Molecular Characterization of a Novel Partitivirus and a Fusarivirus Co-infected in the Nigrospora Sphaerica Fungus


 Here we reported the molecular characterization of two novel mycoviruses co-infected in a plant pathogenic fungus, Nigrospora sphaerica that were designated as Nigrospora sphaerica fusarivirus 1 (NsFV1) and Nigrospora sphaerica partitivirus 1 (NsPV1), respectively. NsFV1 has an undivided genome of 6,147 bp, excluding the polyA tail, and was predicted to contain two nonoverlapping open reading frames (ORF1 and 2). The larger ORF1 encoded a polyprotein containing a conserved RNA-dependent RNA polymerase (RdRp) and a helicase domain that have functions for RNA replication, and the smaller ORF2 encoded a putative protein with an unknown function. The NsPV1 was consists of two genome segments, which were in lengths of 1,796 bp and 1,455 bp, respectively. Each of the two dsRNAs had a single ORF and were deduced to encode proteins with homology to viral RdRp and coat protein (CP), respectively, in the family Partitiviridae. Phylogenetic analysis showed that NsFV1 was placed within the newly proposed family Fusariviridae, while NsPV1 was belonging to the genus Gammapartitivirus in the family Partitiviridae. This was the first description of mycovirses infected the fungus N. sphaerica.


Introduction
Mycoviruses are viruses residing and infecting almost all major taxa of fungal groups, including lamentous fungi, yeasts, and oomycetes [1][2][3][4]. Since the rst report of mycovirus infecting the cultivated button mushroom Agaricus bisporus as a pathogen, a number of mycoviruses have been identi ed, especially via application of high-throughput sequencing technique [5]. Most mycoviruses were con rmed to infect their host latently, but some exerted clearly phenotypic alterations in their host, such as induced hypovirulence. These hypovirulence associated mycoviruses were potential biological agents for control of disease caused by pathogen fungi, as has been exampled by the successfully use of Cryphonectria hypovirus 1 (CHV1) to control chest blight disease in Europe [2,3,6]. In addition, some mycoviruses also have bene cial effects on their host fungus [7,8]. For example, a virus named Curvularia thermal tolerance virus which was identi ed in an endophytic fungus could enhance heat tolerance to the host plant [9]. Recently, some reports suggested the positive interactions between mycoviruses and their fungal hosts. For example, two distinct partitiviruses have been reported to enhance the osmotic tolerance of C. parasitica and mycotoxin production of Aspergillus ochraceus, respectively [10,11]. In addition, a partitivirus infecting an isolate of Trichoderma harzianum could enhance the antagonistic activity of its host [12]. Mycoviruses have aroused wide interests among researchers for biological control against pathogenic fungi and for biotechnological applications, such as in elucidating the pathogenic mechanisms of pathogenic fungi [3,6]. Thus, many mycoviruses have been identi ed during the last few decades, which also in turn expanded our knowledge on mycoviral diversity, evolution and ecology [3,13].
Fusariviridae is a + ssRNA virus family proposed recently to be created with the FgV1 as the prototype [18,19]. Viruses in this family typically have genomes in size ranging from 6 to 10 kb and contain one large open reading frame (ORF) and one to three smaller ORFs. The larger ORF encoded a replicase-related protein containing RdRp and helicase (Hel) domains, while the other smaller ORFs encoded proteins with unknown functions [18,20].
Nigrospora sphaerica is a kind of saprophytic, endophytic and phytopathogenic fungus that distributed widely in nature, even in insects [21,22]. As a plant pathogen, this fungus can infect an amount of plants causing leaf spot, leaf blight or shot hole diseases [23]. As endophytic fungus, some N. sphaerica strains could produce a variety of secondary metabolites with antimicrobial ability [24]. Although a few mycoviruses have been reported in the Nigrospora species [25][26][27][28], however, to date, no virus has been reported in the N. sphaerica.
Genomic organization and phylogenetic analyses indicated that NsPV1 and NsFV1 were novel fusarivirus and partitivirus, respectively.

