Characterization of the Mycovirome from the Plant-Pathogenic Fungus Cercospora beticola

Cercospora leaf spot (CLS) caused by Cercospora beticola is a devastating foliar disease of sugar beet (Beta vulgaris), resulting in high yield losses worldwide. Mycoviruses are widespread fungi viruses and can be used as a potential biocontrol agent for fugal disease management. To determine the presence of mycoviruses in C. beticola, high-throughput sequencing analysis was used to determine the diversity of mycoviruses in 139 C. beticola isolates collected from major sugar beet production areas in China. The high-throughput sequencing reads were assembled and searched against the NCBI database using BLASTn and BLASTx. The results showed that the obtained 93 contigs were derived from eight novel mycoviruses, which were grouped into 3 distinct lineages, belonging to the families Hypoviridae, Narnaviridae and Botourmiaviridae, as well as some unclassified (−)ssRNA viruses in the order Bunyavirales and Mononegavirales. To the best of our knowledge, this is the first identification of highly diverse mycoviruses in C. beticola. The novel mycoviruses explored in this study will provide new viral materials to biocontrol Cercospora diseases. Future studies of these mycoviruses will aim to assess the roles of each mycovirus in biological function of C. beticola in the future.


Introduction
Cercospora leaf spot (CLS) is a destructive foliar disease of sugar beet caused by Cercospora beticola, responsible for severe yield losses in epidemic years worldwide [1]. As a well-known pathogen of sugar beet and most species of the Beta genus, C. beticola has also been reported as a pathogen of other members of the family Chenopodiaceae, Acanthaceae, Apiaceae, Brassicaceae, Malvaceae, Plumbaginaceae, and Polygonaceae [2]. Disease caused by C. beticola is very difficult to control, because cultural and chemical controls provide limited effect due to the increasing fungicide resistance of C. beticola [3][4][5]. Although elevated resistance has been reported in most employed fungicides [6][7][8], application of fungicides with diverse modes of action is still widely used to manage CLS disease. Mycoviruses, as a potential biocontrol agent, have received increasing attention due to their ability to reduce the virulence of host fungi. Therefore, using mycoviruses as a biocontrol agent is a potential way to reduce the economic impact on sugar beet and other crop species.
Mycoviruses are viruses that can infect and replicate in fungi [9]. With appearance of next-generation sequencing (NGS), many mycoviruses have been discovered. For example, Osaki et al. detected a dsRNA sample from a single strain of Fusarium poae by deep sequencing and obtained 14 known mycoviruses and two viral sequences as-yet assigned to species in the genera Ophiovirus and Phlebovirus, respectively [10]; Yao et al. used

Total RNA Extraction
A total of 0.2 g mycelium from each isolate was scratched with a blade and put into a 2 mL tube. Total RNA was extracted by thermal phenol method as follows: the mycelia were grounded into fine powder in liquid nitrogen by Vortex mixer (Vortex-Genie 2, 230V (Model G560E), Scientific industries, Inc.). A total of 350 µL 80 • C preheated water-saturated phenol and equal volume of RNA extraction buffer (100 mM Tris-HCL (pH 8.0), 0.1 M LiCl, 10 mM EDTA (pH 8.0), 1.0% SDS) was immediately added into the powdered tissue and vibrated on the vortex mixer for 20 s. After 5 min at room temperature, the thawed mixture was combined with 350 µL chloroform and shaken vigorously for 20 s, then allowed to stand for 5 min at room temperature. The resulting mixture was centrifuged at 13,000 g for 15 min and the supernatant was transferred to a new tube. RNA in the supernatant was precipitated by equal volume of 4 M lithium chloride. The precipitate Viruses 2021, 13, 1915 3 of 16 was washed two times with chilled 75% ethanol. The total RNA was dissolved in a final volume of 40 µL of diethyl pyrocarbonate-treated water and measured by Nanodrop UV spectrophotometer.
The total RNA of C. beticola isolates with the same colony morphology (color and shape), collection site, or fungicide (difenoconazole or pyraclostrobin)-resistant isolates were mixed as one group. Library 01 included strains with normal phenotype collected from the Inner Mongolia Autonomous Region in 2019, library 02 included strains with normal phenotype collected from the Inner Mongolia Autonomous Region, Heilongjiang Province and the Xinjiang Uygur Autonomous Region in 2020, library 03 contained strains with fanned-out edges collected from the Inner Mongolia Autonomous Region, Heilongjiang Province and the Xinjiang Uygur Autonomous Region of China in 2020, library 04 were strains with red or yellow pigmentation collected from the Xinjiang Uygur Autonomous Region, Beijing and the Inner Mongolia Autonomous Region in 2020, library 05 included difenoconazole-resistant strains from the Xinjiang Uygur Autonomous Region and the Inner Mongolia Autonomous Region in 2020, library 06 were pyraclostrobinresistant strains from the Inner Mongolia Autonomous Region in 2020. Library 07 and 08 were strains with irregular edge (detailed information and colony morphology of each group can be seen in Supplementary Materials Table S1 and Figure S1). Finally, 8 groups, each group with mixed total RNA from 9, 23,20,19,24,17,15, and 12 strains, were used for sequence analysis.

