Genome Characterization and Phylogenetic Analysis of a Novel Endornavirus That Infects Fungal Pathogen Sclerotinia sclerotiorum

Endornaviruses are capsidless linear (+) ssRNA viruses in the family Endornaviridae. In this study, Scelrotinia sclerotiorum endornavirus 11 (SsEV11), a novel endornavirus infecting hypovirulent Sclerotinia sclerotiorum strain XY79, was identified and cloned using virome sequencing analysis and rapid amplification of cDNA ends (RACE) techniques. The full-length genome of SsEV11 is 11906 nt in length with a large ORF, which encodes a large polyprotein of 3928 amino acid residues, containing a viral methyltransferase domain, a cysteine-rich region, a putative DEADc, a viral helicase domain, and an RNA-dependent RNA polymerase (RdRp) 2 domain. The 5’ and 3’ untranslated regions (UTR) are 31 nt and 90 nt, respectively. According to the BLAST result of the nucleotide sequence, SsEV11 shows the highest identity (45%) with Sclerotinia minor endornavirus 1 (SmEV1). Phylogenetic analysis based on amino acid sequence of RdRp demonstrated that SsEV11 clusters to endornavirus and has a close relationship with Betaendornavirus. Phylogenetic analysis based on the sequence of endornaviral RdRp domain indicated that there were three large clusters in the phylogenetic tree. Combining the results of alignment analysis, Cluster I at least has five subclusters including typical members of Alphaendornavirus and many unclassified endornaviruses that isolated from fungi, oomycetes, algae, and insects; Cluster II also has five subclusters including typical members of Betaendornavirus, SsEV11, and other unclassified viruses that infected fungi; Cluster III includes many endorna-like viruses that infect nematodes, mites, and insects. Viruses in Cluster I and Cluster II are close to each other and relatively distant to those in Cluster III. Our study characterized a novel betaendornavirus, SsEV11, infected fungal pathogen S. sclerotiorum, and suggested that notable phylogenetic diverse exists in endornaviruses. In addition, at least, one novel genus, Gammaendornavirus, should be established to accommodate those endorna-like viruses in Cluster III.

Sclerotinia sclerotiorum belongs to ascomycetes, it is a cosmopolitan plant pathogen that can attack more than 700 plant species, mainly dicotyledons [18]. It can destroy lots of economic important crops that belong to crucifer, legume, compositae, Solanaceae and so on, while it could also endophytically grow on wheat, rice, and other Poaceae plants [19]. Hypovirulent viruses infecting S. sclerotiorum was potential biocontrol factors. For instance, the infection of two viruses separately, SsHV1 and SsDAHV-1, reduces the pathogenicity of S. sclerotiorum apparently [20][21][22]. It is very important to mine more valuable biocontrol factors [23]. In addition, about 16 families or genera of +ssRNA, -ssRNA, dsRNA, and ssDNA viruses are reported in S. sclerotiorum [24][25][26][27]. The diversity of viruses is very rich. S. sclerotiorum is an ideal material for studying viral evolution and abundance. Endornaviruses also have been isolated from S. sclerotiorum, among them, Sclerotinia sclerotiorum endornavirus 1 (SsEV1) is a worldwide virus, which has been found in Asia, North American, and Oceania [6,24,[28][29][30].
In order to increase the understanding of the diversity of fungal viruses and explore potential biological control resources, strains which were isolated from sclerotia from infected rapeseed plants in a small field located at Xinyang City, Henan Province, China were used for RNA-Seq analysis to mine novel mycoviruses. After sequencing and assembling, a putative protein encoded by contig 2 (with a length of 11,864 nt) showed high similarity to endornaviral RNA polymerase, inferring that an unknown endornavirus may exist in a debilitating strain XY79. In this study, we determined the genome characteristics of this novel endornavirus and found great phylogenetic diversity of members in the family Endornaviridae.

