Molecular Characterization of the First Alternavirus Identified in Fusarium oxysporum

A novel mycovirus named Fusarium oxysporum alternavirus 1(FoAV1) was identified as infecting Fusarium oxysporum strain BH19, which was isolated from a fusarium wilt diseased stem of Lilium brownii. The genome of FoAV1 contains four double-stranded RNA (dsRNA) segments (dsRNA1, dsRNA 2, dsRNA 3 and dsRNA 4, with lengths of 3.3, 2.6, 2.3 and 1.8 kbp, respectively). Additionally, dsRNA1 encodes RNA-dependent RNA polymerase (RdRp), and dsRNA2- dsRNA3- and dsRNA4-encoded hypothetical proteins (ORF2, ORF3 and ORF4), respectively. A homology BLAST search, along with multiple alignments based on RdRp, ORF2 and ORF3 sequences, identified FoAV1 as a novel member of the proposed family “Alternaviridae”. Evolutionary relation analyses indicated that FoAV1 may be related to alternaviruses, thus dividing the family “Alternaviridae” members into four clades. In addition, we determined that dsRNA4 was dispensable for replication and may be a satellite-like RNA of FoAV1—and could perhaps play a role in the evolution of alternaviruses. Our results provided evidence for potential genera establishment within the proposed family “Alternaviridae”. Additionally, FoAV1 exhibited biological control of Fusarium wilt. Our results also laid the foundations for the further study of mycoviruses within the family “Alternaviridae”, and provide a potential agent for the biocontrol of diseases caused by F. oxysporum.

A collection of F. oxysporum isolates (BH19) were obtained from fusarium wilt-diseased stems of Lilium brownii in the Liaoning province of China. Our preliminary study showed that a similar banding pattern of dsRNA may exist in this isolate. Sequencing and analysis of this dsRNA showed that it corresponded with a novel alternavirus-the first to be described that infects a F. oxysporum strain-which was provisionally named Fusarium oxysporium alternavirus 1 (FoAV1). Accordingly, we conducted genome characterization to elucidate the molecular features of FoAV1, investigate its impact on virulence and understand its biological properties in F. oxysporum.

Strains and Culture Conditions
Seven isolates of F. oxysporium (BH19, BH3, BH19-4V, BH19-3V, MC-1, MC-1-4V and  MC-1-3V) were used in the study. Strains BH19 and BH3 were isolated from a fusarium wilt-diseased stem of Lilium brownii in Liaoning province of China in 2019. Strains BH19-4V and BH19-3V were single-ascospore isolates of strain BH19. MC-1 was a F. oxysporum f. sp. momordicae strain incorporating a hygromycin-resistance gene (hygromycin B phosphotransferase), which has a normal colony morphology and high virulence in its hosts. Strains MC-1-4V and MC-1-3V were derivative strains isolated from MC-1 following confrontation training with BH19-4V and BH19-3V. All strains were stored at −80 • C in glycerol and cultured on potato dextrose agar (PDA) medium at 28 • C. For dsRNA extraction, mycelium was cultured on a PDA plate covered with cellophane membranes at 28 • C for 4-5 days.

DsRNA Extraction and Purification
The extraction of dsRNA was performed as described previously [39]. Strains were grown for 4-5 days on cellophane membranes on PDA medium. Fresh mycelia (1-2 g) were harvested to isolate dsRNA by selective absorption using cellulose powder CF-11 (Sigma-Aldrich, St. Louis, MO, USA), with nucleic acid co-precipitator added to improve the yield of dsRNA. The dsRNA was treated with RNase-free DNase I and S1 nuclease (Takara, Dalian, China). The dsRNAs were electrophoresed on a 1% agarose gel, stained with ethidium bromide, and visualized with a gel documentation and image analysis system (InGenius LhR, Syngene, UK).

cDNA Synthesis, Cloning, and Sequencing
The purified dsRNA samples were used as a template for cDNA synthesis. cDNA synthesis and cloning were conducted according to the methods described previously [41]. A cDNA library was constructed using TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix ( ® TransGen Biotech, Beijing, China) according to the manufacturer's instructions. To obtain initial sequence clones, a random primer (RACE3RT) was used for RT-PCR amplification. Partial gap sequences between the initial sequences were filled by RT-PCR with sequence-specific primers designed from the obtained sequences. For the 5 and 3 terminal sequences cloning, an anchor primer PC3-T7 loop was ligated to the dsRNA using T4 RNA ligase and used for the RT reaction. Then, a PC2 primer (designed based on the corresponding sequence of the PC3-T7 loop) and sequence-specific primers (designed based on the proximal regions sequences) were used for the amplification of terminal sequences. All PCR products were cloned into a pMD18-T vector (Takara, Dalian, China), which was then transformed into E. coli DH5α cells (Takara, Dalian, China) for sequencing. To achieve high-quality consensus sequences, each nucleotide of full-length cDNA was sequenced at least three times. All the primers used for cDNA cloning and sequencing are listed in Supplementary Table S1.