High-Throughput Sequencing and Sequence Analysis
Transcriptome sequencing of samples was performed by Beijing Biamark Biotechnology Co., Ltd. Novaseq 6000 was used for high-throughput sequencing, and the sequencing read length was PE150 to obtain raw data. Clean data with high quality were obtained by filtering low quality (Q ≤ 19 bases accounted for more than 50% of the total bases), joint contamination (> 5 bp) and reads containing more than 5% N in the original data.
Virus identification referred to the process of Wu et al. [19,20]: the genome sequence of C. beticola was filtered out by Hisat2 (version 2.2.1), and then clean data without the host genome were assembled by Mega-Hit (v1.2.9) to obtain primary contigs. Subsequently, Cap3 (Version Date: 02/10/15) and CD-Hit-Est (version 4.8.1) were used for splicing primary contigs and clustering them with 95% homologous data, respectively. Finally, the contigs obtained were then subjected to BLAST against GenBank using BLASTn and BLASTx (the nucleotide sequences of the contigs were converted into amino acid sequences then a BLASTp search was run).

DNA Extraction
Fungal DNA was extracted by the CTAB method as previously described although slightly modified [21]. A total of 0.1 g mycelium from each isolate was scratched with a blade and put into a 2 mL centrifuge tube. The mycelia were ground into fine powder in liquid nitrogen by vortex mixer. A total of 600 µL DNA extraction buffer (50 mM Tris-HCl (pH 8.0); 0.7 M NaCl; 10 mM EDTA; 1% CTAB) was immediately added into the powdered tissue and mixed well. After incubation at 65 • C for 1 h, the mixture was combined with 600 µL chloroform/isopentyl alcohol (24:1) and shaken for 20 s, then allowed to stand for 5 min at room temperature. The resulting mixture was centrifuged at 13,000 g for 20 min and 540 µL supernatant was transferred to a new tube. DNA in the supernatant was precipitated by an equal volume of isopropyl alcohol. The precipitate was washed two times with chilled 75% ethanol. The DNA was dissolved in a final volume of 40 µL ddH 2 O.

Confirmation of the Putative Mycoviruses
To verify the presence of putative mycovirus in strains, assembled contigs that matched viral sequences were used to design detection primers (Supplementary Materials Table S2). The first cDNAs strand was synthesized using Moloney murine leukemia virus (M-MLV) transcriptase M531A (Promega Madison, WI USA). Viral sequences were detected both Viruses 2021, 13,1915 4 of 16 by RT-PCR and DNA PCR with corresponding primer pairs. Internal Transcribed Spacer (ITS) Region for each C. beticola strain was amplified with primers ITS1 and ITS4 (Table S2) as positive control. The specific PCR fragments were purified and sequenced by Tsingke Biotechnology Co., Ltd.

Phylogenetic Analysis
The nucleotide sequences and translated amino acid sequences of contigs with high similarity to known viral nucleic acids and proteins in GenBank were used for phylogenetic analysis. Alignments were performed by MAFFT (v7.037b) [22] and phylogenetic trees were constructed by the maximum likelihood method with a bootstrap value of 1000 replicates through MEGA-X [23]. The JTT model was selected, and the coding parameters were added to replace type selection 4. The initial tree BIONJ was improved to NNI. For some contigs that were incomplete and could not be used for phylogenetic analysis, the relationships were judged based on the result of the BLASTp. Viruses and accession numbers of viral gene(s) which were selected to perform phylogenetic analysis are listed (Supplementary Materials Table S3).

