| Mycoviruses are virus that infect and replicate in fungal cells.Some mycoviruses that infect fungal plant pathogens could induce hypovirulence,and could be used as biological control agents to control plant fungal diseases.Previous studies indicated that mycoviruses are generally transmitted vertically through asexual and / or sexual spores or transmitted horizontally via hyphal anastomosis between vegetatively compatible individuals.So far as we know,there was no transmission vector reported for mycovirus at present.Lycoriella ingenua is a common mycophagous insect,its adult with a small size(1.8-3.2 mm).Female adults lay eggs on fungal colonies,after hatching from eggs,larvae feeding on fungal mycelia.The recipe of L.ingenua is wide and it could also feed on numerous fungi including Sclerotinia sclerotiorum,a notorious plant pathogen.We observed that Sclerotinia sclerotiorum hypovirulence-associated DNA virus 1(SsHADV-1)infected S.sclerotiorum strain exhibited stronger attraction to L.ingenua adults than virus free strain did in the laboratory.We suspected that there are special interactions between SsHADV-1 and L.ingenua.To verify whether there are interactions between SsHADV-1 and L.ingenua,we used SsHADV-1-infected S.sclerotiorum strain DT-8 to rear L.ingenua and then detected whether L.ingenua could acquire SsHADV-1 from strain DT-8 and transmit it to virus free S.sclerotiorum trains,and investigated the possibility of L.ingenua transmitting SsHADV-1 and potential interactions between SsHADV-1 and L.ingenua.The major results and conclusions of this study are presented as bellows:Larvae which reared on colony of strain DT-8 were collected and washed with distilled ddH2 O for 3 times,then extracted DNA for PCR detection.Viral DNA could be successfully detected from larvae,and this result indicated that there was SsHADV-1 either on larvae surface or in the body.Viral DNA even could be detected from larvae which were starved for 7 days after feeding on strain DT-8.Furthermore,SsHADV-1 could be detected from pupae,adults and eggs by PCR amplification and Southern blot detection.SsHADV-1 existed in eggs,larvae,pupae,midguts and oviducts of adult through immunofluorescence observation.Larvae could acquire SsHADV-1 by feeding on strain DT-8 for 0.5 hr.Viruliferous larvae or SsHADV-1 particles injected larvae were transferred to colony of SsHADV-1 non-host fungus Botrytis cinerea strain B05.10 for further feeding to allow larvae pupation,eclosion,mating and oviposition,SsHADV-1 could be detected from more than 80% offspring of these larvae.These results indicated that virus could be transmitted transovarially.We presumed that SsHADV-1 could replicate in insect since the expression of viral Rep and CP in viruliferous L.ingenua were detected by RT-PCR detection successfully.To verify whether SsHADV-1 could replicate in insect,we used purified SsHADV-1 particles to inoculate Spodoptera frugiperda cells(Sf9).Viral Rep and CP were expressed in virus-inoculated Sf9 cells through RT-PCR amplification and Northern blot hybridization.Viral fluorescence signal was observed in virus inoculated Sf9 cells by immunofluorescence observation,and viral genomic DNA was replicated and accumulated in Sf9 cells abundantly by qPCR analysis.Furthermore,we diluted virus particles(300 ng/?l)serially to inoculate Sf9 cells,as a result,SsHADV-1 could still infect Sf9 cells when diluted 1000 folds.When inoculated with virus particles(30 ng/?l)and incubated for 24 hr,almost 60% Sf9 cells were infected through flow cytometry detection.In addition,through passage experiment,we found SsHADV-1 could be released from virus-infected Sf9 cells to infect new insect cells,and the supernatant of virus infected Sf9 cells culture could infect virus free S.sclerotiorum strains extracellularly and confer abnormal colony morphology like strain DT-8.To investigate whether viruliferous L.ingenua larvae could transmit SsHADV-1 to virus-free S.sclerotiorum strain,larvae from colony of strain DT-8 were collected and washed with distilled ddH2 O for 3 times,then transferred to virus free S.sclerotiorum strain DT-8VF for further feeding.Hyphal agars were picked from larvae feeding galleries and transformed to new PDA plates and subcultured for virus detection.The result showed that 8 subcultures out of 39 detected subcultures were infected by SsHADV-1 as examined by PCR amplification.Rolling circle