1

Preprint:Pleasenotethatthisarticlehasnotcompletedpeerreview.

ViromeofriversidephytocommunityecosystemofanancientcanalCURRENTSTATUS:POSTED

ShixingYangJiangsuUniversity

TonglingShanChineseAcademyofAgriculturalSciences

YanWangJiangsuUniversity

JieYangJiangsuUniversity

XuChenJiangsuUniversity

YuqingXiaoJiangsuUniversity

ZhenqiangYouHangzhouMedicalCollege

YuminHeJiangsuUniversity

MinZhaoJiangsuUniversity

JuanLuJiangsuUniversity

ZijunYangJiangsuUniversity

ZiyuanDaiJiangsuUniversity

https://www.researchsquare.com/browse?journal=researchsquare

2

QiLiuJiangsuUniversity

YuxinYaoJiangsuUniversity

XiangLuJiangsuUniversity

HongLiJiangsuUniversity

RuiZhouJiangsuUniversity

WangLiJiangsuTaizhouPeople'sHospital

ChenglinZhouJiangsuTaizhouPeople'sHospital

XiaochunWangJiangsuUniversity

QuanShenJiangsuUniversity

HuiXuTheAffiliatedHospitalofJiangsuUniversity

XutaoDengVitalantResearchInstitute

EricDelwartUniversityofCaliforniaSanFrancisco

WenZhangJiangsuUniversity

z0216wen@yahoo.comCorrespondingAuthorORCiD:https://orcid.org/0000-0002-9352-6153

DOI:

mailto:z0216wen@yahoo.com

3

10.21203/rs.3.rs-25620/v1SUBJECTAREASGeneralMicrobiology

KEYWORDSPlantvirome;Phytocommunity;Virushostswitching;Cross-speciesinfection;co-infection.

https://dx.doi.org/10.21203/rs.3.rs-25620/v1https://www.researchsquare.com/browse?subjectArea=General%20Microbiology

4

AbstractBackground

Theviruscommunityinplantsinalocalplantecosystemhasremainedlargelyunknown.Inthisstudy,

weinvestigatedtheviruscommunityinthesewildandcultivatedplantsinZhenjiangancientcanal

ecosystem.

Results

usingviralmetagenomicapproach,weinvestigatedtheviralcommunityinleaftissuesof161plant

speciesbelongingin38differentordersinalocalriversideplantecosystem.Wediscovered251

differentplant-associatedvirusgenomeswhichincluded88DNAand163RNAvirusesbelongingto27

differentvirusfamilies,ordersorunclassifiedvirusgroups.Theidentifiedvirusesincludesomethat

aresufficientlydivergenttocomprisenewgenera,families,orevenorders.Ourdataindicatedthat

somegroupsofvirusesknowntoinfectnon-plantorganismshadhostswitchingtoinfectingplants.

Cross-speciesinfectionandco-infectionofviruseswerecommoninthisplantecosystem.

Conclusions

thesedatapresentaviewoftheviralcommunityinplantspresentinalocalplantecosystemwhichis

morediversethanthatdepictedincurrentclassificationofplantvirusesandprovideasolid

foundationforstudiesinvirusecologyandevolutioninplants.

BackgroundMuchefforthasbeendevotedtostudyingvirusesassociatedwitheconomicallyimportantor

symptomaticplantswhichonlycompriseaminutefractionofallplantspecies,suggestingthatalarge

gapexistsinouroverallunderstandingofviraldiversity,evolution,andecologyinuncultivated

plants[1].Itisthereforenecessarytostudyvirusesexistinginwildplants,whethersymptomaticor

asymptomatic,togainamoreobjectiveviewofviruspopulationsinplant,whichwillundoubtedly

discovernovelorevenso-callunclassifiedvirusesandprovidemoreinformationonviralevolution

anddiversity.High-throughputDNAsequencingcoupledwithviralmetagenomicsapproachesalso

makesitpossibletoidentifyhighlydivergentviralgenomesinwildplants.Comparingvirusesin

differentplantspecieswithinanaturalplantecosystemcanalsoimproveourunderstandingofvirus

5

transmissionamongstdifferentplantspecies.

TheZhenjiangancientcanalwascreatedduringtheQinDynastyover20centuriesago,andis16

kilometerslongwithanaveragewidthof40meters.TheDingmaosectionofthecanalis2kmlong

withlotsofwildplants,somelandscapeplantsandcropsonbothsides.Inthisstudy,weinvestigated

theviruscommunityinthesewildandcultivatedplantsinthisecosystem.

ResultsOverallviewofthevirome.Weperformedalarge-scaleviralmetagenomicssurveyofpotential

plantleaf-associatedvirusesin161plantspeciesbelongingin38differentorders,7classes

(Coniferopsida,Cycadopsida,Dicotyledoneae,Filicopsida,Ginkgopsida,Magnoliopsida,and

Monocotyledoneae),and4phyla(Angiospermae,Gymnospermae,Pteridophyta,andTracheophyta)

existinginariversideplantecosystem(Fig.1a,SupplementaryTable1,andSupplementaryData1

and2).Amongthe161speciessampled,89belongtowildplant,and72arecultivatedtypes.For

eachspeciesthreeleaftissuesamplesfromthreedifferentindividualplantswerecollected.After

crushingmaterialwithamortarandpestle,supernatantsofthe3leavesfrom3differentindividual

plantsinthesameplantspeciesweremixedintoasamplepoolforviralmetagenomicslibrary

construction.Aftervirusnucleicacidparticlesenrichmentusingfiltration(removingeukaryoticand

bacterialcell-sizedparticles)andDNaseandRNasetreatment(digestingunprotectednucleicacid),

totalnucleicacidwerethenextractedandthenorganizedinto161librariesforIlluminaHiseq2500

sequencing(SupplementaryTable1).Intotal,50,586,188paired-endreadsweregeneratedand

binnedbybarcodesandquality-filtered,leavinghigh-qualitysequencereadswhichwerede

novoassembledwithineachbarcode.Theresultingsequencecontigsandunassembledreadswere

comparedwiththeviralreferencedatabaseandtheGenBanknon-redundantproteindatabaseusing

aBLASTxsearchwithanEvaluecut-offof50%ofviralsequencereads(Fig.1a,

SupplementaryTable1).Fromtheseplants,34differentgroupsofvirusesweredetected,including

virusesbelongingin26families,1genus(Botybirnavirus)unclassifiedinfamily,and7unclassified

6

groupsincludingcircularreplication-associatedproteinencodingsingle-strandedDNAvirus(CRESS

DNAvirus),Parvo-likevirus,Hepe-likevirus,Noda-likevirus,Permutotetra-likevirus,Rhabdo-like

virus,Sobemo-likevirus,unclassifiedmembersofPicornaviralesorder,andunclassifiedmembersof

theRiboviriadomain(Fig.1b,SupplementaryTable1).Comparisonofthepercentageofviralreads

againstthetotaluniquereadsandthenumbervirustypesineachlibraryshowednosignificant

differencebetweenwildandcultivatedplantsamples(SupplementaryData3and4,Supplementary

Table1),suggestingthatinthelocalplantecosystemdifferentcultivationmodesofplantshadno

discernableeffectonsusceptibilitytovirusinfection.Fromtheseviralsequences,251virusstrains

generatedcompetegenome(n=202)ornearlycompletegenomesequences(n=49),including5RNA

virusstrainsbelongingtosegmentedviruses(Fig.1b).BLASTxsearchusingnucleotidesequencesof

the251virusstrainsrevealedthat61ofthemshared

7

strainsweregroupedintothreepreviouslyclassifiedgenerawhiletheother16strainswereclustered

intoaseparategroupgeneticallyfarfromthethreeknowngenera(Fig2,SupplementaryTable1,see

SupplementaryData5).Thesedicistrovirusesintheseparategroupshowedtypicalgenome

organizationsofdicistrovirusesexceptthat10ofthe14strainsshowednocricketparalysisvirus

(CRPV)capsidsuperfamilydomaininthecapsidprotein(SupplementaryData6).BasedonRdRp

proteinsequencesofthe16strainsintheseparatecluster,theyshared

8

withinMarnaviridae,whichindicatedthemarnavirusesgroupincludescloselyrelatedvirusesfrom

plantsandtwostrainsfromfishandmollusk(Fig.2,SupplementaryData9),respectively.

MarnaviridaeisanewlydefinedvirusfamilyinorderPicornavirales,thecurrentlycharacterized

representativememberbeingHeterosigmaakashiwoRNAvirus,isolatedfromHeterosigmaakashiwo

algaeinoceanwater[6].Closelyrelatedviruseshavebeenidentifiedinoceanmarineenvironments

[7].OurdatasuggestthatplantsarecapableofhostingsomemembersinfamilyMarnaviridaeor

theircellularhosts.