Mycoviruses Identification from C. beticola
Fungal viruses have been reported in several phytopathogenic fungi, such as S. sclerotiorum, F. oxysporum, B. cinerea, etc. [24][25][26]. Here, we first showed the presence of various mycoviruses in C. beticola. In this study, 8 RNA sequencing libraries were generated, sequenced to a considerable depth and assembled de novo. The library size of each sample ranged from 2.2-4.9 × 10 7 reads ( Figure 1). Overall, 2.7 × 10 8 reads were generated. After removal of the host genome, a total number of 458,868 primary contigs were assembled by Mega-Hit and 31,416 final contigs were assembled by Mega-Hit and Cap3 were obtained. As a result of sequence analysis, 99 contigs derived from mycoviruses were obtained and the sequences of all the contigs were listed in Supplementary Materials Table S4. These contigs were assigned to 11 putative novel mycoviruses in five different viral families, including Partitiviridae, Hypoviridae, Narnaviridae, Botourmiaviridae, and Metaviridae, as well as some unclassified (−)ssRNA viruses ( Figure 1). Since some putative mycoviruses contained multiple contigs, we first chose the longest contig as the representative one. Then the alignment of nucleotide or amino acid sequence between the representative and other sequences were carried out and the results were shown in Supplementary Materials Table  S5. For example, 33 contigs were all assigned to the virus of the genus Hypovirus in Family Hypoviridae, and the polyprotein of contig1357 had 86.53%−99.79% sequence identity with other contigs at amino acid level. The result suggested that these contigs could be assigned to one virus although variation in the nucleotide sequence existed in different C. beticola strains. Furthermore, contig1357 with the longest length could be selected as representative for further analysis. For each putative novel mycoviruses and their representative contig, provisional names, sequence information and best-matched viruses were listed (Table 1  and Supplementary Materials Table S6).

One Predicted Novel Virus in Family Hypoviridae
The family Hypoviridae, containing only one genus Hypovirus, typically has (+)ssRNA genomes of 9.1-12.7 kb encoding one or two proteins [30]. Here, contig 1357 (accession number MZ546195) showed similarity to members of the family Hypoviridae. Contig 1357 was 12624 nt long and contained one large complete ORF that was predicted to encode a putative polyprotein with 3778 amino acids. The polyprotein had two conserved domains, DUF3525 (PF12039) and DEXDc (SM000487). A phylogenic analysis was conducted using the alignment of RdRp amino acid sequences of contig 1357 with representative members of Hypovirus (Figure 3). Based on BLASTp analysis, the putative protein showed 54%, 51.7%, and 40.8% identity with the polyprotein of Wuhan insect virus 14 (WIV14), Fusarium sacchari hypovirus 1 (FsHV1) and Erysiphe necator associated hypovirus 2, respectively. According to the demarcation criterion for the genus Hypovirus from the ICTV web site, contig 1357 represented a novel hypovirus named Cercospora beticola hypovirus 1 (CbHV1), tentatively.   