Melanconiella theae strains WJB-5 (Supplementary Figure 1), WJB-1-1, WJB-1-2, WJB-1-9, WJB-1-12, WJB-1-20, WJT-1-1, WJT-1-3, WJT-1-7, WJT-1-9, WJT-1-12, and WJT-1-13 were isolated from naturally infected tea plants showing necrotic symptoms (Supplementary Figure 1A) collected from Xuanen county, Hubei province, China, and identified based on the internal transcribed spacer (ITS) region. Of these strains, WJB-1-9 and WJB-1-12 have the same genetic background with WJT-1-9 and WJT-1-12, respectively, since they were derived from the same strain by culturing single mycelium. Fungal mycelia of these strains were grown on PDA for a week at 25°C and then stored in a 25% (v/v) glycerol solution at −70°C until use.

Viral dsRNA was extracted from the fungal mycelial mass using the silica spin column-based method as described previously (Yang et al., 2017). The dsRNAs extracted from M. theae strains WJB-1-1, WJB-1-2, WJB-1-9, WJB-1-12, WJB-1-20, and WJB-5, and those from strain WJB-5 were used for viral genome characterization and treated with RNase-free DNase I and S1 Nuclease to remove any DNA and ssRNA remains, fractionated by agarose gel (1%, w/v) electrophoresis, and detected by UV transillumination after staining with ethidium bromide (0.1 mg/mL).

Purified dsRNA from WJB-5 was used as a template for cDNA synthesis following the previously described method (Xie et al., 2011) using a cDNA synthesis kit (Promega, Madison, WI, United States) and tagged random dN6 primer, 05RACE-3RT (Supplementary Table 1). The resulting cDNA was PCR-amplified using 2 × Gimmico GimiTaq PCR Mix (Gimmico Biotech Co., Wuhan, China) and 05RACE-3 primer (Supplementary Table 1). RT-PCR products were purified and then ligated to the pMD18-T vector (TaKaRa, Beijing, China), then transformed into competent cells of Escherichia coli DH5α. Positive clones with cDNA inserts were selected on Luria-Bertani (LB) agar medium containing ampicillin (50 μg/mL), checked with PCR using M13F-47/M13R-48 primers (Supplementary Table 1), and then submitted for sequencing. Specific primers, MtMV1-F and MtMV1-R (Supplementary Table 1) were designed for rapid amplification of cDNA ends (RACE) to amplify terminal sequences of the genome that were not cloned by the initial random cDNA synthesis. For RACE, the PC3-T7 loop adapter (Supplementary Table 1) was ligated to the 3′ end of each strand in the purified dsRNA using T4 RNA ligase (TaKaRa, Beijing, China) at 16°C for 18 h, as described previously (Ran et al., 2016). The oligonucleotide-ligated dsRNA was purified, denatured in DMSO, and subsequently reverse transcribed. RACE and nested PCR amplification were performed using sets of MtMV1-F/PC2 and PC2/MtMV1-R primers (Supplementary Table 1) to amplify the 3′ - and 5′-termini respectively. PCR amplicons were purified, cloned into a pMD18-T vector, and sequenced. All amplicons were sequenced by Tsingke Biological Technology Co., LTD., Wuhan, China. Sequence homology searches were performed with the BLASTX search tool on the National Center for Biotechnology Information, NCBI². Sequence assemblies from the cDNA and the RACE clones were manipulated using DNAMAN software (version 7.0.2; Lynnon Corp., Vaudreuil-Dorion, QC, Canada) to obtain the complete genome sequence of the dsRNA extracted from the strain WJB-5.

RT-PCR amplification was performed using a specific primer pair (MtMV1-F1: 5′-GTCTTATCCGTGTATTCTGGG-3′; MtMV1-R1: 5′-GGCTTGAGGAACATTGAGA-3′), generating a 1981-bp fragment. An annealing temperature of 56°C was used with a PCR Thermal Cycler (Model A200, LongGene, China).

The viral ORF was identified using Expasy³ website, and the amino acid (aa) sequences encoded by the ORF were deduced. The yeast mitochondrial genetic code was used for the translation of the ORF. The mycovirus sequence was searched using the NCBI BLASTX program to identify similar mycoviruses for phylogenetic analysis. Maximum-likelihood (ML) phylogenetic tree with 1,000 bootstrap replicates was constructed using DNAMAN software (version 7.0.2; Lynnon Corp., Vaudreuil- Dorion, QC, Canada) for the amino acid sequences of RdRp from WJB-5 dsRNA along with 26 mitoviruses and 4 narnaviruses retrieved from the GenBank database. The evolutionary distances were computed using the Poisson correction method (uniform rates between sites). The potential RNA secondary structures of the 5′- and 3′- terminal sequences were predicted, and the free energy (ΔG) was estimated using CLC genomics workbench software (version 21.0.4; QIAGEN Aarhus, Aarhus, Denmark) as described previously (Liu and Di, 2020). Mitoviral RdRp characteristic motifs were searched using the conserved domain database (CDD) search⁴ provided by NCBI, and multiple amino acid alignments.