Fungal Isolates and Biological Characterization
Sclerotia of S. sclerotiorum collected from diseased rapeseed plants growing in a small field in Xinyang City, Henan Province, China, were surface disinfected with 75% alcohol for 45 s and then washed with sterilized water twice. After air drying, sclerotia were incubated on potato dextrose agar (PDA) at 20 • C to perform fungal isolation. A total of 225 S. sclerotiorum isolates were used for virome analysis. A debilitating strain XY79 infected with a novel endornavirus was selected from 225 S. sclerotiorum strains for further study based on virome data. Reference strain 1980 was used as control.
To determine the growth rate, both strains XY79 and 1980 were inoculated on 20 mL PDA medium and incubated at 20 • C. The colony diameter was recorded at 24 and 36 h postinoculation (hpi). Growth rate of each strain was calculated. Colony morphology on PDA was photographed at 3, 5, and 12 days postinoculation (dpi).
To detect the virulence of strain XY79, detached rapeseed (Brassica napus cv. Huashuang No. 4) leaves with similar growth stage were inoculated with activating hyphal agar plug (5 mm diameter), and incubated at 20 • C with 95% relative humidity. The diameter of induced lesions on leaves at 36, 48, and 60 hpi was measured. Strain 1980 was used as control. Each strain had four replications, and the experiment was repeated twice.

Total RNA Extraction and Sequencing
Activated 225 S. sclerotiorum strains isolated from Xinyang City were inoculated onto cellophane-covered PDA plate. After 2-4 days of incubation, 0.1 g of mycelia was harvested and ground to a fine powder in liquid nitrogen for each strain. Total RNA extraction was carried out following the manual of the RNA extraction kit (Newbio industry, Tianjin, China). The concentration and quality of RNA were detected by Thermo Scientific TM NanoDrop 2000 Spectrophotometer (Wilmington, DE, USA) and agarose gel electrophore- sis. Then, RNA samples were mixed and stored at −80 • C before use. RNA-Seq for a mix RNA sample of strains from Xinyang city was performed by GENEWIZ Technology Services (Suzhou, China). After sequencing, viral contigs were assembled as previously described [31]. Clean reads were obtained by filtering out adapter-polluted, contaminated, paired-end reads shorter than 100 bp, low quality, high content of unknown base (N) reads, and the RNA and DNA sequences of S. sclerotiorum from raw data. Then, sequence assembly was conducted using CLC Genomics Workbench (version: 6.0.4). Final UniGenes generated from Primary UniGenes after splicing with CAP3 EST were subjected to BLAST, using BLASTx to search for homology with viral sequences against nonredundant (NR). To generate viral fragments, contigs corresponding to the same viruses were assembled by DNAMAN.

RT-PCR and RACE Cloning of Viral Genome
In order to detect the contigs obtained by RNA-Seq, the total RNA of strain XY79 was used to synthesize cDNA library by reverse transcription kit (Transgen, Beijing, China) with random primers. Viral specific primers were designed to detect viral contigs in the cDNA library (Table S1). PCR products were checked by agarose gel electrophoresis.

Sequence Analysis
BLASTx was performed to find homologous amino acid (aa) sequences of viral contigs on NCBI website (http://blast.ncbi.nlm.nih.gov/Blast.cgi, accessed on 20 May 2021) (Table S2). ORFs were predicted with DNAMAN [33]. MOTIF Search (http://www.genome.jp/tools/ motif, accessed on 20 May 2021) was used to predict putative viral protein. To obtain more classified endorna-like viruses that have similarity to the novel endornavirus found in this study, 500 of Max target sequence for general parameters in Algorithm parameters were selected to perform BLASTx. After BLASTx, endornaviruses and endorna-like viruses which have complete RdRp domain were selected. Alignment of amino acid sequences of viral RdRp domain was conducted using Mattf with Defaults program in software Jalview (Table S3). Identity matrix diagram of aa sequence of RdRp domain was carried out online using Clustal Omega program (https://www.ebi.ac.uk/Tools/msa/clustalo/, accessed on 20 May 2021) and RStudio. Phylogenetic tree based on the aa sequence of RdRp domain of selected viruses was constructed by the maximum likelihood (ML) method using IQ-Tree with 1000 bootstrap replications [34] (Table S3). Software IBS 1.0 was used to construct viral schematic diagram.