In3differentplantspecies,weacquired6virusstainswithcompletegenomesshowingsignificant

sequencesimilaritytoparvovirus-likehybridvirus(PHV)and2virusesshowingcloserelationshipto

densovirus(Fig.2,SupplementaryData10).TheseplantPHVgenomeswerelinearwithlengthof3.6-

4.0-kbcontainingtwomajorforward-directionORFsencodingthereplicationandcapsidproteins

(SupplementaryData11),whichischaracteristicofvirusesinfamilyParvoviridae.The6PHVs

detectedinplantsweregroupedintwodifferentclusters,sharingsequencesimilaritiesof50%-67%

tootherPHVsbasedreplicationproteinsequence.PHVisatypeofhighlydivergentDNAviruswhich

wasrecentlydiscoveredandphylogeneticallylocatedattheinterfacebetweentheParvoviridaeand

Circoviridae[8,9].AlthoughthisviruswasfirstdetectedinChinesepatientswithseronegative(non-A-

E)hepatitisandsubsequentlydiscoveredinawiderangeofclinicalsamples,sharing∼99%

nucleotideandaminoacididentitywitheachother[8],itwaseventuallytracedtocontaminatedsilica-

bindingspincolumnsusedfornucleicacidextraction[9].Thesilicamatrixisgenerallygeneratedby

diatoms(algae),belongingtomicroscopicwaterplants,detectingPHVinsilica-bindingspincolumns

mightbetheinitialevidencethatplantscanserveasthehostsofPHV.Ourdatafurtherconfirmthat

plants(ordiatomswithinthem)arecapableofhostingPHVs.

Besidestheabovefourgroupsofviruseswithmultipledivergentstainsfoundhereinplanttissues,

another4groupsofviruses,notpreviouslyreportedinplants,includingnoda-likevirus,Permutotetra-

likevirus,Yanvirus-likevirus,andChuvirus-likevirus,werealsodetectedhere(Fig.2,Supplementary

Data12-15).Theseviralgroupswererecentlyreportedfrominvertebratesmeta-transcriptomes,and

vertebratesandenvironmentsamples[10–12].Discoveringthesevirusesinplantleafsamples

9

suggeststhatplantsmayalsobethenaturalhostsforsomemembersoftheserecentlydescribed

clades.Bastroviruswaspreviouslyonlydetectedinfecesofmammals(includinghuman)and

mosquito,showsadistantrelationshiptoastroviruses[13,14].Here,aspeciesofplant(Solanum

melongena)waspositiveforvirusgenomesequenceshowing25%RdRpsequencesimilaritytothatof

bastrovirus(Fig.2,SupplementaryData16).Detectingthisdivergentbastrovirus-likevirusinplants

mayimplybastrovirusoriginatesfromplantand/orthatitsdiversememberscaninfectwidely

differenthostsincludingvertebrates,invertebratesandplants.Anotherspeciesofplantwaspositive

forhepe-likevirus,whichhavebeenreportedinmammals,invertebrates,protists,anddifferent

environments[12,15–17].Thishepe-likevirusstrainfromplantwaswellgroupedwithotherhepe-like

virusesfromdifferenttypeoforganismandenvironmentsamplesandsharedsimilargenome

organization(Fig.2,SupplementaryData17),suggestingthistypevirusmayalsoparasitizeplants.

Twotypesofviruses,botybirnavirusandnarna-likevirus,whichwereconsideredtobevirusesof

fungi[18,19]andmorerecentlyCaenorhabditisnematodes[20],weredetectedintwospeciesof

plants,respectively(Fig.2,SupplementaryData18and19).Thebotybirnavirusshowedhigh

sequenceidentity(96.4%)tofungibatybirnavirusbasedonRdRpproteinsequence.Thetwonarna-

likevirusstrainsfrom2differentspeciesofplantsshared99.9%nucleotidesequenceidentityand

identicalbasedonRdRpproteinsequence,andweredivergentfrompreviousnarna-likeviruses.

Divergentvirusesinplants.Forthese12groupsofviruses,firstreportedinplantshere,some

genomesweresodivergentfromtheirclosestidentifiablerelativesusingBLASTxtheymayultimately

qualifyasmembersofnewgeneraorevennewfamilies(Fig.2).Forexample,forthe23dicistrovirus

genomes,7ofwhichgroupedwellintopreviouslydefinedgenera,theother16strainsseemtoforma

separatecladewhichcouldbedesignatedanewgenusintheDicistroviridaefamily.Thesame

conclusioncouldalsoapplytosomegenomesinthegroupsofnoda-like,hepe-like,andbastrovirus-

likevirusesandintheMarnaviridaefamily.

Another23divergentRNAviralgenomeswhoseclosestrelativesareinthePicornaviralesorderwere

characterized.PhylogeneticanalysisbasedonRdRpsequencesofthe6definedfamiliesandthebest

matchesofthe23strainsinGenBankshowedthattheyweregroupedinto8differentclusterswhich

10

weregeneticallydistinctfromthedefined6familiesintheorderPicornavirales(Fig3,Supplementary

Data20).

Tombusviridaeisalargefamilyofplantvirusesthatiscurrentlycomposedofmorethan76species

dividedamong3subfamiliesand16genera.Here,weacquired21genomesshowingsequence

similaritytomembersoftheTombusviridae.Sevengenomesweregeneticallyclosetodefinedgenera

whiletheother14werehighlydivergentandseemedtoformseveraldistinctgenera(Fig3,

SupplementaryData21).FourdifferentvirusstrainsbelongingtofamilyLuteoviridaewerealso

detectedinplantshere,3ofwhichcloselyclusteredwithdifferentdefinedgenera,withtheremaining

formingasingledeeplyrootedseparatebranch,whichmaybelongtoaputativenewgenusclustering

outsidethegenusluteovirus(Fig3,SupplementaryData22).Fourpartitivirusstrainswere

characterizedinthreedifferentspeciesofplants,allofthemwereputativenewspecieswithinthree

differentgeneraofPartitiviridae(Fig3,SupplementaryData23).Sevenvirusgenomesidentifiedhere

alsoshowedsequencesimilaritytosobemo-likeviruseswhichwererecentlydiscoveredfrom

arthropodsusingmeta-transcriptomics[15].Althoughtheseplantssobemo-likeviruses

phylogeneticallygroupedtogetherwithinvertebratesobemo-likevirusestheyweregenetically

distinctandsharing30%-62%aminoacidsequencesimilaritiestoeachother(Fig3,Supplementary

Data24).Twoplantrhabdo-likevirusesalsoshowedacloserelationshiptorecentlydiscovered

invertebratederivedrhabdo-likeviruses(Fig3,SupplementaryData25).LastonedivergentRNA

genomeshowedadistantrelationshiptothreegenomesbelongingtoanunclassifiedmemberofthe

Riboviriadomain,allfromwastewaterorsoilsamples,consistentwithaplantorigin(Fig3,

SupplementaryData26).

PlantCRESSvirus.CRESSDNAvirusistheinformalnameofseveralgroupsofsingle-stranded(ss)

DNAvirusesthathavecircularandreplication-associatedproteinencodinggenome,whichshowhigh

diversityandabundanceinvarioushabitats[21,22].Althoughtherearecurrentlyseveralestablished

CRESSDNAvirusfamiliesincludingBacillidnaviridae,Circoviridae,Geminiviridae,Genomoviridae,

Microviridae,NanoviridaeandSmacoviridae,alargenumberofnovelCRESSDNAviruseshavebeen

discoveredrecentlyandhavenotbeenformallyclassified,forwhichthehostsarecurrentlyunknown

11

[22–24].Amongthesewell-definedCRESSDNAvirusfamilies,GeminiviridaeandNanoviridaearetwo

plant-infectingmembers,whichalsohelpthereplicationandpackageofasatellitevirus:

Alphasatellitidae,anothertypeofcircularssDNAgenome[25].Here,fromplantleavesweacquired79

circulargenomes,amongwhich7weregeneticallyclosetoGeminiviridae,9groupedwellintothe

familyGenomoviridae,7clusteredcloselytoknownsequencesofAlphasatellitidae,15belongtonew

divergentmembersinfamilyMicroviridaepresumablyfrombacteria,withtheremaining41showing

significantsequencesimilaritytounclassifiedCRESSDNAviruses(Fig.4).

