Description, Distribution, and Relevance of Viruses of the Forest Pathogen Gremmeniella abietina

i uses Re iew Desc ip ion, Dis ibu ion, and Rele ance o Vi uses o he Fo es Pa hogen G emmeniella abie ina Le icia Bo ella 1,* and Ja kko Han ula 2 1Phy oph ho a Resea ch Cen e, Depa men o Fo es P o ec ion and Wildli e Managemen , Facul y o Fo es y and Wood Technology, Mendel Uni e si y in B no, Zemˇedˇelská1, 613 00 B no, Czech Republic 2Fo es Heal h and Biodi e si y, Na u al Resou ces Ins i u e Finland (Luke), La oka anonkaa i 9, 00790 Helsinki, Finland; [email p o ec ed] *Co espondence: [email p o ec ed]; Tel.: +420-730-96-1992 Recei ed: 30 Oc obe 2018; Accep ed: 16 No embe 2018; Published: 20 No embe 2018   Abs ac : The Eu opean ace o he ascomyce ous species G emmeniella abie ina (Lage be g) Mo ele includes causal agen s o shoo bligh and s em canke o se e al coni e s in Eu ope and No h Ame ica, which a e known o hos a di e se i ome. GaRV6 is he la es and six h myco i us species epo ed wi hin G. abie ina. Be o e i s desc ip ion, one ic o i i us and one gammapa i i i us species we e desc ibed in bio ype A, wo mi o i uses in bo h bio ypes A and B and a be aendo na i us in bio ype B. Possible pheno ypic changes p oduced by myco i uses on G. abie ina mycelial g ow h ha e been epo ed in Spanish mi o i us- ee and GaRV6-hos ing G. abie ina isola es, which had highe g ow h a es a he op imal empe a u e o 15 ◦ C, bu no o he majo di e ences ha e been obse ed be ween pa i i i us-like dsRNA and dsRNA- ee isola es. In his e iew, we eapp aise he di e si y o i uses ound in G. abie ina so a , and hei ele ance in cla i ying he axonomy o G. abie ina. We also p o ide e idence o he p esence o wo new i uses belonging o he amilies Fusa i i idae and Endo na i idae in Spanish isola es. Keywo ds: B uncho s ia pinea; coni e s; myco i us; dsRNA; ssRNA; phylogeny; e olu ion 1. Taxonomy o G. abie ina and Rele ance in Fo es y G emmeniella abie ina is a i ulen haploid ascomyce e esponsible o shoo dieback and Scle ode is canke on coni e s including sp uces, i s, la ches, pines, and junipe s in No h, Cen al, and Sou h Eu ope, no heas e n No h Ame ica, and Eas Asia [1–6]. The axonomy o G. abie ina and i s ela ion o o es y is a complex issue, since he axon is di ided in o a numbe o a ie ies, aces, and bio ypes. These include wo a ie ies: G. abie ina a . abie ina ha mainly a ec s pines, and G. abie ina a . balsamea ha a acks i s and sp uces [ 7 ]. Wi hin G. abie ina a . abie ina, h ee aces—Asian, No h Ame ican, and Eu opean—we e desc ibed based on se ological analyses [ 8 ]. I has also been p oposed ha hese aces would be conside ed as sepa a e species [ 9 , 10 ]. In he Eu opean ace, h ee bio ypes ha e been iden i ied on he basis o symp oms, sep a numbe s, spo e leng h, and molecula ma ke s; namely, he alpine bio ype, bio ype A (LTT, la ge ee ype), and bio ype B (STT, small ee ype) [ 11 – 14 ]. Fu he mo e, he e is a dis inc i e popula ion o G. abie ina in Spain ha may s em om bio ype A o he Eu opean ace [15,16]. In a axonomic con ex , obliga e pa asi es wi hou an ex acellula phase, such as myco i uses, may be conside ed especially in o ma i e because hey can only sp ead h ough mycelial con ac s [ 17 ]. The e o e, de e mining he p esence and e olu iona y his o y o myco i uses in di e en popula ions o G. abie ina will shed ligh on he o igin and pa hways o i s sp ead. In he li e a u e, he e a e a numbe o examples ha suppo he use o myco i uses as ace s o he o igin and pa hways Vi uses 2018,10, 654; doi:10.3390/ 10110654 www.mdpi.com/jou nal/ i uses Vi uses 2018,10, 654 2 o 14 o di e en plan pa hogens, i.e., Hymenoscyphus axineus [ 18 ], C yphonec ia pa asi ica [ 19 ] and He e obasidion annosum [20]. The s udy o myco i uses may also cla i y he le el o ungal compa ibili y be ween species. The e is g owing e idence sugges ing di e ences in he ungal incompa ibili y a e [ 21 ], and a numbe o cases o myco i us co-speci ici y in phylogene ically sepa a ed ungi ha e been epo ed, no only in he labo a o y, bu also in na u e [20,22–29]. 