Diversity Analysis On Mycoviruses And Molecular Characteristics Of Three Novel Virulence-associated Mycoviruses In Rhizoctonia Solani Isolated From Rice Planting In Hubei Province

Rice sheath blight caused by the world widespread fungal pathogen Rhizoctonia solani is an important rice disease in China,which affect the yield and quality of rice and causes huge economic losses.Mycoviruses are viruses that infect fungi,which is common in fungi.Some mycoviruses confer hypovirulence on fungal plant pathogens,which have potential to control fungal crop diseases.In this study,we analyzed the diversity of R.solani strains isolated from rice in Hubei province and molecular characteristics of three novel virulence-associated mycoviruses,the results were showed as bellow:There are abundant viral resources in R.solani.In this study,the diseased rice samples were collected from 9 cities in Hubei province,and 174 strains were isolated from the diseased samples.To investigate the diversity of viruses and screen mycoviruses resource for virocontrol,metagenomics was applied to analyze virus diversity in R.solani strains,and finally got 10 G transcriptome data.Among all reads,1% reads were annotated to the genomes of viruses.The obtained reads sequences were spliced,and 324 assembled contigs were constructed and further analyzed.The results of analysis showed that these viruses could be divided into four nucleic acid types including single-stranded DNA,double-stranded RNA,positive-stranded RNA and negative-stranded RNA.Among them,1% contigs were related to single-stranded DNA virus,which were mainly belonging to Genomoviridae;35% contigs were related to double-stranded RNA,which were mainly belonging to Partitiviridae and some unclassified virus families;55%contigs were related to positive-stranded RNA,which were mainly belonging to Endornaviridae,Narnaviridae,Hypoviridae and Benyviridae;9% contigs were related to negative-stranded RNA,which were mainly belonging to Mononegavirales,Bunyavirales and some unclassified virus families.It was showed that all four nucleic acid types of virus were discovered in R.solani,and the dsRNA virus Partitiviridae and positive-stranded RNA virus Narnaviridaec was common virus groups in R.solani.Thus,the diversity of virus is very rich in R.solani.Strains HG81 was a hypovirulent strain,and harbors two new partitiviruses,RsPV3(Rizoctonia solani partitivirus 3)and RsPV4(Rizoctonia solani partitivirus 4).Both of RsPV3 and RsPV4 have two dsRNA segments.The complete nucleotide sequences of the two dsRNA segments of RsPV3 were determined as 1993 nts and as 1733 nts in length,each contained one putative large open reading frame(ORF).The ORF1(the large ORF)of RsPV3 potentially encoded a putative RNA-depended RNA polymerase(RdRp)with618 amino acid(AA),and the ORF2(the small ORF)of RsPV3 encoded a putative coatprotein with 486 AA.The complete nucleotide sequences of the two dsRNA segments of RsPV4 were 1981 nts and 1694 nts in length,and each had one putative ORF,the large one encoded putative RdRp with 625 AA,and the small one encoded a putative coat protein 479 AA.According to the RdRp and CP amino acid sequences alanysis,both of RsPV3 and RsPV4 related to RsPV2 of Alphapartitiviridae,which showed 61% and 65%identity,suggesting as a new member of Alphapartitiviridae.Like other partitiviruses,the particles of RsPV3 and RsPV4 were isometric and approximately 30 nm in diameter.The virus particles of RsPV3 and RsPV4 were successfully transfected to the protoplasts of virulent strain 190 by PEG mediated transfection.Two transfectants,T26 and T28,showed abnormal phenotypes including stunt of hyphal growth,delay and poor development of sclerotia,and lower virulence on its host.Thus,either RsPV3 or RsPV4 alone infection,or together co-infection causes hypovirulence on R.solani.In addition,we anylsed the molecular characters of the genome of a negative-stranded RNA virus(RsRhV1),which was detected and isolated from strain XY175.The full genome sequence of RsRhV1 contains 11716 nts with a short 3'UTR(87nts)lacking poly(A)tail and relative long 5' UTR(244 nt)with a cap structure.The G+C content of this genome was 40%,and there was an obvious complementary characteristic between sequence of 3'-UTR and 5'-UTR.Five putative ORFs non-overlapped and linearly arranged were predicted in the RsRhV1 genome.The five gene-junctions were highly conserved and rich A/U with sequence length ranging from 28 nts to 232 nts.The five genes shared the same initiation codon AUG and termination codon UAA with exception of ORF5 using UGA as termination codon.However,RsRhV1 did not have the same strict conservative "AAAACUUAGG" gene transcription termination signal and "UAUUUAAUAAAACU" gene transcription start signal.The ORF1 encoded for a putative nuclear capsid protein(N protein),ORF IV for a glycoprotein(G protein),and ORF V for L protein that contained a RdRp conserved domain with a motif GxxTx(n)HR.GxxTx(n)HR was necessary for the formation of cap structure of mRNA,which is prerequisite to transcription and stability of RNA virus.In known Rhabdoviruses,ORF II and ORF III encoded phosphoprotein(P)and matrix protein(M),however,the function of the proteins encoded by ORF II and ORF III of RsRhV1 were not clearly known despite the ORF III protein has a putative zinc finger structure(C2H2 and C2 HC zinc finger).Based on L-protein phylogenetic analysis showed that RsRhV1 was a novel mononegative RNA virus,which was distantly related to known rhabdoviruses.Therefore,RsRhV1 is a new member in Rhabdoviridae family and is unique.This is the first reportof Rhabdoviruses in R.solani.Purification particles of RsRhV1 are long,filiform,slippy,and are 20-50 nm in diameter and 800-1000 nm nm in length.Derived strains 175A1-VF13,175A1-VF15 and 175A1-VF32 were obtained by protoplast regeneration.In the determination of virulence,the 175A1-VF15 strains(containing RsRhV1 virus)are more virulent than the 175A1-VF13 and 175A1-VF32 strains(no containing RsRhV1 virus),and the statistical analysis is significantly different.It seemed to indicate the RsRhV1 virus help the host to increase virulence.In the present study,the mycoviruses diversity was investigated in rice sheath blight fungus isolated from Hubei province,and three novel mycoviruses were characterized on molecular level.Our discoveries might provide potential mycoviruses to control rice sheath blight and enhance our understanding on the diversity and evolution of mycoviruses.