A framework for the evaluation of biosecurity, commercial, regulatory, and scientific impacts of plant viruses and viroids identified by NGS technologies

PERSPECTIVE published: 24 Janua y 2017 doi: 10.3389/ micb.2017.00045 F on ie s in Mic obiology | www. on ie sin.o g 1Janua y 2017 | Volume 8 | A icle 45 Edi ed by: Da id Gilme , Uni e si y o S asbou g, F ance Re iewed by: D’Ann Rochon, Ag icul u e and Ag icul u e-Food Canada, Canada Ma ie-Line Isk a-Ca uana, CIRAD, F ance *Co espondence: Sebas ien Massa [email p o ec ed] Special y sec ion: This a icle was submi ed o Vi ology, a sec ion o he jou nal F on ie s in Mic obiology Recei ed: 10 No embe 2016 Accep ed: 06 Janua y 2017 Published: 24 Janua y 2017 Ci a ion: Massa S, Cand esse T, Gil J, Lacomme C, P edajna L, Ra nika M, Reyna d J-S, Rumbou A, Salda elli P, Ško i ´ c D, Vainio EJ, Valkonen JPT, Vande schu en H, Va e i C and We zel T (2017) A F amewo k o he E alua ion o Biosecu i y, Comme cial, Regula o y, and Scien i ic Impac s o Plan Vi uses and Vi oids Iden i ied by NGS Technologies. F on . Mic obiol. 8:45. doi: 10.3389/ micb.2017.00045 A F amewo k o he E alua ion o Biosecu i y, Comme cial, Regula o y, and Scien i ic Impac s o Plan Vi uses and Vi oids Iden i ied by NGS Technologies Sebas ien Massa 1*, Thie y Cand esse2, José Gil3, Ch is ophe Lacomme4, Lukas P edajna5, Maja Ra nika 6, Jean-Sébas ien Reyna d7, A emis Rumbou8, Pasquale Salda elli9, Dijana Ško i´ c10, Ee a J. Vainio11, Ja i P. T. Valkonen12, He é Vande schu en13, Ch is ina Va e i14 and Thie y We zel15 1Plan Pa hology Labo a o y, Gembloux Ag o-Bio Tech, Uni e si y o Liège, Gembloux, Belgium, 2Ins i u Na ional de la Reche che Ag onomique (INRA), Uni e si y o Bo deaux, CS20032 UMR 1332 BFP, Villena e d’O non, F ance, 3Plan Biology, Linnean Cen e o Plan Biology, Uppsala BioCen e, Swedish Uni e si y o Ag icul u al Sciences, Uppsala, Sweden, 4Vi ology and Zoology, Science and Ad ice o Sco ish Ag icul u e, Edinbou gh, UK, 5Depa men o Plan Vi ology, Ins i u e o Vi ology, Biomedical Resea ch Cen e , Slo ak Academy o Science (SAS), B a isla a, Slo akia, 6Depa men o Bio echnology and Sys ems Biology, Na ional Ins i u e o Biology, Ljubljana, Slo enia, 7Vi ology, Ag oscope, Nyon, Swi ze land, 8Di ision Phy omedicine Len zeallee, Facul y o Li e Sciences, Alb ech Daniel Thae -Ins i u e o Ag icul u al and Ho icul u al Sciences, Humbold -Uni e si y o Be lin, Be lin, Ge many, 9Na ional Resea ch Council Ins i u e o Sus ainable Plan P o ec ion, Ba i, I aly, 10 Depa men o Biology, Facul y o Science, Uni e si y o Zag eb, Zag eb, C oa ia, 11 Managemen and P oduc ion o Renewable Resou ces, Na u al Resou ces Ins i u e Finland (Luke), Helsinki, Finland, 12 Depa men o Ag icul u al Sciences, Uni e si y o Helsinki, Helsinki, Finland, 13 Plan Gene ics, Gembloux Ag o-Bio Tech, Uni e si y o Liège, Gembloux, Belgium, 14 Depa men o Phy opa hology, Benaki Phy opa hological Ins i u e, A hens, G eece, 15 DLR Rheinp alz, Ins i u e o Plan P o ec ion, Neus ad an de Weins asse, Ge many Recen ad ances in high- h oughpu sequencing echnologies and bioin o ma ics ha e gene a ed huge new oppo uni ies o disco e ing and diagnosing plan i uses and i oids. Plan i ology has undoub edly bene i ed om hese new me hodologies, bu a he same ime, aces now subs an ial bo lenecks, namely he biological cha ac e iza ion o he newly disco e ed i uses and he analysis o hei impac a he biosecu i y, comme cial, egula o y, and scien i ic le els. This pape p oposes a scaled and p og essi e scien i ic amewo k o e icien biological cha ac e iza ion and isk assessmen when a p e iously known o a new plan i us is de ec ed by nex gene a ion sequencing (NGS) echnologies. Fou case s udies a e also p esen ed o illus a e he need o such a amewo k, and o discuss he scena ios. Keywo ds: NGS, pes isk analysis, i us diseases, biological cha ac e iza ion, plan heal h, egula o y agencies INTRODUCTION Un il ecen ly, plan i us disco e y appea ed as a long and as idious ask, mainly d i en by he need o iden i y he e iology o diseases o unknown o igin. In he las ew yea s, howe e , he ad en o nex gene a ion sequencing (NGS) has e olu ionized he s udy o plan i uses by p o iding a powe ul al e na i e o hei de ec ion and iden i ica ion, wi hou any a p io i knowledge. NGS echnologies a e p og essi ely eaching he diagnos ic ield (Massa