Characterization of a Botybirnavirus Conferring Hypovirulence in the Phytopathogenic Fungus Botryosphaeria dothidea

A double-stranded RNA (dsRNA) virus was isolated and characterized from strain EW220 of the phytopathogenic fungus Botryosphaeria dothidea. The full-length cDNAs of the dsRNAs were 6434 bp and 5986 bp in size, respectively. The largest dsRNA encodes a cap-pol fusion protein that contains a coat protein gene and an RNA-dependent RNA polymerase (RdRp) domain, and the second dsRNA encodes a hypothetical protein. Genome sequence analysis revealed that the sequences of the dsRNA virus shared 99% identity with Bipolaris maydis botybirnavirus 1(BmBRV1) isolated from the causal agent of corn southern leaf blight, Bipolaris maydis. Hence, the dsRNA virus constitutes a new strain of BmBRV1 and was named Bipolaris maydis botybirnavirus 1 strain BdEW220 (BmBRV1-BdEW220). BmBRV1-BdEW220 contains spherical virions that are 37 nm in diameter and consist of two dsRNA segments. The structural proteins of the BmBRV1-BdEW220 virus particles were 110 kDa, 90 kDa, and 80 kDa and were encoded by dsRNA1 and 2-ORFs. Phylogenetic reconstruction indicated that BmBRV1 and BmBRV1-BdEW220 are phylogenetically related to the genus Botybirnavirus. Importantly, BmBRV1-BdEW220 influences the growth of B. dothidea and confers hypovirulence to the fungal host. To our knowledge, this is the first report of a botybirnavirus in B. dothidea.


Extraction and Purification of dsRNA
The strains were cultured on cellophane membranes overlaid on the surfaces of PDA plates for 5 days at 28 • C in the dark. Approximately 0.5 g of fresh mycelium from each strain was harvested and ground into a fine powder in liquid nitrogen. DsRNAs were extracted using a patented method (no. ZL201310072994.3). Briefly, the method was developed by using silica columns combined with the application of a tissue lysis solution containing sodium dodecyl sulphate (SDS, 2%), as well as binding and washing with buffers containing highly concentrated guanidinium thiocyanate (GuSCN, 50% w/v). The resulting dsRNA extract was dissolved in a total of 50 µL RNase-free water. The dsRNA samples were digested with DNase I (RNase-free) and S1 nuclease (TaKaRa, Dalian, China) to eliminate any contaminating DNA and ssRNA. The purified dsRNA samples were subjected to electrophoresis in a 1% (w/v) agarose gel and viewed on a UV transilluminator after staining with 0.1 µg/mL ethidium bromide. The separated dsRNA segments were excised from the gel and purified using a DNA gel extraction kit (Axygen Scientific, Inc., Wujiang, China). The purified dsRNAs were dissolved in diethylpyrocarbonate (DEPC)-treated water and kept at −80 • C.

cDNA Synthesis and Molecular Cloning
The purified dsRNAs (1.0 µg) combined with 0.1 µg random hexamer primers mixture d(N)6 (TaKaRa, Dalian, China) were used in the first-strand cDNA synthesis using the Moloney murine leukemia virus (M-MLV) reverse-transcriptase according to the manufacturer's instructions (Fermentas, Madison, WI, USA). The second-strand cDNAs were synthesized as previously described in Reference [34], purified by chloroform and isoamyl alcohol, and A-tailed by co-incubation with Taq DNA polymerase and dNTPs (TaKaRa, Dalian, China). The resulting double-stranded cDNAs were ligated into the pMD18-T vector (TaKaRa, Dalian, China) and transformed into competent cells of Escherichia coli DH5 α. All of the positive clones with inserts of more than 500 bp in size were sequenced. Sequence gaps between clones were determined by reverse transcription-polymerase chain reaction (RT-PCR) using primers designed from the obtained cDNA sequences. The terminal sequence of each of the dsRNAs was obtained according to a ligase-mediated rapid amplification of cDNA ends (RLM-RACE) procedure described in [35]. Sequencing was performed by Genscript Biotechnology Co., Ltd., Nanjing, China. At least three independent clones of each product were determined in both orientations.