Virus Diversity and Biological Characteristics of Strain XY79
Via virome sequencing, a total of 65,989,085 raw reads were obtained. After removal of unqualified reads, sequence assembly and BLAST analysis, 41 contigs representing 41 different mycoviruses were generated from 225 S. sclerotiorum strains. Among them, Contig2 with a length of 11,864 nt, a fragment of a novel endornavirus, which showed the highest nucleotide sequence identity (45%) with Sclerotinia minor endornavirus 1 (SmEV1), was designated as Sclerotinia sclerotiorum endornavirus 11 (SsEV11). Using RT-PCR with contig2 specific primers, SsEV11 was found in a debilitating strain XY79. Thus, this strain was selected for further study. The mapping rates of reads of contig2 in sequencing data was 100%. The depth of sequencing was over 36x. Using RT-PCR with 41 viral specific primer pairs, 4 contigs, which corresponded to 4 different mycoviruses, were detectable in strain XY79. Thus, strain XY79 harbored four viruses, including a novel endornavirus and three reported viruses, Sclerotinia sclerotiorum hypovirus 7, Sclerotinia sclerotiorum deltaflexivirus 2-WX, and Sclerotinia sclerotiorum ourmia-like virus 15 ( Figure 1A). Sclerotial formation and maturation of strain XY79 was delayed on PDA ( Figure 1B). Growth rate of strain XY79 was 8.7 mm/12 h, which was significantly lower than that of strain 1980 (12.5 mm/12 h) ( Figure 1C). Strain XY79 could induce lesions with an average diameter of 28.9 mm on detached rapeseed leaf. However, the lesion size was significantly smaller than those induced by strain 1980, whose average diameter was 43.7 mm ( Figure 1D). The results indicated that strain XY79 was hypovirulent ( Figure 1E).

Virus Diversity and Biological Characteristics of Strain XY79
Via virome sequencing, a total of 65,989,085 raw reads were obtained. After removal of unqualified reads, sequence assembly and BLAST analysis, 41 contigs representing 41 different mycoviruses were generated from 225 S. sclerotiorum strains. Among them, Con-tig2 with a length of 11,864 nt, a fragment of a novel endornavirus, which showed the highest nucleotide sequence identity (45%) with Sclerotinia minor endornavirus 1 (SmEV1), was designated as Sclerotinia sclerotiorum endornavirus 11 (SsEV11). Using RT-PCR with contig2 specific primers, SsEV11 was found in a debilitating strain XY79. Thus, this strain was selected for further study. The mapping rates of reads of contig2 in sequencing data was 100%. The depth of sequencing was over 36x. Using RT-PCR with 41 viral specific primer pairs, 4 contigs, which corresponded to 4 different mycoviruses, were detectable in strain XY79. Thus, strain XY79 harbored four viruses, including a novel endornavirus and three reported viruses, Sclerotinia sclerotiorum hypovirus 7, Sclerotinia sclerotiorum deltaflexivirus 2-WX, and Sclerotinia sclerotiorum ourmia-like virus 15 (Figure 1A). Sclerotial formation and maturation of strain XY79 was delayed on PDA ( Figure  1B). Growth rate of strain XY79 was 8.7 mm/12 h, which was significantly lower than that of strain 1980 (12.5 mm/12 h) ( Figure 1C). Strain XY79 could induce lesions with an average diameter of 28.9 mm on detached rapeseed leaf. However, the lesion size was significantly smaller than those induced by strain 1980, whose average diameter was 43.7 mm ( Figure 1D). The results indicated that strain XY79 was hypovirulent ( Figure 1E).

Genome Characteristics of SsEV11
Based on the sequence of contig 2, the 5'-and 3'-terminal sequences of SsEV11 were amplified by RACE PCR (Figure 2A). The 5' untranslated region (UTR) is 31 nt in length, while the 3' UTR is 90 nt ( Figure 2B). Viral genome was assembled with DNAMAN program. The complete genome sequence of SsEV11 is 11,906 nt of 39.2% GC content with one large ORF putatively encoding a polyprotein of 3928 amino acid residues. The genomic sequence of SsEV11 was submitted to GenBank database and assigned accession number MZ605432.
detached rapeseed leaf. The pictures were taken at 60 h postinoculation (hpi). (E) The difference in pathogenicity was calculated by recording the diameter of lesions on rapeseed leaves at 60 hpi (p < 0.001).