Amongthe7CRESSDNAvirusesbelonginginfamilyGeminiviridae,2ofthemfeltwellintothecluster

ofthegenusbegomovirus,beingcloselytosweetpotatoleafcurlvirus,amonopartitegeminivirus.

Theother5werenotgroupedintoanyknowngenusinfamilyGeminiviriaebutdeeplyclustered

outsideofallknowngeminiviruses,suggestingthese5novelgeminivirusesmightbelongtonew

genus(genera)inGeminiviridae(Fig.4,SupplementaryData27).VirusesinthefamilyGenomoviridae

havebeenfrequentlyfoundtobeassociatedwithavarietyofsamplesrangingfromfungitoanimal

sera[26],indicatingthatgenomovirusesarewidespreadaswellasabundantintheenvironment.

Here,9completegenomesofgenomovirus,divergentfrompreviousknownmembersinthatfamily,

werecharacterizedin7differentplantspecies,whichphylogeneticallyclusteredinto5different

groups,includingtwoidenticalgenomesdetectedintwodifferentplantspecies(Fig4,Supplementary

Data28).Currently,thehostsofthelargemajorityofCRESS-DNAvirusesremainunknownexceptfor

onereplicatinginbothfungi[27]andaninsect[28].Detectinggenomovirusesinleafsamplesfrom

differentspeciesofplantmaysuggestplantsoraninternalplant-dwellingorganism,mayhostsome

membersinthefamilyGenomoviridae.

Wealsodiscovered7divergentcompletecirculargenomesinasinglespeciesofplant,whichshowed

sequenceidentitiesof38%-58%topreviousknowngenomesofmembersinAlphasatellitidaebased

onaminoacidsequenceofencodedRepprotein.The7alphasatelliteshadgenomesizesrangingfrom

1309to1503nucleotides,whichweredivergentfromeachotherandgroupedinto4differentclusters

composedofpreviousdefinedalphasatellitesbasedonphylogeneticanalysisoftheirRepprotein(Fig

4,SupplementaryData29).AlphasatellitesarecircularssDNAcomponentswhicharegenerally

12

associatedwithNanoviridaeorsomemembersinGeminiviridae,however,wedidnotdetect

geminivirusornanovirussequenceinthisspeciesofplant,butdiscoveredadivergentCRESSDNA

virusgenomethatshowedthehighestRepproteinsequencesimilarityof60.7%toanunclassified

CRESSDNAvirus,temperatefruitdecay-associatedvirus[29],suggestingthistypeofCRESSDNA

virusmayinfectsplantandservesashelpervirusforalphasatellites.

Apartfromthe3groupsofviruseswithinclassifiedCRESSDNAvirusfamilies,other41unclassified

CRESSDNAviruseswerealsodiscoveredfromdifferentspeciesofplant.TheseCRESSDNAviruses

weresodivergentfromeachother,wephylogeneticallyanalyzedthemin6differentphylogenetic

trees(Fig4,SupplementaryData30-35),whereeachofthemincludesstrainsidentifiedhere,their

bestmatchesinGenBank,andtherepresentativemembersinknownCRESSDNAvirusfamiliesand

otherunclassifiedCRESSDNAviruses,usingfewersequencesineachsequencealignmentsoasto

includeaslargeaspossiblenumberofconservedaminoacidsitesinthephylogeneticanalysis.Based

onRepproteinssequences,theseunclassifiedCRESSDNAvirusessharedsequencesimilarities

26%-61%totheirbestmatches,where6ofthemgroupedwithCRESSDNAvirusesfromfecesof

mammals,3ofthemwithCRESSDNAvirusesfrominvertebrates,13sequenceswithCRESSDNA

virusesidentifiedfromenvironmentalsamples(mainlywastewater),8strainswithCRESSDNAvirus

fromfishspecies,onewithplant-associatedCRESSDNAvirus,whiletheremaining10sequenceswere

toodivergenttoclusterwithanyknownvirusesandwereincludedinCRESSVgroup6inFig.4(Fig4,

SupplementaryData30-35).ConsideringthatmostoftheCRESSDNAvirusescharacterizedinthe

presentstudybestmatchedunclassifiedCRESSDNAgenomesfromenvironmentalsamples,

mammalianfeces,andarthropods,itispossiblethatmostoftheseunclassifiedCRESSDNAviruses

infectplantsandwerecontaminantsinfecesorthegutcontentofarthropods.

FifteengenomesshowingsequencesimilaritytovirusesinfamilyMicroviridaeweredetectedinthree

differentspeciesofplants,12ofwhichwerefromasinglespeciesofwildplant,Kummerowiastriat

(SupplementaryData36).ManystudieshavedemonstratedtheubiquityofMicroviridaegenomes

acrosshabitats(marine,freshwater,wastewater,sediment)andglobalregions(Antarcticto

subtropical),especiallythoserelatedtotheGokushovirinaelineage[30–33],whichinfectobligate

13

intracellularparasites,membersofthebacterialgeneraChlamydia,BdellovibrioandSpiroplasma

[34].

Cross-speciesinfectionandco-infectionofplantviruses.Otherthanthroughseeddispersal

mostplantsareimmobile;henceplantvirustransmissionisoftenassistedbyothersorganisms

[35,36].Here,weinvestigatedtheviromeinplantleavescollectedinasingleecosystem,which

includesinteractionsamongstplants,water,soil,air,insectsandamultitudeofmicro-organisms

providingfavorableconditionsforcross-speciestransmission.Usingviralmetagenomics,wedetected

theviralnucleicacidsanddetermined251(nearly)completeviralgenomes,allowingustocompare

genomesequencesfromdifferentspeciesofplantsandestimatewhethercross-speciestransmission

mightoccurforsomeviruses.Ourresultsindicatedcross-speciestransmissionmighthaveoccurred

for9groupsofviruses.24genomesbelonginginfamilyPotyviridaewerefoundin17differentspecies

ofplant,allinthegenuspotyvirus(SupplementaryTable1,Supplementarydata37).Amongthe9

groupsofpotyviruses,2groupswerecomposedof10and5genomes,respectively,sharing

99%-100%sequenceRdRpproteinidentitieswithineachgroup,suggestingpossiblecross-species

transmission(Supplementarydata37).Wethencomparedthe10and5genomesequences

respectivelyinthese2groupsandfoundthatthe10genomesshared94.6%-100%andthe5

genomesshared94.8%-100%sequenceidentities(includingseveralpairsofidenticalsequences)

(Fig.5),suggestingsomestrainsofthesepotyvirusesmaybecapableofcross-speciestransmission.

Ourdataalsoshowedthatsomedicistrovirusesmightbeplant-infectingvirus.Hereweacquired22

completegenomesofdicistrovirusfrom10differentspeciesofplants,ofwhich6pairspresented

possiblecross-speciestransmissioninplantsaspairofgenomesequencesshared>94.9%identity,

includingonepairofidenticalsequences(Fig5,Supplementarydata5).Putativecross-species

transmissionswerealsoobservedwithunclassifiedCRESSvirusincluding5pairsofidenticalgenomes

derivedfromdifferentspeciesofplants(SupplementaryData38).Twogroupsofmarnaviruses

showing>99%genomicsequenceidentity,andother5pairsofdifferentvirusesincluding

geminivirus,genomovirus,luteovirus,parvo-likevirus,andsobemo-likevirus,fromdifferentputative

hostspeciesshowed92.5%-99.8%sequenceidentitiesbasedoncompletegenomesequence

14

(SupplementaryData38).

Wemarkedtheaccuratesamplingsitesforeachplantspecieswhichmakesitpossibletomeasurethe

geographicaldistanceofdifferentspeciesofplantsinvolvedinthecross-speciestransmissionofa

certainvirussoastoinferwhethergeographicaldistanceofthehostplantshaveeffectonthecross-

speciestransmission.Ourdataindicatedthatcross-speciestransmissionofpotyvirusesmightbe

associatedwiththeirgeographicaldistanceasthegeneticallyveryclosegenomesweremainlyfrom

thesamesamplingsite(Fig.5).Thesamephenomenawerealsoobservedforthemarnavirus,

unclassifiedCRESSDNAvirusgenomes,luteovirus,andparvo-likevirus.Forexample,allthe5

marnavirusgenomesinvolvedinputativecross-speciestransmissionwerefromasinglesamplingsite

and9ofthe11CRESSDNAgenomeswerefromthesamesamplinglocation(Fig.5,Supplementary

Data38).However,theremainingseveralgroupsofviruseswithpropertiesofcross-species

transmission(closelyrelatedgenomesfromdifferentplants)includingdicistrovirus,geminivirus,

genomovirus,andsobemo-likevirusseemtohavenorelationshiptothegeographicaldistanceof

theirhosts’location(SupplementaryData38).Thedifferenteffectofgeographicaldistanceonthe

cross-speciestransmissionmayreflectthedifferenttransmissionpotentialoftheseviruses,for

example,geographicaldistancehadnoeffectonthecross-speciestransmissionofdicistroviruses

suggestedthatthespreadofthisvirusmightbeassistedbyarthropods.Ourdataalsoindicatedthat

mostoftheputativeviralcross-speciesinfectioninthisecosystemoccurredacrossdifferentlevelsof

plantclassification.Forinstances,the10closelyrelatedpotyvirusgenomeswerecharacterizedfrom

plantsbelongingto7differentorderswithin2differentclasses(Fig.5),suggestingawidehostrange.

