The Virome Of Sclerotinia Sclerotiorum Strains From Australia And Molecular Characterizations Of RNA Viruses In The Hypovirulent Strain SX276

Sclerotinia sclerotiorum is a necrotrophic plant pathogen with worldwide distribution.This pathogen has the widest host range and can infect more than 700 plant species and cause serious economic losses.Mycoviruses are viruses that can replicate in fungi and widespread in S.sclerotiorum.Mycoviruses associated with hypovirulence in S.sclerotiorum have the potential to control sclerotinia disease.Exploring the diversity of mycoviruses in the S.sclerotiorum population and individual,not only provides biocontrol resources for the virocontrol of sclerotinia disease,but also provides more evidence for the evolution of the enrichment of the virus-sphere.This thesis analyzed the diversity of mycoviruses in S.sclerotiorum strains from Australia,and explored the molecular characteristics of nine mycoviruses infected in a hypovirulent strain SX276.The specific research content is as follows:Eighty-four S.sclerotiorum strains from various crops in Australia were is olated and the mycoviruses diversity of these strains were analyzed.Biological characteristics such as growth rate,colony morphology and pathogenicity of we re observed.The results indicated the colony morphology and pathogenicity of these strains showed diversity and 11 strains showed hypovirulence.Studies we re undertaken using RNA?Seq analysis to determine the diversity of mycovirus es in these strains.After RNA sequences were subjected to BLASTp analysis using NCBI database,286 contigs representing partial or complete genomes of 57 mycoviruses were obtained,and the majority of the putative viruses were p redicted with +ss RNA genomes accounting for 75% of the total viruses;then d s RNA viruses accounting for 9% of the total viruses and-ss RNA genomes acc ounting for 14% of the total viruses.Further,a contig which predicted to be t he genome of Sclerotinia sclerotiorum hypovirulence-associated DNA virus 1 w as identified.Twenty-five mitoviruses were determined and mitoviruses were do minant in the isolates tested.These 57 viruses were grouped into 13 distinct li neages,namely Partitiviridae(4%),Hypoviridae(5%),Endornaviridae(11%),Mitoviridae(44%),Botourmiaviridae(4%),Tymoviridae(7%),Deltaflexiviridae(2%)Mononegavirales(14%),Totiviridae(2%),Tombusviridae(4%),Botybirna virus(2%),Polymycoviridae(2%)and Genomoviridae(2%).Thirty-four of thes e mycoviruses(60%)were novel mycoviruses.This is not only the first study to show existence of mycoviruses in S.sclerotiorum in Australia,but highlights how they are widespread and that many novel mycoviruses occur there and e nhance our overall knowledge on viral diversity,ecology and evolution.Strain SX276 is a hypovirulent strain infected with nine different mycoviruses.Strain SX276 showed abnormal colony morphology with a significantly smaller and lower number of sclerotia,the tip of the hyphae is obviously curved and the branching increases.The ability of strain SX276 to produce acidic substances is weak and can cause very small lesions on isolated rape leaves,it is a typical hypovirulent strain.The mycovirome of strain SX276 of S.sclerotiorum were investigated.Nine mycoviruses were discovered in strain SX276 including one ds RNA virus,one-ss RNA virus and seven +ss RNA virus.The complete genomes of the nine mycoviruses were determined.Analyze of the genome showed that the nine mycoviruses share identities to the members of Hypoviridae,Endornaviridae,Tymoviridae,Deltaflexiviridae,Botourmiaviridae and the genus Botybirnavirus.Sclerotinia sclerotiorum deltaflexivirus 3(Ss DFV3)is a member of family Deltaflexiviridae,the genome of Ss DFV3 contains three segments,segment 2 encode a protein with a helicase domain.An unassigned capsidless mycovirus Sclerotinia sclerotiorum mycoalphavirus virus 1(Ss MAV1)were identified.Although mycoviruses that related to Ss MAV1 have been found in fungi,but Ss MAV1 has unique molecular features in its genome with duplications of the helicase domain that are significantly different from the reported related viruses.It is speculated that the helicase gene doubling event occurred during the evolution of this virus.The influence of mycoviruses in strain SX276 was analyzed by transfection method,and two botourmiaviruses were found to infectious in a ds RNA or ss RNA form but did not affect the biological characteristics of S.sclerotiorum.Botybirnavirus is a latent infection,and hypovirus is associated with the hypovirulence of SX276.One novel rhabdovirus was identified in S.sclerotiorum.The genome of Sclerotinia sclerotiorum rhabdovirus 1(Ss Rh V1)is 11,356 nt in length,the terminal sequences of 3? leader and 5? trailer of the Ss Rh V1 showed terminal complementarity.Ss Rh V1 was predicted to have five major ORFs(ORFs I–V),and these non-overlapping ORFs are linearly arranged in the antigenomic strand and are separated by intergenic regions.ORF I encodes the nucleocapsid(N),ORF IV encodes glycoprotein(G),ORF V encodes larger protein(L),the protein encoded by ORF II and ORF III does not have any conserved information.The L protein has a 35 % identity to the L protein of the Piry virus in the genus Vesiculovirus,and N protein and G protein also showed low similarity with other rhabdoviruses(identity=29% and 25%).Ss Rh V1 has relatively conserved transcription initiation(TI)and transcription termination/polyadenylation(TTP)sequences in the gene junction region,which is a hallmark of the members within the family Rhabdoviridae,but no conserved non-transcribed intergenic dinucleotide was found in the TI signal and TTP signal in Ss Rh V1.The phylogenetic trees were constructed based on the alignment of N,G and L protein,the results indicated that the selected rhabdoviruses were distributed in 20 distinct monophyletic groups and Ss Rh V1 independently formed a distinct monophyletic group.Thus,we proposed to establish a new genus,Sclerorhabdovirus,to accommodate Ss Rh V1 and its related but unidentified rhabdovirus.Ss Rh V1 influence the growth rate of S.sclerotiorum,but does not affect the pathogenicity of S.sclerotiorum.Sclerotinia sclerotiorum endornavirus 3(Ss EV3)was associated with hypovirulence of host,and is the hypovirulence associated factor of stain SX276.The genome of Ss EV3 is 12,661 nt in length,and composed of a single ORF.The ORF encodes a 4184 aa polyprotein containing conserved domains of methyltransferase(Mtr),viral helicase(Hel),phytoreo?S7(S7),and RNA-dependent RNA polymerase(Rd Rp).The phylogenetic analysis indicated that Ss EV3 is clustered with members in genus Betaendornavirus,Ss EV3 is a new member of genus Betaendornavirus.The strains infected with Ss EV3 showed abnormal colony morphology with lower growth rate and fewer and smaller sclerotia and no pathogenicity on rapeseed.Besides the strains infected with Ss EV3 were more sensitive to abiotic stress salts,sugar alcohols and sugar;and failed to form infection cushions on rapeseed and the ability of these strains to degrade oxalic acid became weak.In this study,the diversity of mycoviruses about a group of S.sclerotiorum isolated from Australia and one hypovirulent strain SX276 were analyzed,forty-one novel mycoviruses were identified and the hypovirulence associated factor,an endornavirus,for strain SX276 were corroborated,this study not only helps expand the diversity of mycoviruses and potential virocontrol agents,but also provides new insights into mycoviral evolution via recombination and gene duplications.