Sequence and Phylogenetic Analysis
Open reading frames (ORFs) and conserved domains were predicted using an ORF finder and CD-search on the NCBI website (http://www.ncbi.nlm.nih.gov) and motifs scan website (http://www.genome.jp/tools/motif/). Multiple sequence alignments were performed using sequences of known alternavirus and the CLUSTALX (2.0) program [42]. Phylogenetic trees were constructed using MEGA7 software and generated by the maximumlikelihood (ML) method with 1000 bootstrap replicates [43].

Horizontal Transmission of Hypovirulence Traits
To assess the potential horizontal transmission of hypovirulence traits of strain BH19, dual culturing technology was used as described previously [44]. Isolate BH19 and virus-free isolate MC-1, which is hygromycin resistant, were grown separately for 4-5 days on PDA medium, and then co-cultured for a further 4-5 days on PDA fresh plates using mycelium blocks of each isolate kept in close proximity (10-15 mm) to one another. Mixed mycelium blocks were then transferred to fresh PDA plates containing hygromycin, and mycelial growth resulting from co-cultivation were termed the derivative strains. Viral transmission in these strains was evaluated based on dsRNA extraction or RT-PCR detection.

RNA Extraction and RT-PCR Detection
To extract total RNA, strains were grown for 4-5 days on cellophane membrane overlying PDA, and fresh mycelia were harvested and ground to a powder in liquid nitrogen. Total RNA was prepared using an RNA reagent (Newbio Industry, Wuhan, China), according to the manufacturer's instructions.
For RT-PCR detection of virus-transmitted strains, first-strand cDNAs were synthesized using TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix ( ® TransGen Biotech, Beijing, China) according to the manufacturer's instructions. Then, PCR amplification was performed using specific oligonucleotide primers (list in Supplementary Table S1), which were designed based on the sequences of FoAV1 dsRNA. All the amplicons were identified following electrophoresis in 1.5% agarose gels.

Virulence Assay
The virulence of virus-transmitted strains was assessed by pot experiments on bitter gourd plants, as described previously [39]. For these experiments, bitter gourd seedlings were grown in boxes containing sterile soil, and 10 mL spores (10 7 mL −1 ) from the various F. oxysporum strains were inoculated into melon roots when the plants had grown to the second or third leaf stage. Control plants were inoculated with water. All inoculated plants were monitored for the development of typical wilt symptoms, and disease symptoms were photographed. All experiments were repeated twice.

DsRNA Segments in F. oxysporum Strain BH19
The F. oxysporum strains BH19 and BH3 were both isolated from a fusarium wiltdiseased stem of L. brownii in Liaoning Province, China. Compared to BH3, the colony morphology of BH19 was clearly defined with a small aerial mycelium ( Figure 1A). Strain BH19 contained four dsRNA segments (named dsRNA1, dsRNA2, dsRNA3 and dsRNA4) while no dsRNAs were found in strain BH3 ( Figure 1B). The four dsRNA bands had estimated sizes of 3.5 kbp (dsRNA1), 2.5 kbp (dsRNA2), 2.3 kbp (dsRNA3) and 1.8 kbp (dsRNA4), respectively. The segments were confirmed to be dsRNA in nature based on resistance to digestion with DNase I and S1 nuclease.
Viruses 2021, 13, x FOR PEER REVIEW 4 of 14 free isolate MC-1, which is hygromycin resistant, were grown separately for 4-5 days on PDA medium, and then co-cultured for a further 4-5 days on PDA fresh plates using mycelium blocks of each isolate kept in close proximity (10-15 mm) to one another. Mixed mycelium blocks were then transferred to fresh PDA plates containing hygromycin, and mycelial growth resulting from co-cultivation were termed the derivative strains. Viral transmission in these strains was evaluated based on dsRNA extraction or RT-PCR detection.

