| Sclerotinia sclerotiorum is an important plant pathogenic fungus.Stem rot disease caused by S.sclerotiorum leads to serious economic losses every year.Mycoviruses are diverse in S.sclerotiorum,and multiple mycoviruses co-infecting a singular strain is a common phenomenon.In the previous study,S.sclerotiorum strain HC025 with hypovirulence and abnormal colony morphology was thought to be infected by a single mitovirus(Sclerotinia sclerotiorum mitovirus 1,Ss MV1).In this study,the strain HC025 was used as the research material,and the virus species in it were identified by metatranscriptome.Based on transcriptome and metabolome analysis,the mechanism of viruses-induced hypovirulence and antagonistic fungal characteristics of S.sclerotiorum were explored,and the function of one gene responding to virus infection was studied.The results are presented as following:The hypovirulent strain HC025 is co-infected by five mycoviruses.In addition to the previously reported virus Ss MV1,four mycoviruses were newly detected in strain HC025 by virome sequencing,including Ss NV4-HC025,Ss NSRV1-HC025,Ss OLV14-HC025,and Ss OLV22-HC025.Among them,the identities of Ss NV4,Ss NSRV1,and Ss OLV14 with the corresponding previously reported viruses are higher than 95% at replicases,which suggest that they are new viral isolates.The fulllength c DNA sequence of Ss OLV22 was obtained,and it was found that its full-length genome contains 3987 nucleotides,which only encodes an Rd Rp.The Rd Rps of Ss OLV22 and Hubei narna-like virus 10 share the identity of 24.84%.Phylogenetic analysis of Ss OLV22 based on Rd Rp showed that Ss OLV22 is a new member of Botourmiaviridae,and can be grouped with Ourmiaviruses and narna-like viruses into a big cluster.Except for Ss NSRV1,other four positive single-stranded RNA(+ss RNA)viruses can horizontally transmit together from the strain HC025 to the virulent strain Ep-1PNA367 via mycelial fusion.The newly virus-infected strain Ep-1PNA367 V is a hypovirulent strain with an abnormal colony morphology,and with the lower contents of ROS and ATP in mycelium.This suggested that the four +ss RNA viruses are related to the hypovirulence of S.sclerotiorum.The effects of +ss RNA virus co-infection on S.sclerotiorum were systematically analyzed at the transcriptome level.The transcriptomes of strains Ep-1PNA367 and Ep-1PNA367 V in vegetative growth and early interaction stages with rapeseed leaves were sequenced and analyzed,and differentially expressed genes(DEGs)affected by the virus co-infection were screened out.The results showed that the composition of mitochondrion,Golgi apparatus and cytoskeleton were abnormal;the secondary metabolism and transmembrane transport were enhanced;biological process of cell growth,differentiation,and polar growth were inhibited.Among the DEGs,there were 31 previously reported S.sclerotiorum genes involved in mycelial growth,sclerotia development,virulence and ROS regulation;149 genes predicted to be transcription factors;12 genes related to the autophagy process,and 9 of which were down regulated;9 genes were related to the secondary metabolism,and 8 of which were up regulated.At the early stage of infecting rapeseed leaves,the weighted correlation network analysis(WGCNA)was used and 8 candidate hub genes of S.sclerotiorum were screened out.The candidate metabolites antagonizing fungi were screened out via the analysis of transcriptome and metabolome.The strain Ep-1PNA367 V co-infected with the four+ss RNA viruses was found to be antagonistic to the growth of pathogenic fungi including S.sclerotiorum,Botrytis cinerea,Rhizoctonia solani,and Fusarium oxysporum.Combined with the results of transcriptome and metabolome analysis of strains Ep-1PNA367 V and Ep-1PNA367,it was found that the process of secondary metabolism was up-regulated and enriched both at transcriptome level and metabolome level.The up-regulated DEGs(sscle＿02g018200,sscle＿03g031520,sscle＿03g031520,sscle＿06g050730,and sscle＿02g017510)were involved in the synthesis and metabolism of toxins,alkaloids,ketones,and terpenoids.Nine alkaloids,steroids,and their derivatives were also found in the differentially up-regulated metabolites.Among them,two substances with the highest confidence were solanidine and solanine.It was speculated that these 9 substances had the activity of inhibiting fungal growth,and may be involved in the antagonistic fungal process of the strain Ep-1PNA367 V.The biological function of S.sclerotiorum gene Ss SLN1(sscle＿16g108830)responding to virus co-infection was analyzed.The gene Ss SLN1 supposedly encodes a two-component histidine kinase and functions in the MAPK pathway.Knockout of the gene Ss SLN1 in S.sclerotiorum resulting in the growth hindrance of S.sclerotiorum,yield reduction and deformity of sclerotia,abnormal hyphal tips,reduced pathogenicity.It is worth noting that the ability of hyphae fusion between knockout transformants decreased,but this did not block the horizontally transmission of +ss RNA viruses.After horizontally transmitting mycoviruses in strain Ep-1PNA367 V to the knockout transformants,the knockout transformants became malformed and grew slowly.However,compared with strain Ep-1PNA367 V,the growth inhibition rate of knockout transformants infected with the same kind of viruses decreased.In summary,this study clarifies that the hypovirulent strain HC025 is co-infected by five mycoviruses,which provides new virus resource for the biocontrol of S.sclerotiorum.Eight important S.sclerotiorum candidate hub genes in the early stage of S.sclerotiorum-rapeseed interaction,and nine metabolites including solanidine and solanine which may have fungal growth inhibition activity were screened out.Finally,it was proved that the MAPK signal pathway related gene Ss SLN1 responding to virus infection was involved in the growth,sclerotia development and pathogenicity of S.sclerotiorum.Therefore,this study provides new biocontrol resources and potential target genes for green prevention and control of stem rot disease. |
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