A Novel Strain of Fusarium oxysporum Virus 1 Isolated from Fusarium oxysporum f. sp. niveum Strain X-GS16 Influences Phenotypes of F. oxysporum Strain HB-TS-YT-1hyg

A novel strain of Fusarium oxysporum virus 1 (FoV1) was identified from the Fusarium oxysporum f. sp. niveum strain X-GS16 and designated as Fusarium oxysporum virus 1-FON (FoV1-FON). The full genome of FoV1-FON is 2902 bp in length and contains two non-overlapping open reading frames (ORFs), ORF1 and ORF2, encoding a protein with an unknown function (containing a typical −1 slippery motif G_GAU_UUU at the 3′-end) and a putative RNA-dependent RNA polymerase (RdRp), respectively. BLASTx search against the National Center for the Biotechnology Information (NCBI) non-redundant database showed that FoV1-FON had the highest identity (97.46%) with FoV1. Phylogenetic analysis further confirmed that FoV1-FON clustered with FoV1 in the proposed genus Unirnavirus. FoV1-FON could vertically transmit via spores. Moreover, FoV1-FON was transmitted horizontally from the F. oxysporum f. sp. niveum strain X-GS16 to the F. oxysporum strain HB-TS-YT-1hyg. This resulted in the acquisition of the F. oxysporum strain HB-TS-YT-1hyg-V carrying FoV1-FON. No significant differences were observed in the sporulation and dry weight of mycelial biomass between HB-TS-YT-1hyg and HB-TS-YT-1hyg-V. FoV1-FON infection significantly increased the mycelial growth of HB-TS-YT-1hyg, but decreased its virulence to potato tubers and sensitivity to difenoconazole, prochloraz, and pydiflumetofen. To our knowledge, this is the first report of hypovirulence and reduced sensitivity to difenoconazole, prochloraz, and pydiflumetofen in F. oxysporum due to FoV1-FON infection.


Introduction
Mycoviruses are widespread in all major taxa of fungi, including yeasts and mushrooms, as well as plant-, insect-, and human-pathogenic fungi, and other organisms such as oomycetes [1][2][3].According to the latest taxonomy report of the International Committee on Virus Taxonomy (ICTV) and the accepted list of all mycoviruses in the ICTV VMR_MSL38_v2 (https://ictv.global/vmr,(accessed on 17 January 2024), there are 30 families and two genera containing a total of 491 species that infect fungi.The majority of them have an RNA genome, including the positive-sense single-stranded RNA (+ssRNA) genome, negative-sense single-stranded RNA (−ssRNA) genome, double-stranded RNA (dsRNA) genome, and single-stranded reverse-transcribing RNA (ssRNA-RT) genome; furthermore, there are several mycoviruses with a single-stranded DNA (ssDNA) genome [4].
In the present study, a novel strain of Fusarium oxysporum virus 1 (FoV1) was isolated from the F. oxysporum f. sp.niveum strain X-GS16 and tentatively designated as Fusarium oxysporum virus 1-FON (FoV1-FON), which was the causal agent of watermelon Fusarium wilt.An assay on the vertical transmission of FoV1-FON via spores was conducted.The horizontal transmission of FoV1-FON from the F. oxysporum f. sp.niveum strain X-GS16 to the F. oxysporum strain HB-TS-YT-1 hyg , causing potato dry rot through hyphal contact using pairing cultures, was carried out.In addition, the impacts on the colony growth, sporulation, virulence, and sensitivity to three fungicides (difenoconazole, prochloraz, and pydiflumetofen) of the F. oxysporum strain HB-TS-YT-1 hyg induced by FoV1-FON were studied.

Fungal Strains
Three strains (F.oxysporum f. sp.niveum strain X-GS16, F. oxysporum strain HB-TS-YT-1 hyg , and F. oxysporum strain HB-TS-YT-1 hyg -V) were used in this study.X-GS16 was isolated from diseased watermelon roots with the symptom of Fusarium wilt and identified to be F. oxysporum f. sp.niveum, according to the methods described previously [28][29][30], which was collected from Tianshui city, Gansu province of China.HB-TS-YT-1 hyg conferring a hygromycin resistance was confirmed to be FoV1-FON-free based on the reverse transcription-polymerase chain reaction (RT-PCR) detection of FoV1-FON using FoV1-FONspecific primers (FoV1-FON-F and FoV1-FON-R listed in Table 1), which was obtained by transforming the hygromycin B phosphotransferase gene to the F. oxysporum strain HB-TS-YT-1 (the causal agent of potato dry rot) successfully in our previous study [15].HB-TS-YT-1 hyg -V carrying FoV1-FON was obtained through hyphal contact by pairing cultures of the colonies from F. oxysporum f. sp.niveum strain X-GS16 (donor strain) and F. oxysporum strain HB-TS-YT-1 hyg (recipient strain).All the three strains were grown on PDA plates for 7 d at 25 • C in the dark for subsequent use.