RNA Extraction and RT-PCR Detection
To extract total RNA, strains were grown for 4-5 days on cellophane membrane overlying PDA, and fresh mycelia were harvested and ground to a powder in liquid nitrogen. Total RNA was prepared using an RNA reagent (Newbio Industry, Wuhan, China), according to the manufacturer's instructions.
For RT-PCR detection of virus-transmitted strains, first-strand cDNAs were synthesized using TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix ( ® TransGen Biotech, Beijing, China) according to the manufacturer's instructions. Then, PCR amplification was performed using specific oligonucleotide primers (list in Supplementary Table S1), which were designed based on the sequences of FoAV1 dsRNA. All the amplicons were identified following electrophoresis in 1.5% agarose gels.

Virulence Assay
The virulence of virus-transmitted strains was assessed by pot experiments on bitter gourd plants, as described previously [39]. For these experiments, bitter gourd seedlings were grown in boxes containing sterile soil, and 10 mL spores (10 7 mL −1 ) from the various F. oxysporum strains were inoculated into melon roots when the plants had grown to the second or third leaf stage. Control plants were inoculated with water. All inoculated plants were monitored for the development of typical wilt symptoms, and disease symptoms were photographed. All experiments were repeated twice.

DsRNA Segments in F. oxysporum Strain BH19
The F. oxysporum strains BH19 and BH3 were both isolated from a fusarium wiltdiseased stem of L. brownii in Liaoning Province, China. Compared to BH3, the colony morphology of BH19 was clearly defined with a small aerial mycelium ( Figure 1A). Strain BH19 contained four dsRNA segments (named dsRNA1, dsRNA2, dsRNA3 and dsRNA4) while no dsRNAs were found in strain BH3 ( Figure 1B). The four dsRNA bands had estimated sizes of 3.5 kbp (dsRNA1), 2.5 kbp (dsRNA2), 2.3 kbp (dsRNA3) and 1.8 kbp (dsRNA4), respectively. The segments were confirmed to be dsRNA in nature based on resistance to digestion with DNase I and S1 nuclease.

Molecular Characterization of FoAV1 in Strain BH19
Following cDNA cloning and sequencing, the complete sequences of the four dsRNA segments found in strain BH19 were obtained, comprising a virus nominated as Fusarium oxysporum alternavirus 1 (FoAV1). The complete sequence of FoAV1 dsRNA1 (GenBank Accession No. MT659125) was 3447 nucleotides (nt) long with a GC content of 56.6%, and it contained one ORF (ORF1) that initiated at position nt 629 and terminated at position nt 3385. Based on the universal genetic code, this sequence potentially encoded 917 amino acid (aa) residues with a calculated molecular mass (Mr) of 103.4 kDa.

Molecular Characterization of FoAV1 in Strain BH19
Following cDNA cloning and sequencing, the complete sequences of the four dsRNA segments found in strain BH19 were obtained, comprising a virus nominated as Fusarium oxysporum alternavirus 1 (FoAV1). The complete sequence of FoAV1 dsRNA1 (GenBank Accession No. MT659125) was 3447 nucleotides (nt) long with a GC content of 56.6%, and it contained one ORF (ORF1) that initiated at position nt 629 and terminated at position nt 3385. Based on the universal genetic code, this sequence potentially encoded 917 amino acid (aa) residues with a calculated molecular mass (Mr) of 103.4 kDa.  Using the homology search on BLASTP, FoAV1 ORF1 encoded a protein which was closely related to RdRps of alternaviruses, including Aspergillus foetidus dsRNA mycovirus (67.72%), Fusarium poae alternavirus 1 (67.61%) and Alternaria alternata virus 1 (66.63%) ( Table 1); FoAV1 ORF2-and ORF3-encoded proteins were also only closely related to their equivalents. Alternavirus ORF2 and ORF3 proteins showed similar identities to those predicted for ORF1 (Tables 2 and 3). FoAV1 ORF4 encoded a protein that had no significant similarities with any other protein in the NCBI database. These results indicated that FoAV1, which was isolated from F. oxysporum strain BH19, contained four dsR-NAs segments and was thus a new member of the proposed "Alternaviridae" family.  Using the homology search on BLASTP, FoAV1 ORF1 encoded a protein which was closely related to RdRps of alternaviruses, including Aspergillus foetidus dsRNA mycovirus (67.72%), Fusarium poae alternavirus 1 (67.61%) and Alternaria alternata virus 1 (66.63%) ( Table 1); FoAV1 ORF2-and ORF3-encoded proteins were also only closely related to their equivalents. Alternavirus ORF2 and ORF3 proteins showed similar identities to those predicted for ORF1 (Tables 2 and 3). FoAV1 ORF4 encoded a protein that had no significant similarities with any other protein in the NCBI database. These results indicated that FoAV1, which was isolated from F. oxysporum strain BH19, contained four dsRNAs segments and was thus a new member of the proposed "Alternaviridae" family.  Further similarities between FoAV1 and other members of the proposed family "Alternaviridae" were investigated using conserved domain database searches together with the multiple protein alignment of related proteins. The obtained results suggested that FoAV1 ORF1, which putatively encode an RdRp, and equivalent proteins encoded by other members of the proposed family "Alternaviridae", contained several conserved amino acid motifs (Figure 3). Similar conservation of several amino acid motifs was obtained when the proteins predicted from the sequences of FoAV1 ORF2 and ORF3 and related alternaviruses were compared (Supplementary Figure S1A,B). There were no significant similarities between the aa sequence of the protein predicted from FoAV1 ORF4 and the protein sequences predicted from other alternaviruses that contained four dsRNAs (Supplementary Figure S1C).