2. Occu ence o Vi uses in G. abie ina: Desc ip ion o Thei Genome and S uc u e, and Phylogene ic Rela ionships The p esence o pu a i e myco i al dsRNA was i s desc ibed in G. abie ina bio ype A by Tuomi i a e al. [ 30 ]. They ound wo independen dsRNA banding pa e ns in a single G. abie ina isola e, and la e cha ac e ised one o i i us (G emmeniella abie ina RNA i us-lone 1, GaRV-L1) and wo pa i i i uses (G emmeniella abie ina RNA i us mul isegmen ed, GaRV-MS1 and GaRV-MS2), bu could no ind dsRNA in ascospo e isola es [ 31 , 32 ]. Na na i uses we e obse ed la e , and a e now known o be p esen in bo h bio ypes o G. abie ina [ 33 , 34 ]. The ea e , bio ype B has also been shown o hos an endo na i us (G emmeniella abie ina RNA i us XL, GBRV-XL) [ 35 ] and, wi hin he Spanish popula ion, a axonomically unca ego ised myco i us (G emmeniella abie ina RNA i us 6, GaRV6) [36,37]. 2.1. Pa i i i uses Th ee ull-leng h genomes ha e been cha ac e ised in h ee di e en s ains o G. abie ina, wo in Finnish isola es o bio ype A (GaRV-MS1 and 2) [ 31 , 32 ], and one in an isola e o he Spanish popula ion o G. abie ina (GaRV-MS1-3) [ 38 ]. GaMRV-MS1 has i s genome di ided in o h ee segmen s, and he la ges one con ains he ORF (open eading ame) ha codes o an RNA-dependen RNA polyme ase (RdRp) and has a size o ci ca (ca.) 1.7 kb, he medium segmen (ca. 1.5 kb) codes o a capsid p o ein (CP), and he smalles one (III) (ca. 1.1 kb) codes o a p o ein wi h unknown unc ion (Figu e 1A). The compa ison o amino acid sequences o he CP, RdRp, and unknown p o ein e ealed ha he h ee ull-leng h sequences desc ibed belong o he same species o he genus Gammapa i i i us [ 31 , 32 , 38 ]. In e es ingly, GaRV-MS1 appea s o ha e low gene ic a iabili y, and i is highly conse ed no only in Eu ope, bu also in No h Ame ica [ 38 ]. GaRV-MS1 was de ec ed in 28% o 162 in es iga ed isola es. I p ima ily occu s in G. abie ina bio ype A (Table 1) bu is also p esen in bio ype B in Tu key [ 29 ]. When he occu ence o GaRV-MS1 was analysed wi hin each popula ion/bio ype, he highes incidence was ound in he Spanish popula ion (56% o 50 isola es), ollowed by he bio ype A popula ion in No h Ame ica (45% o 11 isola es), bio ype A in mos o Eu ope (16% o 68 isola es), and bio ype B (and he Alpine bio ype; in only 6% o 33 isola es). The i us GaMRV-MS1 e ol es no only h ough pu i ying selec ion bu also, o some ex en , ia ecombina ion. S ain GaRV-MS1-2 seemed o be a ecombinan be ween he comple e CP sequences o GaRV-MS1-1 and GaRV-MS1-3, sugges ing ha GaRV-MS1-2 o one o i s ances o s was a ecombinan . Likewise, ecombina ion was iden i ied in he ull-leng h RdRp sequences o GaRV-MS1-3 and 2, wi h GaRV-MS1-1 indica ed o be a ecombinan [38]. Vi uses 2018,10, 654 3 o 14 Vi uses 2018, 10, x 3 o 14 Figu e 1. Schema ic illus a ion o he genomic o ganisa ion o all ully sequenced G. abie ina myco i uses. ORFs and conse ed domains a e ep esen ed by ec angula colou ed boxes indica ing he coded p o eins and he size o hei amino acid sequences. They a e lanked by 5′- and 3′-UTRs, numbe s ep esen base pai s (bp). The ull-leng h dsRNA segmen size is indica ed on he igh . (A) G emmeniella abie ina RNA i us-MS1; (B) G emmeniella mi o i us 1 (p e iously named G emmeniella abie ina RNA i us-S1); (C) G emmeniella mi o i us 2; (D) G emmeniella abie ina RNA i us-L; (E) G emmeniella be aendo na i us 1 (p e iously G emmeniella abie ina RNA i us- XL) consis s o a polyp o ein o 3249 and i e conse ed egions: i al me hyl ans e ase (VMe ), cys eine- ich egion (CRR), DExH box helicase (DExH), i al RNA helicase 1 (VHel1), and RNA_dep_RNApol2 (RdRp); (F) G emmeniella abie ina RNA i us 6. A) G emmeniella abie ina RNA i us- MS1 (3 dsRNA segmen s) B) G emmeniella mi o i us 1 