Diversity Of Mycoviruses In Botryosphaeria Dothidea And Molecular Biological Characteristics Of Novel Viruses

The cultivation area and yield of pears in China rank first in the world,but the occurrence of pear stem wart disease caused by Botryosphaeria dothidea is more common in the main pear producing areas,which poses a greater threat to the quantity and quality of pears,and the demand for solving this problem is increasingly urgent.Mycoviruses associate host hypovirulence have potential biocontrol values.Here,we studied the diversity of diversity of mycoviruses and the molecular characteristics of the newly identified viruses in B.dothidea strains.It provides useful information for in-depth understanding of the characteristics of mycoviruses derived from woody plants and provides experimental support for improving the classification of mycoviruses,as well as providing new mycovirus resources potential for the further development of biological control of pear diseases.The main results are as follows: 1.Diversity of mycoviruses in Botryosphaeria dothidea289 B.dothidea strains isolated from pear samples collected from 8 provinces in China were used to detect double-stranded RNA(ds RNA)viruses.It was notable that 22 different ds RNA elements showing a wide range of molecular weights from ?0.8 to?10.0 kbp were detected among the infected strains,suggested that ds RNA viruses were diverse in these B.dothidea strains.The carrying ratio and distribution of different ds RNAs were also illustrated,the ds RNAs in the range of 5.0-7.5 kbp shown the highest carrying rate,which was 35.7%.The second highest was the ds RNAs corresponding to Bd CV1 genomic ds RNAs,which was 29.2%,then the two ds RNAs of ? 2.0 kbp and the Bd RV1 genomic ds RNAs.There are 29 different ds RNA patterns were harbored by these strains,indicating that different virus' s coinfention occered in B.dothidea.The ds RNA fragments in the strains of different provinces were counted separately,and it was found that the ds RNA virus strains carried by the B.dothidea strains from different pear producing areas were quite different.A metatranscriptomics approach was used to explore the diversity of mycoviruses in 156 B.dothidea strains.This is the first report of the diversity of mycoviruses carried by the B.dothidea in China.A total of 72 mycoviruses belong to 13 families were found in these strains,of which 55 were new viruses.Of these new mycoviruses,52 were positive singlestranded RNA(+ss RNA)viruses,19 were ds RNA viruses,and only one was negative single-stranded RNA(ss RNA)virus.Phylogenetic analysis based on the Rd Rp amino acid(aa)sequences of 41 viruses indicated 36 were classified into known viral families,including Narnaviridae,Botourmiaviridae,Alphaflexiviridae,Deltaflexiviridae,Partitiviridae,Totiviridae,Ambiguivirida,Fusariviridae and Bipartitiviridae.There were five other viruses that cannot be clearly classified,so a new viral genus Botryosoulivirus in the family Botourmiaviridae and a new viral family Mycobromoviridae were recommended to establish to accommodate these viruses.In this study,a systematic research of the virus diversity in the B.dothidea was performed,and it was clarified that these strains carried a total of 22 ds RNAs of different sizes.These ds RNAs have 29 different infection patterns in different strains,and it was found that the distribution of these ds RNA viruses in different pear production areas have large differences.In addition,the high-throughput sequencing of some strains further discovered the viruses in B.dothidea.A total of 72 viruses were found,55 of which were new viruses,and 41 of them were selected for phylogenetic analysis.It was found that these viruses can be classified under 11 virus families.2.Identification and molecular biological characteristics of novel viruses in B.dothideaThe mycoviruses in strain L153 from Liaoning Province was identified.Strain L153 was found carried by five viruses,which we namely Botryosphaeria dothidea botrexvirus 1(Bd BV1),Botryosphaeria dothidea narnavirus 2(Bd NV2),Bd NV3,Bd NV4 and Botryosphaeria dothidea partitivirus 1(Bd PV1),and a novel virus Bd BV1 was subject for futher analysis.Bd BV1 contains two +ss RNA genomes with the lengths of 5033 bp and 1063 bp,respectively.RNA1 contained four open reading frames(ORFs),of which ORF1 encodes a viral replicase.A search of the conserved domains of ORF1 revealed that three conserved domains exist: viral methyltransferase,helicase and Rd Rp.Other ORFs functions of RNA1 were unknown.RNA2 contained an ORF that encodes the viral CP.It was noting that the 248 nucleotides of the 3? end of the RNA2 were completely identical to the ends of RNA1,which contains the ORF4 and the untranslated region(UTR)of RNA1.At present,no such structure has been reported in fungal viruses.Multiple alignment of the CP of Bd BV1 and other related viruses both showed a salt bridge conserved domain,which is a hydrophobic domain specific to flexuous rod-shaped viruses that infect plants.Biological characteristics analysis of the sub-strain L153-29,which does not carry Bd BV1 alone,suggested that the L153-29 had a greater recovery in colony morphology,mycelium growth rate and virulence than strain L153.Indicating that the abnormal growth and hypovirulence of the strain L153 were mainly caused by Bd BV1.Two different shapes of viral particles were found in strain L153,of which the spherical viral particles potentially belong to Bd PV1,and the other one was filamentous.This is the first report of filamentous virus isolated in B.dothidea,and it can be measured up to 5451.5 nm,which was also the longest virus particle found so far.SDS-PAGE analysis revealed that five structural proteins obtained in the viral particles of strain L153,of which P35,P25 and P15 were unknown.These unknown proteins were identified by peptide mass fingerprinting(PMF)analysis,and the results revealed that P35 was the structural proteins of Bd BV1 encoded by CP.Prokaryotic expression and polyclonal antibody preparation were performed based on the full length of CP encoded by Bd BV1 RNA2.Immunosorbent electron microscopy(ISEM)observation showed that polyclonal antibodies were concentrated around filamentous virions,indicating that the filamentous virions present in strain L153 was Bd BV1.A monopartite ds RNA virus was identified from a B.dothidea strain MSD53 from Shandong Province.Bd VV2 contained spherical virions that were ?38 nm in diameter consisting of a single linear ds RNA genome of 5,090 bp in length.Bd VV2 contained two overlapping ORFs which encoded a putative CP and an Rd Rp,respectively.Phylogenetic analysis,based on the aa sequences of CP and Rd Rp,indicated that Bd VV2 was phylogenetically related to Sphaeropsis sapinea RNA viruses 1(Ss RV1),a member of the genus Victorivirus in the family Totiviridae.To our knowledge,Bd VV2 was a novel victorivirus in the family Totiviridae.A novel +ss RNA mycovirus isolated from a hypovirulent strain G91 from Jiangxi Province,which we name “Botryosphaeria dothidea botourmiavirus 1(Bd BOV1)was characterizated.The complete genome of Bd BOV1 was 2547 nucleotides(nt)long,contained one ORF potentially encoding an Rd Rp,which showed a close relationship to the mycoviruses of the family Botourmiaviridae.Phylogenetic analysis based on the Rd Rp sequence indicated that Bd BOV1 clustered together with Magnaporthe oryzae ourmia-like virus(Mo OLV)and Rhizoctonia solani ourmia-like virus(Rs OLV1)which were officially ratified members of the genus Magoulivirus but independent from it and other genera of the family Botourmiaviridae.To the best of our knowledge,the +ss RNA mycovirus Bd BOV1 in strain G91 is a new member of the genus Magoulivirus or possibly represent a new genus of the family Botourmiaviridae with its closely phylogenetic related mycoviruses.