Discovery Of Novel Viruses In Sclerotinia Sclerotiorum And Construction Of Infectious CDNA Clones Of +ssRNA Viruses

Sclerotinia sclerotiorum is an important pathogenic fungus.It can cause sclerotinia stem rot of various crops such as rape,soybean,and sunflower,and affect plant production seriously.Abundant viruses are harbored in S.sclerotiorum,some of which significantly influence the pathogenicity of S.sclerotiorum and have the potential for biological control.S.sclerotiorum sclerotium sample PX14 was collected in Ande town,Chengdu,Sichuan province,sterilized,sliced,and cultured on PDA.Eleven strains PX14A(1-4),PX14B(1-6),and PX14 D were isolated and found to have different colony morphology on PDA and pathogenic ability on detached rapeseed leaves.Through virome sequencing and ds RNA random cloning,a total of twelve RNA viruses(or virus strains)and 5 full-length sequences of ds RNA fragments without encoding Rd Rp were identified in the eleven strains.The viruses in PX14 series strains belonged to five different taxonomy,Sclerotinia sclerotiorum hypovirus 1(Ss HV1)of family Hypoviridae,Sclerotinia sclerotiorum ourmia-like virus 4(Ss OLV4)and Sclerotinia sclerotiorum ourmia-like virus(Ss OLV5)of family Botourmiaviridae,Sclerotinia sclerotiorum Phenui-like virus 1(Ss Ph LV1)and Sclerotinia sclerotiorum Phenui-like virus 2(Ss Ph LV2)of order Bunyavirales,Sclerotinia sclerotiorum mitovirus 30-PX14(Ss MV30-PX14)of family Mitoviridae,three substrains of Sclerotinia sclerotiorum deltaflexivirus 1(Ss DF1-PX14A1,Ss DFV1-PX14A4,Ss DFV1-G27),Sclerotinia sclerotiorum deltaflexivirus 3(Ss DFV3)and two substrains of Sclerotinia sclerotiorum mycotymovirus 2(SsMTV2 A and SsMTV2B)of order Tymovirales.Among them,Ss OLV4,Ss DFV3,SsMTV2 A,SsMTV2 B,Ss Ph LV1,and Ss Ph LV2 are newly discovered viruses.Ss HV1,Ss OLV5,Ss MV30-PX14,Ss DFV1-PX14A1,Ss DFV1-PX14A4,Ss DFV1-G27 are new strains of known viruses.The five ds RNAs,which do not encode Rd Rp,were only related to some putative proteins through BLASTX with relatively high E values,thus no function was determined.Among them,the putative proteins of PX14-C and PX14-2.5k match the hypothesis proteins of different viruses,and the putative proteins of PX14-D and PX14-1.8k have conserved domain related to bacterial proteins.PX14-F could not match any protein through BLAST.Two novel segmented negative-strand RNA viruses Ss Ph LV1 and Ss Ph LV2 were discovered.Ss Ph LV1 had three fragments(Ss Ph LV1-L,M,S)and Ss Ph LV2 has four segments(Ss Ph LV2-L,M,S,X).Analyses of these fragments revealed that each fragment contained conserved terminal sequences among the fragments,and formed a panhandle-like(panhandle)structure in termini.Each fragment encoded one ORF,L protein encoded by the L fragment contained an Rd Rp conserved domain and the N protein encoded by the S fragment had a tenui-N conserved domain,while the protein encoded by the M fragment had no any information through BLASTX alignment.The N protein of the Ss Ph LV formed a plum-shaped hexamer,the virus particles were irregular spherical with a membrane structure,and the size was between 90-120 nm.Based on the phylogenetic analysis of L protein and N protein,Ss Ph LV1 and Ss Ph LV2 belonged to a new virus group.We propose to establish a new virus family,Mycobunyaviridae,to accommodate these viruses.Four virus strains were identified as members of family Deltaflexiviridae,among which,Ss DFV3 was a novel virus,Ss DFV1-PX14A1,Ss DFV1-PX14A4,and Ss DFV1-G27 were different strains of virus Ss DFV1.The full-length genome of Ss DFV3 was 7424 nt,which could be presumed to be two ORFs.ORF1 encoded a 2070 aa polyprotein,which shared a 40%(761/1920)identity with the polyprotein of Fusarium graminearum deltaflexivirus 1;ORF2 encoded a 192 aa protein,which is 27%(48/176)identical to the putative protein of Ss DFV1.Phylogenetic analysis using the putative polyprotein of Ss DFV3 ORF1 revealed that this virus was closely related to Ss DFV2 and was a new member of Deltaflexiviridae.Ss DFV1-PX14A1,Ss DFV1-PX14A4,and Ss DFV1-G27 could be estimated to encode 4-5 ORFs.Analysis of the sequence of each ORF found that ORF1 encoded replication-related protein(with a conserved domain of Mtr,Hel,and a Rd Rp conserved domain).Compared with Ss DFV1-PX14A4,Ss DFV1-G27 has a partial deletion in the sequence encoding the other ORFs following ORF1,resulting in the deletion of the sequence of the putative protein.At present,it is unclear what function the proteins encoded by these ORFs,but Ss DFV1-G27 can exist in strain G27 alone,indicating that partial deletion of these ORFs will not influence its replication.SsMTV2 A and SsMTV2 B were found to have total genomes of 7502 nt and 7326 nt,respectively,and the nucleotide identity between them was 82%(6010/7300).SsMTV2 A and SsMTV2 B shared a nucleotide identity of 88%(5567/6344)and 82%(5274/6411)with Sclerotinia sclerotiorum mycotymovirus 1(Ss MTV1)(MK530705.1,full-length 6391 nt).SsMTV2 A and SsMTV2 B had significant differences in the genome size and 5' terminal sequence from Ss MTV1.Therefore,the two viruses are regarded as novel viruses closely related to Ss MTV1 or different strains of Ss MTV1.Considering that SsMTV2 A,SsMTV2 B,and Ss MTV1 are significantly different from the known families of Tymovirales,it is recommended to