Complete genome sequence of a novel mitovirus from the phytopathogenic fungus Fusarium oxysporum

Fusarium oxysporum is a cosmopolitan plant pathogen that causes fusarium wilt and fusarium root rot in many economically important crops. There is still limited information about mycoviruses that infect F. oxysporum. Here, a novel mitovirus tentatively named "Fusarium oxysporum mitovirus 1" (FoMV1) was identified in F. oxysporum strain B2-10. The genome of FoMV1 is 2,453 nt in length with a predicted AU content of 71.6% and contains one large open reading frame (ORF) using the fungal mitochondrial genetic code. The ORF putatively encodes an RNA-dependent RNA polymerase (RdRp) of 723 aa with a molecular mass of 84.98 kDa. The RdRp domain of FoMV1 shares 29.01% to 68.43% sequence identity with the members of the family Mitoviridae. Phylogenetic analysis further suggested that FoMV1 is a new member of a distinct species in the genus Mitovirus.

Fusarium oxysporum is one of the most destructive pathogens in agriculture, infecting more than 120 plant species, including many economically important crops such as cotton, tomato, banana, and tobacco [1,2]. This species complex not only causes vascular disease in a large number of economically important crops but also causes fusarium root rot in different Solanaceae species [3,4]. The typical symptoms of fusarium root rot in tobacco include chlorosis of lower leaves, decaying roots, and vascular discoloration. Generally, stems and taproots show reddish to brown vascular discoloration, and the whole plant dies eventually [5]. This pathogen is soil-borne and can be detected both inside and outside of seeds. Moreover, it can interact with parasitic root knot and encysted nematodes to increase disease and facilitate survival in the soil for years in the absence of host plants [6]. Although the use of fumigants is the most consistently effective management strategy for F. oxysporum, methyl bromide and chloropicrin treatment results in poor wrapper leaf quality and burn quality [6]. The use of resistant varieties provides the most effective and economical means of reducing disease [7].
Mycoviruses are viruses that infect fungi, including phytopathogenic and entomopathogenic fungi [8][9][10][11][12]. Most mycoviruses cause cryptic infections and seldom produce symptoms [9]. Mycoviruses belonging to different virus families cause different effects on the morphology of their hosts, including reduced mycelia growth and increased pigmentation [13,14]. Mycovirus-induced hypovirulence is a topic of interest to pathologists. The successful case of applying hypovirulent strains of Cryphonectria parasitica to combat chestnut blight in Europe has provided the impetus for exploiting mycoviruses as virocontrol agents [15]. Other mycoviruses with significant biocontrol effects include Sclerotinia sclerotiorum hypovirulence-associated DNA virus 1 (SsHADV1) and Rosellinia necatrix megabirnavirus 1 (RnMBV1) [16][17][18][19]. With the development of high-throughput sequencing technology, more and more novel mycoviruses have been identified. Hence, there are still opportunities for novel fungal viruses to be used as biological control agents.
Knowledge about mycoviruses in the phytopathogenic species Fusarium oxysporum is still very limited. Four dsRNA elements were detected in a hypovirulent F. oxysporum strain isolated from soybean seedlings, but they were not sequenced or characterized further [20]. Two mycoviruses, viz., Fusarium oxysporum f. sp. dianthi mycovirus 1 1 3 (FodV1), isolated from F. oxysporum-diseased carnation, and Fusarium oxysporum ourmia-like virus 1 (FoOuLV1), isolated from F. oxysporum-diseased bitter gourd, both of which are associated with hypovirulence, have been identified and characterized [21,22]. Subsequently, two more novel mycoviruses have been reported, including Fusarium oxysporum f. sp. dianthi hypovirus 2 (FodHV2) and Fusarium oxysporum f. sp. dianthi mitovirus 1 (FodMV1). FodHV2 belongs to the family Hypoviridae, but it does not alter the vegetative growth or virulence of its host [23,24]. A novel polymycovirus, hadaka virus 1 (HadV1), also isolated from F. oxysporum, has 11(+) RNA genomic segments but lacks a typical structural protein and exemplifies a potential novel lifestyle for multi-segmented RNA viruses [25]. These results suggest that there are mycoviruses harbored by F. oxysporum that still remain to be described, some of which might have potential as biological control agents. Here, we characterized a novel mitovirus from F. oxysporum strain B2-10, which was tentatively named "Fusarium oxysporum mitovirus 1" (FoMV1).

Provenance and sequencing of strains
F. oxysporum strain B2-10 was isolated from the diseased root of a tobacco plant with typical symptoms of fusarium root rot that was collected in the city of Xuchang, Henan province, China, in 2019. The strain was purified by singlespore isolation. Fusarium spp. primers for the translation elongation factor 1 alpha (EF-1α), RNA polymerase II subunit I (RPB1), and RNA polymerase II subunit II (RPB2) genes were used to confirm the identity of the fungus [26,27]. The sequences of EF-1alpha, RPB1, and RPB2 were subjected to a BLAST search of the Fusarium-ID database (fusariumdb.org), respectively. The BLAST results for all three genes indicated that strain B2-10 was Fusarium oxysporum (Supplementary Table S1). Mycelial agar plugs of the fungus were transferred to a 9-cm-diameter potato dextrose agar Petri plate covered with a cellophane membrane and cultured at 25 °C in the dark for four to six days. Mycelial mats were harvested and stored at -70 °C until use. Total RNA extracted from 1.0 g of mycelium using an RNAiso kit (TaKaRa, China) was sent to Shanghai Bohao Biotechnology Co., Ltd. for high-throughput sequencing using an Illumina HiSeq 2500 platform. The rRNA was depleted using a Ribo-ZeroTM rRNA Removal Kit (Illumina, CA, USA). Detailed parameters used in the bioinformatics pipeline were described previously by Wang et al. [28]. Contigs that were identical or complementary to viral genomic sequences were identified as putative viral sequences.
Putative open reading frames (ORFs) were predicted using ORF Finder on the NCBI website (https:// www. ncbi. nlm. nih. gov/ orffi nder/). Potential secondary structures in the 5′-terminal and 3′-terminal nucleotide sequences of FoMV1 (positive strand) were predicted using the RNAfold Web-Server (http:// rna. tbi. univie. ac. at/ cgi-bin/ RNAWe bSuite/ RNAfo ld. cgi). A multiple sequence alignment of the RdRp sequences encoded by FoMV1 and other mitoviruses was performed using DNAMAN software (version 9) and the CLUSTALX program (version 2.1). A phylogenetic tree was constructed using the maximum-likelihood (ML) method with 1000 bootstrap replicates, using the MEGA X program (version 10.1.8).