So far, there are 4 species approved by the ICTV, Cryphonectria hypovirus 1-4 (CHV1-4), and many other hypoviruses reported in the GenBank database. Some hypoviruses have been reported to induce hypovirulence to their host fungi and are considered to be a potential biocontrol resource in plant disease management [15,31]. CHV1 has been successfully applied to chestnut blight control in Europe through altering fungal morphology and reducing virulence on chestnut trees [32]. Based on the CHV1−Cryphonectria parasitica pathosystem, many molecular mechanisms of mycovirology, including replication, pathogenicity, and RNA silencing-associated host immunity, have been extensively revealed [33,34]. Recently, a novel hypovirus named Alternaria alternata hypovirus 1 (AaHV1) isolated from Alternaria alternata f. sp. mali has been identified. Pathogenicity tests suggest that AaHV1 is responsible for the slow growth and hypovirulence of the host, which can be useful for the biocontrol of fungal diseases [15]. Thus, further studies on the molecular characterization and the pathogenicity of CbHV1 make it possible to be used as a biocontrol agent for fungal crop disease management. DUF3525 (PF12039) and DEXDc (SM000487). A phylogenic analysis was conducted using the alignment of RdRp amino acid sequences of contig 1357 with representative members of Hypovirus (Figure 3). Based on BLASTp analysis, the putative protein showed 54%, 51.7%, and 40.8% identity with the polyprotein of Wuhan insect virus 14 (WIV14), Fusarium sacchari hypovirus 1 (FsHV1) and Erysiphe necator associated hypovirus 2, respectively. According to the demarcation criterion for the genus Hypovirus from the ICTV web site, contig 1357 represented a novel hypovirus named Cercospora beticola hypovirus 1 (CbHV1), tentatively.
So far, there are 4 species approved by the ICTV, Cryphonectria hypovirus 1-4 (CHV1-4), and many other hypoviruses reported in the GenBank database. Some hypoviruses have been reported to induce hypovirulence to their host fungi and are considered to be a potential biocontrol resource in plant disease management [15,31]. CHV1 has been successfully applied to chestnut blight control in Europe through altering fungal morphology and reducing virulence on chestnut trees [32]. Based on the CHV1− Cryphonectria parasitica pathosystem, many molecular mechanisms of mycovirology, including replication, pathogenicity, and RNA silencing-associated host immunity, have been extensively revealed [33,34]. Recently, a novel hypovirus named Alternaria alternata hypovirus 1 (AaHV1) isolated from Alternaria alternata f. sp. mali has been identified. Pathogenicity tests suggest that AaHV1 is responsible for the slow growth and hypovirulence of the host, which can be useful for the biocontrol of fungal diseases [15]. Thus, further studies on the molecular characterization and the pathogenicity of CbHV1 make it possible to be used as a biocontrol agent for fungal crop disease management.   (Figure 3). Based on BLASTp analysis, the putative protein showed 54%, 51.7%, and 40.8% identity with the polyprotein of Wuhan insect virus 14 (WIV14), Fusarium sacchari hypovirus 1 (FsHV1) and Erysiphe necator associated hypovirus 2, respectively. According to the demarcation criterion for the genus Hypovirus from the ICTV web site, contig 1357 represented a novel hypovirus named Cercospora beticola hypovirus 1 (CbHV1), tentatively.