Sequence Analysis, Alignment, and Phylogenetic Analysis
The DNAMAN software package (DNAMAN version 6.0; Lynnon Biosoft, Montreal, Canada) was used to detect potential open reading frames (ORFs). The prediction of the stem-loop structures of the terminal sequences of the viral RNAs was conducted using the RNA folding program from the Mfold website, implementing the default parameters (http://mfold.rna.albany.edu/?q=mfold/RNA-Folding-Form2.3) [36]. The CD-search website of the National Center for Biotechnology Information (NCBI) (http://www.ncbi.nlm.nih.gov/) and the motif scan website (http://www.genome.jp/tools/ motif/) were used to identify the conserved domains of the full-length cDNA virus sequences. MAFFT software [37] was used for multiple nucleotide and amino acid sequence alignments, and the results were visualized on the BoxShade website (http://www.ch.embnet.org/software/BOX_ form.html). Phylogenetic trees were constructed using the maximum likelihood method in Molecular Evolutionary Genetics Analysis (MEGA) software 7 [38]. Bootstrap values (relative) were generated based on 1000 replicates. Reference sequences of the viruses used for comparative analyses were obtained from NCBI (http://www.ncbi.nlm.nih.gov/genomes).

Virion Purification
The purification of viral particles was performed as previously described [24]. Strain EW220 was grown at 28 • C for 10 days on sterile cellophane films placed on PDA. About 20 g mycelia was harvested and ground into a fine powder in liquid nitrogen. The powder was transferred to a container with 120 mL of 0.1 M sodium phosphate extraction solution (pH 7.0). The suspension liquid was centrifuged at 10,000× g for 20 min. The supernatant was transferred to another plastic tube and then centrifuged at 10,000× g for 20 min again to remove any remaining hyphal cell debris. The supernatant was centrifuged at 100,000× g at 4 • C for 2 h. The obtained pellet was re-suspended in 0.4 mL of 0.1 M phosphate buffer (PB, pH 7.0). The supernatant containing the virus particles was then overlaid on a centrifuge tube containing sucrose gradient (10-50%, w/v) and centrifuged at 70,000× g at 4 • C for 3 h. Each fraction was individually collected, and total RNA was extracted using phenol-chloroform and chloroform-isoamyl alcohol. Viral dsRNAs were detected by 1% agarose gel electrophoresis. The fractions containing viral dsRNAs were centrifuged at 100,000× g at 4 • C for 2 h, and the precipitate was suspended in 100 µL PB (0.05 M, pH 7.0). Viral particles were stained with 2% (w/v) uranyl acetate and examined by transmission electron microscopy (Model Tecnai G2 20; Field Electron and Ion Company, Hillsboro, OR, USA). The viral particle suspension was loaded onto a 12% polyacrylamide gel amended with 1% (w/v) sodium dodecyl sulfate (SDS), run for 8 h at 20 V/cm, and then stained with Coomassie brilliant blue R-250 (Bio-Rad, Hercules, CA, USA). The separated proteins in the SDS-PAGE were used for polypeptide mass fingerprinting-mass spectrum (PMF-MS) analyses.

Vertical and Horizontal Transmission
Single-conidium sub-strains from the strain EW220 were used for evaluating the vertical transmission of the mycovirus. Conidia from strain EW220 were induced on a PDA plate as previously described [30]. The individual sub-strains were obtained as described previously [24], and 120 individual single-conidium sub-strains were assessed for the presence of dsRNA segments in the mycelia. Horizontal transmission of viral dsRNA segments via hyphal anastomosis was executed according to the previously described method [39]. The strain EW220 served as the donor, and strains JNT1111 and EW220-64, which lacked detectable dsRNAs (Table 1), served as the recipients. The strain EW220 and each virus-free strain were co-cultured on the same PDA plates in the three replicates. After 5 days of co-incubation at 25 • C, the mycelial agar discs most distant from the contact point between the two colonies were obtained from the edge of the recipient strains. The new derivatives from strains JNT1111 (EW220-J11-T1, EW220-J11-T2, and EW220-J11-T3) and EW220 (EW220-64-T1, EW220-64-T2, and EW220-64-T3) were individually purified by single hyphal tip culturing and used to assess the mycovirus content and biological properties.