Co-infectionofhostsbytwoormoreplantvirusesiscommoninbothagriculturalcrops[37,38]and

naturalplantcommunities[39].Inthepresentstudy,apartfromcross-speciesinfection,co-infectionof

plantviruseswasalsocommonlyobserved,where73outof161(45.3%)librariescontained>3

differentvirustypes(orfamilies)(SupplementaryTable1),suggestingco-infectionofvirusesexisted

innearlyhalfoftheplantsinthisecosystemaseachlibraryconsistedofsamplesfromthreedifferent

individualplant.Consideringthesamevirusfamiliesortypeinasinglelibrarymaycontaindifferent

15

virusstrainortype,therateofco-infectionislikelytobehigherthan45.3%.Amongthe251genomes

weacquiredfromtheseplants,somegenomeswerefromthesamelibrarieswhichallowsus

investigatetheco-infectionofcertainvirusesinspecificspeciesofplants.AsshowninFig.6,PCR

screeningofdifferentvirusgenomesin7differentspeciesofplantsrevealedthatmostof(20/21)the

individualplantcontained>2differenttypesofvirus,whereoneplantspeciesofForsythiasuspensab

evencarried16virusesbelongingin12differentfamilies.Thewidepresenceofapparentviralco-

infectionsintheseplantsinasingleecosystemmayleadtointeractionsbetweenvirusesthatcould

influencediseasedevelopmentinindividualplants.

Otherplantviruses.Apartfromthesevirusesmentionedabove,manytypesoftypicalplantviruses

belongingintheBromoviridae,Closteroviridae,Comoviridae,andBotourmiaviridaefamiliesand

Tymoviralesorderwerealsodetectedinseveralspeciesofplants.Theseplantviruseswere

geneticallyclosetopreviouslydescribedviruses(Supplementarydata39-43),indicatingtypicalplant

virusinfectionswerereadilydetectedinthisplantecosystem.

DiscussionThecommonperceptionthatplantvirusesareprimarilypathogensresultsfromthefocusgivento

agriculturalplanthealth.Emergingdiseaseshavegarneredmostattentionbecauseofdamageto

economicallyimportantfoodandornamentalplantspecies.Importantexamplesofvirusesthatare

responsibleforwell-studiedemergingdiseasesincludecassava-infectingbegomoviruses(inthe

Geminiviridaefamily)[40],closterovirusescausinggrapevineleafrolldisease[41],luteovirusessuchas

barleyyellowdwarfvirus[42]andsobemovirusessuchasriceyellowmottlevirus[43].Relativesofall

thesepathogenicvirusesweredetectedinthisstudyinapparentlyhealthyplantsfromdiverse

familiesororders.Therelativelyunbiasedsequencingofviralgenomeswithinentireenvironmentsas

performedhereischangingtheperspectiveofvirusesfromagentsofdiseasetocommoncomponents

ofecosystems,astheplanttissuesamplesstudiedwereallfromapparentlyhealthyplants.

Thedatainthepresentstudyalsorevealedthatseveralvirusessuchasdicistrovirus,iflavirus,

marnavirus,noda-like,andparvo-likeviruses,whichhavenotbeenreportedinplantsweredetected

hereinleaftissues.Amongtheseviruses,dicistrovirus,iflavirus,andnoda-likevirusaregenerally

16

hostedbyarthropods[44,45].Detectionofthesegenomesinplantsindicatedthatinsectsmaymight

vectoredthembetweenplants.Theclosestnon-plant-infectingrelativesofsomegenomesfromplants

reportedheretendedtoinfectarthropodsorfungi.Currentlyplant-infectingvirusesmaytherefore

haveevolvedfromvirusesthatonceinfectednon-plantorganisms(orvideversa).Further,the

hypothesisthatmanyplantandvertebratevirusesmayhaveoriginatedfromarthropodvirusesisalso

plausibleassomevirusesinfectarthropodscanalsoinfectplants.Forexample,flockhousevirus(in

theNodaviridaefamily)infectsarthropodsbutcanalsosystemicallyinfectplantswhenitis

complementedwiththemovementproteinsofeithertobaccomosaicvirusorredclovernecrotic

mosaicvirus(bothofwhichareplantviruses)[46].

Thecross-speciestransmissionofvirusesfromonehostspeciestoanotherisresponsibleforthe

majorityofemerginginfections,bothinanimalandplantpopulations[47–49].Decadesof

inventorying,trackingandanalyzingofplantvirusesshowedthattheemergenceofnewdiseasesis

drivenbyadaptiveviralevolutioninresponsetonovelecologicalconditions[50,51],includingthe

introductionofvirusesandvectorstonewareas,theintensificationofagricultureandurbanization,

andecologicalchangesinresponsetochangingclimaticconditions.Ourdatashowedthatanumber

ofgenomesfromviralfamiliesnotknowntoinfectplantsareindeedpresentinplants.Furthermore

severalgeneticallycloseoridenticalvirusesweredetectedinplantsfromindifferentspecies,

suggestingcross-plantspeciestransmission.Inaddition,itisnoteworthythatasmallnumberof

virusesshowinggeneticrelationshiptovirusespreviouslyfoundinmammalianfeces(e.g.bastrovirus

andhepe-likevirus)werealsodetectedinplanttissues,possiblyindicatingtheirplanttropism.

ConclusionsOurstudypresentsanoverviewoftheviruscommunityexistinginleaftissuesfromplantsinalocal

plantecosystem,whichismorediversethanthatdepictedincurrentclassificationofplantviruses.

Virustypesandviralreadsinwildandcultivatedplantswerecompared,showingnodifferent

betweentwogroups.Cross-speciesinfectionandco-infectionofviruseswerecommoninthisplant

ecosystem.251differentplant-associatedvirusgenomeswerefullycharacterizedand

phylogeneticallyclassifiedinto27differentvirusfamilies,ordersorunclassifiedvirusgroups.This

17

studyprovidesasolidfoundationforstudiesinvirusecologyandevolutioninplantsandwillbe

helpfulforidentificationofnewlyemerging,possiblypathogenic,virusesofplants.

MaterialsAndMethodsSamplecollectionandpreparation

Thegoalofthisstudywastoinvestigatetheviromeofplantspeciesinanancientcanalecosystemin

ZhenjiangCity,JiangsuProvince,China.TheZhenjiangAncientCanalhasahistoryofmorethan2000

years.ItrunsthroughthewholetownofZhenjiangfromsoutheasttonorthwestandis16kilometers

longwithanaveragewidthof40meters.Byinvestigatingtheriparianvegetationoftheancient

canal,theDingmaosectionofthecanalwaschosentostudyasarepresentativesection.Itis2km

longwithlotsofwildplants,somelandscapeplantsandcropsonbothriversides.Intotalof161plant

speciesbelongingto38differentorder,6classes(Coniferopsida,Cycadopsida,Dicotyledoneae,

Filicopsida,Ginkgopsida,andMonocotyledoneae),and3phyla(Angiospermae,Gymnospermae,

andPteridophyta)werecollectedinthisareaforthisstudy.Thesamplingsitesforeachplantspecies

arelabeledonthemapwithnumberscorrespondingtoplantlibrarynumbers(SupplementaryTable1,

SupplementaryData1and2).Amongthoseplantspecies,72arewildplantsand89arecultivated

plantsincludinglandscapeplantsandcrops.Duringsampling,3leavesfromthree

differentindividualplantsbelongingtothesamespecieswererespectivelycollectedintodisposable

materials,beforethisstep,distilledwater(ddH2O)wasusedtocleanthedustandothernon-plant

organismsontheleafsurface.Beforeviralmetagenomicanalysis,about0.1gleaftissuesampleof

eachplantwasgroundedusingsteelballsandre-suspendedin1mLofphosphate-bufferedsaline

(PBS)andvigorouslyvortexedfor5min.Thegroundedsampleswerethenfrozenandthawedthree

timesondryice.Thesupernatantswerethencollectedaftercentrifugation(10min,15,000×g)and

storedat-80℃untiluse.HostspeciesidentificationwasinitiallyidentifiedusingAPP“PictureThis”

whichisonlineplantencyclopediaandplantidentifier,andfutureconfirmedbyexperiencedfield

biologists.

