Population Structure of a Novel Putative Mycovirus Infecting the Conifer Root-Rot Fungus Heterobasidion Annosum Sensu Lato

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Virology 422 (2012) 366–376

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Virology

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Population structure of a novel putative mycovirus infecting the conifer root-rot fungus Heterobasidion annosum sensu lato

Eeva J. Vainio a,⁎,Raﬁqul Hyder a, Gülden Aday b, Everett Hansen c, Tuula Piri a,Tuğba Doğmuş-Lehtijärvi b, Asko Lehtijärvi b, Kari Korhonen a, Jarkko Hantula a

a Finnish Forest Research Institute, Jokiniemenkuja 1, P.O. Box 18, 01301 Vantaa, Finland b Süleyman Demirel University, Faculty of Forestry, Department of Botany, 32260 Isparta, Turkey c Oregon State University, Department of Botany and Plant Pathology, 2082 Cordley Hall, Corvallis, OR 97331, USA

article info abstract

Article history: We describe a novel putative mycovirus infecting the conifer root-rot fungus Heterobasidion annosum sensu lato. Received 8 June 2011 This virus, designated as Heterobasidion RNA virus 6 (HetRV6), is taxonomically distant from all previously Returned to author for revision 21 July 2011 known viruses of Heterobasidion species, but somewhat related to the Curvularia thermal tolerance virus and Accepted 28 October 2011 the Fusarium graminearum virus 4. Based on a population analysis including 35 virus strains from Heterobasidion Available online 3 December 2011 abietinum, Heterobasidion parviporum, Heterobasidion annosum sensu stricto and Heterobasidion occidentale,

Keywords: HetRV6 showed a considerable degree of geographical and host-related differentiation. The North American dsRNA and Eurasian virus populations were clearly separated. In Eurasia, we observed cases of discrepancy between Mycovirus virus and host taxonomy, suggesting interspecies virus transfer. HetRV6 was also successfully transmitted Horizontal transmission between the three European species H. abietinum, H. annosum and H. parviporum. Based on growth rate tests Co-speciation on agar plates and spruce stem pieces, HetRV6 seemed to be cryptic or slightly mutualistic to its host. Anastomosis © 2011 Elsevier Inc. All rights reserved. Cryptic virus Basidiomycota Heterobasidion annosum Heterobasidion insulare

Introduction debilitation-associated RNA virus resemble positive-strand RNA plant viruses in the family Flexiviridae (Howitt et al., 2006; Kwon et al., 2007; Viruses occur commonly in all major groups of the true fungi Xie et al., 2006), while the Diaporthe ambigua RNA virus has a distant (Ghabrial and Suzuki, 2009; Pearson et al., 2009). Fungal viruses relationship to the plant virus family Tombusviridae (Preisig et al., (mycoviruses) have genomes composed of single-stranded (ss) or 2000). On the other hand, the reovirus Cryphonectria parasitica 9B21 double-stranded (ds) RNA, or rarely DNA (Yu et al., 2010). Currently, and the S. sclerotiorum RNA virus L are related to animal pathogenic mycoviruses are classified according to their genomic composition viruses (Hillman et al., 2004; Liu et al., 2009). into four dsRNA virus families (Chrysoviridae, Partitiviridae, Reoviridae Most mycoviruses are cryptic and seem to have no phenotypic and Totiviridae)andfive ssRNA virus families (Barnaviridae, Hypoviridae, effects on their host fungi. However, some are detrimental to their Narnaviridae, Pseudoviridae and Metaviridae; International Committee host and can mediate hypovirulence (reduction of virulence) in on Taxonomy of Viruses, ICTV; www.ictvonline.org). plant pathogenic fungal species. This phenomenon was originally de- During recent years, several novel mycovirus taxa have been described for hypoviruses of C. parasitica, and many other examples scribed. Some of these new virus species, like the Curvularia thermal have since been found (Anagnostakis and Day, 1979; Deng et al., 2003; tolerance virus, the Rosellinia necatrix megabirnavirus 1, and the Huang and Ghabrial, 1996; Lakshman et al., 1998; Preisig et al., 2000; Gremmeniella abietina type B RNA virus XL, are distant from all previously Yu et al., 2010). Recently, also mutualistic associations have been de- known mycoviruses (Chiba et al., 2009; Márquez et al., 2007; Tuomivirta scribed: the Nectria radicicola virus L1 enhances the virulence of its et al., 2009). Many also resemble plant viruses: the Botrytis virus X, the plant pathogenic host fungus (Ahn and Lee, 2001), and the Curvularia Fusarium graminearum virus DK21 and the Sclerotinia sclerotiorum thermal tolerance virus is involved in a three-way symbiosis with an endosymbiont fungus and its host grass (Márquez et al., 2007). The fungal genus Heterobasidion consists of two species com- plexes: Heterobasidion annosum (Fr.) Bref. sensu lato and Heterobasidion ⁎ Corresponding author. Fax: +358 10 211 2206. insulare (Murrill) Ryvarden sensu lato. The H. insulare complex includes E-mail address: eeva.vainio@metla.fi (E.J. Vainio). mostly saprotrophic wood-decay fungi that occur in eastern Asia (Dai