C) G emmeniella mi o i us 2 D) G emmeniella abie ina RNA i us-L (1 dsRNA segmen ) E) G emmeniella Be aendo na i us (1 dsRNA segmen ) F) G emmeniella abie ina RNA i us 6 (1 dsRNA segmen ) Figu e 1. Schema ic illus a ion o he genomic o ganisa ion o all ully sequenced G. abie ina myco i uses. ORFs and conse ed domains a e ep esen ed by ec angula colou ed boxes indica ing he coded p o eins and he size o hei amino acid sequences. They a e lanked by 5 0 - and 3 0 -UTRs, numbe s ep esen base pai s (bp). The ull-leng h dsRNA segmen size is indica ed on he igh . ( A ) G emmeniella abie ina RNA i us-MS1; ( B ) G emmeniella mi o i us 1 (p e iously named G emmeniella abie ina RNA i us-S1); ( C ) G emmeniella mi o i us 2; ( D ) G emmeniella abie ina RNA i us-L; ( E ) G emmeniella be aendo na i us 1 (p e iously G emmeniella abie ina RNA i us-XL) consis s o a polyp o ein o 3249 and i e conse ed egions: i al me hyl ans e ase (VMe ), cys eine- ich egion (CRR), DExH box helicase (DExH), i al RNA helicase 1 (VHel1), and RNA_dep_RNApol2 (RdRp); (F) G emmeniella abie ina RNA i us 6. Vi uses 2018,10, 654 4 o 14 Table 1. Desc ibed i uses in G. abie ina. Vi us Name Vi us Abb e ia ion Full-Leng h Vi us S ains GenBank Accessions * Vi us Genus Genome Fungal Popula ion Popula ion S udies Coun ies De ec ed Re e ences G emmeniella abie ina RNA i us mul i segmen ed 1 GaRV-MS1 3 KJ786411- KJ786413, AY089993-5, AY615211-13, Gammapa i i i us dsRNA A, B, SP YES Canada, USA, Finland, Spain, Mon eneg o, I aly, Tu key [31,32,38] G emmeniella abie ina mi o i us 1 GMV1 3 HE586988, AF534641, AY615209 Mi o i us (+) ssRNA A, SP YES Finland, Spain [32–34] G emmeniella abie ina mi o i us 2 GMV2 1 JN654496 Mi o i us (+) ssRNA B, SP YES Finland, Spain [34] G emmeniella abie ina RNA i us lone GaRV-L 2 AF337175, AY615210 To i i us dsRNA A NO Finland [31,32] G emmeniella abie ina RNA i us XL GaBRV-XL 2 DQ399289-90 Be aendo na i us dsRNA B NO Finland [35] G emmeniella abie ina RNA i us 6 GaRV6 1 KJ742567.1 Unassigned †dsRNA A, SP YES Finland, Canada, I aly, Spain [36] †Vi us belonging o a new amily no de ined and submi ed in he ICTV ye . * GenBank accession numbe s o ull-leng h i us sequences. Vi uses 2018,10, 654 5 o 14 We eanalysed he axonomic s a uses o G. abie ina i uses as he sequence in o ma ion in GenBank is inc easing quickly. This was done simply by using a BLAST sea ch (amino acid sequences) o ind he mos simila RdRp genes, and MAFFT alignmen using BLOSUM62 cos ma ix o de e mine iden i ies [ 39 ]. The eanalysis was ca ied ou in Oc obe 2018. Based on his analysis, he closes ela i es o GaRV-MS1 (and o he pa i i i uses o G. abie ina) we e 20 s ains o Pseudogymnoascus des uc ans pa i i i uses (PdPV-pa) [ 40 ] and Valsa malicola pa i i i us (VmPV; (GenBank accession numbe AIS37554) wi h 77–78% iden i ies. The closes s ain o PdPV-pa (APG38267.1) wi h a pa ial sequence co e ing amino acid posi ions 53–362 o GaRV-MS1 RdRp had 78% iden i y o GaRV-MS1. The VmPV sequence was e y sho , and co e ed only amino acid posi ions 198–331 o GaRV-MS1 RdRP and had 76% iden i y wi h i . 2.2. Na na i uses Two sepa a e na na i us popula ions o he genus Mi o i us ha e been cha ac e ised and epo ed in G. abie ina: G emmeniella mi o i us 1 (GMV1) (o iginally named as GaMRV-S) and G emmeniella mi o i us 2 (GMV2) (Table 1) [ 32 – 34 ]. They sha e 94% iden i y a aa-le el, and ha e a ypical mi o i us monopa i e genome o a ound 2.5 kb and GC con en o 30% (Figu e 1B,C). Using he mi ochond ial ansla ion able, bo h o hem code o a single la ge ORF o ca. 2 kb. Based on he sc eening o he 2.5 kb band in 353 isola es o G. abie ina [ 34 ], he e was no e idence o mi o i uses in he six Swiss (Alpine bio ype), wo Tu kish (bio ype B), and six No h Ame ican isola es (EU ace o bio ype A in No h Ame ica). Howe e , 68 o he 91 Spanish isola es ha bou ed he dsRNA band. Simila ly, among he 211 bio ype A Finnish isola es