e al., 2014), impac ing also qua an ine egula ions (Ma in e al., 2016). Thei use allows he con inuous disco e y o new plan Massa e al. P oposal: F amewo k o Cha ac e isa ion o New Vi uses i uses, he obse a ion o an inc easing di e si y o a ian s o known i uses and he equen exis ence o a complex o di e en i uses. Downs eam o hese indings, he main challenge o be add essed is he biological signi icance (pa hogenici y, hos s, ansmission, epidemiology...) o he disco e y o a no el i us species o s ain in single plan s, pa icula ly in asymp oma ic plan s and o i uses ha a e he ounding membe s o new i us gene a o amilies and sha e li le o no sequence simila i y wi h known i uses (Wu e al., 2015). Plan i ologis s a e now challenged o do he wo k backwa ds, namely o cha ac e ize new i uses, wi h genome sequence in o ma ion as he unique s a ing poin . Fu he mo e, he abili y o NGS o de ec c yp ic (symp omless) i uses in cul i a ed and wild plan s, and he epo ed mu ualis ic in e ac ions challenging he adi ional dogma ha all i uses a e pa hogens (Roossinck, 2011), a e adding new di icul ies o he p edic ion o he impac o new i uses. Undoub edly, hese new scien i ic ou comes will ha e o be handled wi h ex eme ca e in e ms o dissemina ion om basic esea ch o applied ag onomy, and o plan heal h, ag icul u e and o es y policy make s and egula o s. Indeed, he amoun o in o ma ion needed o assess he isk posed by a new i us species o a ce ain commodi y o egion is huge. Scien is s may indeed ha e o wo k o yea s o p o ide he answe s needed o conduc a ho ough Pes Risk Analysis (PRA) acco ding o in e na ional phy osani a y s anda ds (ISPM 2 and ISPM 11) (FAO, 2004, 2007). The e o e, he main challenge o scien is s a ising om he disco e y o a new i al sequence is o e icien ly cha ac e ize he biological p ope ies h ough e icien sho , mid-, and long- e m s a egies while c ea ing app op ia e communica ion channels wi h he egula o y au ho i ies. In his pape , we aim o discuss he new challenges aised by NGS as illus a ed by case s udies, and o sugges guidelines o esea che s, policy make s, plan heal h au ho i ies and plan inspec ion se ices by desc ibing he necessa y s eps, he app op ia e in e ac ions and he inhe en p io i iza ion o be ollowed a e disco e ing a new i us sequence [o a new isola e o a ian , o (a) new hos (s)]. O e all, he ad en o new NGS echnologies wisely complemen ed by adi ional o classical i us s udy me hods is expec ed o globally p omo e plan heal h and plan p o ec ion, bu may also cause mayhem in ade and ag icul u e i challenges and ques ions a e no add essed p ope ly. CURRENT SITUATION WITH KNOWN VIRUSES Qua an ine and ce i ica ion lis s a e egula ly upda ed ollowing new PRAs. They a e based on 6 ca ego ies o in o ma ion: (i) knowledge o he iden i y o he pes (and he e o e being able o di e en ia e i om o he i al agen s); (ii) da a on i s dis ibu ion and (iii) hos ange; (i ) in o ma ion on he modes and e iciency o sp ead and on he iden i y o any ec o (s); ( ) sui abili y o he local ag o-en i onmen al condi ions o he pes [and ec o (s)]; and ( i) he abili y o cause a disease and impac he de elopmen , ep oduc ion, o p oduc i i y o cul i a ed o wild hos plan s. Fu he , e inemen o he isk assessmen can be based on addi ional in o ma ion such as he a ailabili y o e icien and easy- o-implemen con ol me hods. The PRA p o ides he isk assesso wi h a isk assessmen ool whose de ails will depend bo h on da a a ailabili y conce ning he six poin s lis ed abo e and on he needs o he isk manage , whose ole is o conside he a ailable scien i ic in o ma ion ( isk assessmen ) as well as o he ac o s (economical, accep able poli ical isk, easibili y, and impac o measu es) in eaching a decision on whe he o egula e o add ess he pes in any speci ic way. EMERGING CHALLENGES FOR SCIENTISTS AND AUTHORITIES When a known i us is de ec ed by NGS echnologies, he main ques ions a e ela ed o i s legal s a us (see Figu e 1). The decision scheme o be applied is based on exis ing qua an ine o ce i ica ion egula ions and is simila o he p ocess ou inely ca ied ou wi h PCR o ELISA-based me hodologies. Ne e heless, NGS may also e eal addi ional complexi y and e en eo ien disease in es iga ion o known species (see he S udy Case N◦1 in Supplemen a y Ma e ial). I should be s essed ha isk assessmen is some imes needed