Complete genome sequence of the first chrysovirus from the phytopathogenic fungus Alternaria solani on potato in China

The complete genome sequence of a double-stranded RNA (dsRNA) mycovirus that was isolated from Alternaria solani strain DT-10 causing potato foliar disease was determined. The virus, designated as "Alternaria solani chrysovirus 1" (AsCV1), has four dsRNA segments (dsRNA 1-4) with a length of 3600 bp, 3128 bp, 2996 bp, and 2714 bp, respectively. The RNA-dependent RNA polymerase (RdRp, 1084 amino acids [aa]), putative capsid protein (905 aa), alphachryso-P3 (835 aa), and alphachryso-P4 (729 aa) were encoded by dsRNA1, dsRNA2, dsRNA3, and dsRNA4, respectively, which had the highest sequence identity of 41.77%-72.38% to their counterparts in Helminthosporium victoriae virus 145S (HvV145S) of the genus Alphachrysovirus, family Chrysoviridae. Moreover, the 5′-untranslated regions (UTRs) of AsCV1 dsRNA 1-4, which contained several unique inserts (3-37 bp) and deletions (5-64 bp), shared 51.65%-68.01% identity with those of HvV145S. Phylogenetic analysis based on RdRp sequences showed that AsCV1 clustered the most closely with HvV145S. Considering its distinct host specificity, the low sequence similarity of its encoded proteins to those of other viruses, the unusual features of the 5′-UTRs of its dsRNA 1-4, and the phylogenetic position of its RdRp gene, AsCV1 should be considered a member of a new species in the genus Alphachrysovirus. To the best of our knowledge, this is the first alphachrysovirus identified from phytopathogenic A. solani.

Recently, an ssRNA mycovirus named Alternaria solani fusarivirus 1 (AsFV1) was isolated from A. solani [17]. In our previous study, A. solani was documented to cause potato early blight in China [18,19]. In this study, A. solani strain DT-10, which was recovered from diseased leaves of potato with the symptoms of early blight in the city of Datong, Shanxi province, China, was identified using methods described previously [18,19]. A new alphachrysovirus was found to be associated with A. solani strain DT-10 and was designated as "Alternaria solani chrysovirus 1" (AsCV1).

Provenance of the virus material
Alternaria solani strain DT-10 was cultured on potato dextrose agar (PDA) plates overlaid with cellophane membranes for 7 days in the dark at 25°C. Multiple dsRNAs were extracted from the mycelia by binding to CF-11 cellulose powder in 15% ethanol as described by Morris and Dodds [20]. After treatment with S1 nuclease and DNase I (TaKaRa, Dalian, China), the dsRNAs were analyzed by 1.0% agarose gel electrophoresis (Fig. 1A) and purified using a gel extraction kit (Aidlab Biotechnologies, Beijing, China). Partial complementary DNA (cDNA) sequences of the dsRNA segments were obtained and assembled as described previously [21]. Using specific primers designed based on the cDNA sequences (Supplementary Table S1), the terminal sequences were determined by the RNA-ligasemediated rapid amplification of cDNA ends (RLM-RACE) method as described previously [22,23]. The full genome sequence of AsCV1 was obtained by assembling all of the partial cDNA and terminal sequences using DNAMAN 7.0 (Lynnon Biosoft, USA) and was deposited in the Gen-Bank database with the accession numbers MW656210, MW656211, MW656212, and MW656213 for dsRNA1, dsRNA2, dsRNA3, and dsRNA4, respectively.
The RdRp sequences of AsCV1 and 24 members of the family Chrysoviridae were analyzed using ClustalX 2.0 and MEGA 6.0 [23]. A phylogenetic tree constructed by the neighbor-joining algorithm with 1000 replicates based on the RdRp sequences showed that AsCV1 formed a wellsupported taxonomic cluster with the members of the genus Alphachrysovirus and was closely related to HvV145S-A9 and BmCV1 (Fig. 1C).
According to the ICTV, the host of isolation, nucleotide and deduced amino acid sequence data, the length of genome segments, and 5′-UTRs are useful species demarcation criteria for the genus Alphachrysovirus [5]. Based on the host fungus A. solani, the low sequence similarity of the encoded proteins, the unusual characteristics of the 5′-UTRs, and the phylogenetic relationships to known members of the genus Alphachrysovirus, AsCV1 should be considered a novel member of the genus Alphachrysovirus. This is the first report of the complete genome sequence of a new chrysovirus infecting A. solani.

Conflict of interest
The authors declare that they have no conflict of interest.
Ethical approval This study did not include experiments with human participants or animals performed by any of the authors. Dark blue and green represent 100% and 67% nucleotide sequence identity, respectively. The red stars indicate AsCV1