026amp;D Program of Henan Province (232301420117), China Agriculture Research System of MOF and MARA (CARS-14), The Key Project of Science and Technology of Henan Province (251100110100), and Major Science and Technology Project of China National Tobacco Corporation (110202101051). The funder had no role in the study design, data collection and analysis, decision to publish or preparation of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of Interest:\u0026nbsp;\u003c/strong\u003eThe authors declare no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval:\u0026nbsp;\u003c/strong\u003eThis article does not contain any studies with human participants or animals performed by any of the authors.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eGhabrial SA, Cast\u0026oacute;n JR, Jiang D, Nibert ML, Suzuki N (2015) 50-plus years of fungal viruses. Virology 479\u0026ndash;480:356\u0026ndash;368\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eJiang D, Fu Y, Guoqing L, Ghabrial SA (2013) Viruses of the plant pathogenic fungus Sclerotinia sclerotiorum. 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ISME J 14:3120\u0026ndash;3135\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":"Fusarium root rot, Partitiviridae, Gammapartitivirus, Hypovirulence, Biological control","lastPublishedDoi":"10.21203/rs.3.rs-8194396/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8194396/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eFusarium root rot, a persistent soil-borne disease, pose a serious threat to crop production, quality, and ultimately to food security. We identified two double-stranded RNA viruses co-infecting the phytopathogenic fungus \u003cem\u003eFusarium oxysporum\u003c/em\u003e strain 3S-18: Fusarium oxysporum partitivirus 1 isolate 3S-18 (FoPV1/3S18) and Fusarium oxysporum virus 1 (FoV1). The genome of FoPV1/3S18 consists of two segments. dsRNA1 is 1,761 nt in length with a large open reading frame (ORF) encoding an RNA-dependent RNA polymerase (RdRp) of 539 amino acids (aa). dsRNA2 is 1,556 nt in length with an ORF encoding a putative coat protein (CP) of 430 aa. Phylogenetic analysis based on both RdRp and CP amino sequences indicated that FoPV1/3S18 clusters with the members of the genus G\u003cem\u003eammapartitivirus\u003c/em\u003e within the family \u003cem\u003ePartitiviridae\u003c/em\u003e. FoV1 was identified as a new monopartite dsRNA virus with 2,944 nt, containing two ORFs which encode a encoding a protein of 590 aa RdRp and 134 aa nucleocapsid protein, respectively. Its belonging to the genus \u003cem\u003eUnirnavirus.\u003c/em\u003e Furthermore, we demonstrated that both FoPV1/3S18 and FoV1 can be successfully transmitted via hyphal anastomosis to a virus-free strain. Co-infection with FoV1 and FoPV1/3S18 reduced conidial production but did not attenuate fungal virulence. In contrast, Infection by FoV1 alone not only reduced conidial production but also induced hypovirulence.\u003c/p\u003e","manuscriptTitle":"Effect of two double-strand RNA viruses on the virulence of the phytopathogenic fungus Fusarium oxysporum","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-05 15:50:54","doi":"10.21203/rs.3.rs-8194396/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major Revision - English Corrections","date":"2026-01-06T02:34:44+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2025-12-03T13:13:17+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-12-01T10:27:02+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-11-26T14:17:40+00:00","index":"","fulltext":""},{"type":"submitted","content":"Archives of Virology","date":"2025-11-24T09:29:16+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":"cb8bfd41-058e-4abc-95a5-23c8a8cdb5c1","owner":[],"postedDate":"December 5th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-03-23T06:48:57+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-05 15:50:54","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8194396","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8194396","identity":"rs-8194396","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}