Biological Testing
Using previously described procedures [30], the mycelial growth rate, colony morphology, and virulence of the virus-infected (EW220, EW220-64-T1, EW220-64-T2, and EW220-64-T3) and virus-free (EW220-64 and JNT1111) strains of B. dothidea were assessed (Table 1). Briefly, mycelial growth rates of the strains were measured on fresh PDA plates at 28 • C. Each of the strains was assessed using six replicates. To evaluate the virulence of strain EW220, 12 one-year-old shoots on five-year-old pear (P. pyrifolia cv. 'Hohsui') plants grown in the field were used. The strains EW220 and JNT1111 were tested for their pathogenicity on unwounded shoots by directly placing colonized agar plugs (5 mm in diameter) on the surface of the lenticels (shoots disinfected with 75% ethanol). Wart and canker symptoms were monitored, and disease incidence was recorded at 60 days post inoculation (dpi). To assess the virulence of all the tested strains, actively growing mycelial plugs from each strain were inoculated onto four detached pear fruits (P. bretschneideri cv. 'Huangguan'). A PDA plug lacking the fungi was used as the non-inoculated control. Inoculated fruits were maintained in a 28 • C incubator for 4 days, following which the average lesion size was measured. The experiment was repeated twice. The biological properties were investigated by one-way analysis of variance (ANOVA) using the SAS 9.0 program, and differences with p-values < 0.05 were considered statistically significant.

Biological Characteristics of Strain EW220 and the Detection of dsRNA
Strain EW220, which was cultured on PDA plates at 28 • C in the dark, exhibited abnormal growth. The mycelia grew slowly and exhibited a few aerial hyphae ( Figure 1A). The developing colonies covered the entire dish (90-mm diameter) at 10 dpi. In contrast, the virus-free strain JNT1111 grew rapidly and developed numerous hyphae. The colony of strain JNT1111 could cover the entire culture dish at 3 dpi. The growth rate of the strain EW220 was only 30% of that of the strain JNT1111 ( Figure 1A). The annual branches of the pear tree (P. pyrifolia cv. 'Hohsui') inoculated with strain EW220 showed no symptoms at 60 dpi ( Figure 1B). However, the strain JNT1111 induced typical warts and canker symptoms ( Figure 1B), and the disease incidence reached 80% (10 out of 12 inoculate sites). These results suggested that strain EW220 was a weak virulent strain.
DsRNA was extracted from strains EW220 and JNT1111. The results of the 1.0% agarose gel electrophoresis showed that strain EW220 harbored dsRNA segments with sizes ranging from 7.0 kb to 7.5 kb. No dsRNAs were detected in the preparation from strain JNT1111 using the same treatment conditions (Figure 2A). with strain EW220 showed no symptoms at 60 dpi ( Figure 1B). However, the strain JNT1111 induced typical warts and canker symptoms ( Figure 1B), and the disease incidence reached 80% (10 out of 12 inoculate sites). These results suggested that strain EW220 was a weak virulent strain. DsRNA was extracted from strains EW220 and JNT1111. The results of the 1.0% agarose gel electrophoresis showed that strain EW220 harbored dsRNA segments with sizes ranging from 7.0 kb to 7.5 kb. No dsRNAs were detected in the preparation from strain JNT1111 using the same treatment conditions (Figure 2A). with strain EW220 showed no symptoms at 60 dpi ( Figure 1B). However, the strain JNT1111 induced typical warts and canker symptoms ( Figure 1B), and the disease incidence reached 80% (10 out of 12 inoculate sites). These results suggested that strain EW220 was a weak virulent strain. DsRNA was extracted from strains EW220 and JNT1111. The results of the 1.0% agarose gel electrophoresis showed that strain EW220 harbored dsRNA segments with sizes ranging from 7.0 kb to 7.5 kb. No dsRNAs were detected in the preparation from strain JNT1111 using the same treatment conditions (Figure 2A).

Figure 2.
Double-stranded RNAs extracted from strain EW220 and the genomic organization of Botryosphaeria dothidea botybirnavirus 1 (BmBRV1-BdEW220). (A) One percent agarose gel electrophoretic profiles of dsRNA preparations extracted from strains EW220 and JNT1111 after digestion with DNase I and S1 nuclease; (B) schematic diagrams of the genomic organization of BmBRV1-BdEW220.

