Study On The Identification And Characterization Of A Novel Chrysovirus Isolated From Pestalotiopsis Theae

Tea grey blight is an important disease of tea trees [Camellia sinensis(L.)O.Kuntze],results in serious defoliation,and leads to big losses of the yield and the quality as it severely occurs.Pestalotiopsis theae is the dominant species causing tea leaf spot disease.We isolated a P.theae strain LI-41 a tea leaf,whcih showed abnormal colonial morphology and lower growth rates,than the normal one.This study has uncovered the reason for the changes of the biological traits of P.theae strain LI-41,and the results are as follows:Four double-stranded RNA(ds RNA)strands were identified in strain LI-41 as it was subjected to ds RNA extraction.Random and terminal sequencing revealed that they are 3387,2831,2599 and 932 bp,respectively.Each of the ds RNAs was concluded to encode a single open reading frame(ORF),serially termed ORF1,ORF2,ORF3 and ORF4.Of which,ORF1 was concluded to encode a putative proteinase,and ORF4 unknown function protein BLASTp searches of these ORF-coding proteins revealed that they had the highest similarities with those of Colletotrichum gloeosprioides chrysovirus 1(Cg CV1),and shared the identities of 65%,48%,54%,respectively,whereas the ORF4-coding protein had no detectable proteins.The virus was concluded as a novel member in the Chrysovirus genus,tentatively termed Pestalotiopsis theae chrysovirus 1(Pt CV-1).Extractionof nucleic acid from each of the sucrose fractions revealed that the four ds RNAs present in all the fractions(10% to 50%)but with the highest concentraction in the 50%,and their positions match those from the mycelia.The virion in 50% sucrose fraction were transfered onto copper mesh and subjected totransmission electron microscopy after negatively stained,it revealed the diameter is 30.1~39.8 nmLI-41 was induced to produce conidia,and fifty conidia were cultured for ds RNA extraction.The results showed that 67% of the conidium-generating colonies were infected by Pt CV-1.As contact cultured between strains LI-41 and LI-41-1(derived from conidium-generating colony free of Pt CV-1),and fifty subisolates were excized from the edges of LI-41-1 colony for ds RNA extraction after cultured.The results showed six of the subisolates positive for Pt CV-1,indicating that Pt CV-1 can be horizontally transed to the strain LI-41-1s.Using PEG-mediated viral particles or nucleic acid transfection of the protoplasts of Pt CV-1-free strains(LI-41-1),150 protoplast generating progenies were subjected to ds RNA detection.The results showed that Pt CV-1 was detected in five progenary colonies that transfected by particlaes,suggesting that Pt CV-1 can be transmited to other strains by particles but not by their nuclic acids.Comparison of the biological characteristics of the virus-free strains(LI-41-1,LI-41-2,LI-41-3),virus-infected parental strain(LI-41),virus–infected conidium-generating progeny strains(LI-41-V1,LI-41-V2),virus-transfected strains(LI-41-1T1,LI-41-1T2,LI-41-1T3)and virus-transfected strains derived form contact-cultues(LI-41-1P-1,LI-41-1P-2.LI-41-1P-3)showed that they had significantly different morphologies and virulences.The virus-infected strains presented more pigments,absent of hyphal rings,abnormal colonies decreased growth rates(the virus-free strains are 6.5-6.7 mm/d,while the virus-infected strains 2.0-2.8 mm/d),and attenuated virulences(the lesions induced by virus-free strains being 11.9-14.1 mm,while by virus-infected strains 1.7-4.1 mm).It suggested that Pt CV-1 infection can cause abnormal colonies,lower growth rates and attenuated virulences of P.theae.In this study,we report a novel chrysovirus Pt CV-1,which infects P.theae resulting in abnormal morphologies,decrease of the growth rates,and attenuated virulences,and to our knowledge,this is the first report of a mycovirus infecting P.theae.Pt CV-1 can significantly attenuate the host virulence,which expected for further application in biocontrolment of the fungal disease.The discovery of Pt CV-1 is also the first fungus-virus system for P.theae,which may be used in studying the genomic functions of this fungus as reverse genetic approach.