o eques ed by Na ional Au ho i ies in si ua ions whe e ha dly any in o ma ion is a ailable, a seen in ecen examples in 2016 o Peppe ein yellow i us (PeVYV) o G ape ine pino g is i us (Ge many). In such a case, isk assesso s canno wai un il enough da a is a ailable and i s conclusions ha e o be eached based on wha e e in o ma ion is a hand. This mus be done cau iously, while simul aneously aking in o accoun he unce ain ies associa ed o o caused by he lack o da a and making hem clea o he isk manage . In his con ex , he inc easing iden i ica ion o new i al sequences by NGS echnologies may complica e he decision- making p ocess o ce i ica ion p og ams, qua an ine p ocesses, and mo e gene ally he ading o plan ma e ials (see he S udy Case N◦2 in Supplemen a y Ma e ial). The only in o ma ion a ailable may be he ull o pa ial genome sequence wi h he addi ional complexi y ha i may ha e been de ec ed in asymp oma ic samples o in combina ion wi h o he i uses. I is he e o e inc easingly impo an o unde s and he biology o any new i al sequence o p o ide a basis o assessing he isk hey pose and ake scien i ically-based decisions. Scien is s a e he e o e now challenged o p o ide biological da a on hese newly desc ibed i al sequences, in a sho ime ame and wi h limi ed unding. The ollowing chap e s p opose a amewo k o scaled ac ions o e icien ly add ess he need o PRA o egula o y au ho i ies. EARLY STEPS OF BIOLOGICAL CHARACTERIZATION The de ec ion o a new i al sequence should di ec ly be ollowed by se e al immedia e ac ions o i s ea ly biological cha ac e iza ion o each a i s decision le el o no i ica ion o au ho i ies (see Figu e 2). F on ie s in Mic obiology | www. on ie sin.o g 2Janua y 2017 | Volume 8 | A icle 45 Massa e al. P oposal: F amewo k o Cha ac e isa ion o New Vi uses FIGURE 1 | F amewo k o known i uses. Y means posi i e esponse and N nega i e esponse (do ed lines co espond o op ional analyses o non qua an ine pa hogens depending on he eques o he cus ome ). Con i ma ion o De ec ion To dispel a labo a o y o bioin o ma ics e o o con amina ion, NGS-based iden i ica ion o pu a i e new i al sequence o new s ains o exis ing i al species equi es u he con i ma ion by complemen a y me hods, usually by (RT)-PCR using speci ic p ime s. I he concen a ion is e y low, he sample can be en iched in i al pa icles h ough homogeniza ion, il a ion, and/o ul acen i uga ion p io o nucleic acid ex ac ion. En iched i al pa icles can also be obse ed by elec on mic oscopy, e en i elec on mic oscopy lacks sensi i i y and i al pa icles can be missed. Fo DNA i uses, especially Caulimo i idae, addi ional esea ch is also needed o e alua e whe he he de ec ed i al sequences exis in an episomal o m and no only as he ace o a “ ossilized” i us in eg a ed in he plan genome (Liu e al., 2010; Aiewsakun and Ka zou akis, 2015). P o isional Taxonomy Assignmen The assembled con ig will gene ally allow o p o isional axonomic assignmen o he i al sequence. Ne e heless, his can u n ou o be a e y di icul ask, made e en mo e complex by (i) he equen incomple eness o he genome sequence da a ob ained, (ii) he a iable and i egula co e age o i al genomes, (iii) he incomple eness o i al axonomy, and (i ) he a iabili y o species, genus, o amily molecula disc imina ion c i e ia. Speci ic guidelines can be ound in he la es ICTV epo (h p://www.ic online.o g). The de ec ed i al sequences may also co espond o a bona ide i us in ec ing o he o ganisms associa ed wi h he sample, including bac e ia, ungi o a h opods (Al Rwahnih e al., 2011; Ma zano and Domie , 2015) o o i al sequences in eg a ed in o he plan genome. The u u e solu ion o his p oblem is o es ablish he baseline o i us p esence h ough an imp o ed knowledge o he i omes o ag onomically impo an plan s as well as i omes o na u al ecosys ems, including he in eg a ed i al sequences. Bibliog aphy O e all, p o isional axonomic assignmen should always be conside ed wi h cau ion bu can gi e i s clues on he physical and/o chemical p ope ies o a i us as well as on i s biology, including (i) he pu a i e hos ange o he en a i e iden i ica ion o al e na i e hos s, (ii) he p edic ion o he pa hogenic po en ial in hose hos s, (iii) he modes o ho izon al and/o e ical ansmission, including he iden i ica ion o candida e ec o s. These hypo heses ha e p ac ical consequences such as he