Exploration Of Novel Mycoviruses In Sclerotinia Sclerotiorum And Interaction Researches Between Mycoviruses And S.sclerotiorum

A necrotrophic pathogon Sclerotinia sclerotiorum causes Sclerotinia stem rot(SSR)known as white mold disease.The quality and safety of crop production in a number of commercial crops,including rapeseed,sunflower,and soybean,are at risk from this destructive disease.Mycoviruses,or fungal viruses,are viruses replicating inside fungal hosts.Some of them have the ability to reduce their hosts pathogenicity and are thus known as viruses associated with hypopervirulence.Hypovirulent mycoviruses in nature obsess great research value for that they are potential resources for the biocontrol of fungal disease.The study about mycoviruses in S.sclerotiorum and their interactions contributes to a better understanding of viral variety and effects on this fungus,and additionally,offers theoretical supports and biological resources for SSR management.In this paper,through transcriptome sequencing,we investigated the viral composition in diseased sunflower fields as well as the interactions between various mycoviruses and S.sclerotiorum,and yielded the following major points:37 different mycoviruses,including nine novel viruses,were discovered in 124 S.sclerotiorum strains collected from diseased sunflower fields in Washington State,USA.According to genomic classification,+ss RNA viruses were the most prevalent species that comprised 64.9% of the total,followed by ds RNA viruses at 29.7% and a very low degree,5.4% by-ss RNA viruses.Mitoviruses and ourmia viruses were the most common +ss RNA viruses,comprising 75% of the species,and were ecologically dominant viruses in S.sclerotiorum under local natural circumstances.Up to 73% of all ds RNA viruses were partitiviruses,containing 6 new viruses,and some of which shared great sequence identity with plant viruses.These results illustrate abundant viral resources exist in S.sclerotiorum strains from Washington,USA,and provide new materials for study on taxonomy and evolutionary of mycoviruses,and exploration of biocontrol agents for SSR disease.The first alphapartitivirus associated with hypovirulence in S.sclerotiorum was isolated from strain AHS232,which was characterized and designated as Sclerotinia sclerotiorum alphapartitivirus 1(Ss APV1).The genome of Ss APV1 encompasses two ds RNA segments,ds RNA1(1983 bp)and ds RNA2(1803 bp),encoding Rd Rp and CP severally.Two segments share conserved 5’-untranslated region and ploy(A)domain at the 3’ terminus.Defective product,ds RNA1-S(1757 bp)was perceived during laboratory cultivation derived from ds RNA1,featured with 226 bp omission in the middle of Rd Rp domain.Multiple alignments and phylogenetic analysis indicated that Ss APV1 was a new member in the genus Alphapartitivirus.In addition,the CP heatmap and dendrogram also revealed the evolutionary relationship with plant gene products and the occurrences of horizontal gene transfer between two organisms.It is observed that the virions of Ss APV1 were icosahedral symmetry with a diameter of approximately 26 nm.Moreover,S.sclerotiorum strain transfected with purified virions of Ss APV1 presented debilitated pathogenicity compared with the original strain.Hence,it is of great potentiality to apply Ss APV1 in biocontrol of S.sclerotiorum in the field.A novel fungal flexivirus with a special genomic structure,named Sclerotinia sclerotiorum alphaflexivirus 1(Ss AFV1),was cloned from a hypovirulent strain AHS31.Strain AHS31 was also co-infected with two botourmiaviruses and two mitoviruses,and its colony morphology was similar to strain Ep-1PNA367 but growth rate and pathogenicity was significantly lower than those of the control strain.The complete genome of Ss AFV1/AHS31 comprised 6939 bp with four open reading frames(ORFs);the ORF1 encodes a replicase while ORF3 encodes a coat protein(CP),and the function of the proteins encoded by ORF2 and ORF4 was unknown.The virion of Ss AFV1/AHS31 is flexuous filamentous 480-510 nm in length and 9-10 nm in diameter.The results of the alignment and the phylogenetic analysis showed that Ss AFV1/AHS31 is related to allexivirus and botrexvirus,such as Garlic virus X of the genus Allexivirus and Botrytis virus X in the genus Botreviruse.In addition,both the replicase and CP sequences formed separate branches.Thus,Ss AFV1/AHS31 is a novel virus and a new genus,Sclerotexvirus,is proposed to accommodate this novel alphaflexivirus.In the same genetic background,the impacts of 12 distinct species of mycoviruses with different genome type on host gene expression levels were examined for the first time.Based on transcriptome analysis of virus-infected Ep-1PNA367,differentially expressed genes(DEGs)in each strain were screened.A total of 24 up-regulated DEGs and 2 down-regulated DEGs were discovered in all virus-infected strains.Among the 24 up-regulated DEG,2 genes possess domains associated with host signal transduction;3 genes have conserved domains connected to transmembrane transport;as for 2 down-regulated DEGs,gene sscle＿09g068880holds methionine synthase domain connected with methionine cycle while sscle＿04g036610 holds a domain belonged to fatty acyltransferase-like subfamily involved in phospholipids metabolism.These genes are essential for further research for that they have great potential to act a crucial role in interaction between viruses and S.sclerotiorum.Besides,there are extra 25 up-regluated and 3 down-regluated host genes specially influenced by 7 +ss RNA viruses.In which,a half of up-regluated genes involved in transmembrane transport while 2 genes for each domains of oxidoreductase,hydrolase and protein kinase;2 of 3 down-regluated genes related to methionine cycle and the other contained mannose hydrolase domain.By comparison,the DNA virus,-ss RNA viruses and 3 ds RNA viruses had greater impact on host gene expression with much more differentially expressed genes.Furthermore,based on the result of differentially expressed genes,the KEGG and GO enrichment analysis elucidated that the majority of viral infections led to increased the level of sugar metabolism,transmembrane transport,and signaling in Ep-1PNA367.In contrast,the pathways involved in amino acid metabolism,aerobic metabolism,the methionine cycle,and pantothenic acid production were suppressed.Notably,the cysteine and methionine metabolic pathway was enriched in down-regulated pathways in all RNA virus-infected cultivars.Moreover,analysis based on the gene expression linked to these pathways showed that,on the one hand,infection of different viruses led to varying degrees of up-regulated genes involved in sugar metabolism and transport,suggesting that viruses may increase host sugar metabolism in order to gather more energy;in addition,virtually all viruses disrupt the expression of host genes that are engaged in RNAi-associated methionine cycle pathway and the antiviral vitamin B-associated ubiquitin synthesis pathway to promotes their own replication.On the other hand,host genes associated to ROS and RNAi pathways were triggered by viral infection,indicating that S.sclerotiorum activated RNAi pathway and ROS-associated enzymes to prevent viral multiplication.In conclusion,we explorated mycovirus resources of S.sclerotiorum isolates in diseased sunflower fields in Washington,USA,and further characterized two novel viruses Ss APV1 and Ss AFV1/AHS31.Meanwhile,using 12 virus-infected Ep-1PNA367 cultivars as materials,we analyzed similarities and differences of various mycovirus impacts on S.sclerotiorum during their interaction,and mined key genes and pathways involved.This research is essential for improving our knowledge on mycovirus diversity,and ascertaining novel viral materials for biocontrol of SSR disease.Moreover,it is helpful to further elucidate the mechanism of mycovirus-fungus interactions from new research dimensions.