analysed, 51 isola es (24%) ca ied a 2.5 kb dsRNA segmen . Only h ee pu a i e mi o i uses we e ound among he 37 Finnish bio ype B isola es es ed (8%). The popula ion gene ic pa ame e s calcula ed o he wo popula ions sugges ha GMV1 is gene ically mo e a iable han GMV2. The e olu ion o bo h GMV1 and GMV2 is mainly d i en by mu a ion and selec ion, as no ecombina ion e en s we e de ec ed [ 34 ]. Based on he compa ison o sequences in he GenBank, GMV1 is mos closely ela ed o soybean lea -associa ed mi o i us 4 (SlaMV4) [ 41 ], soybean lea -associa ed mi o i us 2 (SlaMV2) [ 41 ], Al e na ia a bo escens mi o i us 1 (AaMV1) [ 42 ], and Al e na ia b assicicola mi o i us 1 (AbMV1) [ 43 ]. The highes simila i y was obse ed o he pa ial sequence o SSlaMV4, which was aligned wi h amino acid posi ions 1–529 o GMV1 RdRp wi h 44% iden i y. Fo he ull sequences o SlaMV2, AaMV1, and AbMV1, RdRp had 43%, 42% and 42% iden i ies wi h ha o GMV1, espec i ely. GMV1 is only obse ed in he Finnish bio ype A and Spanish s ains, whe eas GMV2 in ec s he Finnish bio ype B and he Spanish popula ion. The eby, he Spanish popula ion o G. abie ina ha bou s mi o i us s ains ha , in Finland, occu sepa a ely in bio ype A and B s ains. The e o e, he Spanish popula ion is he i s one hos ing dis an ly ela ed myco i uses o a single genus in one popula ion o G. abie ina. This may sugges ha ho izon al ansmission o i uses could ha e occu ed be ween bio ype B and he Spanish popula ion (A ype o igin) in Spain, al hough bio ype B has ne e been obse ed he e. Fu he mo e, GMV2 has been obse ed only in one o he ou s udied locali ies in Spain, sugges ing a ce ain local di e en ia ion o i us popula ions among he Spanish G. abie ina [ 34 ]. 2.3. To i i uses GaRV-L1 and GaRV-L2 a e pu a i e membe s o he same species belonging o he genus Vic o i i us. They we e sequenced om wo Finnish isola es belonging o bio ype A [ 31 ]. Thei genome leng hs a e ca. 5 kb, and show 90% o e all iden i y (RdRp 98%). GaRV-L1 and 2 con ain wo la ge pa ially o e lapping ORFs (Figu e 1D). The i s ORF s a s a app oxima ely nucleo ide 270 om he 5’ end o he coding s and. S a ing nucleo ides o he second ORF we e loca ed in posi ions loca ed 2.6 kb om he 5’ end in bo h cases. The p o ein encoded by he second ORF con ained all eigh conse ed mo i s o RdRps o i uses in ec ing lowe euka yo es [ 44 ]. Based on BLAST analysis using he RdRp amino acid sequence, he closes ela i es o hese i uses a e Penicillium au an iog iseum Vi uses 2018,10, 654 6 o 14 o i i us 1 (PaTV1) [ 45 ], Penicillium digi a um i us 1 (PdV1) [ 46 ], and Aspe gillus myco i us 178 (AMV178) [ 47 ]. PaTV1 and PdV1 co e ed he ull RdRp sequence o GaRV-L1 wi h iden i ies o 60% and 59%, whe eas AMV178 co e ed posi ions 53–825 o GaRV-L1 RdRp sequence wi h an iden i y o 60%. The i s ORF in bo h isola es coded o a pu a i e CP, as BLAST sea ches indica ed high simila i y wi h analogous p o eins o he i uses desc ibed abo e. Al hough he e a e no popula ion s udies a ailable on GaRV-L, i should be no ed ha hese i uses ha e only been obse ed in bio ype A [31]. 2.4. Endo na i uses The i us wi h he la ges genome ound o da e is GaBRV-XL, desc ibed in wo Finnish isola es o G. abie ina B ype (Table 1) [ 35 ]. I is a linea , monopa i e dsRNA i us o ca. 11 kb (Figu e 1E), and belongs o he genus Be aendo na i us. The GaBRV-XL genomic s uc u e has i e conse ed si es, and encodes o a pu a i e 3249 aa polyp o ein wi h ou egions showing high simila i y o pu a i e i al me hyl ans e ases, DExH box helicases, RNA helicase 1 o i uses, and RNA-dependen RNA polyme ases. The closes ela i es o GaBRV-XL, acco ding o he BLAST sea ch wi h RdRp mo i o he polyp o ein, a e Discula des uc i a i us 3 (DdV3) [ 48 ] and se e al endo na i uses om Scle o inia scle o io um (including SsEV2-A) [ 49 ]. The DdV3 sequence co e ed only amino acid posi ions 107–165 in GaBRV-XL sequence wi h 72% iden i y, whe eas SsEV2-A sequence co e ed he comple e RdRp mo i wi h 69% iden i y. In o de o ollow he In e na ional Commi ee on Taxonomy o Vi uses (ICTV) ules, we p opose enaming his i us as G emmeniella be aendo na i us 1 (GBEV1). 