selec ion o app op ia e hos s and expe imen al modes o inocula ion, a i s assessmen o he possible epidemic po en ial o a new i al agen , o a en a i e iden i ica ion o possible al e na i e hos s in ecosys ems. Al oge he , his ea ly s ep, which equi es expe ise in molecula biology, in plan i ology, and basic bioin o ma ics skills, migh lead o he elabo a ion o a d a epidemiological scena io (Loconsole e al., 2012; Adams e al., 2014). Ne e heless, his app oach can be biased as o example in he genus Clos e o i idae, which includes economically impo an i uses wi h simila i ion mo phology, genome size, and o ganiza ion bu wi h con as ed biological p ope ies (Ma elli e al., 2012). Sample Documen a ion In addi ion, all he me ada a abou he sample should be collec ed p ecisely o eed he isk assessmen p ocess. I is impo an o documen symp oms (i any) and ime o appea ance, o iden i y he plan species and cul i a , o desc ibe he sampled plan F on ie s in Mic obiology | www. on ie sin.o g 3Janua y 2017 | Volume 8 | A icle 45 Massa e al. P oposal: F amewo k o Cha ac e isa ion o New Vi uses FIGURE 2 | F amewo k p oposal ollowing disco e y o new i uses. Y means posi i e esponse and N nega i e esponse. The numbe s be ween b acke s co espond o he chap e s in he ex . *The absence o i al pa icle should be eco ded and u he expe imen s ca ied ou i new cases a e disco e ed. pa (lea es, ui , seeds, e c.), he geog aphical o igin o he sample, i s in ended use e c... Any missing in o ma ion should be comple ed a his s age so as o be included in he i s no i ica ion. Full Genome Sequencing and Anno a ion The assembly o i al sequence(s) based on NGS eads can esul in h ee scena ios o genome co e age: (i) comple e, (ii) incomple e bu con inuous, o (iii) a se o sca olds and con igs. The econs uc ion o he ull genome sequence o a candida e new i us is mainly based on a ge ed (RT)-PCR ampli ica ion and sequencing o ill he sequence gaps be ween de no o gene a ed con igs, e i y hei exclusi e i al o igin and he absence o hos -de i ed sequences. Taxonomic assignmen can also be ine- uned hanks o he whole genome sequence; bu in a wo s case si ua ion, e en he a ailabili y o a comple e genomic sequence may be insu icien o se le such a ques ion (Ma ais e al., 2016). F on ie s in Mic obiology | www. on ie sin.o g 4Janua y 2017 | Volume 8 | A icle 45 Massa e al. P oposal: F amewo k o Cha ac e isa ion o New Vi uses De elopmen o a Diagnos ic P o ocol Iden i ying a new i us species, e en om a pa ial genome sequence, igge s he immedia e need o de elop a diagnos ic p o ocol. This is a undamen al s ep owa d he managemen o i al diseases in cul i a ed c ops o he un eiling o a new i us eco-epidemiological ole in wild plan s. The diagnos ic o newly iden i ied and con i med i uses is usually done using PCR-based me hods ( he mos popula ones o new i uses) o wi h he LAMP echnology, as a ecen al e na i e (Boonham e al., 2014). Nucleic acid-based me hods a e cu en ly p e e ed o diagnose new i uses because hey a e quicke , easie and cheape o de elop han an ibody p oduc ion o ELISA. Field/Ba ch Obse a ion Small-scale epidemiological su eys a he disco e y loca ion can be unde aken. Such su eys will also ake in o accoun he hypo heses ha may ha e a isen a e bibliog aphical esea ch and should be ocused on he plan species whe e he i us was de ec ed; bu hey could also ake in o accoun , whene e possible, he po en ial hos (s) and ec o (s), hei geog aphical sp ead and seasonali y. Likewise, sampling me hods enabling o he s a is ical linking o indings and ield obse a ions should be selec ed. Se e al scena ios a e possible depending on he o igin o he sample: ield o ade/qua an ine/ce i ica ion. The p esence o symp oms in he ini ial ield hos has o en been pe cei ed as a s a ing poin o u he in es iga ions abou he p esence o a i us, bu asymp oma ic plan s mus also be included in he sampling because symp oma ology e lec s he complex in e play o in ec ious agen (s), he hos plan me abolism, i s de ense sys ems and a ie al speci ici ies, as well as abio ic ac o s. The ad en o wide me agenomic s udies o en i onmen al i omes (K is ensen e al., 2010; Mohiuddin and Schellho n, 2015) allows o he iden i ica ion o new i uses om any sample, including asymp oma ic ield samples. Thus, e en i ocused symp om obse a ions in he ield canno be neglec ed, a