establish a new virus family in this order,namely Mycotymoviridae,to accommodate these viruses.Viruses Ss OLV4 and Ss OLV5 were identified to belong to family Botourmiaviridae.Ss OLV5 shared an identity of 98%(670/684)with Sclerotinia minor botoulivirus 1 and thus was considered a virus strain in different hosts.Ss OLV4 was a novel virus,had a genome of 2892 nt and encoded a protein with a conserved Rd Rp domain.It had five consecutive guanines at the 5' terminus and five consecutive cytosines at the 3' terminus.Both termini could form stable stem-loop structures.Based on the phylogenetic analysis of Rd Rp,Ss OLV4 had a clear difference in the genetic relationship from the four established genera of family Botourmiaviridae and represented a new virus genus in this family.Ss OLV4 had a close relationship with the member of the genus Penoulivirus which was proposing to establish and can be used as a model species of the genus.An infectious c DNA clone of virus Ss OLV4 was constructed.Ss OLV4 viral RNA was synthesized in vitro and used to transfect the protoplasts of S.sclerotiorum strains N39,N69,and Ep-1PNA367.Multiple transfected strains were obtained successfully and named N39 P,N69P,and Ep-1PNA367 P.Viruses could be transmitted to S.sclerotiorum strain Ep-1PNA367 hyg through the hyphal anastomosis,proving that the rescued Ss OLV4 had an ability of transmission.The sequence of Ss OLV4 in strains Ep-1PNA367 P,Ep-1PNA367 SPE,and Ep-1PNA367 RA were re-sequenced.Compared with the full-length c DNA,the sequence of the rescued Ss OLV4 was different at 2-3 bases,which also indicated that the virus replicated stably.Therefore,the results proved that ourmia-like viruses in fungi are different from ourmiaviruses in plants in that a single segment of Ss OLV4 can complete the process of replication propagation and transmission.The successful infect probability of the synthesized Ss OLV4 RNA to protoplasts was 87%(27/31).With the PEG buffer,viral RNA could infect S.sclerotinia colonies at a lower probability of 24%(5/21).For the first time,we proved that naked RNA viruses can also successfully infect colonies with the help of PEG.The biological characteristics of the strains harboring Ss OLV4 were verified,and it was found that most of the virus-containing strains(except the original strain and strain N39P)showed no significant difference in growth rate from the original strains,indicating that Ss OLV4 has little or no impact on host growth and virulence.Strain Ep-1PN is a hypovirulent strain.The previous reports have verified that Ep-1PN was infected by Sclerotinia sclerotiorum debilitation-associated RNA virus(Ss DRV)and Sclerotinia sclerotiorum RNA virus-L(Ss RV-L),while no results proved that the two known viruses were directly related to the pathogenetic decrease of S.sclerotiorum.Virome sequencing was used to identify the viruses in strain Ep-1PN,and four viruses were discovered,including two known viruses Ss RV-L and Ss DRV,and two newly discovered Sclerotinia sclerotiorum mitochondrial virus 6(Ss MV6/A367),and a novel virus similar to narna-like viruses,temporarily named Sclerotinia sclerotiorum nake-like virus 1(Ss Na LV1).The full length genome of Ss Na LV1 gene was 2912 nt.Based on the phylogenic analysis,Ss Na LV1 was close to Beihai narna-like virus 10(Beihai narna-like virus 10),Wenzhou shrimp virus 10(Wenzhou shrimp virus 10),and other viruses found in animals.Ss Na LV1 protein sequence alignment revealed that it was 28% identical to the protein of narna-like virus 10(127/454).Therefore,it is recommended to use Ss Na LV1 as a model species to establish a genus,namely Quasinakevirus.Total RNA of Ep-1PN was used to transfect the strains N69 and Ep-1PNA367,and the resulting strains N69 EPT and Ep-1PNA367 EPT were found with abnormal colony morphology and declined pathogenicity.The phenotype of Ep-1PNA367 EPT was very similar to strain Ep-1PN.Through ds RNA gel electrophoresis and total RNA Northern blot,Ss DRV and Ss Na LV1 could be detected in the transfected strains N69 EPT and Ep-1PNA367 EPT,indicating that these two viruses could be transfected along with total RNA.Combined with previous reports,it was determined that Ss RV-L and Ss MV6-A367 had no effect on the host.We sequenced the full-length genome of Ss DRV and Ss Na LV1 and constructed infectious clones of these two viruses.The rescued viral RNA could successfully infect strains N69 and Ep-1PNA367,and could transmit from the transfected strains to other strain(Ep-1PNA367hyg).The strains infected by Ss DRV and/or Ss Na LV1 had a slower colony growth rate and lesion size than the control strains,but the debilitation was far from that of strains transfected with total RNA of strain Ep-1PN.This indicates that Ss DRV and/or Ss Na LV1 can affect the phenotype of S.sclerotiorum,however strain Ep-1PN may also have other factors that affect the colony morphology and pathogenicity.In summary,in this study,we cloned thirteen viruses from one sclerotium,seven of which were novel viruses.Infectious clones of Ss OLV4,Ss DRV,and Ss Na LV1 were constructed,and the effects of these three viruses on the morphology and pathogenicity of S.sclerotiorum hosts were verified.This research enriches the diversity of fungal viruses and provides new materials for the study of virus evolution.