One Strain of Previously Reported Narnaviruses
So far, there are 4 species approved by the ICTV, Cryphonectria hypovirus 1-4 (CHV1-4), and many other hypoviruses reported in the GenBank database. Some hypoviruses have been reported to induce hypovirulence to their host fungi and are considered to be a potential biocontrol resource in plant disease management [15,31]. CHV1 has been successfully applied to chestnut blight control in Europe through altering fungal morphology and reducing virulence on chestnut trees [32]. Based on the CHV1− Cryphonectria parasitica pathosystem, many molecular mechanisms of mycovirology, including replication, pathogenicity, and RNA silencing-associated host immunity, have been extensively revealed [33,34]. Recently, a novel hypovirus named Alternaria alternata hypovirus 1 (AaHV1) isolated from Alternaria alternata f. sp. mali has been identified. Pathogenicity tests suggest that AaHV1 is responsible for the slow growth and hypovirulence of the host, which can be useful for the biocontrol of fungal diseases [15]. Thus, further studies on the molecular characterization and the pathogenicity of CbHV1 make it possible to be used as a biocontrol agent for fungal crop disease management.

One Strain of Previously Reported Narnaviruses
) represents viruses which can infect insects. Virus identified in this work is indicated by black arrows.

One Strain of Previously Reported Narnaviruses
Narnaviridae is a family of non-encapsidated (+)ssRNA of 2300-2900 nucleotides that encode a single protein of 80-140 kDa with amino acid sequence motifs characteristic of RdRp [35]. Narnavirus is a simple (+)ssRNA virus, since they only encode a single protein, RdRp, without capsid protein or extracellular transmission [36]. They form a ribonucleotide complex with RdRp in a 1:1 stoichiometry and reach a high copy number when under stress conditions, such as nitrogen starvation [37]. There is no apparent phenotype caused by narnaviruses in their host. In addition, due to the difficulty of generating virus-free strains and the fact that there is a complex and highly diverse viral pool in their fungal hosts, it remains difficult to study narnaviruses [38][39][40].
According to the latest demarcation criterion from the ICTV, there is only one genus, Narnavirus, containing two approved species, Saccharomyces 20S RNA narnavirus (ScNV-20S) and Saccharomyces 23S RNA narnavirus (ScNV-23S). However, there are 130 tentative narnaviruses reported in the GenBank database. Some contigs have been reported to have multi-segmented genomes, which will be classified into different taxa [41][42][43][44]. In this study, contig k141-72534 (accession number MZ546196) contained a complete ORF encoding a putative protein of 805 amino acid. According to the sequence and phylogenetic analysis of conserved RdRp domains of narnaviruses, k141-72534 shared 77.1% amino acid identity with Erysiphe necator associated narnavirus 13 (EnNV13) and clustered together into a single branch with EnNV13 ( Figure 4). The species demarcation criteria for Narnavirus are generally less than 50% identity at protein level. Thus, it is likely that k141-72534 is an isolate of EnNV13. We named Cercospora beticola narnavirus 1 (CbNV1) tentatively. After new taxonomic groups are established, CbNV1 will be classified into an exact taxonomic group.
According to the latest demarcation criterion from the ICTV, there is only one genus, Narnavirus, containing two approved species, Saccharomyces 20S RNA narnavirus (ScNV-20S) and Saccharomyces 23S RNA narnavirus (ScNV-23S). However, there are 130 tentative narnaviruses reported in the GenBank database. Some contigs have been reported to have multi-segmented genomes, which will be classified into different taxa [41][42][43][44]. In this study, contig k141-72534 (accession number MZ546196) contained a complete ORF encoding a putative protein of 805 amino acid. According to the sequence and phylogenetic analysis of conserved RdRp domains of narnaviruses, k141-72534 shared 77.1% amino acid identity with Erysiphe necator associated narnavirus 13 (EnNV13) and clustered together into a single branch with EnNV13 ( Figure 4). The species demarcation criteria for Narnavirus are generally less than 50% identity at protein level. Thus, it is likely that k141-72534 is an isolate of EnNV13. We named Cercospora beticola narnavirus 1 (CbNV1) tentatively. After new taxonomic groups are established, CbNV1 will be classified into an exact taxonomic group.

Three Predicted Novel Viruses in Family Botourmiaviridae
Botourmiaviridae is a new family approved by the ICTV, containing six genera: Ourmiavirus, Botoulivirus, Magoulivirus, Penoulivirus, Rhizoulivirus and Scleroulivirus [45,46]. In addition, numerous ourmia-like mycoviruses have been reported and clustered into an unclassified clade which do not belong to five mycovirus genera. In this work, three contigs, contig 965 (accession number MZ568927), contig 1024 (accession number MZ568928), and k141-5161 (accession number MZ568929), showed similarity to members of the family Botourmiaviridae. Contig 965 was 2273 nt long and contained one complete ORF encoding a putative protein of 617 amino acid. BLASTp analysis showed that this putative protein

