Charicterization And The Biocontrol Potential Of Sclerotinia Sclerotiorum Hypovirulence-Associated DNA Virus1

Sclerotinia sclerotiorum (Lib.) de Bary is a notorious fungal pathogen with worldwide distribution. It could cause severe diseases in Brassica and affect the yield and quality of crop severely. Mycoviruses are known to infect and multiply in all major taxa belonging to the kingdom Fungi. Typically, mycoviruses have either double-stranded (ds) or single-stranded (ss) RNA genomes. They are thought not to be infectious as free particles and to lack an extracellular phase in their life cycles. Mycovirus-mediated hypovirulence is a phenomenon in which the virulence of fungal pathogens is reduced or even completely lost as a consequence of virus infection, thus these kinds of mycovirus are considered to be promising biocontrol agents against plant pathogenic fungi.Strain DT-8was isolated from the sclerotia in the diseased rapeseed plant stem. It developed abnormal colony morphology on potato dextrose agar (PDA) and exhibited hypovirulent phenotype when inoculated onto Arabidopsis thaliana or detached leaves of Brassica napus. The total DNA of strain DT-8was extracted and besides the fungal genomic DNA, there were two additional DNA bands with sizes of about2kb--large DNA element (LDE) and0.5kb-small DNA element (SDE), respectively, which were hypovirulence related. The genome of this circular ssDNA virus, named Sclerotinia sclerotiorum hypovirulence-associated DNA virus1(SsHADV-1), is2166nt, coding for a replication initiation protein (Rep) and a coat protein (CP). ORFs are separated by a large intergenic region (LIR) and a small intergenic region (SIR). There are geminivirus related bi-promoter and conserved sequences which are important for virus replication in the LIR.Although phylogenetic analysis of Rep showed that SsHADV-1is related to geminiviruses, it is notably distinct from geminiviruses both in genome organization and particle morphology. Surprisingly, we could not find any CP-related proteins in the known protein databases. Negatively stained viral particles observed with an electron microscope were nontwinned isometric particles,20～22nm in diameter, which are clearly different from the geminivirus. The molecular mass of CP is about35kDa by SDS/PAGE analysis. The0.5kb SDE, which is also circular single-stranded, is proved to be subgenomic DNA of SsHADV-1according to the sequence analysis. Thus, this is the first time that we demonstrated that a DNA virus could replicate in a fungus in nature and reported an ssDNA virus infecting fungi.We found that this virus was surprisingly easy to transmit from strain DT-8to strains belonging to other vegetative compatibility groups (VCGs). Furthermore, Polyethylene glycol (PEG) mediated transfection of fungal protoplasts was successful with either purified SsHADV-1particles or viral DNA isolated directly from infected mycelium. Then we continued to test the infectivity of SsHADV-1towards S. sclerotinia. We demonstrated that purified particles of SsHADV-1are infectious when applied extracellularly to its fungal host S. sclerotiorum. Virus particles isolated from infected host can infect the hyphae of virus-free S. sclerotiorum directly when applied to hyphae grown on PDA plates or spread on leaves of Arabidopsis thaliana and Brassica napus.When applied on leaves, virus infection could suppress development of lesions induced by S. sclerotiorum. Although SsHADV-1can infect host strains belonging to different VCGs when applied externally, it has a narrow host range. Virus particles are likely to be very stable on the leaves of Arabidopsis plants since viral DNA could be detected at15dpi on unwounded leaves and at10dpi on wounded leaves, respectively; however, this virus could not infect and move in plant cells. Our findings may prompt a change in the generalization that mycoviruses lack an extracellular phase in their life cycles; and the potential of using DNA mycoviruses as natural fungicides appear to be promising and may stimulate the search for other DNA mycoviruses.To advance the exploration of SsHADV-1and its interaction with the fungal host, we constructed the infectious clones of SsHADV-1that contain tandemlytandemly repeated viral sequences with at least one full length SsHADV-1and two LIRs. Different repeated lengh of SsHADV-1were used to construct the vectors which are2units,1.8units,1.3units and1unit. When introduced into virulent S. sclerotinia strains by PEG-mediated transfection, a resurrected, cytoplasmically replicating DNA form is generated from the infectious copy except for the1unit vector. Comparision of the successful transformation efficiency among different infectious clone vectors showed the longer the repeated sequences, the higher the successful transformation efficiency. It suggested that the homologous recombination may play the major role in unit-length SsHADV-1DNA release from the vector.Efforts to test whether the CP deleted SsHADV-1could exist in S. sclerotiorum cells has been made, however, we could only detect low level of viral DNA which were easily lost during subculture, strains containing stable replicating CP deleted SsHADV-1could not be abtained. eGFP was used to subsititute for CP to construct the infectious clone vector and although the efficient and stable expression constructs were not observed, transient expression of introduced foreign genes was confirmed. Thus, the coat protein of SsHADV-1plays an essential role in virus maintenance and accumulation.