Extraction and Purification of RNA
The dsRNA was extracted using the CF-11 cellulose (Sigma-Aldrich, St. Louis, MI, USA) chromatography method [31], then purified with a gel extraction kit (Aidlab Biotechnologies, Beijing, China) after being treated with DNase I (TaKaRa, Dalian, China) and S1 Nuclease (TaKaRa, Dalian, China) [15].TRIpure Reagent (Aidlab Biotechnologies) was used for the total RNA extraction according to the manufacturer's instructions.

Synthesis and Molecular Cloning of Complementary DNA (cDNA)
The synthesis and molecular cloning of cDNA were performed as previously described [32].Briefly, the purified dsRNA was coupled with a tagged random primer (RACE3RT: 5 ′ -CGATCGATCATGATGCAATGCNNNNNN-3 ′ ) for reverse transcription, and the obtained random cDNA products were amplified using a single specific primer (RACE3: 5 ′ -CGATCGATCATGATGCAATGC-3 ′ ).Then, the resulting DNA segments were purified (TaKaRa, Dalian, China) and cloned into the pTOPO-TA vector (Aidlab Biotechnologies, Beijing, China).After transforming into Trelif ™ 5α chemically competent cells (Tsingke Biotech Co., Ltd.Beijing, China), positive clones with inserts of >500 bp in length were sent to Tsingke Biotech Co., Ltd. for sequencing.Final sequences were determined using at least three clones, and assembled using DNAMAN 7.0 (Lynnon Biosoft, CA, USA).Gaps between non-overlapping cDNA clones were determined using RT-PCR amplification with specific primers, FoV1-FON-GAP1-F and FoV1-FON-GAP1-R, which were listed in Table 1.

Sequence Analysis, Alignment, and Phylogenetic Analysis
Putative open reading frames (ORFs) in dsRNA were identified using the ORF Finder program on the NCBI website (https://www.ncbi.nlm.nih.gov/,(accessed on 17 January 2024) with the standard genetic code.The Conserved Domain Database (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi, (accessed on 17 January 2024) was utilized to identify conserved motifs.CLUSTAL_X 1.8 was used to conduct multiple alignment [34].A phylogenetic tree was constructed based on the deduced amino acid (aa) sequence of RNA-dependent RNA polymerase (RdRp) using the maximum-likelihood (ML) method in MEGA version 6.0 software with a bootstrap value of 1000 [34].Hypothetical RNA secondary structures of 3 ′ -terminal sequence of dsRNA were generated using the RNAfold webserver (http://rna.tbi.univie.ac.at/cgi-bin/RNAWebSuite/RNAfold.cgi,(accessed on 17 January 2024).
The collected mycelia of the strains derived from X-GS16 were subjected to the extraction of total RNA, which was used as a template to conduct RT-PCR using FoV1-FON-specific primers (FoV1-FON-F and FoV1-FON-R listed in Table 1).Gel electrophoretic profiles of RT-PCR products were performed to confirm whether FoV1-FON was eliminated from X-GS16 successfully [15,36].

Vertical and Horizontal Transmission of FoV1-FON
The vertical transmission of FoV1-FON via spores was assessed using the single-spore isolation method mentioned above [35,36].
The horizontal transmission of FoV1-FON through hyphal contact using pairing cultures was evaluated according to the methods described previously [15,36,39].ZnCl 2amended PDA plates (1.5 mM) were used to overcome mycelial incompatibility between the donor strain (X-GS16) and recipient strain (HB-TS-YT-1 hyg ).The mycelia of each derivative strain were collected to extract total RNA.The extracted total RNA was used as a template to conduct RT-PCR using FoV1-FON-specific primers (FoV1-FON-F and FoV1-FON-R listed in Table 1).Gel electrophoretic profiles of RT-PCR products were performed to confirm the positive presence of total RNA in the derivative strains, and, thus, determined whether FoV1-FON had been horizontally transmitted from X-GS16 to HB-TS-YT-1 hyg or not.In addition, gel electrophoretic profiles of the dsRNA of derivative strains were carried out to further confirm the existence of FoV1-FON in HB-TS-YT-1 hyg .The derivative strain verified to carry FoV1-FON was designated as HB-TS-YT-1 hyg -V.