Three Predicted Novel Viruses in Family Botourmiaviridae
Botourmiaviridae is a new family approved by the ICTV, containing six genera: Ourmiavirus, Botoulivirus, Magoulivirus, Penoulivirus, Rhizoulivirus and Scleroulivirus [45,46]. In addition, numerous ourmia-like mycoviruses have been reported and clustered into an unclassified clade which do not belong to five mycovirus genera. In this work, three contigs, contig 965 (accession number MZ568927), contig 1024 (accession number MZ568928), and k141-5161 (accession number MZ568929), showed similarity to members of the family Botourmiaviridae. Contig 965 was 2273 nt long and contained one complete ORF encoding a putative protein of 617 amino acid. BLASTp analysis showed that this putative protein was most similar to the RdRp of Erysiphe necator associated ourmia-like virus 10, a member of the Ourmiavirus genus, with 68.1% identity. Contig 1024 was 1440 nt and contained one complete ORF, which encode a putative protein of 383 amino acid. The predicted amino acid sequence of this protein was similar to the RdRp of Plasmopara viticola lesion-associated ourmia-like virus 42 with 82% identity. k141-5161 was 2108 nt and the amino acid sequence was most similar to the RdRp of soybean thrips ourmia-like virus 1 (STOLV1) with 82.8% identity. The species demarcation criteria for the four genera in Botourmiaviridae is less than 90% amino acid identity in the RdRp or below 70% amino acid identity in CP. Therefore, contig 965, contig 1024, and k141-5161 represented three novel botourmiaviruses that we named Cercospora beticola ourmia-like virus 1 (CbOV1), Cercospora beticola ourmia-like virus 2 (CbOV2), and Cercospora beticola ourmia-like virus 3 (CbOV3), respectively.
To elucidate the evolutionary history of CbOV1-3, a phylogenetic analysis was conducted using the conserved RdRp domain encoded by CbOV1-3 and other selected viral sequences, including members of Penoulivirus, Botoulivirus, Scleroulivirus, Magoulivirus, and Ourmiavirus ( Figure 5). The phylogenetic tree revealed that CbOV1 and CbOV2 were grouped with the ourmia-like viruses, suggesting that these ourmia-like viruses might represent a separate taxonomical group in the family Botourmiaviridae, and CbOV3 were in the group of Scleroulivirus. grouped with the ourmia-like viruses, suggesting that these ourmia-like viruses might represent a separate taxonomical group in the family Botourmiaviridae, and CbOV3 were in the group of Scleroulivirus.
In recent years, many ourmia-like viruses were found in plant-pathogenic fungi [47][48][49]. For example, the synthesized RNA of Sclerotinia sclerotiorum ourmia-like virus 4 (SsOLV4) can replicate in and transfer among strains of S. sclerotiorum via hyphal anastomosis, suggesting that a single RNA encoding an RdRp is sufficient for replication, infection, and transmission of ourmia-like viruses in fungi [47]. These phenomena indicate that ourmia-like viruses might have originated from Ourmiavirus by gene-loss events during their adaption to the fungal host [48,50]. Consequently, increasingly more ourmia-like viruses identified can help us have a better understanding of evolution and taxonomy of mycovirus.  grouped with the ourmia-like viruses, suggesting that these ourmia-like viruses might represent a separate taxonomical group in the family Botourmiaviridae, and CbOV3 were in the group of Scleroulivirus. In recent years, many ourmia-like viruses were found in plant-pathogenic fungi [47][48][49]. For example, the synthesized RNA of Sclerotinia sclerotiorum ourmia-like virus 4 (SsOLV4) can replicate in and transfer among strains of S. sclerotiorum via hyphal anastomosis, suggesting that a single RNA encoding an RdRp is sufficient for replication, infection, and transmission of ourmia-like viruses in fungi [47]. These phenomena indicate that ourmia-like viruses might have originated from Ourmiavirus by gene-loss events during their adaption to the fungal host [48,50]. Consequently, increasingly more ourmia-like viruses identified can help us have a better understanding of evolution and taxonomy of mycovirus. ) represents viruses which can infect insects, and the leaf pattern ( grouped with the ourmia-like viruses, suggesting that these ourmia-like viruses might represent a separate taxonomical group in the family Botourmiaviridae, and CbOV3 were in the group of Scleroulivirus. In recent years, many ourmia-like viruses were found in plant-pathogenic fungi [47][48][49]. For example, the synthesized RNA of Sclerotinia sclerotiorum ourmia-like virus 4 (SsOLV4) can replicate in and transfer among strains of S. sclerotiorum via hyphal anastomosis, suggesting that a single RNA encoding an RdRp is sufficient for replication, infection, and transmission of ourmia-like viruses in fungi [47]. These phenomena indicate that ourmia-like viruses might have originated from Ourmiavirus by gene-loss events during their adaption to the fungal host [48,50]. Consequently, increasingly more ourmia-like viruses identified can help us have a better understanding of evolution and taxonomy of mycovirus. In recent years, many ourmia-like viruses were found in plant-pathogenic fungi [47][48][49]. For example, the synthesized RNA of Sclerotinia sclerotiorum ourmia-like virus 4 (SsOLV4) can replicate in and transfer among strains of S. sclerotiorum via hyphal anastomosis, suggesting that a single RNA encoding an RdRp is sufficient for replication, infection, and transmission of ourmia-like viruses in fungi [47]. These phenomena indicate that ourmia-like viruses might have originated from Ourmiavirus by gene-loss events during their adaption to the fungal host [48,50]. Consequently, increasingly more ourmia-like viruses identified can help us have a better understanding of evolution and taxonomy of mycovirus.