su ey canno be led by symp oma ology alone. Hence, i us sp ead o o he ag o-ecological niches whe e hey may encoun e new hos s, co-in ec ing i uses o ec o s, may cause a shi in hei pa hogenic po en ial and esul in a se ious disease. IN-DEPTH BIOLOGICAL CHARACTERIZATION The in-dep h biological cha ac e iza ion o a new i us is en isioned as a mid- o long- e m goal in o de o g adually educe he unce ain ies associa ed wi h he ea ly isk assessmen and decision-making p ocesses. Mo e speci ically, in- dep h biological cha ac e iza ion aims o build comp ehensi e knowledge on he symp oma ology/e iology, epidemiology and ecology o a new i us by ga he ing in o ma ion on i s hos ange, i s symp oma ology on a ious cul i a s and hos species, i s ec o s and modes o ansmission, i s geog aphical dis ibu ion, and i s in e ac ions wi h o he i uses. Ful illing Koch’s pos ula es can be conside ed as a co ne s one and is pi o al in his in- dep h biological cha ac e iza ion. An addi ional di icul y a ises in he case o a i us iden i ied om asymp oma ic issue o i uses wi h po en ially neu al o bene icial in e ac ions wi h he plan s (Xu e al., 2008; an Molken e al., 2012). In hese cases, Koch’s pos ula es may p o e useless, because hey p esume he de elopmen o disease symp oms ollowing in oduc ion o he pa hogen in o a heal hy hos . Bu labo a o y and g eenhouse expe imen s as well as ield su eys will b ing mo e in o ma ion and migh b ing he p oo o mu ualis ic o syne gis ic in e ac ions be ween he i us and he plan . These da a will be necessa y o isk assessmen o make u he p og ess by de e mining whe he he i us is p esen (locally o widesp ead) o no in a gi en geog aphical a ea, i s po en ial o sp ead ( ype o ansmission, hos ange), and ul ima ely o e alua e i s economic impac (symp oms alone o in associa ion). In u n, local au ho i ies may de ine app op ia e egula o y measu es in ela ion o Plan Heal h p o ec ion and con ol i equi ed. The S udy Case N◦3 on G ape ine Pino G is i us (see Supplemen a y Ma e ial) exempli ies his complex app oach. T ansmission Expe imen s The inocula ion o a new i us o es plan s in labo a o y/g eenhouse condi ions is he basis o any biological cha ac e iza ion and may be pa icula ly complex. Depending on he objec i e, he i us can be ansmi ed o indica o plan s, hos plan candida es o o he cul i a s o a same species. The modes o e ical and ho izon al ansmission o i uses a e di e se, so ha he in eg a ion o axonomic (Figu e 2 and Sec ion P o isional Taxonomy Assignmen ), bibliog aphical (Figu e 2 and Sec ion Bibliog aphy), and ield su ey da a (Figu e 2 and Sec ion Field Su eys) is o u mos impo ance o he p ope design o expe imen al ansmission p ocedu es in he labo a o y o he g eenhouse. Plan s can be inocula ed by se e al echniques, he mos common ones being mechanical inocula ion, g a ing, ec o inocula ion o he use o dodde , bu all hese echniques ha e po en ial limi a ions as hey may no sepa a e i uses in case o a mix u e o i uses. A e y in e es ing and uni e sal echnique o an in-dep h cha ac e iza ion o a i us is he p epa a ion o in ec ious clones. The in ec ious clones, equi ing he comple e i al genome sequence, o e aluable in o ma ion abou (i) symp oma ology o indi idual i uses and o mix u e o i uses, (ii) hos ange s udies and ansmissibili y, (iii) he assessmen o na u al o induced mu a ion/ ecombina ion a es in hos plan s, and (i ) he e ec s o a ge ed modi ica ions on i us-hos in e ac ions and symp oma ology. Mas e ing i us ansmission will also be key o he de elopmen o esea ch e o s o unde s and he po en ially posi i e e ec s o i uses on plan s. Inocula ed plan s om a ious species o cul i a s can be submi ed o bio ic o abio ic s esses, and hei eac ion can be s udied in labo a o y and/o in g eenhouse expe imen s o p o ide e idence o bene icial in e ac ions. Field Su eys La ge-scale su eys, o ganized on a na ional o in e na ional scale and based on he diagnos ic echnique(s) de eloped o he new agen , should be unde aken o e alua e i s p e alence and dis ibu ion wi hin a gi en ecosys em (K enz e al., 2014). These s udies will aim o moni o he candida e/demons a ed hos plan s/ ec o s. They should be ca ied ou in ag icul u al F on ie s in Mic obiology | www. on ie sin.o g 5Janua y 2017 | Volume 8 | A icle 45 Massa e al. P oposal: F amewo k o Cha ac e isa ion o New Vi uses