Genetic Analysis of dsRNAs
Based on the ds-cDNAs library, the 7.5-kb dsRNA generated 23 random fragments with lengths ranging from 800-2200 bp. BLASTn analysis of these fragments was highly similar to segment 1 of BmBRV1 (sequence identities ranging from 99% to 100%). The 7-kb dsRNA generated a total of 18 random fragments with lengths ranging from 800 bp to 2000 bp. BLASTn analysis revealed that these sequences were most similar to segment 2 of BmBRV1 (sequence identities ranging from 99% to 100%). By combining the sequences obtained from the RT-PCR and RLM-RACE, we obtained the cDNA sequences of the two segments of the dsRNAs. The full-length cDNAs of dsRNAs were 6434 bp, and 5986 bp, respectively ( Figure 2B). The corresponding sequences were deposited in GenBank under accession numbers MH684534 and MH684535.
The cDNA length of dsRNA1 was 6434 bp with a GC content of 49.4%. BLASTn searches revealed that the dsRNA1 sequence shared high similarity (E-value = 0.0; 99% identity; 6381/6434 nt) with the dsRNA1 of BmBRV1 ( Figure S1). A single large ORF from positions 546 nt to 6323 nt was discovered, and this ORF encodes a tentative protein (P1) of 1925 amino acid (aa) residues with a mass of approximately 217 kDa. Motif scanning revealed that the P1 contained a conservative RdRp functional domain (RdRp_4 Pfam02123) at its C-terminal ( Figure 2B). BLASTp analysis indicated that the protein was closely related to the cap-pol fusion protein of the members of genus Botybirnavirus, exhibiting 30% to 99% identity. In particular, P1 showed significant similarity to the cap-pol fusion protein of BmBRV1, which showed 99% identity (1906/1925 aa). The protein P1 was also most closely related to the cap-pol fusion protein of SsBRV1 with an identity of 82% ( Figure S2). However, two amino acid fragments from 1 to 240 and from 901 to 1080 of BmBRV1-BdEW220 exhibited low similarity with SsBRV1 (56% and 50% identities, respectively) ( Figure S2). Interestingly, an amino acid sequence (107 aa) encoded by the nucleotide sequence of dsRNA1 (from 3462 to 3785 nt; Figure S1) showed only 39% identity with SsBRV1 ( Figure S2). In addition, two bipartite nuclear localization signal profiles from 1128 to 1142 aa (KRAVYTIGTLLRKLK, E value = 2.1 × 10 −4 ) and from 1417 to 1431 aa (KRNSQLLEEKEERRR, E value = 2.1 × 10 −4 ), a phosphatase tensin-type domain profile from 1264 to 1453 aa (E value = 1.7 × 10 −2 ), a glutamine cyclotransferase from 1333 to1346 aa (KLKSLGLKVDGHAN, E value = 0.0026), and an immunoreceptor tyrosine-based activation motif from 420 to 440 aa (TDLYNSIGDRAIAERYYDHVVTA, E value = 0.69) were also detected in the P1.
According to the International Committee on Taxonomy of Viruses (ICTV), a sequence identity >90% is indicative that the compared sequences belong to the same virus species [40]. Therefore, we can conclude that the viral sequence analyzed in this study is a strain of BmBRV1. It was described as Bipolaris maydis botybirnavirus 1 strain BdEW220 (BmBRV1-BdEW220).
According to the International Committee on Taxonomy of Viruses (ICTV), a sequence identity >90% is indicative that the compared sequences belong to the same virus species [40]. Therefore, we can conclude that the viral sequence analyzed in this study is a strain of BmBRV1. It was described as Bipolaris maydis botybirnavirus 1 strain BdEW220 (BmBRV1-BdEW220).