Viralmetagenomicanalysis

About300μLsupernatantfromeachofthethreedifferentplantsamplesinthesamespecieswas

18

mixedintoonesamplepoolandfilteredthrougha0.45-μmfilterandcentrifugedat120,000gfor20

minutesat4℃toremoveeukaryoticandbacterialcell-sizedparticles.Un-encapsidatednucleicacids

werethendigestedbyDNaseandRNaseat37°Cfor60min[52–55].Totalnucleicacidswere

extractedasamixedRNA/DNAsolutionusingQiaAmpMiniViralRNAkit(Qiagen)accordingtothe

manufacturer’sprotocol.161librarieswereconstructedusingNexteraXTDNASamplePreparationKit

(Illumina).Forbioinformaticsanalysis,paired-endreadsof250bpgeneratedbyMiSeqwere

debarcodedusingvendorsoftwarefromIllumina.Anin-houseanalysispipelinerunningona32-node

Linuxclusterwasusedtoprocessthedata.Readswereconsideredduplicatesifbases5to55were

identicalandonlyonerandomcopyofduplicateswaskept.Clonalreadswereremovedandlow

sequencingqualitytailsweretrimmedusingPhredqualityscoretenasthethreshold.Theuniqueread

numberofeachlibrarywasshowninTable1.Adaptorsweretrimmedusingthedefaultparametersof

VecScreenwhichisNCBIBLASTnwithspecializedparametersdesignedforadapterremoval.The

cleanedreadsweredenovoassembledwithineachbarcodeusingtheENSEMBLEassembler[56].

Contigsandsingletsreadsarethenmatchedagainstacustomizedviralproteomedatabaseusing

BLASTxwithanEvaluecutoffof

19

usingthesoftwareGeneiousv11.1.2andprimersbridgecontigswerethendesigned[61].Gapswere

filledby(RT–)PCRandSangersequencing.Toconfirmtheassemblyresultsofafullgenome,reads

weredenovoassemblebacktothefulllengthgenomeusingthelowsensitivity/fastestparameterin

Geneious11.1.2.Forgenomeswithnovelstructures,weverifiedthecompleteornearcompleteviral

genomebydesigningoverlappingprimersbasedontheassembledsequences.Forthosevirusesthat

firstlyisolatedfromplants,weusedPCRandSangersequencingtoverifyit’saccuratebasedonthe

assembledsequences.

Confirmationofviralco-infection

In7librariesincludingpt065,pt067,pt110,pt111,pt112,pt119andpt151,whichhavefarmorethan

threedifferentvirusstrains,showedevidentco-infectioninindividualplant.Toinvestigatethe

presencestatusofdifferentviralstraininthreeindividualplantsfromthesamelibrary,PCRand

Sangersequencingwereperformedusingspecificprimersdesignedbasedontheconserveddomain

sequencesoftheseviruses.

Phylogeneticanalysisofviruses

Throughanalyzedtheproteinsequencesobtainedinthisstudy,wedividethemintothreecategories

includingRNAviruses,Parvovirus-likevirusesandCRESSDNAviruses.Toinferthephylogenetic

relationships,proteinsequencesofreferencestrainsbelongingtoRNAviruses,Parvovirus-like

viruses,andCRESSDNAvirusesweredownloadedfromtheNCBIGenBankdatabase.ForRNAviruses,

thephylogenetictreewasconstructedbasedontheRNA-dependentRNApolymerase(RdRp),for

parvovirus-likeviruses,thephylogenetictreewasconstructedbasedonnonstructuralprotein(NS),

fortheCRESSDNAviruses,thephylogenetictreewasconstructedbasedonthereplication-associated

protein(Rep)exceptforMicroviridaeviruseswhosemajorcapsidproteinwasusedforthe

phylogenetictreeconstruction.Therelatedproteinsequenceswerefirstlyalignedusingalignment

programimplementedintheCLCGenomicsWorkbench10.0,thealignmentresultwasfurther

optimizedusingMUSCLEinMEGAv7.0[62]andMAFFTv7.3.1employingtheE-INS-Ialforithm[63].

Sitescontainingmorethan50%gapsweretemporarilyremovedfromalignments.Bayesianinference

treeswerethenconstructedusingMrBayesv3.2[64].TheMarkovchainwasrunforamaximumof1

20

milliongenerations,inwhichevery50generationsweresampledandthefirst25%ofMarkovchain

MonteCarlo(mcmc)sampleswerediscardedasburn-in.Theapproximatefamily/genusofvirusesthat

obtainedinthisstudywasdeterminedthroughtheabovetree,furtherconstructedthedetailedtrees

pointateachvirusfamilythatarerelativelycloselyrelatedtothevirusesdiscoveredhereusingthe

samemethod.MaximumLikelihoodtreeswerealsoconstructedtoconfirmedalltheBayesian

inferencetreesusingsoftwareMegav7.0[62]orPhyMLv3.0[65].

Virusgenomeannotation

Putativeviralopenreadingframes(ORFs)werepredictedbyGeneiousv11.1.2withbuilt-in

parameters(Minimumsize:300;Geneticcode:Standard;Startcodons:ATG)[61],furtherwere

checkedthroughcomparingtorelatedvirusesbyBlastpinNCBI.TheannotationsoftheseORFswere

basedoncomparisonstotheConservedDomainDatabase.PotentialexonandintronofGenomovirus

werepredictedbyNetgenes2athttp://www.cbs.dtu.dk/services/NetGene2/.

Qualitycontrolinthenucleicacidmanipulation

Standardprecautionswereusedforallprocedurestopreventthecross-samplecontaminationand

nucleicaciddegradation.Mainly,aerosolfilterpipettipswereusedtoreducethepossibilityofsample

crosscontamination,andallthematerials(includingmicrocentrifugetubes,pipettips,etc.)which

directlycontactedwithnucleicacidsampleswereRNaseandDNasefree.Thenucleicacidsamples

weredissolvedinDEPCtreatedwater.Inordertoexcludethepossibilityofcontaminationwithnucleic

acidsofparvovirus-likehybridvirus(PHV)andmiciroviruspresentinthelaboratoryorfromQiagen

nucleicacidextractionkits,thesamplespositiveforthetwotypesofviruswerechosenandthe

nucleicacidwerere-extractedusingTrizolreagent(Invitrogen).PCRusingprimersspecifictothose

virusesconfirmedtheirpresenceintheoriginalbiologicalsamples.Asacontrol,alibrarywasalso

constructedusingddH2Oassamplewhichgenerated13,228rawreadsandcontainednoviralreads

basedonBLASTxsearching.

AbbreviationsCRESSDNAvirus:circularreplication-associatedproteinencodingsingle-strandedDNAvirus;RdRp:

RNA-dependentRNApolymerase;Rep:replicationproteins;NS:non-structuralprotein;CRPV:cricket

http://www.cbs.dtu.dk/services/NetGene2/

21

paralysisvirus;PHV:parvovirus-likehybridvirus

DeclarationsAcknowledgements

WethankYimingLiuatTropicalCropsGeneticResourcesInstituteinChineseAcademyofTropical

AgriculturalSciencesforidentificationofplantspecies.ThisworkwassupportedbyNationalKey

ResearchandDevelopmentProgramsofChinaNo.2017YFC1200201,JiangsuProvincialKeyResearch

andDevelopmentProjectsNo.BE2017693andIndependentProjectofChengduResearchBaseof

GiantPandaBreedingNo.2020CPB-C11.

Authors'contributions

WZconceived,designedandsupervisedthestudy.SY,YH,MZ,JL,ZY,ZD,andXLcollectedplant

samples.SY,YW,JY,andXCperformedexperiments.SY,XD,andTSanalyseddata.YW,JY,XC,YX,

HL,RZ,QL,andWLtookpartindatasortingandanalysis.WZandSYwrotethemanuscript.WZ,ED,

ZY,CZ,XW,QS,andHXrevisedandeditedthemanuscript.Allauthorsreadthemanuscriptand

agreedtoitscontents.

