Study On The Interaction And Transmission Of Mycoviruses In Sclerotinia Sclerotiorum

Fungal viruses or mycoviruses are kind of viruses that replicate and propagate in fungal cells.Mycoviruses are widely spread in all groups of fungi.Different mycoviruses can affect their host via various forms of interaction,and mycoviruses related to hypovirulence can be applied to control fungal disease.Sclerotinia sclerotiorum is an important plant pathogenic fungus,and is responsible for the huge loss in the quality and production of rapeseed,soybean,sunflower and many other important crops.Mycoviruses associated with hypovirulence have become a potential approach to combat Sclerotinia disease of crops.S.sclerotiorum habors rich diversity mycoviruses and some of them have been characterized in molecular level.The system of S.sclerotiorum-virus interaction has been becoming a model for research on interaction between fungi and mycoviruses.However,the application with mycoviruses for biocontrol diseases under field conditons and the exploration on ecological diversity of mycovirus is still limited.In the our reseach,the interactions between virus and virus or virus and host,and the types of virus transmission were investigated based on three hypovirulent S.sclerotiorum strains SX10,DT47,SH051 and their associated mycoviruses.These results facilitate the virus-host interaction system to establish and to understand the ecological behavior of mycovirus.These studies also will provide rich virocontrol resources for control Sclerotinia disease.S.sclerotiorum strain SX10 infected by a hypovirulence-associated mycoreovirus,and temporarily named Sclerotinia sclerotiorum mycoreovirus 4(SsMYRV4).SsMYRV4 is very similar to the previously reported mycoreovirus Rn MYRV3 isolated from phytopathogenic fungus Rosellinia necatrix.The SsMYRV4 consists of 12 dsRNA segments ranging from 1000 bp to 4474 bp in size,and the terminal sequences of each dsRNA segement are highly conserved(5’-ACAAUUU---UGCAGAC-3’).The virus particles were spherical virions with 65-70 nm in diameter.It was found that SsMYRV4 could suppress host non-self recognition and facilitate horizontal transmission of heterologous viruses.We found that cell death in intermingled colony regions between SsMYRV4-infected S.sclerotiorum strain and other tested vegetatively incompatible strains was markedly reduced and inhibition barrage lines were not clearly observed.Vegetative incompatibility,which involves heterotrimeric guanine nucleotide-binding proteins(G proteins)signaling parthway,is controlled by specific loci termed het(heterokaryon incompatibility)loci.Reactive oxygen species(ROS)plays a key role in vegetative incompatibility-mediated PCD.The expression of G protein subunit genes,het genes,and ROS-related genes were significantly down-regulated,and cellular production of ROS was suppressed in the presence of SsMYRV4.Furthermore,SsMYRV4-infected strain could easily accept other viruses through hyphal contact and these viruses could be efficiently transmitted from SsMYRV4-infected strain to other vegetatively incompatible individuals.Thus,we concluded that SsMYRV4 is capable of suppressing host non-self recognition and facilitating heterologous virus transmission among host individuals.These findings may enhance our understanding of virus ecology,and provide a potential strategy to utilize hypovirulence-associated mycoviruses to control fungal diseases.The complicated interactions between different mycoviruses were found in S.sclerotiorum.When dual-cultured with strains Ep-1PN(infected by SsDRV and SsRV-L)and Ep-1PNA367T1(infected by SsMYRV4),SsDRV and SsRV-L were rapidly introduced into the strain Ep-1PNA367T1,However,SsMYRV4 could not be detected in strain Ep-1PNA367T1 after 20 days’ culture.This result indicated that the two(+)ssRNA viruses SsDRV and SsRV-L could eliminate the mycoreovirus SsMYRV4 in specific strain.Furthermore,DNA virus SsHADV1,dsRNA virus SsRe V1,(+)ssRNA virus SsDRV/SsRV-L and mitovirus SsMV1/HC025 were also able to inhibit or even eliminate SsMYRV4 when co-infected with SsMYRV4.However,mitovirus SsMV1/051 that is very similar to previously reported SsMV1,can coexist with SsMYRV4.In the same way,the fungal DNA virus SsHADV1 can inhibit the replication of RNA virus SsDRV/SsRV-L and SsMV1.Thus,the antagonism interaction between mycoviruses is very universal phenomenon in S.sclerotiorum.We also found that SsMYRV4 could inhibit the expression genes related to RNAi pathway,and the mitovirus SsMV1/HC025 could induce upregulation,but the infection of SsHADV1,(+)ssRNA virus SsDRV and SsRV-L would’t change the expression level of genes related to RNAi pathway.Thus,the results indicated that antagonism interaction between heterologous mycoviruses does not entirely rely on activating the antiviral mechanism of host fungi RNAi.The interaction between hypovirulent-associate viruses can also lead to the recovery of recession of fungi.S.sclerotiorum strain DT47 habors three mycoviruses include a negative-stranded virus(SsNSRV2/DT47),an unclassified positive-stranded virus(SsRV/DT47),a mitovirus SsMV6/DT47,and an unknown small dsRNA fragment(S-RNA)via combination two techniques of molecular cloning and high-throughput sequencing.The Rd Rp of SsNSRV2/DT47 shares 93% identity to SsNSRV2,and the virus particles are similar to SsNSRV1.Three mycoviruses can be successfully transfected into the protoplasts of S.sclerotiorum strain Ep-1PNA367 and 1980 via the virus particle extraction solution,and also can horizontally transmitted through hyphal contact.Based on analysis of the biological characteristics of the strains DT47 and its derivatives,it was found that neither SsMV6/DT47 or SsRV/DT47 alone infection could cause significant changes in the biological characteristics of S.sclerotiorum.The time of mature Sclerotia formation was one or two days ahead when strain was infected by SsNSRV2/DT47.Strains were co-infected with SsNSRV2/DT47 and SsRV/DT47 shows hypovirulence phynotypes including slow growth and lower pathogenicity.Thus,the present results suggested that synergistic effect exists between those two latend-infected mycoviruses,which leads to hypovirulence on S.sclerotiorum.In addition,the three mycoviruses were able to enter the ascospores with high frequency,which was further confirmed via fluorescence in situ hybridization.This result supplied an evidence that mycoviruses can vertically spread by ascospores of filamentous fungi,and synergies interaction between mycoviruses can promote the hypovirulence on S.sclerotiorum.Hypovirulence-associated strain SH051 was co-infected by 15 different mycoviruses.The genome types of these viruses are DNA,dsRNA,(+)ssRNA and(-)ssRNA virus,and the morphology of the extracted virus particles are linear,long rod and spherical,which showed rich diveristy.To study the effects of different viruses on S.sclerotiorum,several viruses were associated with hypovirulence and these viruses were easily eliminated under specific culture conditions.Thus,interactions among and the widespread of viruses are complex,which revealed that mycoviruses are in a dynamic balance in a particular strain under nature conditions.