2.5. G emmeniella abie ina RNA Vi us 6 GaRV6 consis s o a polyme ase segmen o ca. 2.1 kb wi h 54.7% GC con en (Figu e 1F) [ 36 ]. I seems o be pa o an unclassi ied myco i us g oup ha is likely o be p oposed as a no el i us amily [ 37 ]. I s membe s a e hos ed by impo an plan pa hogens, such as Fusa ium g aminea um [ 50 ], Rhizoc onia solani [ 51 ], C. pa asi ica (GenBank accession numbe s KC549809 and KC549810), H. annosum [ 20 ], and he endophy e Cu ula ia p o ube a a [ 52 ]. The closes ela i es o GaRV6 based on BLAST sea ch using RdRp gene a e Fusa ium g aminea um dsRNA myco i us 5 (FgV5) [ 53 ] and Fusa ium g aminea um dsRNA myco i us-4 (FgV4) [ 50 ]. The FgV-5 and FgV4 sequences co e ed amino acid posi ions 95–638 and 60–638 o GaRV6 RdRp wi h iden i ies o 48% and 46%, espec i ely. This i us g oup is closely ela ed o he Pa i i i idae amily [ 20 , 37 , 50 ]. Mos o hese i uses ha e bipa i e genomes wi h sizes esembling hose o pa i i i uses (1700 o 2400 bp). The la ge segmen encodes he RdRp, whe eas he smalle segmen appea s o ha e one o wo ORFs coding p o eins wi h unknown unc ions. Howe e , he smalle genome segmen o Us ilaginoidea i ens RNA i us 4 (U RV4) [54] and He e obasidion RNA i us 6 (He RV6) [20] ha e no been de ec ed. GaRV6 p ima ily appea ed in he Spanish popula ion o G. abie ina, whe e i s gene ic di e si y was minimal, despi e i s ela i ely high abundance (46% o 50 isola es sc eened) [ 36 ]. GaRV6 has also been de ec ed by RT-qPCR in h ee o he isola es belonging o bio ype A in I aly, Canada, and Finland [55]. 2.6. Mul iple Vi us In ec ions and E idence o he Exis ence o No el Vi uses G. abie ina commonly hos s mo e han one i us in a single isola e [ 30 – 32 , 36 ]. In bio ype A, up o i e dsRNAs a e ound in some isola es [ 30 ], in B ype h ee [ 35 ] and, in he Spanish popula ion, he e a e up o eigh di e en dsRNA-banding pa e ns desc ibed, some o which include up o eigh bands (Figu e 2), sugges ing he exis ence o o he G emmeniella i uses no epo ed ye . No hing is known abou he in e ac ions o mul iple i uses co-inhabi ing he same mycelia, no o hei e ec s on he hos pheno ype. He e, we p o ide u he in o ma ion abou he excep ional ichness o he i us communi y hos ed by Spanish isola es o G. abie ina no ed by Bo ella e al. [ 36 ]. Two dsRNA bands o pu a i e i uses we e de ec ed in se e al isola es, including P3-7 and 06P (Figu e 2) which we e u he analysed Vi uses 2018,10, 654 7 o 14 as p e iously (Supplemen a y Da a). These isola es o G. abie ina ha e been included in he p e iously men ioned s udies abou GaRV-MS1, GMV1 and GaRV6 [34,36,38]. Vi uses 2018, 10, x 7 o 14 analysed as p e iously (Supplemen a y Da a). These isola es o G. abie ina ha e been included in he p e iously men ioned s udies abou GaRV-MS1, GMV1 and GaRV6 [34,36,38]. Figu e 2. dsRNA banding pa e n o he isola es 003P, P3-7, and 06P o G emmeniella abie ina a e DNase I and S1 nuclease ea men . M, DNA ma ke (GeneRule 1 kb Plus DNA Ladde , 75–20,000 bp, The mo Scien i ic); F, dsRNAs co esponding o a pu a i e usa i i us; V, dsRNA co esponding o a pu a i e ic o i i us, U, unknown band, Mi, dsRNA co esponding o G emmeniella mi o i us 1; 6, dsRNA band co esponding o GaRV6; P, dsRNA co esponding o G emmeniella abie ina RNA i us MS1. 