Phylogenetic Analysis of FoAV1
To examine the phylogenetic relationship between FoAV1 with other mycoviruses, a phylogenetic tree was constructed using a maximum likelihood method based on the RdRp (ORF1) sequences of FoAV1, proposed "Alternaviridae" family members and other related viruses from the families Ourmiaviridae, Partitiviridae, Megabirnaviridae, Totiviridae and Chrysoviridae. The results indicated that FoAV1 clustered with members of the proposed "Alternaviridae" family but that it was not a strain or isolate of a previously described alternavirus. The proposed "Alternaviridae" family is itself clustered into four clades (clades I, II, III and IV) ( Figure 4A). Phylogenetic trees based on all the alternavirus ORF2 and ORF3 sequences, including those from FoAV1, showed similar clustering into four clades, as illustrated in Figure 4B,C, respectively.

Phylogenetic Analysis of FoAV1
To examine the phylogenetic relationship between FoAV1 with other mycoviruses, a phylogenetic tree was constructed using a maximum likelihood method based on the RdRp (ORF1) sequences of FoAV1, proposed "Alternaviridae" family members and other related viruses from the families Ourmiaviridae, Partitiviridae, Megabirnaviridae, Totiviridae and Chrysoviridae. The results indicated that FoAV1 clustered with members of the proposed "Alternaviridae" family but that it was not a strain or isolate of a previously described alternavirus. The proposed "Alternaviridae" family is itself clustered into four clades (clades Ⅰ, Ⅱ, Ⅲ and Ⅳ) ( Figure 4A). Phylogenetic trees based on all the alternavirus ORF2 and ORF3 sequences, including those from FoAV1, showed similar clustering into four clades, as illustrated in Figure 4B,C, respectively.

Untranslated Regions Sequences Analysis of FoAV1
The coding sequences of all four FoAV1 dsRNA segments were flanked by two untranslated regions (UTRs) at the 5 -end and 3 -termini, excluding poly (A) tails. The FoAV1 5 -and 3 -UTR sequences of all four genomic dsRNAs were strictly conserved but shared little conservation with equivalent sequences of other alternaviruses which, within the clades, shared limited conservation with each other (Figure 5A,B).

Elimination of FoAV1 dsRNA from F. oxysporum Strain BH19 during Subculture
During passage of the F. oxysporum strain BH19, it was discovered that some isolates (nominated BH19-3V), though not all (nominated BH19-4V), spontaneously lost the FoAV1 dsRNA4 genomic segment. The absence of FoAV1 dsRNA4 in BH19-3V compared to BH19-4V was confirmed following dsRNA extraction and agarose gel electrophoresis ( Figure 6C), as well as by RT-PCR amplification of a fragment of FoAV1 dsRNA4 in BH19-4V ( Figure 6D). The colony morphology and growth rate of F. oxysporum strains BH19-3V and BH19-4V were not significantly different ( Figure 6A,B).  Maxim likelihood phylogenetic tree based on the FoAV1 ORF3 sequence and other "Alternaviridae" fam members. All phylogenetic analyses generated using MEGA7 with 1000 bootstrap replicates. Ⅰ to represent clades of the proposed "Alternaviridae" family. Different colors represent clustering in ferent families or clades.Ⅰ, Ⅱ, Ⅲ and Ⅳ represent different clades of "Alternaviridae" family, resp tively.