Phylogenetic Analysis of the dsRNA Virus
Multiple amino acid alignments of the predicted RdRp indicated the existence of the motifs I-VIII in BmBRV1-BdEW220 and other members of the family Botybirnaviridae ( Figure 4A). The phylogenetic tree was constructed based on the amino acid sequences of RdRp encoded by members of the families Totiviridae, Partitiviridae, Chrysoviridae, Quadriviridae, and Megabirnaviridae, as well as some unassigned dsRNA viruses. The results indicated that the members of the family Botybirnaviridae grouped into two distinct clusters. BmBRV1-BdEW220 clustered together with BmBRV1, SsBRV1, ABRV1, and SlBRV1 to form a separate evolutionary clade from the other cluster, which contained BpRV1 (Botybirnaviridae mode) and SsBRV2 ( Figure 4B).

Phylogenetic Analysis of the dsRNA Virus
Multiple amino acid alignments of the predicted RdRp indicated the existence of the motifs I-VIII in BmBRV1-BdEW220 and other members of the family Botybirnaviridae ( Figure 4A). The phylogenetic tree was constructed based on the amino acid sequences of RdRp encoded by members of the families Totiviridae, Partitiviridae, Chrysoviridae, Quadriviridae, and Megabirnaviridae, as well as some unassigned dsRNA viruses. The results indicated that the members of the family Botybirnaviridae grouped into two distinct clusters. BmBRV1-BdEW220 clustered together with BmBRV1, SsBRV1, ABRV1, and SlBRV1 to form a separate evolutionary clade from the other cluster, which contained BpRV1 (Botybirnaviridae mode) and SsBRV2 ( Figure 4B).

Virus Particles
Virus particles were purified from the mycelia of strain EW220 using sucrose gradient (10-50% sucrose gradient fractions) centrifugation. Agarose gel electrophoresis of the nucleic acids extracted from the sucrose fractions showed that the dsRNA segments were mostly recovered from the 40% fraction (data not shown). To examine the morphology of the viral particles of BmBRV1-BdEW220, the fraction containing viral dsRNAs was centrifuged and re-suspended. The viral particles were isometric and approximately 37 nm in diameter as observed under TEM ( Figure 5A). Furthermore, the result of the agarose gel electrophoresis showed that the dsRNAs directly extracted from the purified viral particles and extracted from the mycelia of strain EW220 had similar migration rates ( Figure 5B). In addition, SDS-PAGE electrophoresis of the viral particles revealed three major protein bands with approximate sizes of 110 kDa (p110), 90 kDa (p90), and 80 kDa (p80) ( Figure 5C).

Virus Particles
Virus particles were purified from the mycelia of strain EW220 using sucrose gradient (10%-50% sucrose gradient fractions) centrifugation. Agarose gel electrophoresis of the nucleic acids extracted from the sucrose fractions showed that the dsRNA segments were mostly recovered from the 40% fraction (data not shown). To examine the morphology of the viral particles of BmBRV1-BdEW220, the fraction containing viral dsRNAs was centrifuged and re-suspended. The viral particles were isometric and approximately 37 nm in diameter as observed under TEM ( Figure  5A). Furthermore, the result of the agarose gel electrophoresis showed that the dsRNAs directly extracted from the purified viral particles and extracted from the mycelia of strain EW220 had similar migration rates ( Figure 5B). In addition, SDS-PAGE electrophoresis of the viral particles revealed three major protein bands with approximate sizes of 110 kDa (p110), 90 kDa (p90), and 80 kDa (p80) ( Figure 5C).

Structural Proteins of the Virus
To further assess the virus structural proteins, the proteins of the BmBRV1-BdEW220 viral particle preparations were loaded onto SDS-PAGE gels and separated ( Figure 5C). These were individually assessed, and their corresponding genes were determined using PMF-MS. A total of 13, 8, and 11 peptide fragments were discovered in p110, p90, and p80, respectively (Tables S1-S3). The 110 kDa protein matched the partial sequence at amino acid 132-685 of P2 encoded by dsRNA2, accounting for 31.0% of the entire coverage (1767 amino acids). The 90 kDa protein matched the partial sequence at amino acid 323-865 of P1 encoded by dsRNA1, accounting for 28.0% of the entire coverage (1925 amino acids). The 11 peptides from p80 corresponded to an ORF1-encoded protein at the amino acid position of 383-865, accounting for 25.0% of the complete coverage (1925 amino acids). Based on the PMF-MS results, p110 was confirmed to correspond to the deduced 110-kDa proteins encoded by the ORF2 of dsRNA2, and p90 and p80 were encoded by the ORF1 of dsRNA1