2.6.1. The ca. 6 kb dsRNA Band A pa ial 2309 bp sequence o he ca. 6 kb dsRNA agmen (GenBank accession numbe LR031261; Figu e 2) om isola e P3-7 was de e mined as p e iously desc ibed (Supplemen a y Da a) [21,23,27,29]. The compa ison in BLASTX (Table 2), esul ed in he highes simila i y (65%) wi h he RdRp o Mac ophomina phaseolina single-s anded RNA i us 1 (MpSRV1) ollowed by Neo usicoccum lu eum usa i i us 1 (62%). These i uses a e unclassi ied ssRNA i uses belonging o Fusa i i idae, which is a ecen ly p oposed amily no ye o icially accep ed by he ICTV. The sequence included a 332 bp conse ed domain wi h an E- alue o 3.34 × 10 −3 , belonging o a hypo he ical ATP-dependen RNA helicase H pB o Haemophilus in luenzae (Accession numbe PRK11664). Table 2. BLASTX compa ison o a 2228 bp RdRp sequence ob ained om cloning 6 kb dsRNA o P3- 7 G. abie ina isola e. GenBank BLASTX Da abase Sequence ID Family Iden i ies Ma ching Region 1 Mac ophomina phaseolina single-s anded RNA i us 1 ALD89094.1 Fusa i i idae * 217/335 (65%) 512 o 845 Neo usicoccum lu eum usa i i us 1 ARO52688.1 Fusa i i idae * 205/333 (62%) 527 o 856 Penicillium au an iog iseum usa i i us 1 YP_009182154.1 Fusa i i idae * 198/328 (60%) 511 o 838 Penicillium oque o i ssRNA myco i us 1 YP_009052456.1 Fusa i i idae * 177/313 (57%) 500 o 812 Fusa ium g aminea um dsRNA myco i us-1 YP_223920.2 Fusa i i idae * 179/334 (54%) 530 o 863 Sodiomyces alkalinus usa i i us 1 ATP75827.1 Fusa i i idae * 183/326 (56%) 445 o 770 Rosellinia neca ix usa i i us 1 YP_009047147.1 Fusa i i idae * 80/329 (55%) 522 o 850 Pleospo a yphicola usa i i us 1 YP_009182158.1 Fusa i i idae * 178/329 (54%) 521 o 849 Fusa ium poae usa i i us 1 YP_009272906.1 Fusa i i idae * 172/326 (53%) 496 o 818 Aga icus bispo us i us 11 AQM49938.1 Fusa i i idae * 151/314 (48%) 592 o 901 Figu e 2. dsRNA banding pa e n o he isola es 003P, P3-7, and 06P o G emmeniella abie ina a e DNase I and S1 nuclease ea men . M, DNA ma ke (GeneRule 1 kb Plus DNA Ladde , 75–20,000 bp, The mo Scien i ic); F , dsRNAs co esponding o a pu a i e usa i i us; V , dsRNA co esponding o a pu a i e ic o i i us, U , unknown band, Mi , dsRNA co esponding o G emmeniella mi o i us 1; 6 , dsRNA band co esponding o GaRV6; P , dsRNA co esponding o G emmeniella abie ina RNA i us MS1. 2.6.1. The ca. 6 kb dsRNA Band A pa ial 2309 bp sequence o he ca. 6 kb dsRNA agmen (GenBank accession numbe LR031261; Figu e 2) om isola e P3-7 was de e mined as p e iously desc ibed (Supplemen a y Da a) [ 21 , 23 , 27 , 29 ]. The compa ison in BLASTX (Table 2), esul ed in he highes simila i y (65%) wi h he RdRp o Mac ophomina phaseolina single-s anded RNA i us 1 (MpSRV1) ollowed by Neo usicoccum lu eum usa i i us 1 (62%). These i uses a e unclassi ied ssRNA i uses belonging o Fusa i i idae, which is a ecen ly p oposed amily no ye o icially accep ed by he ICTV. The sequence included a 332 bp conse ed domain wi h an E- alue o 3.34 × 10 −3 , belonging o a hypo he ical ATP-dependen RNA helicase H pB o Haemophilus in luenzae (Accession numbe PRK11664). Table 2. BLASTX compa ison o a 2228 bp RdRp sequence ob ained om cloning 6 kb dsRNA o P3-7 G. abie ina isola e. GenBank BLASTX Da abase Sequence ID Family Iden i ies Ma ching Region 1 Mac ophomina phaseolina single-s anded RNA i us 1 ALD89094.1 Fusa i i idae * 217/335 (65%) 512 o 845 Neo usicoccum lu eum usa i i us 1 ARO52688.1 Fusa i i idae * 205/333 (62%) 527 o 856 Penicillium au an iog iseum usa i i us 1 YP_009182154.1 Fusa i i idae * 198/328 (60%) 511 o 838 Penicillium oque o i ssRNA myco i us 1 YP_009052456.1 Fusa i i idae * 177/313 (57%) 500 o 812 Fusa ium g aminea um dsRNA myco i us-1 YP_223920.2 Fusa i i idae * 179/334 (54%) 530 o 863 Sodiomyces alkalinus usa i i us 1 ATP75827.1 Fusa i i idae * 183/326 (56%) 445 o 770 Rosellinia neca ix usa i i us 1 YP_009047147.1 Fusa i i idae * 80/329 (55%) 522 o 850 Pleospo a yphicola usa i i us 1 YP_009182158.1 Fusa i i idae * 178/329 (54%) 521 o 849 Fusa ium poae usa i i us 1 YP_009272906.1 Fusa i i idae * 172/326 (53%) 496 o 818 Aga icus bispo us i us 11 AQM49938.1 Fusa i i idae * 151/314 (48%) 592 o 901 * P oposed amily no o icial in he ICTV ye ; 1Ma ching egion in he aa sequence o he da abase sequences. Vi uses 2018,10, 654 8 o 14 An ORF wi h a leng h o 987 bp, coding o a p o ein o 328 aa, was