Effect of FoV1-FON on Phenotypes of F. oxysporum Strain HB-TS-YT-1 hyg
The effect of FoV1-FON on the colony growth, sporulation, and mycelial biomass of HB-TS-YT-1 hyg and HB-TS-YT-1 hyg -V was determined as described in our previously studies [13,15,36].The mycelial biomass was evaluated by the dry weight of the mycelia collected 7 d after incubation.Three biological replicates and three technical replicates were applied in the experiment.The statistical differences were determined by a t-test through the Graphpad Prism software (version 9.5).

Pathogenicity Test of HB-TS-YT-1 hyg and HB-TS-YT-1 hyg -V
A pathogenicity test of the two strains, HB-TS-YT-1 hyg and HB-TS-YT-1 hyg -V, was conducted as the method described previously [15,40].Three biological replicates and three technical replicates were applied in the experiment.The statistical differences were determined by a t-test through the Graphpad Prism software (version 9.5).

Complete Sequence and Phylogenetic Analysis of FoV1-FON
The electrophoresis of the F. oxysporum f. sp.niveum strain X-GS16 dsRNA extracts resulted in an approximately 3 kb single band (Figure 1A).The complete nucleotide sequence of the dsRNA was determined to be 2902 nt in length with a G+C content of 53.4%.
The genome sequence was deposited in GenBank under the accession number PP404034.BLASTx search against National Center for the Biotechnology Information (NCBI) nonredundant database (accessed on 17 January 2024) showed that it was the most closely related to Fusarium oxysporum virus 1 (FoV1) (identity 97.46%; E value 0; query cover 60%), and designated as FoV1-FON.The genome of FoV1-FON harbors two discontinuous ORFs (ORF1 and ORF2) on the positive strand (Figure 1B).ORF1 encodes an unknown function protein of 314 aa with an estimated mass of 34.27 kDa.The typical −1 slippery motif G_GAU_UUU [42,43] was identified immediately before the stop codon UAA of ORF1 (between 950 nt and 956 nt), suggesting that a programmed ribosomal frameshifting (PRF) was utilized for ORF2 translation.In the case of PRF, the hypothetical fusion protein ORF1+2p contains 926 aa, with an estimated mass of 104.18 kDa.ORF2 sequence contains a predicted RdRp domain, and the palm motifs A, B, and C (Figure 2) were identified within the RdRp domain of FoV1-FON and ten reference Unirnaviruses, whose full name and GenBank accession number were listed in Table S1.
The genome sequence was deposited in GenBank under the accession number PP404 BLASTx search against National Center for the Biotechnology Information (NCBI) n redundant database (accessed on 17 January 2024) showed that it was the most clo related to Fusarium oxysporum virus 1 (FoV1) (identity 97.46%; E value 0; query c 60%), and designated as FoV1-FON.The genome of FoV1-FON harbors two discont ous ORFs (ORF1 and ORF2) on the positive strand (Figure 1B).ORF1 encodes an unkn function protein of 314 aa with an estimated mass of 34.27 kDa.The typical −1 slip motif G_GAU_UUU [42,43] was identified immediately before the stop codon UA ORF1 (between 950 nt and 956 nt), suggesting that a programmed ribosomal frameshif (PRF) was utilized for ORF2 translation.In the case of PRF, the hypothetical fusion pro ORF1+2p contains 926 aa, with an estimated mass of 104.18 kDa.ORF2 sequence cont a predicted RdRp domain, and the palm motifs A, B, and C (Figure 2) were ident within the RdRp domain of FoV1-FON and ten reference Unirnaviruses, whose full n and GenBank accession number were listed in Table S1.