Structural Proteins of the Virus
To further assess the virus structural proteins, the proteins of the BmBRV1-BdEW220 viral particle preparations were loaded onto SDS-PAGE gels and separated ( Figure 5C). These were individually assessed, and their corresponding genes were determined using PMF-MS. A total of 13, 8, and 11 peptide fragments were discovered in p110, p90, and p80, respectively (Tables S1-S3). The 110 kDa protein matched the partial sequence at amino acid 132-685 of P2 encoded by dsRNA2, accounting for 31.0% of the entire coverage (1767 amino acids). The 90 kDa protein matched the partial sequence at amino acid 323-865 of P1 encoded by dsRNA1, accounting for 28.0% of the entire coverage (1925 amino acids). The 11 peptides from p80 corresponded to an ORF1-encoded protein at the amino acid position of 383-865, accounting for 25.0% of the complete coverage (1925 amino acids). Based on the PMF-MS results, p110 was confirmed to correspond to the deduced 110-kDa proteins encoded by the ORF2 of dsRNA2, and p90 and p80 were encoded by the ORF1 of dsRNA1 ( Figure 2B, Tables S1-S3). Therefore, the structural proteins of BmBRV1-BdEW220 are derived from two ORFs encoded by dsRNA1 and dsRNA2.

Vertical and Horizontal Transmission of BmBRV1-BdEW220
One hundred and twenty single-conidium strains were derived from strain EW220. Detection of dsRNAs revealed that strain EW220-64 was not infected by BmBRV1-BdEW220 (Figure 6), whereas others harbored dsRNA segments. This result revealed that BmBRV1-BdEW220 in strain EW220 was vertically transmitted to the conidia sub-strains. Furthermore, the dsRNA-free strains JNT1111 and EW220-64 were used as the recipients in the horizontal transmission. For each strain, three derivative sub-strains were obtained. The dsRNAs of BmBRV1-BdEW220 were extracted from the derivatives of the recipient strains. These results suggest that BmBRV1-BdEW220 from strain EW220 was horizontally transmitted to strains EW220-64T1, EW220-64T2, and EW220-64T3 via hyphal contact ( Figure 6). However, BmBRV1-BdEW220 from strain EW220 was not horizontally transmitted to the sub-strains from strain JNT1111 (data not shown). These results revealed that dsRNAs of BmBRV1-BdEW220 can be successfully transmitted into the single-conidium strain EW220-64 but not into strains with different of origins, such as JNT1111, as earlier proposed for BpRV1 and SsBRV1 [11,12].

Influence of BdBRV1 on the Biological Properties of B. dothidea
Biological assessment of the two BmBRV1-BdEW220-free and four BmBRV1-BdEW220-infected strains (Figure 6), including colony morphology, growth rate, and virulence, was performed. On PDA plates, the colonies of strains JNT1111 and EW220-64 exhibited radial growth and thick hyphae ( Figure 7A). Compared to strains JNT1111 and EW220-64, the derivative strains (EW220-64T1, EW220-64T2, and EW220-64T3) grew slower on PDA at 28 • C and formed abnormally whitish compact colonies ( Figure 7A). The average growth rate of each tested strain varied from 14.6 mm/day to 37.5 mm/day. The average growth rate of the BmBRV1-BdEW220-infected strains EW220 (14.6 mm/day), EW220-64T1 (23.0 mm/day), EW220-64T2 (19.4 mm/day), and EW220-64T3 (20.7 mm/day) were significantly lower than that of EW220-64 (32.6 mm/day), which was the single-conidium strain from EW220 ( Figure 7C). These results revealed that BmBRV1-BdEW220 exerted greater influence on the growth rate of B. dothidea. The results of the pathogenicity tests on the pear fruit revealed that all of the tested strains caused rot lesions on the pear fruits. The average diameter of the lesions induced by strain EW220-64 (37.6 mm) was significantly larger than those induced by the strains EW220 (12.3 mm), EW220-64T1 (24.3 mm), EW220-64T2 (12.9 mm), and EW220-64T3 (18.8 mm) ( Figure 7C). Furthermore, the largest lesion was observed in JNT1111 (44.4 mm) ( Figure 7B,C). In combination, these findings indicate that BmBRV1-BdEW220 confers hypovirulence to the fungal host B. dothidea.
Viruses 2019, 11, x FOR PEER REVIEW 11 of 17 compact colonies ( Figure 7A). The average growth rate of each tested strain varied from 14.6 mm/d to 37.5 mm/d. The average growth rate of the BmBRV1-BdEW220-infected strains EW220 (14.6 mm/day), EW220-64T1 (23.0 mm/day), EW220-64T2 (19.4 mm/day), and EW220-64T3 (20.7 mm/day) were significantly lower than that of EW220-64 (32.6 mm/day), which was the single-conidium strain from EW220 ( Figure 7C). These results revealed that BmBRV1-BdEW220 exerted greater influence on the growth rate of B. dothidea. The results of the pathogenicity tests on the pear fruit revealed that all of the tested strains caused rot lesions on the pear fruits. The average diameter of the lesions induced by strain EW220-64 (37.6 mm) was significantly larger than those induced by the strains EW220 (12.3 mm), EW220-64T1 (24.3 mm), EW220-64T2 (12.9 mm), and EW220-64T3 (18.8 mm) ( Figure 7C). Furthermore, the largest lesion was observed in JNT1111 (44.4 mm) ( Figure 7B,C). In combination, these findings indicate that BmBRV1-BdEW220 confers hypovirulence to the fungal host B. dothidea.