amplification-Southern blot analysis further verified these results.The virus infected subcultures exhibited significant hypovirulence and other abnormal morphology,but the virulence of these infected strains were not significantly strong than that of strain DT-8 and viral DNA content of SsHADV-1 in these strains was too low to be observed by electrophoresis.When fed on virus free strain DT-8VF for 0.5 hr,viruliferous larvae could transmit SsHADV-1 successfully.Furthermore,viruliferous eggs,pupae and adults could also transmit SsHADV-1.Adults could transmit virus on diseased rapeseed plants by simulating transmission experiment on rapeseed in the laboratory.To reduce the possibility of virus transmission by virus infected mycelia adhering on insect body,SsHADV-1 particles injected larvae were starved for 1 day and then transferred to a colony of stain DT-8VF for further feeding,as a result,virus transmission occurred.Adults emerged from virus particles injected-pupae then layed eggs and eggs were transferred to colony of strain DT-8VF.Larvae hatched from these eggs could also transmit virus to strain DT-8VF successfully.L.ingenua adults exhibited a preference of laying eggs on colony of strain DT-8 than strain DT-8VF significantly,and this preference could be disturbed by putting activated carbon between the two colonies.L.ingenua adults showed strong preference to volatiles collected from strain DT-8 than that of strain DT-8VF by “Y”-type tube test.This phenomenon indicated that SsHADV-1 may suppress or promote production of some special volatile substrates.Then volatiles released by these two strains were collected for Gas Chromatography-Mass Spectrometer analysis,the results showed no significant different elements for the volatiles,but the content of Octen-3-ol and 3-Octanone released by strain DT-8 was lower than that of strain DT-8VF significantly.Further analysis by “Y”-type tube test with pure Octen-3-ol and 3-Octanone displayed that they exhibited strong repel effect on female adults.Furthermore,several Octen-3-ol and 3-Octanone synthetic related genes in virus infected S.sclerotiorum strain DT-8 were down-regulated than that in strain DT-8VF.All these results indicated that SsHADV-1 infected S.sclerotiorum and reduced the production of Octen-3-ol and 3-Octanone to attract L.ingenua adults to lay eggs on its colony.Meanwhile,egg production of viruliferous female adults was evidently higher by 28.9% than that of virus free female adults.These results suggested that there were mutualistic interactions between SsHADV-1 and L.ingenua.The natural occurrence of L.ingenua was investigated in the rapeseed field which located in Huazhong Agricultural University,Xianning and Ezhou city of Hubei Province.And we discovered that L.ingenua could occur in naturally sclerotinia-diseased rapeseed field frequently.Larvae and pupae showed preference to live at the inner parts of diseased rapeseed stems,while adults were observed flying on leaves and petals of rapeseed.SsHADV-1 genomic DNA could be detected in adults captured from both strain DT-8-sprayed rapeseed field and non-sprayed field,ratio of viruliferous adults in the strain DT-8 sprayed field(81.4%)was much higher than that in the non-sprayed filed(22.4%).These results indicated that viruliferous L.ingenua adult could occurred in natural rapeseed field and spraying virus-infected strain in rapeseed field could enhance the ratio of viruliferous adults.Furthermore,other mycovirus-like viruses were successfully detected in L.ingenua adults which captured from naturally sclerotinia diseased rapeseed field through high throughput sequencing analysis.These results suggested that L.ingenua may also transmit other mycoviruses.In brief,we first report that mycovirus SsHADV-1 could infect insect(L.ingenua)transkindomly,replicate in L.ingenua and utilize it as transmission vector to transmit virus among S.sclerotiorum strains transovarially,and there are reciprocal interactions between SsHADV-1 and L.ingenua.Our findings may facilitate the exploration of mycoviruses for fungal plant diseases controlling and enhance our understanding of the ecology of SsHADV-1 and other newly emerging SsHADV-1-like viruses,which were recently found to be widespread in various niches including human HIV-infected blood,human and animal feces,insects,plants,even sewage. |
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