Genome Characteristics of SsEV11
Based on the sequence of contig 2, the 5'-and 3'-terminal sequences of SsEV11 were amplified by RACE PCR (Figure 2A). The 5' untranslated region (UTR) is 31 nt in length, while the 3' UTR is 90 nt ( Figure 2B). Viral genome was assembled with DNAMAN program. The complete genome sequence of SsEV11 is 11,906 nt of 39.2% GC content with one large ORF putatively encoding a polyprotein of 3928 amino acid residues. The genomic sequence of SsEV11 was submitted to GenBank database and assigned accession number MZ605432.

Phylogenetic Analysis Exhibits Multiple Lineages of Endornaviruses
Polyprotein sequence of SsEV11 was used as seed to blast NCBI database. Then, aa sequences of endornaviruses and endorna-like viruses that had completed RdRp domain were downloaded. RdRp domain of these viruses (Table S3) was used to perform phylogenetic analysis with IQ-Tree. Three large clusters, Cluster I, Cluster II, and Cluster III, were observed in phylogenetic tree (Figure 4). The result of multiple sequence alignment analysis also confirmed that three large lineages present in endornavirus ( Figure 5).
Cluster I includes typical species of Alphaendornavirus, unclassified viruses that isolated from fungi, oomycetes, and insects, such as Hubei endorna-like virus 1 and Shahe endornalike virus 1 [14]. It showed highly phylogenetical diversity. Combining the results of multiple sequence alignment analysis ( Figure 5), Cluster I could be divided into five subclusters, designated as Cluster Ia, Ib, Ic, Id, and Ie. Cluster Ia had 13 members, which were isolated from plants (mostly), oomycetes, brown algae, and insects; Cluster Ib had 12 members, all of which were isolated from fungi of basidiomycetes and ascomycetes; Cluster Ic had 4 members, all of them were associated with plants; Cluster Id had 4 members isolated from fungi, oomycetes, or associated to plants; and Cluster Ie had 2 members, which were isolated from fungi of ascomycetes. Diplodia seriata endoranvirus 1 and Alternaria brassicicola betaendornavirus 1 [42,43]; and Cluster Ⅱe had only one member, Morchella impotuna endornavirus 1 [44].  Table S3. Viruses written in blue are those infecting or associated with insects, written in red is newly identified virus.
Cluster Ⅲ included endorna-like viruses, which were isolated from insects, nematodes, and mites. Viruses in Cluster Ⅲ were relatively distant to these of Cluster Ⅰ and Cluster Ⅱ. Multiple alignment analysis showed that these viruses were closely related to each other ( Figure 5). In addition, RdRp domains of SsEV11 and these viruses were similar (Table 2). Thus, we proposed to construct a novel genus, Gammaendornavirus, in the family Endornaviridae to accommodate the viruses in the Cluster Ⅲ.   Table S3. Viruses written in blue are viruses that infect insects. Newly identified endornavirus is written in red. Alignment analysis was carried out on website https://www.ebi.ac.uk/Tools/msa/clustalo/, accessed on 20 May 2021.
Cluster III included endorna-like viruses, which were isolated from insects, nematodes, and mites. Viruses in Cluster III were relatively distant to these of Cluster I and Cluster II. Multiple alignment analysis showed that these viruses were closely related to each other ( Figure 5). In addition, RdRp domains of SsEV11 and these viruses were similar (Table 2). Thus, we proposed to construct a novel genus, Gammaendornavirus, in the family Endornaviridae to accommodate the viruses in the Cluster III.