Availabilityofdataandmaterials

AllcompleteorpartialviralgenomeobtainedinthisstudyweredepositedinGenBankwiththe

accessionnumbersMN722411-MN722420,MN723593-MN723599,MN729612-MN729623,MN728806-

MN728814,MN724250-MN724258,MN814305-MN814321,MN831436-MN831448,MN823661-

MN823692,MN841281-MN841303,MN832441-MN832474,MN862333-MN862357,MN891787-

MN891825,MT067617-MT067623andMT134328(SeedetailedinformationinSupplementaryTable

2).TherawsequencereadsgeneratedhereweredepositedintotheSequenceReadArchiveof

GenBankdatabaseandtheaccessionnos.areshownSupplementaryTable2.

Ethicsapprovalandconsenttoparticipate

Notapplicable.

Consentforpublication

Notapplicable.

Competinginterests

22

Theauthorsdeclarethattheyhavenocompetinginterests.

References1. RoossinckMJ.Symbiosisversuscompetitioninplantvirusevolution.Nat.Rev.

Microbiol.2005;3:917–24.

2. VallesSM,ChenY,FirthAE,GuérinDMA,HashimotoY,HerreroS,etal.ICTVVirus

TaxonomyProfile:Dicistroviridae.J.Gen.Virol.2017;98:355–6.

3. BonningBC,MillerWA.Dicistroviruses.Annu.Rev.Entomol.2010;55:129–50.

4. GengP,LiW,deMirandaJR,QianZ,AnL,TereniusO.Studiesonthetransmission

andtissuedistributionofAntheraeapernyiiflavirusintheChineseoaksilkmoth

Antheraeapernyi.Virology.2017;502:171–5.

5. RyabovEV.,FannonJM,MooreJD,WoodGR,EvansDJ.TheIflavirusesSacbroodvirus

andDeformedwingvirusevokedifferenttranscriptionalresponsesinthehoneybee

whichmayfacilitatetheirhorizontalorverticaltransmission.PeerJ.2016;4:e1591.

6. TaiV,LawrenceJE,LangAS,ChanAM,CulleyAI,SuttleCA.CHARACTERIZATIONOF

HaRNAV,ASINGLE-STRANDEDRNAVIRUSCAUSINGLYSISOFHETEROSIGMA

AKASHIWO(RAPHIDOPHYCEAE)1.J.Phycol.JohnWiley&Sons,Ltd;2003;39:343–52.

7. CulleyAI,LangAS,SuttleCA.Highdiversityofunknownpicorna-likevirusesinthe

sea.Nature.2003;424:1054–7.

8. XuB,ZhiN,HuG,WanZ,ZhengX,LiuX,etal.HybridDNAvirusinChinesepatients

withseronegativehepatitisdiscoveredbydeepsequencing.Proc.Natl.Acad.Sci.

2013;110:10264–9.

9. NaccacheSN,GreningerAL,LeeD,CoffeyLL,PhanT,Rein-WestonA,etal.ThePerils

ofPathogenDiscovery:OriginofaNovelParvovirus-LikeHybridGenomeTracedto

NucleicAcidExtractionSpinColumns.J.Virol.2013;87:11966–77.

10. ShiM,LinX-D,TianJ-H,ChenL-J,ChenX,LiC-X,etal.Redefiningtheinvertebrate

23

RNAvirosphere.Nature.2016;540:539–43.

11. ZhangY-Z,ShiM,HolmesEC.UsingMetagenomicstoCharacterizeanExpanding

Virosphere.Cell.2018;172:1168–72.

12. ShiM,LinX-D,ChenX,TianJ-H,ChenL-J,LiK,etal.Theevolutionaryhistoryof

vertebrateRNAviruses.Nature.2018;556:197–202.

13. OudeMunninkBB,CottenM,CanutiM,DeijsM,JebbinkMF,vanHemertFJ,etal.A

NovelAstrovirus-LikeRNAVirusDetectedinHumanStool.VirusEvol.2016;2:vew005.

14. BauermannFV.,HauseB,BuysseAR,JoshiLR,DielDG.Identificationandgenetic

characterizationofaporcinehepe-astrovirus(bastrovirus)intheUnitedStates.Arch.

Virol.2019;164:2321–6.

15. ShiM,LinX-D,TianJ-H,ChenL-J,ChenX,LiC-X,etal.Redefiningtheinvertebrate

RNAvirosphere.Nature.2016;540:539–43.

16. WuN,ZhangP,LiuW,WangX.Sogatellafurciferahepe-likevirus:Firstmemberofa

novelHepeviridaecladeidentifiedinaninsect.VirusRes.2018;250:81–6.

17. PurdyMA,HarrisonTJ,JameelS,MengX-J,OkamotoH,VanderPoelWHM,etal.ICTV

VirusTaxonomyProfile:Hepeviridae.J.Gen.Virol.2017;98:2645–6.

18. ZhaiL,YangM,ZhangM,HongN,WangG.CharacterizationofaBotybirnavirus

ConferringHypovirulenceinthePhytopathogenicFungusBotryosphaeriadothidea.

Viruses.2019;11:266.

19. SahinE,AkataI.Virusesinfectingmacrofungi.VirusDisease.2018;29:1–18.

20. RichaudA,FrézalL,TahanS,JiangH,BlatterJA,ZhaoG,etal.Verticaltransmission

inCaenorhabditisnematodesofRNAmoleculesencodingaviralRNA-dependentRNA

polymerase.Proc.Natl.Acad.Sci.U.S.A.2019;116:24738–47.

21. ZhaoL,RosarioK,BreitbartM,DuffyS.EukaryoticCircularRep-EncodingSingle-

StrandedDNA(CRESSDNA)Viruses:UbiquitousVirusesWithSmallGenomesanda

24

DiverseHostRange.Adv.VirusRes.2019;103:71–133.

22. KazlauskasD,VarsaniA,KooninEV,KrupovicM.Multipleoriginsofprokaryoticand

eukaryoticsingle-strandedDNAvirusesfrombacterialandarchaealplasmids.Nat.

Commun.2019;10:3425.

23. KrupovicM.Networksofevolutionaryinteractionsunderlyingthepolyphyleticorigin

ofssDNAviruses.Curr.Opin.Virol.2013;3:578–86.

24. TiszaMJ,PastranaDV,WelchNL,StewartB,PerettiA,StarrettGJ,etal.Discoveryof

severalthousandhighlydiversecircularDNAviruses.Elife.eLifeSciences

Publications,Ltd;2020;9.

25. BriddonRW,MartinDP,RoumagnacP,Navas-CastilloJ,Fiallo-OlivéE,MorionesE,et

al.Alphasatellitidae:anewfamilywithtwosubfamiliesfortheclassificationof

geminivirus-andnanovirus-associatedalphasatellites.Arch.Virol.2018;163:2587–

600.

26. KrupovicM,GhabrialSA,JiangD,VarsaniA.Genomoviridae:anewfamilyof

widespreadsingle-strandedDNAviruses.Arch.Virol.2016;161:2633–43.

27. YuX,LiB,FuY,JiangD,GhabrialSA,LiG,etal.Ageminivirus-relatedDNA

mycovirusthatconfershypovirulencetoaplantpathogenicfungus.Proc.Natl.Acad.

Sci.U.S.A.2010;107:8387–92.

28. LiuS,XieJ,ChengJ,LiB,ChenT,FuY,etal.FungalDNAvirusinfectsa

mycophagousinsectandutilizesitasatransmissionvector.Proc.Natl.Acad.Sci.U.

S.A.2016;113:12803–8.

29. BassoMF,daSilvaJCF,FajardoTVM,FontesEPB,ZerbiniFM.Anovel,highly

divergentssDNAvirusidentifiedinBrazilinfectingapple,pearandgrapevine.Virus

Res.2015;210:27–33.

30. RouxS,KrupovicM,PouletA,DebroasD,EnaultF.Evolutionanddiversityofthe

25

Microviridaeviralfamilythroughacollectionof81newcompletegenomesassembled

fromviromereads.DutilhBE,editor.PLoSOne.2012;7:e40418.

31. QuaiserA,DufresneA,BallaudF,RouxS,ZivanovicY,ColombetJ,etal.Diversity

andcomparativegenomicsofMicroviridaeinSphagnum-dominatedpeatlands.Front.

Microbiol.2015;6:375.

32. TuckerKP,ParsonsR,SymondsEM,BreitbartM.Diversityanddistributionofsingle-

strandedDNAphagesintheNorthAtlanticOcean.ISMEJ.2011;5:822–30.

33. HopkinsM,KailasanS,CohenA,RouxS,TuckerKP,ShevenellA,etal.Diversityof

environmentalsingle-strandedDNAphagesrevealedbyPCRamplificationofthe

partialmajorcapsidprotein.ISMEJ.2014;8:2093–103.