Untranslated Regions Sequences Analysis of FoAV1
The coding sequences of all four FoAV1 dsRNA segments were flanked by two translated regions (UTRs) at the 5′-end and 3′-termini, excluding poly (A) tails. T FoAV1 5′-and 3′-UTR sequences of all four genomic dsRNAs were strictly conserved shared little conservation with equivalent sequences of other alternaviruses which, wit the clades, shared limited conservation with each other (Figure 5A,B).  Virulence testing of F. oxysporum on L. brownii plants proved difficult, so FoAV1 was transmitted from the BH19-3V and BH19-4V strains to F. oxysporum f.sp. momordicae MC-1 to determine any effects on virulence. Using dual culture, two derivative strains, MC-1-3V and MC-1-4V, were generated, and virus infection with, respectively, three and four genomic segments, was verified ( Figure 7B). Additionally, RT-PCR amplification was conducted ( Figure 7C). The colony morphology of strains MC-1-3V and MC-1-4V did not significantly differ from the wild-type strain MC-1 ( Figure 7A).

Elimination of FoAV1 dsRNA from F. oxysporum strain BH19 during subculture.
During passage of the F. oxysporum strain BH19, it was discovered that some isolates (nominated BH19-3V), though not all (nominated BH19-4V), spontaneously lost the FoAV1 dsRNA4 genomic segment. The absence of FoAV1 dsRNA4 in BH19-3V compared to BH19-4V was confirmed following dsRNA extraction and agarose gel electrophoresis ( Figure 6C), as well as by RT-PCR amplification of a fragment of FoAV1 dsRNA4 in BH19-4V ( Figure 6D). The colony morphology and growth rate of F. oxysporum strains BH19-3V and BH19-4V were not significantly different ( Figure 6A,B). Fusarium wilt, caused by F. oxysporum, results in systemic infection and is characterized by a typical symptom: complete plant wilt. In order to determine whether the presence of FoAV1 reduced the virulence in F. oxysporum f. sp. momordicae strains, the strains MC-1, MC-1-3V and MC-1-4V were inoculated into bitter gourd seedlings at the stage of two-or three-leaf growth. Virulence was assessed before, during, and after the presentation of typical wilt symptoms (Figure 8). The results showed that bitter gourd plants inoculated with the virus-free MC-1 strain exhibited obvious dryness and wilting, while the plants inoculated with the MC-1-3V or MC-1-4V strains remained generally healthy with few or no symptoms (Figure 8).

Effects of FoAV1 on the virulence of F. oxysporum f. sp. momordicae
Virulence testing of F. oxysporum on L. brownii plants proved difficult, so Fo transmitted from the BH19-3V and BH19-4V strains to F. oxysporum f.sp. momord 1 to determine any effects on virulence. Using dual culture, two derivative strain 3V and MC-1-4V, were generated, and virus infection with, respectively, three genomic segments, was verified ( Figure 7B). Additionally, RT-PCR amplification ducted ( Figure 7C). The colony morphology of strains MC-1-3V and MC-1-4V did nificantly differ from the wild-type strain MC-1 ( Figure 7A).

Effects of FoAV1 on the virulence of F. oxysporum f. sp. momordicae
Virulence testing of F. oxysporum on L. brownii plants proved difficult, so F transmitted from the BH19-3V and BH19-4V strains to F. oxysporum f.sp. momo 1 to determine any effects on virulence. Using dual culture, two derivative stra 3V and MC-1-4V, were generated, and virus infection with, respectively, thre genomic segments, was verified ( Figure 7B). Additionally, RT-PCR amplificatio ducted ( Figure 7C). The colony morphology of strains MC-1-3V and MC-1-4V d nificantly differ from the wild-type strain MC-1 ( Figure 7A).  ence of FoAV1 reduced the virulence in F. oxysporum f. sp. momordicae strains, the strains MC-1, MC-1-3V and MC-1-4V were inoculated into bitter gourd seedlings at the stage of two-or three-leaf growth. Virulence was assessed before, during, and after the presentation of typical wilt symptoms (Figure 8). The results showed that bitter gourd plants inoculated with the virus-free MC-1 strain exhibited obvious dryness and wilting, while the plants inoculated with the MC-1-3V or MC-1-4V strains remained generally healthy with few or no symptoms (Figure 8).