de ec ed. This sequence was ob ained om a con ig o 3 o e lapping clones (682, 743 and 438 bps) and 5 e e se ansc ip ion (RT) PCR p oduc s. Speci ic p ime s (Table S1) we e designed based on he i s con ig achie ed by cloning he 6 kb dsRNA band (Supplemen a y Da a). The aa sequence had 61% iden i y wi h he MpSRV1 RdRp gene. We p opose he name G emmeniella usa i i us 1 (GFV1) o his myco i us. A simila banding pa e n has also been ound in o he isola es o G. abie ina [ 56 ], such as 003P (Figu e 2). Howe e , he equency o GFV1 in Spanish o o he G. abie ina popula ions has no been de e mined, wi h mo e speci ic me hods such as e e se ansc ip ion (RT) PCR. 2.6.2. The ca. 5 kb dsRNA Band The 1852 bp and 1261 bp sequences (accession numbe s LR030278 and LR030279) we e de e mined om a 43-clone con ig om a ca. 5 kb dsRNA agmen o Spanish isola e 06P (Figu e 2). The BLASTX analyses wi h he wo nucleo ide sequences showed ha hey code o RdRp and CP p o eins o a pu a i e new i us o he genus Vic o i i us. The BLASTX compa ison o he pa ial RdRp sequence had he highes simila i y (78%) wi h Scle o inia ni alis ic o i i us 1, ollowed by Aspe gillus oe idus slow i us 1 (52%) (Table 3), bu was dis an ly ela ed o he p e iously known ic o i i uses, GaRV-MS1 and GaRV-MS2 (39% and 38% iden i ies in Blas x, espec i ely), om G. abie ina bio ype A. The pa ial RdRp agmen con ains an ORF o 1557 bp ha codes o a 512 aa p o ein. Table 3. BLASTX compa ison o a 1852 bp RdRp sequence ob ained om cloning 5 kb dsRNA o 06P G. abie ina isola e. GenBank BLASTX da abase Sequence ID Family Iden i ies Ma ching Region 1 Scle o inia ni alis ic o i i us 1 YP_009259368.1 To i i idae 456/587 (78%) 247 o 833 Aspe gillus oe idus slow i us 1 YP_009508249.1 To i i idae 306/584 (52%) 254 o 837 Beau e ia bassiana ic o i i us 1 AMQ11131.1 To i i idae 302/585 (52%) 258 o 841 Sphae opsis sapinea RNA i us NP_047558.1 To i i idae 314/587 (53%) 254 o 837 Rosellinia neca ix ic o i i us 1 BAM36400.1 To i i idae 397/587 (67% 107 o 692 Beau e ia bassiana ic o i i us NZL/1980 YP_009032633.1 To i i idae 301/585 (51%) 258 o 841 Soybean-associa ed double-s anded RNA i us 1 ALM62239.1 To i i idae 311/587 (53%) 255 o 840 Us ilaginoidea i ens RNA i us 1 AGO04407.1 To i i idae 302/584 (52%) 243 o 825 Bipola is maydis ic o i i us 1 AXB26764.1 To i i idae 289/588 (49%) 253 o 835 Bo yo inia uckeliana o i i us 1 YP_001109580.1 To i i idae 291/581 (50%) 266 o 836 1Ma ching egion in he aa sequence o he da abase sequences. In he case o he pa ial CP agmen , he consensus sequence de i ed om a se en-clone con ig o 1261 bp also showed he highes simila i y (81%) wi h Scle o inia ni alis ic o i i us 1, ollowed by Tolypocladium cylind ospo um i us 1 (68%) (Table 4). Acco ding o he 60% maximum iden i y c i e ion o he ICTV 9 h epo (2011) o he genus Vic o i i us, his i us should be conside ed a s ain o Scle o inia ni alis ic o i i us 1 because i shows 78% RdRp and 81% CP simila i y o Scle o inia ni alis ic o i i us 1. The e o e, we designa e he i us as Scle o inia ni alis ic o i i us 1—s ain 1 om G emmeniella abie ina (SnVV1-Ga1) A simila dsRNA banding pa e n has also been ound in o he isola es o Spanish G. abie ina [ 56 ], such as 003P and P3-7 (Figu e 2), bu i s p esence among G. abie ina isola es has no been de e mined wi h speci ic me hods. Vi uses 2018,10, 654 9 o 14 Table 4. BLASTX compa ison o a 1261 n capsid p o ein (CP) sequence ob ained om cloning 5 kb dsRNA o 06P G. abie ina isola e. GenBank BLASTX Da abase Sequence ID Family Iden i ies Ma ching Region 1 Scle o inia ni alis ic o i i us 1 YP_009259367.1 To i i idae 293/363 (81%) 1 o 363 Tolypocladium cylind ospo um i us 1 YP_004089629.1 To i i idae 247/362 (68%) 1 o 362 Beau e ia bassiana ic o i i us NZL/1980 YP_009032632.1 To i i idae 237/361 (66%) 1 o 361 Helmin hospo ium ic o iae i us 190S NP_619669.2 To i i idae 240/363 (66%) 1 o 363 Bipola is maydis