Discovery And Genome Analysis Of Three RNA Mycovirus Isolated From Alternaria Brassicicola, The Causal Agent Of Alternaria Blackspot Disease Of Rapeseed

The alternaria blackspot of rapeseed is one of the most prominent diseases of rapeseed which is caused by Alternaria brassicae, Alternaria brassicicola and Alternaria japonica.The influence caused by this diseaseis becaming a seriousproblem since its first outburst in Hunan, in 2012.Thus to understand the fungal pathogenicityand to explore environment friendly biological control strateges is the critical measurefor control of this disease. Mycoviruses or fungal viruses are prevalent among the major taxonomic groups of fungi, some of which can produce serious phenotypic alterations in their fungal hosts.These kinds of mycovirusescan be employed for biological control of the fungal disease.Moreover, the establishment of some host-mycovirus systems is beneficial for studying virus-host interactions to elucidate in the aim of elucidating fungus pathogenicity. In this study, we screened dsRNAs from A.brassicicola strains, cloned and sequenced the genome sequences of three mycoviruses, and probedthe effects of virus infection in their host fungus.(1) We screened dsRNA distributionfrom a collection oftwentyA. brassicicola strains.DRNAs were extracted from six fungal strains and showed as three banding pattern examined by agarose gel electrophoresis:two dsRNAs in co-existed in molecμLar weight of 10kb and 7kb; only one dsRNA segmentof 2.5kb was presented; another single dsRNA banding inmolecμLar size of 5kb. According to the following-up research, these dsRNA areprovedto belong to at least three novel viruses, whichare distinguishable from other Alternariaisolated mycoviruses.(2) We obtained the fuLl-length cDNA sequences, the genome of two mycoviruses, from thetwo dsRNAs in strain YY817-14.Among the dsRNAs, the 10kb dsRNA was an endornavirus named as Alternaria brassicicola endornavirus 1(AbEV1). The AbEV1 genome is 10290 nt long, resembling those associated with the viruses belonging to the family Endornaviridae Genome analysis revealed presence of a single open reading frame coding for a polyprotein of 3400 aa containing conserved domains of viral methyltransferase (MTR), viral RNA helicase 1(Hel-1), and RNA-dependent RNA polymerase (RdRp). In addition, a cysteine-rich region (CRR) with conserved CXCC motifs, shared among several endornaviruses, was also identified between MTR and Hel-1 domains. Phylogenentic analysis based on the RdRp strongly suggested that the virus infecting A. brassicicola shoulμLd be considered representative of a novel endornavirus species.Thes parts of resμLts have been published in Archives of Virology.(3) Here we report a novel positive-sense RNA virus from an A. brassicicola strainYY817-14, which was named as Alternaria brassicicola fusarivirus 1" (AbFV1).The virus has a 6639 nucleotide (nt) long genome, excluding a poly (A)-tail, and was predicted to contain three putative open reading frames (ORF1, ORF2, and ORF3). The large ORF1 encoded a 174-kDa polyprotein (composed of 1522 amino acid residues) containing a conserved RNA-dependent RNA polymerase (RdRp) domain and a helicase domain. The other two smaller ORFs encoded polypeptides with unknown function. Homology search and phylogenetic analysis, based on the RdRp and helicase domains, suggest that this virus is related to and grouped with Sclerotinia sclerotiorum fusarivirus 1(SsFV1), Rosellinia necatrix fusarivirus 1 (RnFV1), Fusarium graminearum virus-DK21 (FgV1).and Penicillium roqueforti RNA mycovirus 1 (PrRV1), all of which belong to a newly proposed family Fusariviridae.Virus elimination by the combination of protoplast regenerationand chemical treatmentrevealed that AbFV1 has no conspicuous impact on the biological properties of its host.These resμLts have been published in the journal of Virus Research.(4) A novel mitovirus named Alternaria brassicicola mitovirus 1(AbMV1) was firstlt identified from strain HXY-12-15. The genome of AbMV1 was in length of 2506 nt, with G+C content of 69.4%. The5’act on the boil 224 and122nt long, respectively.A ORF which was predicted to encode a 83.5kDa protein was found when fungal mitochondrial codon usage wasinvoked. BLASTp homology search and mμLtiple aa sequences alignments were performed and demonstrated that a conserved RdRP region containing the characteristic motifs of the genus mitovirus was existed, and the Soybean-associated mitovirus 2 was similar to AbMV1 with aa identity of 59% followed by Gremmeniella mitovirus with aa identity of 40%. Potentialstablestem-loopstructures were predicted atthe 5wasinvoked. BLASTp homology search and muLtiple aa sequences alignments were performed and demon AbMV1 is a novel mitovirus, this is the first report of mitovirus isolated from A.brassicicola.