Mycelium plugs of the strain WJB-5 were grown on an autoclaved cellophane membrane placed on media for 8 days (d) at 25°C. Minor modifications were adopted to isolate and purify viral particles in the previously described method (Ran et al., 2016). In brief, 35 g mycelia of WJB-5 strain were harvested and crushed in the presence of liquid nitrogen. After that, the crushed mycelia were transferred into new sterile tubes containing 3 volumes of cold phosphate buffer (0.1 M sodium phosphate, pH 7.0, containing 0.2 M KCl and 0.5% β-mercaptoethanol) and gently mixed. The homogenate was centrifuged at 10,000 rpm and 4°C for 15 min to remove the hyphal debris. After three ultracentrifugation cycles, virus particles were purified using a stepwise sucrose gradient of 20–50% (W/V). Following sucrose gradient ultracentrifugation, sucrose fractions were collected, diluted, and subjected to ultracentrifugation to pellet virus particles, and the pellet was resuspended again in 150 μL of phosphate buffer (0.1 mol/L sodium phosphate, pH 7.0). Viral dsRNA was recovered from purified viral particles by a phenol-chloroform-based technique as described previously (Toni et al., 2018), fractionated by agarose gel (1%, w/v) electrophoresis and detected by UV transillumination. For negative staining, 10 μL purified suspension of the virus was loaded on a hydrophobic parafilm surface, and subsequently copper grids with a carbon-formvar coating (200-mesh) were floated onto them for 3 min. After drying with a filter paper, the grids plated with virus particles were immediately refloated for 3 min in a drop of 2% (W/V) phosphotungstic acid solution. The excess suspension was removed using filter paper, and the grids were then air-dried for a few min. Finally, transmission electron microscopy (TEM) was used to visualize the samples.

To eliminate the mycovirus from WJB-5 and five other (WJB-1-1, WJB-1-2, WJB-1-9, WJB-1-12, and WJB-1-20) strains of Melanconiella theae, hyphal tipping method together with thermal/dark treatment were conducted. All strains were separately incubated on PDA-T [potato dextrose agar medium containing 0.4% (w/v) green-leaf tea powder] (Yang and Zhang, 2019) media in the dark at 20°C for 3 days. The hyphal tips in the colony margin were individually cut using a fine needle, transferred again to PDA-T in new Petri dishes (1 hyphal tip per dish), and incubated simultaneously. The colonies were subcultured on PDA-T at 20°C in dark conditions for three consecutive subculturing. Finally, the subculturing was achieved on PDA through incubation at 20°C and then used to detect MtMV1 dsRNA. To emphasize that the M. theae strains got cured from MtMV1, total RNA was extracted and RT-PCR amplification was performed using specific primers (MtMV1-F1: 5′-GTCTTATCCGTGTATTCTGGG-3′; MtMV1-R1: 5′-GGCTTGAGGAACATTGAGA-3′), as previously described.

Conidia of WJB-5 strain (harvested from a 30-d-old culture grown on PDA at 20°C in the dark) were suspended in sterile distilled water to the final concentration of 3 × 10⁷ conidia/mL. The resulting conidial suspension was poured on PDA plates and incubated at 20°C in darkness, and used for culturing of single-conidium isolates. A total of 100 single-conidium isolates derived from WJB-5 were obtained and separately incubated on PDA plates at 20°C for mycelial growth. After the colonies had grown, they were subjected to dsRNA extraction to detect the described virus infection.

Morphological traits of virus-infected strains (WJT-1-1, WJT-1-3, WJT-1-7, WJT-1-9, WJT-1-12, and WJT-1-13) and virus-eliminated strains (WJB-5, WJB-1-1, WJB-1-2, WJB-1-9, WJB-1-12, and WJB-1-20) of M. theae were assessed as previously described (Jia et al., 2017). Briefly, freshly grown mycelial discs (5 mm in diameter) were transferred onto PDA and incubated in darkness at 25°C. Colony diameter was measured daily for 9 days to calculate growth rates (mm/d).

To check whether Melanconiella fungi harbor mycoviruses, six strains (WJB-1-1, WJB-1-2, WJB-1-9, WJB-1-12, WJB-1-20, and WJB-5) from M. theae isolated from Hubei province were randomly chosen from the fungal collection and subjected to dsRNA extraction. It revealed that all strains of the phytopathogenic fungus M. theae contain mycovirus-like dsRNA segments (Figure 1). After digestion with DNase I and S1 nuclease enzymes, for the strain WJB-5, a clear band of dsRNA approximately 2.40 kb was detected (Figure 1).