Discussion
This study presents the first report of a molecular and morphological characterization of a botybirnavirus isolated from the plant phytopathogenic fungus B. dothidea. Based on the sequence analysis of the virus, it constitutes a strain of BmBRV1 and infects B. maydis. It was named Bipolaris Figure 7. Colony morphology and virulence of strain EW220, strain JNT1111, and derived sub-strains on pear fruit (P. bretschneideri cv. Huangguan). Strains EW220-64-T1, EW220-64-T2, and EW220-64-T3 were derived from EW220-64 in the pairing cultures of EW220/EW220-64. (A) Colony morphology in PDA medium (28 • C, 3 days); (B) pear fruits wound-inoculated with colonized plugs of tested strains and photographed at 4 dpi; (C) statistical analysis of the growth rate and the lesion size. The error bars indicate the standard deviations from different sample means. The letter indicates a significant difference at the p < 0.05 level of confidence based on a multiple range test.

Discussion
This study presents the first report of a molecular and morphological characterization of a botybirnavirus isolated from the plant phytopathogenic fungus B. dothidea. Based on the sequence analysis of the virus, it constitutes a strain of BmBRV1 and infects B. maydis. It was named Bipolaris maydis botybirnavirus 1 strain BdEW220 (BmBRV1-BdEW220). The largest dsRNA of BmBRV1-BdEW220 encodes a cap-pol fusion protein that contains a coat protein gene with an RNA-dependent RNA polymerase (RdRp) domain, and the second dsRNA encodes a hypothetical protein. BmBRV1-BdEW220 contains spherical virions that are 37 nm in diameter. The viral particle of BmBRV1-BdEW220 is composed of different structural proteins. BmBRV1-BdEW220 influences the growth of B. dothidea and confers hypovirulence to the fungal host.
Our research is the first to show that botybirnavirus occurs naturally in two taxonomically distinct fungi. The host of BmBRV1 is B. maydis [16], which is the causal agent of corn southern leaf blight [41]. In fact, B. dothidea, the host of BmBRV1-BdEW220, usually infects woody plants [24,25]. They have different hosts and biotopes. Even though B. maydis and B. dothidea belong to Dothideomycetes, it is obvious that these two fungi are not phylogenetically closely related. For instance, B. dothidea belongs to Botryosphaeriales, whereas B. maydis belongs to Pleosporales. In another report, a conspecific mycovirus that naturally occurs in two taxonomically distinct fungi, namely, Ophiostoma novo-ulmi mitovirus 3a-Ld (OnuMV3a-Ld), was found in Sclerotinia homoeocarpa and Ophiostoma novo-ulmi [42]. The natural occurrence of BmBRV1 in these two fungi suggests that the horizontal transmission of this virus may have occurred between B. dothidea and B. maydis. However, the horizontal transmission experiments in this study indicated that BmBRV1-BdEW220 was successfully transmitted from strain EW220 into EW220-64, which is the single-conidium strain from EW220, but it did not transmit into a strain with different origins (strain JNT1111), as proposed before for BpRV1 [11]. These results indicated that the virus might not overcome the vegetative incompatibility in different strains. There might be another important mechanism that allows for the successful transmission of BmBRV1-BdEW220 between B. dothidea and B. maydis. In contrast to plant and animal pathogenic viruses, natural vectors are largely unknown in fungal viruses, which typically have no extracellular phase [2]. However, one of the best-known examples, SsHADV-1, infects a mycophagous insect and utilizes it as a transmission vector [43]. Further studies are needed to elucidate if there is potential insect vector transmission of BmBRV1-BdEW220 in nature.