34. BrentlingerKL,HafensteinS,NovakCR,FaneBA,BorgonR,McKennaR,etal.

Microviridae,afamilydivided:isolation,characterization,andgenomesequenceof

phiMH2K,abacteriophageoftheobligateintracellularparasiticbacterium

Bdellovibriobacteriovorus.J.Bacteriol.2002;184:1089–94.

35. RoossinckMJ.Plants,virusesandtheenvironment:Ecologyandmutualism.Virology.

2015;479–480:271–7.

36. ChenY,EvansJ,FeldlauferM.Horizontalandverticaltransmissionofvirusesinthe

honeybee,Apismellifera.J.Invertebr.Pathol.AcademicPress;2006;92:152–9.

37. BernardoP,Charles-DominiqueT,BarakatM,OrtetP,FernandezE,FillouxD,etal.

Geometagenomicsilluminatestheimpactofagricultureonthedistributionand

prevalenceofplantvirusesattheecosystemscale.ISMEJ.2018;12:173–84.

38. MuthukumarV,MelcherU,PierceM,WileyGB,RoeBA,PalmerMW,etal.Non-

cultivatedplantsoftheTallgrassPrairiePreserveofnortheasternOklahoma

frequentlycontainvirus-likesequencesinparticulatefractions.VirusRes.

2009;141:169–73.

26

39. NasirA,Caetano-AnollésG.Aphylogenomicdata-drivenexplorationofviralorigins

andevolution.Sci.Adv.2015;1:e1500527.

40. McCallumEJ,AnjanappaRB,GruissemW.Tacklingagriculturallyrelevantdiseasesin

thestaplecropcassava(Manihotesculenta).Curr.Opin.PlantBiol.2017;38:50–8.

41. AlmeidaRPP,DaaneKM,BellVA,BlaisdellGK,CooperML,HerrbachE,etal.Ecology

andmanagementofgrapevineleafrolldisease.Front.Microbiol.2013;4:94.

42. WallsJ,RajotteE,RosaC.ThePast,Present,andFutureofBarleyYellowDwarf

Management.Agriculture.MultidisciplinaryDigitalPublishingInstitute;2019;9:23.

43. Pinel-GalziA,TraoréO,SéréY,HébrardE,FargetteD.Thebiogeographyofviral

emergence:riceyellowmottlevirusasacasestudy.Curr.Opin.Virol.2015;10:7–13.

44. JunglenS,DrostenC.Virusdiscoveryandrecentinsightsintovirusdiversityin

arthropods.Curr.Opin.Microbiol.2013;16:507–13.

45. CalisherCH,HiggsS.TheDiscoveryofArthropod-SpecificVirusesinHematophagous

Arthropods:AnOpenDoortoUnderstandingtheMechanismsofArbovirusand

ArthropodEvolution?Annu.Rev.Entomol.AnnualReviews;2018;63:87–103.

46. DasguptaR,GarciaBH,GoodmanRM.SystemicspreadofanRNAinsectvirusin

plantsexpressingplantviralmovementproteingenes.Proc.Natl.Acad.Sci.U.S.A.

2001;98:4910–5.

47. WuF,ZhaoS,YuB,ChenY-M,WangW,SongZ-G,etal.Anewcoronavirus

associatedwithhumanrespiratorydiseaseinChina.Nature.2020;579:265–9.

48. NgLFP,HiscoxJA.Coronavirusesinanimalsandhumans.BMJ.2020;368:m634.

49. OhshimaK,AkaishiS,KajiyamaH,KogaR,GibbsAJ.Evolutionarytrajectoryofturnip

mosaicviruspopulationsadaptingtoanewhost.J.Gen.Virol.2010;91:788–801.

50. FargetteD,KonatéG,FauquetC,MullerE,PeterschmittM,ThreshJM.Molecular

EcologyandEmergenceofTropicalPlantViruses.Annu.Rev.Phytopathol.

27

2006;44:235–60.

51. JonesRAC.Plantvirusemergenceandevolution:origins,newencounterscenarios,

factorsdrivingemergence,effectsofchangingworldconditions,andprospectsfor

control.VirusRes.2009;141:113–30.

52. ZhangW,LiL,DengX,KapusinszkyB,PesaventoPA,DelwartE.Faecalviromeof

catsinananimalshelter.J.Gen.Virol.2014;95:2553–64.

53. ZhangW,LiL,DengX,BlümelJ,NüblingCM,HunfeldA,etal.Viralnucleicacidsin

humanplasmapools.Transfusion.2016;56:2248–55.

54. SiqueiraJD,Dominguez-BelloMG,ContrerasM,LanderO,Caballero-AriasH,XutaoD,

etal.ComplexviromeinfecesfromAmerindianchildreninisolatedAmazonian

villages.Nat.Commun.NaturePublishingGroup;2018;9:4270.

55. ZhangW,YangS,ShanT,HouR,LiuZ,LiW,etal.Viromecomparisonsinwild-

diseasedandhealthycaptivegiantpandas.Microbiome.2017;5:90.

56. DengX,NaccacheSN,NgT,FedermanS,LiL,ChiuCY,etal.Anensemblestrategy

thatsignificantlyimprovesdenovoassemblyofmicrobialgenomesfrom

metagenomicnext-generationsequencingdata.NucleicAcidsRes.2015;

57. Skewes-CoxP,SharptonTJ,PollardKS,DeRisiJL.ProfilehiddenMarkovmodelsfor

thedetectionofviruseswithinmetagenomicsequencedata.TseH,editor.PLoSOne.

2014;9:e105067.

58. JohnsonLS,EddySR,PortugalyE.HiddenMarkovmodelspeedheuristicanditerative

HMMsearchprocedure.BMCBioinformatics.2010;11:431.

59. EddySR.Anewgenerationofhomologysearchtoolsbasedonprobabilistic

inference.GenomeInform.2009;23:205–11.

60. FinnRD,ClementsJ,EddySR.HMMERwebserver:interactivesequencesimilarity

searching.NucleicAcidsRes.2011;39:W29-37.

28

61. KearseM,MoirR,WilsonA,Stones-HavasS,CheungM,SturrockS,etal.Geneious

Basic:anintegratedandextendabledesktopsoftwareplatformfortheorganization

andanalysisofsequencedata.Bioinformatics.OxfordUniversityPress;

2012;28:1647–9.

62. KumarS,StecherG,TamuraK.MEGA7:MolecularEvolutionaryGeneticsAnalysis

Version7.0forBiggerDatasets.Mol.Biol.Evol.2016;33:1870–4.

63. KurakuS,ZmasekCM,NishimuraO,KatohK.aLeavesfacilitateson-demand

explorationofmetazoangenefamilytreesonMAFFTsequencealignmentserverwith

enhancedinteractivity.NucleicAcidsRes.2013;41:W22-8.

64. RonquistF,TeslenkoM,vanderMarkP,AyresDL,DarlingA,HöhnaS,etal.MrBayes

3.2:efficientBayesianphylogeneticinferenceandmodelchoiceacrossalargemodel

space.Syst.Biol.OxfordUniversityPress;2012;61:539–42.

65. GuindonS,GascuelO.ASimple,Fast,andAccurateAlgorithmtoEstimateLarge

PhylogeniesbyMaximumLikelihood.RannalaB,editor.Syst.Biol.OxfordAcademic;

2003;52:696–704.

Figures

29

Figure1

Identificationofvirusesindifferentspeciesofplants.a,Theabundanceofplant-associated

virusesindifferentspeciesofplant.Thetopgraphshowsthetotalnumberofuniquereads

ineachlibrary.ThelibraryIDsareshownontopofeachbar,whilethehostOrdersare

shownabovethebargraph.ThebottomgraphshowsthenumberofvirushitspassedNR

filterinviralmetagenomicbioinformaticanalysis.Theredasteriskshowsthoselibraries

fromwhichwehaveacquiredcompleteornearlycompletegenomeofviruses.b,The

numberanddiversityofplant-associatedviruses.Thelefthistogramshowsthenumbersof

DNAviruses(bluebar)andRNAviruses(redbar).Therightpiechartsshowthevirus

classificationidentifiedinthisstudy.c,Theaminoacidsequenceidentityandcoverageof

plant-associatedviruseswiththebestmatchedvirusstrainsinBLASTxsearchingbasedon

the251completegenomesequenceacquiredinplantspecies.