Sequences Related to (−)ssRNA Viruses
The existence of (−)ssRNA mycoviruses were first identified in 2013 [51]. Many mycoviruses belonging to the order Mononegavirales were first reported [52][53][54]. Subsequently, segmented (−)ssRNA mycoviruses within Bunyavirales were reported in 2019 [55]. We identified seven (−)ssRNA viruses in this study. Six contigs were related to members of unclassified families in the order Bunyavirales, one contig was related to viruses in the order Mononegavirales.
The order Mononegavirales includes 11 families and some unclassified viruses, and Mymonaviridae is the only classified viral family related to mycoviruses in the order Mononegavirales [57,58]. Here, we found that one contig has similarity with (−)ssRNA viruses in the order Mononegavirales. k141-3378 (accession number MZ599588) has 6191 nt and contains a complete ORF, which share 62.8% identity with the RdRp of Plasmopara viticola lesion-associated mononegaambi virus 5, representing a new mononega-like virus named Cercospora beticola negative-stranded virus 3 (CbNSV3). Viruses in the order Mononegavirales are mostly monopartite with multiple ORFs. A few viruses in the Mononegavirales have been reported, such as Sclerotinia sclerotiorum negative-stranded RNA virus 1 (SsNSRV-1) and Fusarium graminearum negative-stranded RNA virus 1 (FgNSRV-1) [53,54]. The infection of SsNSRV-1 leads to attenuated symptoms of the fungal host, including defective growth rate, abnormal colonial morphology, curled hyphal tips, and hypovirulence [53]. Later, it has also been proved that the integrity of SsNSRV-1 genome may be necessary to protect viral mRNA from splicing and inactivation by the host fungi [59]. However, to date there are still many unknown (−)ssRNA viruses and the properties of most reported (−)ssRNA viruses remains unknown.
A phylogenic analysis based on multiple alignments of the RdRp amino acid sequences of four (−)ssRNA viruses and other members in the order Bunyavirales and Mononegavirales was conducted ( Figure 6). In phylogenic analysis, CbNSV1, CbBYV1 (k141-6617), and CbBYV2 clustered together with members in Betamycobunyaviridae, a new proposed family in Bunyavirales [46]. CbNSV3 grouped with members of the newly proposed genus Betasclerotimonavirus in family Mymonaviridae [46]. CbBYV1 (k141-67601 and k141-47347) were not included in the phylogenetic tree, because it did not encode a conserved RdRp domain.

Sequence Related to ssRNA-RT Viruses
Metaviridae (TY3/Gypsy) is a family of retrotransposons and reverse-transcribing viruses with long terminal repeats (LTRs) that are widely distributed in eukaryotes [60]. There are two genera in this family: Metavirus and Errantivirus. The RNA genomes of Metaviridae are two of the same copies of linear positive-stranded RNA, encoding two polyproteins, Gag and Pol. Some metaviruses, such as Cladosporium fulvum T-1 virus [29], encode an Env protein displaying characteristics of typical transmembrane (TM) and surface (SU) proteins. In this study, k141-31340 has 1247 nt and the predicted amino acid sequence of putative protein was similar to env homologue of Cladosporium fulvum T-1 virus with 64.3% identity. The species demarcation criteria set by the ICTV for Metavirus is less than 50% identity in their Gag proteins. However, k141-31340 only carries an ORF for a potential env-like gene in this study, which cannot be used for demarcation. Thus, we preliminarily supposed that k141-31340 might be a new metavirus. Due to a lack of a gag homologue gene, further study is needed to confirm whether k141-31340 represented a new metavirus. s 2021, 13, x FOR PEER REVIEW 5 of 17 6617), and CbBYV2 clustered together with members in Betamycobunyaviridae, a new proposed family in Bunyavirales [46]. CbNSV3 grouped with members of the newly proposed genus Betasclerotimonavirus in family Mymonaviridae [46]. CbBYV1 (k141-67601 and k141-47347) were not included in the phylogenetic tree, because it did not encode a conserved RdRp domain.