The natural host ranges of mycoviruses have been considered to be limited to individuals within the same or closely related vegetative compatibility groups [18]. Generally, viral transmission is described in the same species by hyphal anastomosis horizontally and via spores vertically [44]. The protoplasts of several fungi have been successfully transfected with purified virus particles mediated by polyethylene glycol (PEG); the assay allowed for the extension of the host range of some mycoviruses. For instance, Sclerotinia sclerotiorum hypovirulence-associated DNA virus 1 (SsHADV-1) can infect Sclerotinia minor and S. nivalis, which are the sister species of S. sclerotiorum [23]. Another study showed that Aspergillus thermomutatus chrysovirus 1 (AthCV1), isolated from Aspergillus thermomutatus, can infect A. fumigatus, A. nidulans, and A. niger [45]. In vitro experiments have shown that mycoviruses are transmitted horizontally between fungal species via hyphal anastomosis [46,47]. Melzer et al. [46] described that a dsRNA virus was transmitted from isolates of S. sclerotiorum to S. minor. Cryphonectria hypovirus 1 (CHV-1) could transmit from Cryphonectria parasitica to an unidentified Cryphonectria species in vitro [47]. In nature, interspecies fungi in some genera could be infected by the same mycovirus. CHV-1 was detected in the strains of C. parasitica and Cryphonectria species [47], while Heterobasidion RNA virus 1 (HetRV1) occurred in five different Heterobasidion species [48]. In these studies, conspecific viruses have occurred during transmission in different fungal host species belonging to the same genus. Interestingly, OnuMV3a-Ld and BmBRV1-BdEW220 were found in different taxonomically-distinct fungi. Considering the high probability of co-infection of plants with phytopathogenic fungi in nature, we believe that the cross-infections of conspecific mycoviruses to fungi are not merely isolated cases and could occur more frequently.
In the genus Botybirnavirus, BpRV1, SsBRV1, SsBRV2, ABRV1, BmBRV1, and BmBRV1-BdEW220 infect filamentous fungi [11][12][13][14]16], whereas SlaBRV1 was detected in a soybean phyllosphere via a metatranscriptomics technique [15]. Five mycoviruses in the family Botybirnaviridae have been found to infect phytopathogenic fungi, but only two were involved in conferring hypovirulence to their host fungi [11,12]. The results of the present study indicated that BmBRV1-BdEW220 is related to hypovirulence to B. dothidea. Similarly, BpRV1 and SsBRV2 could alter colony morphology and reduce the virulence of their host fungi [11,13]. Interesting, S. sclerotiorum strain infected with SsBRV1, which exhibits satellite-like RNA, showed hypovirulence [12]. The satellite-like RNA (dsRNA 3) of SsBRV1 might participate in modulating the virulence of the virus [12]. Moreover, the ABRV1-infected fungal strains maintained active growth and a normal colony morphology but exhibited attenuated virulence [14]. Furthermore, in addition to the significant differences between the BmBRV1-BdEW220-infected and BmBRV1-BdEW220-free strains, there were also significant differences between the virus-infected strains. This might be associated with the use of the sectored or normal region of the mycelial agar plugs for assaying the virulence. In combination, these findings suggest that BmBRV1-BdEW220 is closely associated with the hypovirulence of the phytopathogenic fungus B. dothidea.