The untranslated regions (UTRs) flanking the ORF at the 5′-and 3′-end were dete to be 14 nt and 108 nt, respectively.Interestingly, stable stem-loop structures were dicted to present in the 3′-UTR of FoV1-FON (Figure 1C, with minimum optimal en ∆G= −44.22 kcal/mol).This prediction was also evidenced in 3′-UTR of another Uni virus, Fusarium culmorum virus 1 (FcV1) [43].Stem loop structures were common in v UTRs, and might be biologically important in replication and translation [43].The untranslated regions (UTRs) flanking the ORF at the 5 ′ -and 3 ′ -end were detected to be 14 nt and 108 nt, respectively.Interestingly, stable stem-loop structures were predicted to present in the 3 ′ -UTR of FoV1-FON (Figure 1C, with minimum optimal energy ∆G= −44.22 kcal/mol).This prediction was also evidenced in 3 ′ -UTR of another Unirnavirus, Fusarium culmorum virus 1 (FcV1) [43].Stem loop structures were common in viral UTRs, and might be biologically important in replication and translation [43].
A maximum-likelihood phylogenetic analysis based on the aa sequence of the RdRp of FoV1-FON and 48 representative members in the five families (Amalgaviridae, Curvulaviridae, Partitiviridae, Totiviridae, and the proposed family Unirnaviridae) and the proposed genus Ustivirus revealed that FoV1-FON clustered together with members of the proposed family Unirnaviridae and was the most closely related to FoV1 (Figure 3), whose full name and GenBank accession number were listed in Table S2.Collectively, all the results indicated that FoV1-FON is a novel strain of FoV1 belonging to the genus Unirnavirus within the proposed family Unirnaviridae.A maximum-likelihood phylogenetic analysis based on the aa sequence of the RdRp of FoV1-FON and 48 representative members in the five families (Amalgaviridae, Curvulaviridae, Partitiviridae, Totiviridae, and the proposed family Unirnaviridae) and the proposed genus Ustivirus revealed that FoV1-FON clustered together with members of the proposed family Unirnaviridae and was the most closely related to FoV1 (Figure 3), whose full name and GenBank accession number were listed in Table S2.Collectively, all the results indicated that FoV1-FON is a novel strain of FoV1 belonging to the genus Unirnavirus within the proposed family Unirnaviridae.S1.A maximum-likelihood phylogenetic analysis based on the aa sequence of the RdRp of FoV1-FON and 48 representative members in the five families (Amalgaviridae, Curvulaviridae, Partitiviridae, Totiviridae, and the proposed family Unirnaviridae) and the proposed genus Ustivirus revealed that FoV1-FON clustered together with members of the proposed family Unirnaviridae and was the most closely related to FoV1 (Figure 3), whose full name and GenBank accession number were listed in Table S2.Collectively, all the results indicated that FoV1-FON is a novel strain of FoV1 belonging to the genus Unirnavirus within the proposed family Unirnaviridae.S2.

Vertical and Horizontal Transmission of FoV1-FON
Gel electrophoretic profiles of RT-PCR products from 24 single-spore cultures derived from X-GS16 were evaluated, and the results indicated that FoV1-FON could vertically transmit via spores (Figure 4A).Table S2.

Vertical and Horizontal Transmission of FoV1-FON
Gel electrophoretic profiles of RT-PCR products from 24 single-spore cultures derived from X-GS16 were evaluated, and the results indicated that FoV1-FON could vertically transmit via spores (Figure 4A).
The attempts to eliminate FoV1-FON from X-GS16 failed.The F. oxysporum strain HB-TS-YT-1hyg-V carrying FoV1-FON was obtained successfully by pairing cultures and verified by gel electrophoretic profiles of the detection of FoV1-FON using RT-PCR (Figure 4B) with the specific primers (FoV1-FON-F and FoV1-FON-R listed in Table 1) and dsRNA of HB-TS-YT-1hyg-V (Figure 1A).The attempts to eliminate FoV1-FON from X-GS16 failed.The F. oxysporum strain HB-TS-YT-1 hyg -V carrying FoV1-FON was obtained successfully by pairing cultures and verified by gel electrophoretic profiles of the detection of FoV1-FON using RT-PCR (Figure 4B) with the specific primers (FoV1-FON-F and FoV1-FON-R listed in Table 1) and dsRNA of HB-TS-YT-1 hyg -V (Figure 1A).