Provenance Of The Virus Material
The N. sphaerica strain Ns2-3 was collected from diseased pepper plant and its parasite bemisia tabaci from Hunan Province of China, in 2018. It was cultured on potato dextrose agar plates at 26°C. The mycelial mass was harvested after 7 days of culture in PD broth with shaking (170 rpm) at 26°C. DsRNA was extracted using the CF cellulose chromatography method as described by Morris and Dodds [29].
The dsRNA extractions were treated with S1 nuclease and DNase I for elimination of any contaminated DNA and ssRNA molecules, and then separated in a 1% (w/v) agarose gel. DsRNA fragments were individually puri ed and applied to cDNA cloning. The cDNA libraries were constructed using reverse transcriptase and random hexanucleotide primers. Sequences were completed by lling the gaps using RT-PCR ampli cation and by obtaining the dsRNA terminals using adaptor ligation-mediated PCR ampli cation [30]. All the PCR products were ligated into the pMD18-T vector (TaKaRa, Dalian, China), cloned and sequenced. Every base of the sequences was determined in at least three independent clones. The resulting sequences were nally assembled and subjected for sequence analysis.
Potential open reading frames (ORFs) and conserved domain(s) were found on the National Center for  Fig. 1A and Fig. 2A of the ORF2 encoded protein, which was predicted to be associated with cell cycle control, cell division and chromosome partitioning. The SMC proteins were found in bacteria, archaea and eukaryotes. However, some viruses including fusariviruses also contained the SMC domain as described previously [33]. Its occurrence in mycovirus is noteworthy and might reveal the horizontal gene transfer between viruses and other eukaryotes or prokaryotes. In the other hand, the presence of SMC in different viruses might also indicate the common origin of these viruses. The true function and evolution of the SMC domain in the NsFV1 ORF2 needed further study.
In order to estimate the phylogenetic relationships between NsFV1 and other mycoviruses, we conducted phylogenetic analysis based on aa alignments of the RdRp and Hel domains. The phylogenetic tree generated based on the RdRp domain alignment showed that NsFV1 was clustered in a fusarivirus-clade, including the prototype FgV1, and distinct from members of the family Hypoviridae (Fig. 1C). A phylogenetic tree of the Hel domain also showed that NsFV1 was grouped in a clade and exhibited a similar tree topology with the RdRp based phylogenetic analysis (Fig. S1). Overall, based on the similarities in conserved RdRp and Hel domains and phylogenetic analysis, we can suggest the NsFV1 as a novel member of the proposed family Fusariviridae.
Sequences analysis indicated that the NsPV1 was composed of two dsRNA segments, which were designated as dsRNA 1 and dsRNA 2, respectively. NsPV1 dsRNA 1 was 1,796 bp in length, with a G + C content of 47.6%, while the dsRNA 2 was 1,455 bp long, containing a 51.2% G + C content. The 5′ and 3′ untranslated regions (UTRs) were 104 bp and 75 bp in dsRNA 1, 92 bp and 100 bp in dsRNA 2, respectively. The 5' and 3'UTRs between the dsRNA 1 and dsRNA 2 were conserved, possessing an identical stretch of 5'-CGTGAAATAC-3' in the 5'-terminal and a stretch of 5'-TAAACCAAAA-3' in the 3'terminal (Fig. 2B), which were considered to be important for replication of viruses with multicomponent RNA genome [34].  (Fig. 2C). It revealed that NsPV1 was placed in a branch including members of the genus Gammapartitivirus, as expected from the homology search result.
The species demarcation criteria of partitiviruses are that aa identities in the RdRp and CP is fewer than 90% and 80%, respectively [16]. Since the aa identities of RdRp and CP between NsPV1 and the closely related MoPV1 were lower than the cutoff values, we can suggest the classi cation of NsPV1 as a novel species of the genus Gammapartitivirus in the family Partitiviridae.
In conclusion, we identi ed two novel mycoviruses from a N. sphaerica strain Ns2-3. Sequence similarity, genome organization and phylogenetic analysis supported the a liation of NsFV1 and NsPV1 as novel members of the family Fusariviridae and Partitiviridae, respectively. As far as we know, this was the rst report of mycoviruses co-infected in the fungus N. sphaerica. The biological effects of these viruses on the fungal host and the functions of the virus genes in terms of virus-host interactions remained further study.