The entire cDNA sequence of the dsRNA fragment of the strain WJB-5 was obtained through random priming cDNA synthesis, RT-PCR, and RACE cloning. Sequence assembly of thirty random cDNA clones revealed that the genome was composed of a single dsRNA segment with a molecular size of 2,461 bp and with an AU-rich content of 62.37%. Searching of the full-length genome and the deduced amino acid sequences using BLASTX and BLASTP, the coding protein shared 37.77–48.65% identities with the RdRp sequences of known mitoviruses belonging to the family Mitoviridae, and the highest identities are 48.65 and 48.35% with those of grapevine-associated mitovirus 20 (GaMV20) and Colletotrichum fructicola mitovirus 1 (CfMV1), respectively (Supplementary Table 2). Thus, the dsRNA component is proposed as a novel mitovirus and tentatively named Melanconiella theae mitovirus 1 (MtMV1). The complete nucleotide sequence of MtMV1 has been deposited in GenBank with the accession number MW802251.

Based on the yeast mitochondrial code, the genomic organization analysis revealed that the MtMV1 genome contains a single large ORF on the positive sense strand of the dsRNA (Figure 2A).

The ORF starts and terminates at nucleotide positions 46 and 2,310, respectively, of 2,265 bp in size, and potentially codes for an RdRp protein with 754 aa in size. The ORF contains multiple codons (10 UGA and 2 UGG) encoding Trp, and the UAG codon stops the translation. Moreover, the 5′- and 3′-untranslated regions (UTRs) are 45 and 151 bp long, respectively (Table 1). The phylogenetic analysis separated the known mitoviruses into two distinct clusters and placed MtMV1 together with CfMV1 and GaMV20 in cluster I, which is distantly separated from other members (Figure 2B). Based on the conserved domain database (CDD) search and multiple amino acid alignments, the six typical amino acid motifs (I–IV) characterized for the mitoviral RdRp domain superfamily were found to be conserved in MtMV1 (Figure 3). However, MtMV1 contains many unique amino acids as compared with the other members in the conserved motifs (e.g., positions 12 and 20 in motif I, 4, and 19 in motif II, 18 in motif III, 3 and 4 in motif IV), which were composed by threonines (Thr) for MtMV1 while leucines (Leu), isoleucines (Ile) or valines (Val) for the others (Figure 3). Moreover, an unique serine (Ser) in position 16 in motif IV and a unique histidine (His) in position 7 in motif V were observed for MtMV1, while alanine in motif IV and aspartic acids, asparagines or threonines in motif V in these positions for the others (Figure 3).

The 5′- and 3′-terminus sequences of the MtMV1 genome were subjected to the prediction of their secondary structures using the CLC genomic workbench software. The results showed that both 5′- and 3′-UTRs could be folded into potential stem-loop structures that stabilize the RNA genome, with ΔG values of −14.3 and −56.5 kcal/mol, respectively (Figures 4A,B). Additionally, the partial regions of both terminal sequences are reverse complementary, with positions 1 to 27 of 5′-UTR paired with positions 100 to 126 of 3′-UTR, and potentially fold into a panhandle structure with ΔG values of −105 kcal/mol (Figure 4C).

To determine whether virions are associated with MtMV1, possible viral proteins were extracted from the mycelia of strain WJB-5 and subjected to ultracentrifugation in stepwise sucrose gradients. The sucrose fraction containing the MtMV1 dsRNAs was further purified and subjected to observation with a transmission electron microscope (TEM). No virus-like particles were observed under TEM, confirming that MtMV1 dsRNAs are naked like other mitoviruses.

To cure the MtMV1 from the WJB-5 strain and the other five strains of M. theae (WJB-1-1, WJB-1-2, WJB-1-9, WJB-1-12, and WJB-1-20), the hyphal tipping method combined with antiviral extracts (incorporating green-leaf tea powder in the culture medium) was used. After three sequential rounds of subculturing, MtMV1 was detected by dsRNA extraction. The results showed that MtMV1 was cured from all detected strains (Figure 5B), which was further confirmed by RT-PCR identification resulting in no target bands (1.98 kb in size) (Supplementary Figure 2).

To investigate whether MtMV1 can be vertically transmitted to the next generation through fungal conidia, conidial suspension prepared from the strain WJB-5 were smeared on PDA, and a total of 100 single-conidium generated colonies were subjected to dsRNA extraction. The results showed that MtMV1 was retained in all these subisolates, suggesting that MtMV1 has efficiently transmitted through the conidia of M. theae (Figure 5A).

To access the effect of MtMV1 on the host fungus, six MtMV1-infected and six MtMV1-free strains of Melanconiella spp. were cultured on PDA in darkness to access their growth rates and morphologies. Most of these strains had similar morphologies and growth rates, ranging from 4.4 to 6.1 mm/d for the MtMV1-infected strains and 4.1 to 5.4 mm/d for the MtMV1-free ones, suggesting that no obvious effects were observed for MtMV1 related to the morphologies and growth rates of their host strains (Supplementary Figure 3).