Discussion
In the present study, we identified and characterized a novel strain of FoV1 associated with the F. oxysporum f. sp.niveum strain X-GS16 causing watermelon Fusarium wilt, which was designated as FoV1-FON.FoV1-FON could vertically transmit efficiently via spores, and horizontally transmit from X-GS16 to HB-TS-YT-1 hyg .No significant differences were observed in sporulation and mycelial biomass between HB-TS-YT-1 hyg and HB-TS-YT-1 hyg -V.FoV1-FON significantly increased the mycelial growth of HB-TS-YT-1 hyg -V but decreased its virulence to potato tubers and sensitivity to difenoconazole, prochloraz, and pydiflumetofen.To our knowledge, this is the first report of an Unirnavirus affecting the virulence and sensitivity to fungicides of F. oxysporum.
The proposed genus Unirnavirus and the proposed family Unirnaviridae were suggested to be established in 2015 [44] and in 2017 [45], respectively.Later, several mycoviruses discovered in different fungal species also supported the establishment of Unirnaviridae, such as FcV1 infecting F. culmorum [43] and Penicillium citrinum non-segmented dsRNA virus 1 infecting Penicillium citrinum [46].In previous studies, it was reported that the members of the proposed family Unirnaviridae were related to monopartite viruses with a similar genome organization; these belong to the family Amalgaviridae or the proposed genus Ustivirus [42,43,46].In this study, phylogenetic analysis showed that FoV1-FON formed a well-supported independent clade together with members of the genus Unirnavirus within Unirnaviridae, and presented a distant relation with amalgaviruses or ustiviruses.Therefore, the results of our study also supported that Unirnaviridae should be established as a new family, thus broadening the diversity of the mycoviruses belonging to this taxon.
The function of the protein encoded by ORF1 in Unirnaviruses is currently not clear.Conserved α-helical coiled-coil structures were predicted in the proteins encoded by ORF1, which might play a vital role in Unirnaviruses [43].Campo et al. [47] predicted that the putative capsid protein encoded by the ORF1 of Colletotrichum higginsianum nonsegmented dsRNA virus 1 (ChNRV1) was present at very low amounts in the Colletotrichum higginsianum wild-type strain; however, repeated attempts to observe ChNRV1 virions using electron microscopy were unsuccessful.In the present study, the attempt to extract and purify virus particles from X-GS16 also failed.
The classical slippery sequence for −1 PRF is X_XXY_YYZ, where XXX is any three of the same nucleotides, although several deviations such as GGA are tolerated; YYY is AAA or UUU (do not confuse with the use of Y for pyrimidine in other cases); Z is A, C, or U [42].The nucleotides located immediately or soon before the stop codon in ORF1 were observed in the Unirnaviruses reported previously.Among them, Penicillium miczynskii RNA virus 1 (PmRV1) and Ustilaginoidea virens unassigned RNA virus HNND-1 (UvURV-HNND1) contain the U_UUA_AAC heptamer, while the remaining Unirnaviruses have a −1 PRF slippery motif consisting of G_GAU_UUZ (Z stands for A, C or U) (Table S3).As described by Mahillon et al. [43], the −1 PRF slippery motif immediately or soon before the stop codon of ORF1 is one of the features of Unirnaviruses.Similar to ChNRV1, FcV1 was believed to produce a fusion protein due to the −1 PRF, which is likely to be associated with viral replication [43,47].In the current study, the typical −1 slippery motif G_GAU_UUU was present at the 3 ′ -end of ORF1 of FoV1-FON, further supporting that FoV1-FON was a new strain of FoV1, an Unirnavirus within the proposed family Unirnaviridae.
Virus-infected and virus-free isogenic lines are required for an effective investigation of the impact of a virus on its host, and the common method is to eliminate the virus from infected strains or introduce the virus into virus-free fungal isolates [48].In this study, we used five methods (single-spore isolation, hyphal tipping, protoplast regeneration, cycloheximide treatment, and ribavirin treatment) to eliminate FoV1-FON from X-GS16, but we failed.In the previous study, FcV1 was not eliminated from the F. culmorum strain A104-1 successfully [43].It seemed that Unirnaviruses had evolved efficient strategies to be vertically retained in their respective hosts.Fortunately, FoV1-FON was successfully horizontally transmitted from X-GS16 to HB-TS-YT-1 hyg by hyphal contact, and, thus, HB-TS-YT-1 hyg -V carrying FoV1-FON was obtained, which made it possible to study the effect of FoV1-FON on the phenotypes of its host fungus.
When one Monilinia fructicola isolate, which is a fundamental agent causing brown rot on Prunus species, was co-infected by three mycoviruses, its mycelial growth rate increased, but there were no visible effects on lesions growth patterns on plums [49].FoMyV1 infection reduced vegetative growth and spore production of its fungal host, F. oxysporum, but did not alter its virulence [26].RsPV2-BJ-1H infected Rhizoctonia solani and did not significantly affect the hyphal width, sclerotia dry weight, and radial growth rate of its host fungus, but significantly decreased the virulence of its host [13].FaAV1 could not alter the colony morphology and spore production of its fungal host, but increased the mycelial biomass and virulence of its host fungus [15].In this study, FoV1-FON could reduce the virulence of F. oxysporum to potato tubers and increase the mycelial growth rate of its host fungus; however, no significant differences were observed between HB-TS-YT-1 hyg and HB-TS-YT-1 hyg -V in terms of spore production and the dry weight of the mycelial biomass.
Difenoconazole, a demethylation inhibitor (DMI) fungicide, has been widely utilized to control black spot disease caused by Alternaria [50,51], as well as Fusarium wilt incited by F. oxysporum [52,53].Prochloraz is also a DMI fungicide, which is used to manage spot disease caused by A. tenuissima [54] and the Fusarium head blight incited by F. graminearum [55] or Fusarium wilt induced by F. oxysporum [53].Pydiflumetofen, as a succinate dehydrogenase inhibitor (SDHI) fungicide, can effectively control Alternaria leaf spot caused by A. alternata [56] and Fusarium wilt incited by F. oxysporum f. sp.niveum [57].In our previous studies, the infection of Alternaria alternata chrysovirus 1-AT1 (AaCV1-AT1) could reduce the sensitivity of its host fungus, A. tenuissima, to difenoconazole [58]; Alternaria alternata botybirnavirus 1-AT1 (AaBRV1-AT1) also decreased the sensitivity of its host fungus, A. tenuissima, to difenoconazole [36].Similarly, in the present study, FoV1-FON reduced the sensitivity of F. oxysporum to difenoconazole; moreover, FoV1-FON lowered the sensitivity of F. oxysporum to prochloraz and pydiflumetofen.This is the first report of a mycovirus affecting the sensitivity of its host fungus to pydiflumetofen.