ic o i i us 1 AXB26763.1 To i i idae 240/363 (66%) 1 o 363 Bo yo inia uckeliana o i i us 1 YP_001109579.1 To i i idae 227/363 (63%) 1 o 363 Aspe gillus oe idus slow i us 1 YP_009508248.1 To i i idae 221/360 (61%) 1 o 360 Fusa ium asia icum ic o i i us 1 AYD49681.1 To i i idae 225/364 (62%) 1 o 363 Us ilaginoidea i ens RNA i us L YP_009094184.1 To i i idae 223/363 (61%) 1 o 363 Rosellinia neca ix ic o i i us 1 BAM36399.1 To i i idae 227/362 (63%) 1 o 361 Scle o inia scle o io um ic o i i us 1 (pa ial) AWY10937.1 To i i idae 208/263 (79%) 1 o 233 1Ma ching egion in he aa sequence o he da abase sequences. 3. Myco i uses and Thei Cla i ying Message on he Complex Di e si y o G. abie ina One o he undamen al ques ions in e olu iona y biology is he deg ee o which he di e si ica ion o pa asi es and/o symbion s is associa ed wi h he di e si ica ion o hei hos s [ 57 ]. Myco i uses wi h RNA genomes p oduce mos ly c yp ic in ec ions, eplica e only in he ungal cy oplasm, and a e ans e ed only h ough anas omosis and/o ia spo al dispe sal [ 58 ]. Such mechanisms would indica e ha e olu iona y pa e ns o myco i uses can be expec ed o ollow hose o hei hos s [59,60] . The di e si ica ion o he Eu opean ace o G. abie ina in o di e en bio ypes, and possible species [ 9 ], is mos ly he ou come o geog aphical sepa a ion and ecological adap a ion. Fu he mo e, i s i us communi ies p o ide insigh s abou his his o y o dispe sion, wi h a special ele ancy o he es ablishmen o new popula ions in Spain and Tu key. The Eu opean ace o G. abie ina is known o hos only one species o gammapa i i i us (GaRV-MS1) [ 38 ]. In no he n Eu ope, i has only been ound in bio ype A isola es, bu i occu s also in Tu kish isola es, which belong o bio ype B (Table 1) [ 16 , 34 ]. On he o he hand, he lack o GaRV-MS1 in bio ype B in no he n Eu ope and i s occu ence in Tu key sugges s ha he i us ansmission mus ha e occu ed be ween bio ypes A and B du ing hei e olu iona y his o y. This i us ansmission may ha e happened p io o he in oduc ion o G. abie ina o Tu key o , al e na i ely, in Tu key wi h an un ecognised bio ype A popula ion. In he i s scena io, bio ype B isola es a i ed in Tu key wi h GaRV-MS1 i uses and, in he second scena io, hey would ha e ecei ed he i uses in Tu key. A hi d possibili y is ha GaRV-MS1 i uses occu ed in bo h bio ypes du ing hei e olu iona y his o y, bu we e la e los in B- ype wi h he excep ion o he Tu kish popula ion. The p esence o GaRV-MS1 in he Eu opean ace (bio ype A) in No h Ame ica, in oduced om Eu ope in 1977 [ 61 ], indica es ha he i us a elled wi h i s hos , and has no changed much o e hese yea s. The i us communi y inhabi ing he Spanish G. abie ina popula ion suppo s i s o igin as bio ype A [ 16 ] because (i) ecombina ion e en s ha e been de ec ed be ween h ee s ains o GaRV-MS1 p esen in Finland and Spain and (ii) GaRV6 p ima ily appea s in Spain, bu was also ound in single isola es o bio ype A om Canada, I aly and Finland, and (iii) he Spanish popula ion also ha bou s bo h mi o i us species GMV1 and GMV2, associa ed wi h bio ype A and B s ains in no he n Eu ope, espec i ely. The occu ence o GMV2 in bo h bio ype B and he Spanish popula ion, oge he wi h i s ex emely low gene ic a iabili y, would be in acco dance wi h a ecen hos swi ch and subsequen adap a ion o a new hos [ 34 ]. Na u al in e species ansmission o i uses has been shown o C yphonec ia sp. and C yphonec ia pa asi ica [ 22 ], Scle o inia homoeoca pa and Ophios oma ulmi [ 23 ], O. ulmi and Ophios oma no o-ulmi [ 24 ], and wi hin species o he gene a He e obasidion [ 20 , 25 , 26 ], Scle o inia [ 27 , 28 ], and Rhizoc onia [ 29 ]. Howe e , bio ype B o G. abie ina has ne e been shown o exis in Spain and, he e o e, he iming and place o his possible hos jump emains open. I could ha e occu ed in he pas , o e en oday, in he moun ains o no he n Spain, whe e he snow co e in win e ime is enough