ecosys ems ( ields, comme cial o cha ds o ineya ds, nu se ies, and ge mplasm collec ions), and in na u al ecosys ems, o enable a holis ic cha ac e iza ion o he pa hosys ems wi h pa icula a en ion o symp oma ology. They will complemen he p elimina y in o ma ion gained a a smalle scale (See Sec ion Field/Ba ch Obse a ion) o in he labo a o ies and g eenhouses (See Sec ion T ansmission Expe imen s). The in e play be ween ield su eys and labo a o y expe imen s is pa icula ly impo an a his s age because i will allow a ge ing he mos app op ia e hos plan (s) and ec o (s), and hus inc eases knowledge on i us biology. La ge-scale su eys will also p o ide insigh s in o he gene ic a iabili y o a new i us wi hin he su eyed e i o ies. This knowledge will be key o e o s o op imize diagnos ic echniques by selec ing p ime s co e ing he gene ic di e si y o he species (Massa e al., 2014). In addi ion, he iden i ica ion o new a ian s may b ing new undamen al hypo heses on he pa hogenici y o he i uses, and hese new a ian s could be cha ac e ized unde labo a o y and g eenhouse condi ions (See Sec ion T ansmission Expe imen s). The Speci ic Challenge o Vi us Complexes The biological cha ac e iza ion o new i uses may be pa icula ly challenging wi h complex o di e en i uses, whe e he combina ion o wo o mo e i uses can signi ican ly modi y hei pa hogenic po en ial h ough syne gis ic o an agonis ic in e ac ions (Ma in and Elena, 2009; Sylle and G upa, 2016). Mix u e o i us species is indeed e y equen , especially in woody plan s like g ape ine (Joos e e al., 2015), o en leading o unp edic able a ia ions in symp oms, in ec i i y, accumula ion, and/o ec o ansmissibili y. Biological and echnological solu ions o isola ing and de e mining he ole o indi idual i uses a e some imes possible using selec i e expe imen al hos s ollowed by back-inocula ion o “pu i ied” isola es o he o iginal hos (Ali and Roossinck, 2008), pa ial sani a ion (Maliogka e al., 2015), in ec ious clones (Nagyo á and Šub , 2007), o he use o ion-exchange ch oma og aphy on monoli hic suppo s o sepa a e i al pa icles (Rušˇ ci´ c e al., 2015). The biological p ope ies o i uses alone o in mix u e can he e o e be compa ed in dep h (see Sec ion Field Su eys). The complexi y o disease e iology in ield condi ions migh pose addi ional challenges, as exempli ied in he Case S udy N◦4 on ca o (See Supplemen a y Ma e ial). CONCLUSIONS NGS echnologies pose new challenges o scien is s and o plan heal h au ho i ies when i comes o analyzing he isks associa ed wi h a new i us o pa hogen, i s po en ial o sp ead, o i s economic impac and when ying o ake a imely decision on whe he o le go o des oy con amina ed plan ma e ials. As sequencing h oughpu and bioin o ma ics analyses a e less and less limi ing, downs eam epidemiology and disease e iology s udies will be he ob ious bo lenecks in abili y o conclude on he biological signi icance and impac o no el i uses o o complex o di e en i uses. While exhaus i e knowledge on he e iology o a disease o he epidemiology o a i us is e y di icul i no impossible o achie e, we p opose a amewo k o expe imen s and in es iga ions o cha ac e ize hese newly disco e ed i uses and o unde ake ele an ac ions in a imely ashion in ela ion o he con ex o he inding. In he ame o he isks posed by g owing ade and clima e change, he aim is o p og essi ely educe he unce ain ies linked wi h isk managemen and o help plan heal h p o ec ion au ho i ies decide on he impo ance o a new i us in a qua an ined ma e ial o a ce i ied seed s ock. Howe e , his decision will also always be in luenced by local legisla ion and he socio-economic and poli ical in e p e a ion o he p og essi ely gene a ed in o ma ion. Once adop ed by au ho i ies and ade, his amewo k should help o ake a ionalized decisions on he mos ele an ac ions o ake (i.e., con i ma ion using speci ic me hods, ways o assess impac o a i us in i s en i onmen ) and po en ially p e en “con lic s” be ween impo /expo pa ne s. AUTHOR CONTRIBUTIONS All he au ho s signi ican ly con ibu ed o he w i ing and edi ing o he manusc ip . SM c ea ed he igu es. SM and TW coo dina ed he w i ing and edi ing o he manusc ip . FUNDING This a icle is based upon wo k om COST Ac ion FA1407 (DIVAS), suppo ed by COST (Eu opean Coope a ion in Science and Technology). ACKNOWLEDGMENTS We hank F ancoise