Virome Analysis Of Rhizoctonia Solani Isolated From Hubei Province And Characterization Of Three Novel Mycoviruses Displaying Specific Features

The plant pathogenic fungus Rhizoctonia solani is the causal agent of numerous crop diseases leading to significant economic losses,with a broad host range.To date,no cultivars are displaying complete resistance to R.solani,and it is imperative to develop an approach,such as using hypovirulence-associated mycoviruses,to control this disease.Mycoviruses(also called fungal viruses)are widespread in all significant clusters of fungi.In this study,518 R.solani strains were collected from a rice field in four counties and one region of Hubei Province,including Enshi(ES),Huanggang(HG),Suizhou(SZ),Xianning(XN),and Xiangyang(XY),and used for mycoviruses examination through next-generation sequencing(NGS)approach.The isolates were first grouped into four groups Ⅰ(ES),Ⅱ(HG&SZ),Ⅲ(XN),andⅣ(XY)and subjected to total-RNA extraction.Subsequently,the sequences data(40G)were assembled and analyzed.The analysis revealed 74%ssRNA viral contigs following with 25%dsRNA viral contigs,and 1%dsDNA viral contig.150 partial or complete genome was analyzed and detected through RT-PCR.One hundred thirty-two viral sequences represented viruses belong to described families whereas eighteen were unclassified viruses.The unclassified families displayed close similarity to singlestranded RNA(ssRNA)viruses(3 viruses),double-stranded RNA(dsRNA)viruses(14 viruses),and double-stranded DNA(dsDNA)virus(1 virus).Furthermore,the encoded protein of viral contig of this dsDNA virus showed close similarity(46%)to dTDP-Dglucose 4,6-dehydratase domains of Hokovirus and more viral DNA sequences have been acquired.Hence,the prevalence of these viruses in novel families is most likely to exist.Of the 132 viruses related to described order,family or proposed family are grouped in 13 distinct lineages.Eighteen viruses are negative-stranded RNA viruses that belong to Bunyavirales(1 virus),Serpentovirales(4 viruses)and Mononegavirales(13 viruses),among them,14 viruses share 26 to 68%identity to that of Serpentovirales(2 viruses),Mononegavirales(12 viruses).Seventy-five viruses belong to Mitoviridae,among them,seventy-two novel viruses share 28 to 80%identity to the members of mitoviruses.Eight viral contigs displayed RNA-dependent RNA polymerase(RdRp)domain of Partitiviridae while twelve viral contigs contained Coat-Protein(CP)domain belong to Partitiviridae,which are highly related to alphapartiviruses.Six viruses belong to Hypoviridae,all of them shared 48 a 59%identity to the members of Hypoviridae,among which First_contig133 represented a virus that can be used to establish a new genus within Hypoviridae family.Four viruses belong to Botourmiaviridae,they share 31 to 39%identity to botourmiaviruses,among which the First_contig5162 represented new virus shared close similarity to Magoulivirus,the other three viruses are significantly different the four genera in the Botourmiaviridae,may represent a novel genus.Three viruses belong to Endornaviridae,they show 35 to 40%identity to alphaendornaviruses;importantly,a novel contig6356 represented virus separated the two genera Alphaendornavirus and Betaendornavirus,suggesting that the establishment of a new genus within the family Endornaviridae.Three viruses belong to Totiviridae,they show 29 to 50%identity to the member of Totiviridae,the first_contig3225 represented virus displayed high similarity to Giardiavirus that infects protist.Four viruses belong to the families Megabirnaviridae,proposed Fusariviridae,proposed Mycoalphaviridae and Narnaviridae,respectively;among them,Contig32423 represented virus shares 73.5%identity with Pterostylis megabirnavirus-like,suggesting likely to be a novel Megabirnavirus;Contig 17739 represented virus shares 70.0%identity with Rhizoctonia solani fu sari virus 1,contig30143 represented virus shares 58.0%identity with Rhizoctonia solani alphavirus-like 3,and Contig927 represented narna-like virus shares 28.3%identity with Changjiang narna-like virus.An alphaendornavirus was identified from R.solani virulent strain XY74 and tentatively named Rhizoctonia solani alphaendornavirus 1 XY74(RsAEV1/XY74).RsAEV1/XY74 genome is 16747 nucleotides(nts)long and contains a single ORF.The ORF encodes a poly protein of 5539 amino acid(aa)that codes for D123 protein,Alphavirus-like methyltransferase(Met),RNA dependent RNA Polymerase(RdRP),Cysteine-rich region(CRR)and a Viral_helicase1(Hel)domains.D123 is a protein reported