Discussion
In this study, we identified and characterized a novel mycovirus, FoAV1, from F. oxysporium strain BH19, which is a causal agent of fusarium wilt disease in Lilium brownii. The genome of FoAV1 contains four dsRNA segments (dsRNA1, dsRNA2, dsRNA3 and dsRNA4) and a single open reading frame (ORF) in each dsRNA segment, namely: ORF1, ORF2, ORF3 and ORF4. BLAST searches revealed that ORF1, ORF2 and ORF3 were all similar in sequence to the ORFs predicted for several alternaviruses. We proposed that FoAV1 be considered a novel member of the proposed family "Alternaviridae".
With the advent of low cost, next-generation sequencing, there has been a dramatic increase in the generation of complete mycovirome sequences [27,45]. Numerous mycoviruses have been described from different families, infecting many phytopathogenic fungi and entomopathogenic fungi [2,8,46]. However, among the hundreds of mycoviruses described so far, only six mycoviruses have been identified in F. oxysporum, an important phytopathogenic species [34][35][36][37][38][39], with no reports of any viruses belonging to the proposed family "Alternaviridae". FoAV1 is the first alternavirus described to infect F. oxysporum.
The members of the proposed family "Alternaviridae" were all isolated from fungi, and clustered mainly according to homology BLAST, multiple alignment and phylogenetic analysis [24,27,28]. Until now, only eight mycoviruses have been proposed as alternaviruses of the family "Alternaviridae"-and these were clustered into one branch, with no classification at the genus level [21,[23][24][25][26][27][28]. The RdRp sequence of FoAV1 was closely

Discussion
In this study, we identified and characterized a novel mycovirus, FoAV1, from F. oxysporium strain BH19, which is a causal agent of fusarium wilt disease in Lilium brownii. The genome of FoAV1 contains four dsRNA segments (dsRNA1, dsRNA2, dsRNA3 and dsRNA4) and a single open reading frame (ORF) in each dsRNA segment, namely: ORF1, ORF2, ORF3 and ORF4. BLAST searches revealed that ORF1, ORF2 and ORF3 were all similar in sequence to the ORFs predicted for several alternaviruses. We proposed that FoAV1 be considered a novel member of the proposed family "Alternaviridae".
With the advent of low cost, next-generation sequencing, there has been a dramatic increase in the generation of complete mycovirome sequences [27,45]. Numerous mycoviruses have been described from different families, infecting many phytopathogenic fungi and entomopathogenic fungi [2,8,46]. However, among the hundreds of mycoviruses described so far, only six mycoviruses have been identified in F. oxysporum, an important phytopathogenic species [34][35][36][37][38][39], with no reports of any viruses belonging to the proposed family "Alternaviridae". FoAV1 is the first alternavirus described to infect F. oxysporum.
In past studies, an interesting phenomenon was discovered; namely, that several segments of viruses are dispensable for replication, and these dispensable segments are considered satellite-like RNAs [47]. Satellite RNAs may have no effect at all, while some satellite RNAs are able to play important roles in biological functions [48]. Interestingly, according to the above classification of proposed alternavirus members, all members (AaV1 and SlV) in clade I have four segments [23], two members (AfV and AsV341) in clade II have four segments [21,24] and all members (FiAV 1, FpAV 1 and FgAV 1) in clade IV have three segments [25,26,28]. As a member of clade III, FoAV1 has four segments, but dsRNA4 can eliminate these during subculture, with no significant difference in the effect (colony morphology, growth rate and virulence) on the host regarding the presence or absence of dsRNA4. Therefore, the dsRNA4 may be a satellite-like RNA with no known biological function, but it may also be related to the evolution of alternaviruses and used as evidence for the proposed family "Alternaviridae".
Fusarium wilt caused by F. oxysporum is an important disease in a wide variety of agricultural crop species [29]. Biological control is an effective method for combatting this disease [30,31]. In recent years, a large number of hypovirulent mycoviruses, such as CHV1 [13] and SsHADV1 [49], have been identified and explored as potential biocontrol agents against fungal diseases [4,32,33]. However, until now, only two mycoviruses, FodV1 and FoOuLV1, isolated from F. oxysporum, exerted hypovirulent effects [34][35][36][37][38][39]. FoAV1 exhibited a good biological control effect for Fusarium wilt, suggesting that it may be a good resource for controlling Fusarium wilt.
In conclusion, we reported herein that FoAV1 is a new alternavirus in F. oxysporum, and that FoAV1 infection can cause hypovirulence in a host. Our results not only lay a foundation for the further study of mycoviruses within the proposed family "Alternaviridae", but also provide a potential agent for the biocontrol of diseases caused by F. oxysporum.

Conflicts of Interest:
The authors declare no conflict of interest.