Sequence properties
The complete genome sequence of FoMV1 was obtained by assembling partial-length cDNAs amplified from total RNA and the 5′-and 3′-terminal sequences, which were determined using rapid amplification of cDNA ends. The complete sequence of FoMV1 has been submitted to the GenBank database with the accession number MW690927.
The full-length cDNA sequence of FoMV1 is 2,453 nt long and has a relatively high A+U content of 71.6% which is higher than that of the AU-rich regions of Fusarium andiyazi mitovirus 1 (FaMV1) and Fusarium oxysporum f. sp. dianthi mitovirus 1 (FodMV1), which have an A+U content of 70.5% and 58.8%, respectively [24,29]. AU-richness is a common characteristic of fungal and plant mitochondrial mitoviruses [30]. The nucleotide sequence of FoMV1 was examined using ORF Finder with fungal mitochondrial codon usage. The positive strand was found to contain one major large ORF (nt 241-2,412) and one small ORF (nt 83-172). The untranslated regions (UTRs) at the 5′ and 3′ ends are 240 nt and 41 nt in length, respectively (Fig. 1A). The large ORF encodes a 723-aa protein with a molecular mass of 84.98 kDa and a predicted pI of 9.83. A total of 13 UGA-encoded tryptophan residues were found in this ORF [31]. The small ORF near the 5′ terminus encodes a protein of 29 aa with a molecular mass of 3.31 kDa and a predicted pI of 8.94. The potential secondary structures of the FoMV1 5′-and 3′-UTRs were predicted, indicating that the 5′-terminal sequence (nt 4-47) could be folded into three stem-loop structures with a ΔG value of -15.60 kcal/mol (Fig. 1B). The 3′-terminal sequence (nt 2,415-2,453) could be folded into a stable stem-loop structure with a ΔG value of -8.70 kcal/mol (Fig. 1B). Moreover, the 5′-and 3′-UTRs of the positive strand of FoMV1 had inverted complementarity, from which a potentially stable panhandle structure could be predicted with a ΔG value of -11.00 kcal/mol (Fig. 1B).
A BLASTx search of the NCBI database indicated that the internal 2,150-nt region (nt 241-2,391) of the FoMV1 sequence is 74.41% identical (E-value 0.0) to the full-length genome sequence (nt 229-2,386) of Fusarium andiyazi mitovirus 1 (GenBank accession no. QIQ28423). A homology search using BLASTp showed that the FoMV1 RdRp aa sequence is most closely related to those of Fusarium andiyazi mitovirus 1, Fusarium circinatum mitovirus 2, Fusarium sacchari mitovirus 1, and Fusarium poae mitovirus 1, with 45.19%-68.43% identity (E-value 0.0). A conserved domain database (CDD) search confirmed that nt 925-2,136 of the FoMV1 RdRp contained a conserved Mitovir_RNA_pol domain (pfam05919). Furthermore, multiple alignment of aa sequences of FoMV1 RdRp and other mitoviruses showed that it contained six conserved motifs (Fig. 2A). This is a characteristic of mitochondrial virus RdRps [32,33]. Therefore, according to the rules for species demarcation of mitoviruses defined by the ICTV (https:// talk. ictvo nline. org/), our results suggest that FoMV1 should be considered a novel tentative member of a new species in the family Mitoviridae. However, there were no proteins or polypeptides homologous to the putative protein sequence of the small ORF. In addition, the presence of small ORFs coding for polypeptides with unknown functions near the 5′-terminal region has been reported previously for other mycovirus, including Botrytis cinerea mitovirus 1 [34], Helminthosporium   [35], Macrophomina phaseolina victorivirus 1 [36], and Rosellinia necatrix megabirnavirus 1 [18].
A phylogenetic tree based on the complete FoMV1 RdRp aa sequence and 25 selected mitovirus RdRp sequences was constructed using the maximum-likelihood (ML) method (Fig. 2B). FoMV1 clustered with Fusarium sacchari mitovirus 1, FaMV1, and Fusarium circinatum mitovirus 2-1 to form a clade that clustered with Fusarium circinatum mitovirus 1, Fusarium globosum mitovirus 1, Fusarium coeruleum mitovirus 1, and Fusarium poae mitovirus 2, all of which are mitochondrial viruses that infect Fusarium spp. Fusarium oxysporum f. sp. dianthi mitovirus 1 (FodMV1) was the first mitovirus identified in F. oxysporum, but it is distantly related to FoMV1 and is more closely related to mitoviruses that infect Rhizoctonia solani and Sclerotinia sclerotiorum [24]. Phylogenetic analysis of RdRp sequences suggested that FoMV1 is a novel member of the family Mitoviridae. Funding This research work was supported by the Science and Technology Project of Henan Provincial Tobacco Company (2020410000270012). The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Availability of data and material The datasets used or analyzed during the current study are available from the corresponding author on reasonable request.

Conflict 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.