Figure 2 .
Figure 2. Multiple alignment of the amino acid (aa) sequence of RdRp of Fusarium oxysporum virus 1-FON (FoV1-FON) and ten reference viruses of the genus Unirnavirus.Three conserved motifs (motif A, motif B, and motif C) were identified to be present in FoV1-FON and the 10 representative members of the genus Unirnavirus.Asterisks represent identical amino acid residues, colons represent amino acids with a high level of chemical similarity, and dots represent amino acid residues with a low level of chemical similarity.The red box indicates the highly conserved GDD tripeptide.The names and GenBank accession numbers of these ten representative members were listed in Table S1.

Figure 2 .
Figure 2. Multiple alignment of the amino acid (aa) sequence of RdRp of Fusarium oxysporum virus 1-FON (FoV1-FON) and ten reference viruses of the genus Unirnavirus.Three conserved motifs (motif A, motif B, and motif C) were identified to be present in FoV1-FON and the 10 representative members of the genus Unirnavirus.Asterisks represent identical amino acid residues, colons represent amino acids with a high level of chemical similarity, and dots represent amino acid residues with a low level of chemical similarity.The red box indicates the highly conserved GDD tripeptide.The names and GenBank accession numbers of these ten representative members were listed in TableS1.

Figure 2 .
Figure 2. Multiple alignment of the amino acid (aa) sequence of RdRp of Fusarium oxysporum virus 1-FON (FoV1-FON) and ten reference viruses of the genus Unirnavirus.Three conserved motifs (motif A, motif B, and motif C) were identified to be present in FoV1-FON and the 10 representative members of the genus Unirnavirus.Asterisks represent identical amino acid residues, colons represent amino acids with a high level of chemical similarity, and dots represent amino acid residues with a low level of chemical similarity.The red box indicates the highly conserved GDD tripeptide.The names and GenBank accession numbers of these ten representative members were listed in Table S1.