Discussion
In this study, we characterized a novel endornavirus, Sclerotinia sclerotiorum endornavirus 11 (SsEV11), that infected S. sclerotiorum strain XY79. Complete nucleotide sequence of SsEV11 had no significant identity to other viruses, even at the most similar parts (such as sequence coding for RdRp). Nucleotide identity between SsEV11 and other viruses were less than 75% ( Table 1), suggesting that SsEV11 represents a novel species in the genus Betaendornavirus. This species, SsEV11, was tentatively designated as Sclerotinia sclerotiorum betaendornavirus 2. Phylogenetically, Sclerotinia sclerotiorum betaendornavirus 2 is closely related to Sclerotinia minor betaendornavirus 1.
The members of genus Alphaendornavirus were originally identified from plants, and later, were also found to infect fungi and oomycetes. Here, we found an endornavirus that infects brown algae (Brown algae endornavirus 2) and two viruses associated with insects (Shahe endorna-like virus 1 and Hubei endorna-like virus 1) [14,46] are members of Alphaendornavirus. Brown algae endornavirus 2 are closely related to Shahe endornalike virus 1 [14]. Hubei endorna-like virus 1 and Yerba mate alphaendornavirus have a close evolutionary relationship. Shahe endorna-like virus 1 is closely related to Oryza sativa endornavirus and Oryza rufipogon endornavirus (Figure 4) [4,47]. Thus, these endornaviruses should be members of Alphaendornavirus. In addition, this conclusion was also supported by alignment analysis (Figures 3 and 5). Therefore, the host range of viruses in the genus Alphaendornavirus can be expanded to algae and insects.
Previously, a novel genus in the family Endornaviridae was proposed based on a novel Rhizoctonia solani endornavirus [48]. As aa identity of RdRp domain of this virus share quite high identity to alphaendornavirus, we suggested that this virus is actually an alphaendornavirus (Figures 4 and 5). However, the members in Alphaendornavirus are phylogenetically diverse. At least, five lineages (Cluster Ia-Ie) could be grouped. Endornaviruses that infect plants distribute in three subclusters (Cluster Ia, Ic, and Id), suggesting that these plant infecting endornaviruses have different origins. The aa identity of RdRp domains of viruses in Cluster Id and Cluster Ie are considerably low compared to those of viruses in other subclusters. For example, the aa identities are less than 40% for Cluster Ie, and less than 42% for Cluster Id ( Figure 5). Therefore, it is likely that both Cluster Id and Cluster Ie may represent novel genera which are phylogenetically closely related to Alphaendornavirus.
A similar diverse situation has also been observed in the genus Betaendornavirus. In Cluster II, viruses which belong to Cluster IIc, Cluster IId, and Cluster IIe may represent three novel genera, which are related to betaendornaviruses, respectively (Figure 4). It is worth noting that all viruses in Cluster II were isolated from ascomycetes. However, it does not mean that ascomycetes could only be infected by betaendornavirus. For example, S. sclerotiorum actually could be infected by either alphaendornaviruses or betaendornaviruses (Table S4).
Viruses in Cluster III are phylogenetically closely related to typical alphaendornaviruses and betaendornaviruses (Figure 4 and Table 2). These viruses are mainly isolated from various animals, such as nematodes, mites, and insects (  [44,46]. Therefore, we proposed a novel genus Gammaendornaviruses in the family Endornaviridae. The establishment of Gammaendornavirus may deepen our understanding on the diversity and evolution of endornaviruses, and may facilitate to investigate the connection among endornaviruses which originally isolated from fungi, plants, oomycetes, and animals. Endornaviruses are usually not associated with hypovirulence and do not affect the phenotype of their host [6,51,52]. However, so far, more and more endornaviruses with biocontrol potential have been reported. Horizontal transmission of the Rhizoctonia solani endornavirus 1 (RsEV1) can lead to attenuation of the derivative isogenic strain of the virulent strain GD-118P [36]. Further, Yang et al. found that Sclerotinia minor endornavirus 1 (SmEV1) has horizontal and vertical transmission characteristics, and the mycelium fragments of strain LC22 can attenuate the virulence of S. minor [37]. Recent studies have found that plants infected by Bell pepper endornavirus (BPEV) have alterations of organelles and other cell components, which are believed to be caused by parasitic effects between the endornavirus and host [53]. Additionally, the coinfection of Phytophthora endornavirus 2 (PEV2) and Phytophthora endornavirus 3 (PEV3) also affects the sensitivity of oomycetes to fungicides [54]. Here, we found that the SsEV11-infecting strain XY79 has some abnormal phenotypes, such as lower growth rate on PDA medium, delayed sclerotial formation and maturation, and decreased virulence on detached rapeseed leaves. However, weather these attenuated traits were contributed by SsEV11 is unknown since XY79 is co-infected by other viruses.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/10 .3390/v14030456/s1. Table S1: The information of primers used in this study. Table S2: Information of four viral contigs in strain XY79. Table S3: The information of RdRp domain of viruses selected for multiple sequence alignment analysis and phylogenetic analysis in this study.