Pe e and Baldisse a Gio anni (EPPO) o hei inpu and c i ical eading o he manusc ip . SUPPLEMENTARY MATERIAL The Supplemen a y Ma e ial o his a icle can be ound online a : h p://jou nal. on ie sin.o g/a icle/10.3389/ micb. 2017.00045/ ull#supplemen a y-ma e ial REFERENCES Adams, I. P., Skel on, A., Maca hu , R., Hodges, T., Hinds, H., Flin , L., e al. (2014). Ca o yellow lea i us is associa ed wi h ca o in e nal nec osis. PLoS ONE 9:e109125. doi: 10.1371/jou nal.pone.0109125 Aiewsakun, P., and Ka zou akis, A. (2015). Endogenous i uses: connec ing ecen and ancien i al e olu ion. Vi ology 479–480, 26–37. doi: 10.1016/j. i ol.2015.02.011 Ali, A., and Roossinck, M. J. (2008). A simple echnique o sepa a ion o Cowpea chlo o ic mo le i us om Cucumbe mosaic i us in na u al mixed in ec ions. J. Vi ol. Me hods 153, 163–167. doi: 10.1016/j.j i ome .2008. 07.023 Al Rwahnih, M., Daube , S., U bez-To es, J. R., Co de o, F., Rowhani, A., Rwahnih, M. A., e al. (2011). Deep sequencing e idence om single g ape ine plan s e eals a i ome domina ed by myco i uses. A ch. Vi ol. 156, 397–403. doi: 10.1007/s00705-010-0869-8 F on ie s in Mic obiology | www. on ie sin.o g 6Janua y 2017 | Volume 8 | A icle 45 Massa e al. P oposal: F amewo k o Cha ac e isa ion o New Vi uses Bag, S., Al Rwahnih, M., Li, A., Gonzalez, A., Rowhani, A., Uyemo o, J. K., e al. (2015). De ec ion o a new lu eo i us in impo ed nec a ine ees: a case s udy o p opose adop ion o me agenomics in pos -en y qua an ine. Phy opa hology 105, 840–846. doi: 10.1094/PHYTO-09-14-0262-R Boonham, N., K euze, J., Win e , S., an de Vlug , R., Be ge oe , J., Tomlinson, J., e al. (2014). Me hods in i us diagnos ics: om ELISA o nex gene a ion sequencing. Vi us Res. 186, 20–31. doi: 10.1016/j. i us es.2013.12.007 FAO (ed.). (2004). “ISPM No. 11. Pes isk analysis o qua an ine pes s including analysis o en i onmen al isks and li ing modi ied o ganisms,” in In e na ional S anda ds o Phy osani a y Measu es 1 o 29,2007 Edn. (Rome: Food and Ag icul u e O ganiza ion o he Uni ed Na ions), 135–160. FAO (ed.). (2007). “ISPM No. 2. F amewo k o pes isk analysis,” in In e na ional S anda ds o Phy osani a y Measu es 1 o 29,2007 Edn. (Rome: Food and Ag icul u e O ganiza ion o he Uni ed Na ions), 27–41. Giampe uzzi, A., Roumi, V., Robe o, R., Malossini, U., Yoshikawa, N., La No e, P., e al. (2012). A new g ape ine i us disco e ed by deep sequencing o i us- and i oid-de i ed small RNAs in C Pino g is. Vi us Res. 163, 262–268. doi: 10.1016/j. i us es.2011.10.010 Jalkanen, R., Bü ne , C., and VonBa gen, S. (2007). Che y lea oll i us, CLRV, abundan on Be ula pubescens in Finland. Sil a Fenn. 41, 755–762. A ailable online a : www.me la. i/sil a ennica Joos e, A. E. C., Molenaa , N., Ma ee, H. J., Bes e , R., Mo ey, L., de Koke , W. C., e al. (2015). Iden i ica ion and dis ibu ion o mul iple i us in ec ions in G ape ine lea oll diseased ineya ds. Eu . J. Plan Pa hol. 142, 363–375. doi: 10.1007/s10658-015-0620-0 K enz, B., Thompson, J. R., McLane, H. L., Fuchs, M., and Pe y, K. L. (2014). G ape ine ed blo ch-associa ed i us is Widesp ead in he Uni ed S a es. Phy opa hology 104, 1232–1240. doi: 10.1094/PHYTO-02-14- 0053-R K is ensen, D. M., Mushegian, A. R., Dolja, V. V., and Koonin, E. V. (2010). New dimensions o he i us wo ld disco e ed h ough me agenomics. T ends Mic obiol. 18, 11–19. doi: 10.1016/j. im.2009.11.003 Liu, H., Fu, Y., Jiang, D., Li, G., Xie, J., Cheng, J., e al. (2010). Widesp ead ho izon al gene ans e om double-s anded RNA i uses o euka yo ic nuclea genomes. J. Vi ol. 84, 11876–11887. doi: 10.1128/JVI.00955-10 Loconsole, G., Önelge, N., Po e e, O., Giampe uzzi, A., Bozan, O., Sa a , S., e al. (2012). Iden i ica ion and cha ac e iza ion o Ci us Yellow Vein Clea ing Vi us, a pu a i e new membe o he genus Manda i i us. Phy opa hology 102, 1168–1175. doi: 10.1094/PHYTO-06-12-0140-R Maliogka, V. I., Ma elli, G. P., Fuchs, M., and Ka is, N. I. (2015). Con ol o i uses in ec ing g ape ine. Ad . Vi us Res. 91, 175–227. doi: 10.1016/bs.ai i .2014.11.002 Ma ais, A., Fau e, C., and Cand esse, T. (2016). New insigh s in o Asian p unus i uses in he ligh o NGS-based ull genome sequencing. PLoS ONE 11:e0146420. doi: 10.1371/jou nal.pone.0146420 Ma elli, G. P., Abou Ghanem-Sabanadzo ic, N., Ag ano sky, A. A., Al Rwahnih, M., Dolja, V. V., Do as, C. I., e al., (2012). Taxonomic e ision o he amily Clos e o i idae wi h special e e ence o he G ape ine lea oll-associa ed membe s o he Genus Ampelo i us and he pu a i e species unassigned o he amily. J. Plan Pa hol. 94, 7–19. doi: 10.4454/jpp. a.2012.022 Ma in, R. R., Cons able, F., and Tzane akis, I. E. (2016). Qua an ine egula ions and he impac o mode n de ec ion me hods. Annu. Re . Phy opa hol. 54, 189–205. doi: 10.1146/annu e -phy o-080615-100105 Ma in, S., and Elena, S. F. (2009). Applica ion o game heo y o he in e ac ion be ween plan i uses du ing mixed in ec ions. J. Gen. Vi ol. 90, 2815–2820. doi: 10.1099/ i .0.012351-0 Ma zano, S.