30

Figure1

Identificationofvirusesindifferentspeciesofplants.a,Theabundanceofplant-associated

virusesindifferentspeciesofplant.Thetopgraphshowsthetotalnumberofuniquereads

ineachlibrary.ThelibraryIDsareshownontopofeachbar,whilethehostOrdersare

shownabovethebargraph.ThebottomgraphshowsthenumberofvirushitspassedNR

filterinviralmetagenomicbioinformaticanalysis.Theredasteriskshowsthoselibraries

fromwhichwehaveacquiredcompleteornearlycompletegenomeofviruses.b,The

numberanddiversityofplant-associatedviruses.Thelefthistogramshowsthenumbersof

DNAviruses(bluebar)andRNAviruses(redbar).Therightpiechartsshowthevirus

classificationidentifiedinthisstudy.c,Theaminoacidsequenceidentityandcoverageof

plant-associatedviruseswiththebestmatchedvirusstrainsinBLASTxsearchingbasedon

the251completegenomesequenceacquiredinplantspecies.

31

Figure2

Phylogeniesofviralgenomesidentifiedfromplants.TwelveBayesianinferencetreeswere

constructedusingMrBayesv3.2basedonvirusRdRpdomainofRNAvirusesorNSproteinof

parvovirus-likeviruses,withineachtree,thevirusesfoundinthisstudyaremarkedwithred

32

line.Hostsareindicatedwithdifferentsilhouetteofmammal,bird,arthropod,plantleaf,or

wavesstandingforvirusenvironmentalsource.Thenameofthevirusfamilyorgenusis

shownontherightsideofeachcluster.Eachscalebarindicates0.5aminoacid

substitutionspersite.

33

Figure2

Phylogeniesofviralgenomesidentifiedfromplants.TwelveBayesianinferencetreeswere

constructedusingMrBayesv3.2basedonvirusRdRpdomainofRNAvirusesorNSproteinof

parvovirus-likeviruses,withineachtree,thevirusesfoundinthisstudyaremarkedwithred

line.Hostsareindicatedwithdifferentsilhouetteofmammal,bird,arthropod,plantleaf,or

wavesstandingforvirusenvironmentalsource.Thenameofthevirusfamilyorgenusis

shownontherightsideofeachcluster.Eachscalebarindicates0.5aminoacid

substitutionspersite.

34

Figure3

Thephylogeniesofpotentiallynewviruses.SevenBayesianinferencetreeswere

constructedusingMrBayesv3.2basedonvirusRdRpdomains,withineachtree,theviruses

foundinthisstudyaremarkedwithredline.InthephylogenetictreeofPicornaviralesthe

bestmatchedvirusbasedBLASTpsearchingusingRdRpsequenceofeachnovelvirusare

labeledwithbluecolor.Thenameofthevirusfamilyorgenusisshownontherightsideof

eachcluster.Eachscalebarindicates0.2aminoacidsubstitutionspersite.

35

Figure3

Thephylogeniesofpotentiallynewviruses.SevenBayesianinferencetreeswere

constructedusingMrBayesv3.2basedonvirusRdRpdomains,withineachtree,theviruses

foundinthisstudyaremarkedwithredline.InthephylogenetictreeofPicornaviralesthe

bestmatchedvirusbasedBLASTpsearchingusingRdRpsequenceofeachnovelvirusare

labeledwithbluecolor.Thenameofthevirusfamilyorgenusisshownontherightsideof

eachcluster.Eachscalebarindicates0.2aminoacidsubstitutionspersite.

36

Figure4

ThephylogeniesofpotentiallynewCRESSDNAviruses.NineBayesianinferencetreeswere

establishedusingMrBayesv3.2basedonREPproteins,withineachtree,thevirusesfound

inthisstudyaremarkedwithredline.Hostsareindicatedwithdifferentsilhouetteof

mammal,arthropod,plantleaf,orwavesstandingforvirusenvironmentalsource.Thename

ofthevirusfamilyorgenusisshownontherightofeachcluster.Eachscalebarindicates

0.2aminoacidsubstitutionspersite.

37

Figure4

ThephylogeniesofpotentiallynewCRESSDNAviruses.NineBayesianinferencetreeswere

establishedusingMrBayesv3.2basedonREPproteins,withineachtree,thevirusesfound

inthisstudyaremarkedwithredline.Hostsareindicatedwithdifferentsilhouetteof

mammal,arthropod,plantleaf,orwavesstandingforvirusenvironmentalsource.Thename

ofthevirusfamilyorgenusisshownontherightofeachcluster.Eachscalebarindicates

0.2aminoacidsubstitutionspersite.

38

Figure5

Potentialcross-speciesinfectionofvirusesamongdifferentspeciesofplants.Neighbor-

39

joiningtreesbasedonnucleotidesequenceofcompletegenomesofvirusesbelongingin

thefamilyPotyviridaeorDicistroviridaeareshownhere.Virusstrainnamesarelabeledon

eachbranchfollowedbypercentageofviralreadsnumberagainsttotaluniquereads

numberincorrespondinglibrary.Virushostplantspeciesnameareshown,dottedlinesare

usedheretoindicatethesamplingsitesofdifferentspeciesofplant.Thecollectingplants

anditsgeographicallocationareconnectedbydottedlinesofdifferentcolors.Inthesame

phylogeneticcluster,thecolorofdottedlinesbehindvirushostsarethesame.Sequence

identitybasedoncompletegenomebetweenneighboringsequencesinthesameclusterare

labeledbetweenbranchesinphylogenetictree.Thephylogenyandclassificationofhost

speciesofdicistrovirusstrainsinthelargerclusterareshownbelowthedicistrovirustreeto

indicatedthelevelsoverwhichthecross-speciesinfectionoccurred.

40

Figure5

Potentialcross-speciesinfectionofvirusesamongdifferentspeciesofplants.Neighbor-

41

joiningtreesbasedonnucleotidesequenceofcompletegenomesofvirusesbelongingin

thefamilyPotyviridaeorDicistroviridaeareshownhere.Virusstrainnamesarelabeledon

eachbranchfollowedbypercentageofviralreadsnumberagainsttotaluniquereads

numberincorrespondinglibrary.Virushostplantspeciesnameareshown,dottedlinesare

usedheretoindicatethesamplingsitesofdifferentspeciesofplant.Thecollectingplants

anditsgeographicallocationareconnectedbydottedlinesofdifferentcolors.Inthesame

phylogeneticcluster,thecolorofdottedlinesbehindvirushostsarethesame.Sequence

identitybasedoncompletegenomebetweenneighboringsequencesinthesameclusterare

labeledbetweenbranchesinphylogenetictree.Thephylogenyandclassificationofhost

speciesofdicistrovirusstrainsinthelargerclusterareshownbelowthedicistrovirustreeto

indicatedthelevelsoverwhichthecross-speciesinfectionoccurred.

42

Figure6

Co-infectingvirusesinplants.Piechartsdescribetheviruseswithcompletegenomein

thoselibrariescontainingmorethan3differentviruses.Sectorareaineachpiechart

representstheproportionofthenumberofreadsmappedtothecompleteviralgenomein

thelibrary.ThreeindividualplantinthesameplantspeciesaremarkedwithS1,S2andS3,

respectively.Checkmarksbelowvirusstrainsnamesshowpositiveofvirusin(RT-)PCR

screening.

43

Figure6

Co-infectingvirusesinplants.Piechartsdescribetheviruseswithcompletegenomein

thoselibrariescontainingmorethan3differentviruses.Sectorareaineachpiechart

representstheproportionofthenumberofreadsmappedtothecompleteviralgenomein

thelibrary.ThreeindividualplantinthesameplantspeciesaremarkedwithS1,S2andS3,

respectively.Checkmarksbelowvirusstrainsnamesshowpositiveofvirusin(RT-)PCR

screening.

SupplementaryFilesThisisalistofsupplementaryfilesassociatedwiththispreprint.Clicktodownload.

44

SupplementaryTable1.xlsxSupplementaryTable2.xlsxSupplementaryinformation.pdfSupplementaryinformation.pdfSupplementaryTable1.xlsxSupplementaryTable2.xlsx

https://assets.researchsquare.com/files/rs-25620/v1/SupplementaryTable1.xlsxhttps://assets.researchsquare.com/files/rs-25620/v1/SupplementaryTable2.xlsxhttps://assets.researchsquare.com/files/rs-25620/v1/Supplementaryinformation.pdfhttps://assets.researchsquare.com/files/rs-25620/v1/Supplementaryinformation.pdfhttps://assets.researchsquare.com/files/rs-25620/v1/SupplementaryTable1.xlsxhttps://assets.researchsquare.com/files/rs-25620/v1/SupplementaryTable2.xlsx