to be responsible for Temperature-Sensitive G1-Phase Arrest in animals(such as rat).This result provides evidence of a horizontal transfer gene between mycoviruses and animals.Furthermore,RsAEV1/XY74 does not contain a nick-site and a poly-C tail.Phylogeny investigation clustered RsAEV1/XY74 in Alphaendornavirus,a new member of the Endornaviridae family.A novel virus with a size of 22 kbp,the largest mycovirus genome ever reported so far,was isolated from R.solani virulent strain XN84 and is tentatively named Rhizoctonia solani megabasidiovirus 1(RsMbV1).Motif analysis of RsMbV1 revealed two putative open reading frames(ORFs)with no junction,separated with 825 nts untranslated region(UTR),and localized on the positive strand.The largest ORF B(13379 nts)encodes Protease(PRO;aa positions 244-352),RNA-dependent RNA polymerase(RdRp;aa positions 2802-3239),and DEAD-like helicases(DEXDc;aa positions 3671-3984)superfamily 2(SF2;nt position from 7679 to 21058)domains;The multiple alignments of RdRp displayed five domains motifs of the positive ssRNA viruses;ORFB-Hel comprises six conserved motifs(motif Ⅰ,Ⅱ,Ⅲ,Ⅳ,Ⅴ,and Ⅵ),among which motif Ⅰ and Ⅲaccommodates GKST box and DExH(DETH)signature,respectively,which are found in members of Hypoviridae.The alignment of PRO showed the conserved Cysteine(C)and histidine(H)residues necessary for the autoproteolytic activity and Glycine(G)residue.Based on the protein encoded by ORF B,RsMbV1 is phylogenetic ally related to Sclerotinia sclerotiorum hypovirus 2(SsHV2;Identity 27.85%,E-value 3e-97)and Cryphonectria hypovirus 1(CHV1;Identity 25.97%,E-value 8e-49).Whereas,ORF A(6029 nts)encodes Helicase(Hel;aa positions 317-554)superfamily 1(SF1)domain which has also six conserved motifs(motif Ⅰ,Ⅱ,Ⅲ,Ⅳ,Ⅴ,and Ⅵ).Interestingly,homologs of ORF A Hel of RsMbV1 are also found in the members of human viruses,such as Human coronavirus,and bacteria,such as Pseudomonas spp.ORF A Hel of RsMbV1 is phylogenetically related to the helicase of viruses,such as coronaviruses,within Nidovirales and bacterial proteins,these results provide clear evidence of a horizontal transfer gene between viruses or between viruses and prokaryotic cellular organism.Some viruses may have two Hel domains of SF1,but RsMbV1 has two Hels domains appertaining to different superfamilies(SF1 and SF2),suggesting RsMbV1 has a very different genome structure.RsMbV1 was able to transmit horizontally into the host vegetative compatible strain.RsMbV1 is associated with the alleviation of virulence,but has no significant effect on the growth rate.A novel positive sense(+)single-stranded(ss)RNA was identified from R.solani virulent strain XY74,and tentative termed Rhizoctonia solani fusarivirus 4/XY74(RsFV4/XY74).RsFV4/XY74 viral genome consists of 10838 nucleotides(nts)except the poly(A)tail and encodes four ORFs(ORF 1 to 4).The largest putative ORF3,1549 aa,encodes an RNA-dependent RNA polymerase(RdRp)and contains a conserved helicase domain(Hel2)domain whereas that of ORF1,786 aa,encodes another Hell domain.The hypothetical ORF2 and 4(503 and 542 aa,respectively)encode proteins with unknown functions.Homology search of RsFV4/XY74 revealed the closest similarity to Rhizoctonia solani AG2-2LP fusarivirus 1.However,the genome organization of RsFV4/XY74 is significantly different from that of other known fusariviruses,it has the largest fusarivirus genome size so far,more important,its genome encodes two helicase domains,this genome organization has not been reported in any known viruses,as far as we know,except for Rhizoctonia solani fusariviruses.Phylogenetic relatedness of the putative domains clustered RsFV4/XY74 in the newly proposed Fusariviridae family thus indicated RsFV4/XY74 as a new member of Fusariviridae,and we proposed a new genus Megafusarivirus in this family.Summarily,this study reported 150 viruses identified from infecting rice sheath blight pathogen(R.solani)from five cities of Hubei Province,China,most of them are novel viruses that revealed various diversity of mycoviruses.This study also finely investigated the characterization of three viruses(hypovirus,alphaendornavirus and fusarivirus),which are significantly different from known viruses.Our findings provide novel insight into the diversity,taxonomy,evolution and ecology of mycoviruses,and reveal horizontal gene transfer between viruses or between viruses and cellular organisms.Furthermore,our discovery of RsMbV1 which can transmit among host vegetative incompatible strains may have the potential to control rice sheath blight.