-Y. L., and Domie , L. L. (2015). No el myco i uses disco e ed om me a ansc ip omics su ey o soybean phyllosphe e phy obiomes. Vi us Res. 213, 332–342. doi: 10.1016/j. i us es.2015.11.002 Massa , S., Olmos, A., Jijakli, H., and Cand esse, T. (2014). Cu en impac and u u e di ec ions o high h oughpu sequencing in plan i us diagnos ics. Vi us Res. Else ie 188, 90–96. doi: 10.1016/j. i us es.2014.03.029 Mohiuddin, M., and Schellho n, H. E. (2015). Spa ial and empo al dynamics o i us occu ence in wo eshwa e lakes cap u ed h ough me agenomic analysis. F on . Mic obiol. 6:960. doi: 10.3389/ micb.2015.00960 Nagyo á, A., and Šub , Z. (2007). In ec ious ull-leng h clones o plan i uses and hei use o cons uc ion o i al ec o s. Ac a Vi ol. 51, 223–237. Roossinck, M. J. (2011). The good i uses: i al mu ualis ic symbioses. Na . Re . Mic obiol. 9, 99–108. doi: 10.1038/n mic o2491 Rumbou, A., on Ba gen, S., Demi al, R., Lange , J., Ro , M., Jalkanen, R., e al. (2016). High gene ic di e si y a he in e -/in a hos le el o Che y lea oll i us popula ion associa ed wi h he “bi ch lea - oll disease” in Fennoscandia. Scand. J. Fo . Res. 31, 546–560. doi: 10.1080/02827581.2016.1165283 Rušˇ ci´ c, J., Gu ié ez-Agui e, I., Tušek Žnida iˇ c, M., Kolundžija, S., Slana, A., Ba u , M., e al. (2015). A new applica ion o monoli hic suppo s: he sepa a ion o i uses om one ano he . J. Ch oma og . A. 1388, 69–78. doi: 10.1016/j.ch oma.2015.01.097 Salda elli, P., Giampe uzzi, A., Mo elli, M., Malossini, U., Pi olo, C., Bianchedi, P., e al. (2015). Gene ic a iabili y o G ape ine Pino g is i us and i s associa ion wi h G ape ine lea mo ling and de o ma ion. Am. Phy opa hol. Soc. 105, 555–563. doi: 10.1094/PHYTO-09-14-0241-R Sylle , J., and G upa, A. (2016). An agonis ic wi hin-hos in e ac ions be ween plan i uses: molecula basis and impac on i al and hos i ness. Mol. Plan Pa hol. 17, 769–782. doi: 10.1111/mpp.12322 an Molken, T., de Caluwe, H., Ho dijk, C. A., Leon-Reyes, A., Snoe en, T. A. L., an Dam, N. M., e al. (2012). Vi us in ec ion dec eases he a ac i eness o whi e clo e plan s o a non- ec o ing he bi o e. Oecologia 170, 433–444. doi: 10.1007/s00442-012-2322-z Villamo , D. E., Meku ia, T. A., Pillai, S. S., and Eas well, K. C. (2016). High- h oughpu sequencing iden i ies no el i uses in nec a ine: insigh s o he e iology o s em-pi ing disease. Phy opa hology 106, 519–527. doi: 10.1094/PHYTO-07-15-0168-R Wu, Q., Ding, S. W., Zhang, Y., and Zhu, S. (2015). Iden i ica ion o i uses and i oids by nex -gene a ion sequencing and homology-dependen and homology-independen algo i hms. Annu. Re . Phy opa hol. 53, 425–444. doi: 10.1146/annu e -phy o-080614-120030 Xu, P., Chen, F., Mannas, J. P., Feldman, T., Sumne , L. W., and Roossinck, M. J. (2008). Vi us in ec ion imp o es d ough ole ance. New Phy ol. 180, 911–921. doi: 10.1111/j.1469-8137.2008.02627.x Con lic o In e es S a emen : The au ho s decla e ha he esea ch was conduc ed in he absence o any comme cial o inancial ela ionships ha could be cons ued as a po en ial con lic o in e es . Copy igh © 2017 Massa , Cand esse, Gil, Lacomme, P edajna, Ra nika , Reyna d, Rumbou, Salda elli, Ško i´ c, Vainio, Valkonen, Vande schu en, Va e i and We zel. This is an open-access a icle dis ibu ed unde he e ms o he C ea i e Commons A ibu ion License (CC BY). The use, dis ibu ion o ep oduc ion in o he o ums is pe mi ed, p o ided he o iginal au ho (s) o licenso a e c edi ed and ha he o iginal publica ion in his jou nal is ci ed, in acco dance wi h accep ed academic p ac ice. No use, dis ibu ion o ep oduc ion is pe mi ed which does no comply wi h hese e ms. F on ie s in Mic obiology | www. on ie sin.o g 7Janua y 2017 | Volume 8 | A icle 45