Figure 3 .
Figure 3. Phylogenetic tree constructed based on amino acid (aa) sequence of RNA-dependent RNA polymerase (RdRp) of Fusarium oxysporum virus 1-FON (FoV1-FON) and 48 representative members in the five families (Amalgaviridae, Curvulaviridae, Partitiviridae, Totiviridae, and the proposed family Unirnaviridae) and the proposed genus Ustivirus using the maximum-likelihood (ML) method with 1000 bootstrap replicates.The bar scale represents a genetic distance of 0.5 aa substitutions per site.Bootstrap values <50% are hidden.The red star represents the position of FoV1-FON.The name and GenBank accession number of these 48 representative members were listed in TableS2.

Figure 4 .
Figure 4. Vertical and horizontal transmission of Fusarium oxysporum virus 1-FON (FoV1-FON), and the effect of FoV1-FON on the phenotypes of F. oxysporum strain HB-TS-YT-1hyg.(A) Confirmation of the presence of FoV1-FON in the 24 single-spore cultures derived from X-GS16 by 1.0% (w/v) agarose gel electrophoretic profiles of reverse transcription-polymerase chain reaction (RT-PCR) products.Lane M: DNA molecular marker DL 2000; Lane 1-24: Products of RT-PCR using viralspecific primers (Table 1) designed based on the nucleotide sequence of FoV1-FON.(B) Confirmation of the presence of FoV1-FON in HB-TS-YT-1hyg-V derived from HB-TS-YT-1hyg by 1.0% (w/v) agarose gel electrophoretic profiles of RT-PCR products.Lane M: DNA molecular marker DL 2000; Lane 1-8: Products of RT-PCR using viral-specific primers (Table 1) designed based on the nucleotide sequence of FoV1-FON.(C) Colony diameter of the two strains, HB-TS-YT-1hyg and HB-TS-YT-1hyg-V, cultured on potato dextrose agar (PDA) plates at 25 °C in darkness for 6 d. (D) Spores produced by the two strains, HB-TS-YT-1hyg and HB-TS-YT-1hyg-V, cultured on PDA plates at 25 °C for

Figure 4 .
Figure 4. Vertical and horizontal transmission of Fusarium oxysporum virus 1-FON (FoV1-FON), and the effect of FoV1-FON on the phenotypes of F. oxysporum strain HB-TS-YT-1 hyg .(A) Confirmation of the presence of FoV1-FON in the 24 single-spore cultures derived from X-GS16 by 1.0% (w/v) agarose gel electrophoretic profiles of reverse transcription-polymerase chain reaction (RT-PCR) products.Lane M: DNA molecular marker DL 2000; Lane 1-24: Products of RT-PCR using viralspecific primers (Table 1) designed based on the nucleotide sequence of FoV1-FON.(B) Confirmation of the presence of FoV1-FON in HB-TS-YT-1 hyg -V derived from HB-TS-YT-1 hyg by 1.0% (w/v) agarose gel electrophoretic profiles of RT-PCR products.Lane M: DNA molecular marker DL 2000; Lane 1-8: Products of RT-PCR using viral-specific primers (Table 1) designed based on the nucleotide sequence of FoV1-FON.(C) Colony diameter of the two strains, HB-TS-YT-1 hyg and HB-TS-YT-1 hyg -V, cultured on potato dextrose agar (PDA) plates at 25 • C in darkness for 6 d. (D) Spores produced by the two strains, HB-TS-YT-1 hyg and HB-TS-YT-1 hyg -V, cultured on PDA plates at 25 • C for 7 d in the dark.(E) Dry weight of mycelial biomass generated by the two strains, HB-TS-YT-1 hyg and HB-TS-YT-1 hyg -V, cultured in potato dextrose broth (PDB) for 7 d at 25 • C in the dark.Asterisks indicate different levels of significant difference (**** p < 0.0001) between HB-TS-YT-1 hyg and HB-TS-YT-1 hyg -V as determined by the t-test using GraphPad Prism version 9.5 software.

Table 1 .
Primers used to determine the complete genome sequence of the Fusarium oxysporum virus 1-FON (FoV1-FON) in this study.