Sequence Related to ssRNA-RT Viruses
Metaviridae (TY3/Gypsy) is a family of retrotransposons and reverse-transcribing viruses with long terminal repeats (LTRs) that are widely distributed in eukaryotes [60]. There are two genera in this family: Metavirus and Errantivirus. The RNA genomes of Metaviridae are two of the same copies of linear positive-stranded RNA, encoding two polyproteins, Gag and Pol. Some metaviruses, such as Cladosporium fulvum T-1 virus [29], encode an Env protein displaying characteristics of typical transmembrane (TM) and surface (SU) proteins. In this study, k141-31340 has 1247 nt and the predicted amino acid sequence of putative protein was similar to env homologue of Cladosporium fulvum T-1 virus with 64.3% identity. The species demarcation criteria set by the ICTV for Metavirus is less than 50% identity in their Gag proteins. However, k141-31340 only carries an ORF for a potential env-like gene in this study, which cannot be used for demarcation. Thus, we preliminarily supposed that k141-31340 might be a new metavirus. Due to a lack of a gag homologue gene, further study is needed to confirm whether k141-31340 represented a new metavirus.
Ty3/Gypsy elements represent a major class of LTR retrotransposons. As a representative of Ty3/Gypsy group, Saccharomyces cerevisiae Ty3 virus has been extensively characterized at molecular level and studied as a model of targeted integration. The study of Ty3 provide us a relatively comprehensive, albeit incomplete picture of the ongoing remodeling of a eukaryotic genome by an LTR retrotransposon [61]. Although several related viruses have been discovered, most of them are not formally classified to update and revise classification of metavirids. Therefore, further studies are needed to understand Metaviridae and retrotransposon.  [46]. CbNSV3 grouped with m genus Betasclerotimonavirus in family Mymonaviridae [46]. C 47347) were not included in the phylogenetic tree, because RdRp domain.

Sequence Related to ssRNA-RT Viruses
Metaviridae (TY3/Gypsy) is a family of retrotransposon ruses with long terminal repeats (LTRs) that are widely d There are two genera in this family: Metavirus and Erran Metaviridae are two of the same copies of linear positivepolyproteins, Gag and Pol. Some metaviruses, such as Cl [29], encode an Env protein displaying characteristics of typ surface (SU) proteins. In this study, k141-31340 has 1247 nt sequence of putative protein was similar to env homologue virus with 64.3% identity. The species demarcation criteria is less than 50% identity in their Gag proteins. However, k for a potential env-like gene in this study, which cannot b we preliminarily supposed that k141-31340 might be a new gag homologue gene, further study is needed to confirm wh a new metavirus.
Ty3/Gypsy elements represent a major class of LTR sentative of Ty3/Gypsy group, Saccharomyces cerevisiae T characterized at molecular level and studied as a model of t of Ty3 provide us a relatively comprehensive, albeit incom remodeling of a eukaryotic genome by an LTR retrotranspo lated viruses have been discovered, most of them are not for revise classification of metavirids. Therefore, further stud Metaviridae and retrotransposon.
) represents viruses which can infect insects. Only bootstrap values above 50% are indicated. Viruses identified in this work are indicated by black arrows.
Ty3/Gypsy elements represent a major class of LTR retrotransposons. As a representative of Ty3/Gypsy group, Saccharomyces cerevisiae Ty3 virus has been extensively characterized at molecular level and studied as a model of targeted integration. The study of Ty3 provide us a relatively comprehensive, albeit incomplete picture of the ongoing remodeling of a eukaryotic genome by an LTR retrotransposon [61]. Although several related viruses have been discovered, most of them are not formally classified to update and revise classification of metavirids. Therefore, further studies are needed to understand Metaviridae and retrotransposon.

Conclusions
Next-generation sequence (NGS) technologies have been widely used for detection and discovery of both known and novel viruses in animals, plants, and fungi [62][63][64]. Here, the sequencing results reveal that mycoviruses are widespread in C. beticola in China and some of them are likely to offer significant potential for mobile genetic elements (MGEs) in C. beticola and innovative biocontrol of CLS. In the future, characterization and biological function of these mycoviruses will facilitate biocontrol of CLS and expand our understanding of the diversity, ecology, evolution, and taxonomy of mycoviruses.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/ 10.3390/v13101915/s1, Figure S1: Phenotypes of Cercospora beticola isolates from different groups used for high-throughput sequencing. Table S1: Cercospora beticola isolates from China used in this study. Table S2: Primer pairs used to confirm the exitance of viral sequences in Cercospora beticola, Table S3: Viruses selected for phylogenetic analysis, Table S4: The sequences of 99 primary contigs assembled from clean data of 139 strains of Cercospora beticola, Table S5: Pairwise alignments of sequences assigned to the same virus.