0042-6822/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.virol.2011.10.032 E.J. Vainio et al. / Virology 422 (2012) 366–376 367 and Korhonen, 2009). The H. annosum complex has a global distribution stem pieces to reveal whether the virus affects the growth rate of its and includes some of the most destructive forest pathogens in the bore- host. al forest region (Woodward et al., 1998). Heterobasidion parviporum Nie- melä & Korhonen infects mainly Norway spruce (Picea abies), and has a Results wide distribution in Europe and northern Asia. H. annosum (Fr.) Bref. sensu stricto prefers pines (Pinus spp.), but infects also other conifers Sequence statistics and conserved motifs and several deciduous trees in Europe. Heterobasidion abietinum Nie- melä & Korhonen occurs in central and southern Europe, and infects Complete putative viral polymerase sequences were determined mainly firs (Abies spp.). Also the two North American species, Heteroba- from the H. abietinum isolates 04188 and 07052, hosting virus strains sidion irregulare Otrosina & Garbelotto and Heterobasidion occidentale designated as HetRV6-ab6 and HetRV6-ab10 (see subsection ‘Sequence Otrosina & Garbelotto, have different host tree preferences (Pinus, Juni- polymorphism’ for virus nomenclature). In addition, primer walking perus and Libocedrus versus Abies, Sequoiadendron, Tsuga, Pseudotsuga was used to determine a partial sequence comprising the first 1556 nu- and Picea; Otrosina and Garbelotto, 2010). cleotides from the 5′-end of the viral polymerase gene from H. occiden- The three species, H. parviporum, H. abietinum and H. occidentale tale Het6 (host for HetRV6-oc1). The two complete RdRp sequences are considered to be closely related, and form the ‘fir/spruce clade’ were both 2050 bp long and contained a single AUG-initiated open read- of the H. annosum complex, while H. annosum and H. irregulare consti- ing frame (ORF) of 606 aa (Mr 69,320 and 69,343 for HetRV6-ab6 and tute the ‘pine clade’ (Otrosina and Garbelotto, 2010). In North America, HetRV6-ab10, respectively). The GC-contents of the sequences were H. irregulare and H. occidentale have been observed to hybridize in 56.8% and 56.2%, respectively. The sequences shared a notably high nature (Garbelotto et al., 1996), but no natural hybrids between the level of sequence similarity in their 3′ UTR regions (100% nucleotide three European species have been described, although they have been identity over 154 bp), whereas the similarity of the 5′ UTRs resembled shown to be capable of forming hybrids in the laboratory (Stenlid and that of the coding region (96% similarity over 75 bp). The sequence Karlsson, 1991). However, H. occidentale and H. annosum seem to hy- from H. occidentale Het6 showed 86% 5′ UTR similarity compared to bridize in Italy (D'Amico et al., 2007; Gonthier et al., 2007), where the the two sequences from H. abietinum. North American species was introduced during the Second World War. The 5′- and 3′-terminal UTRs within HetRV6-ab6 and HetRV6- Approximately 15% of European and western Asian Heterobasidion ab10 showed inverted complementarity and could be folded into spp. isolates are infected by dsRNA viruses (Ihrmark, 2001). Thus far, potential panhandle structures (Fig. S5). In addition, each of the 3′ several virus species have been described from Heterobasidion species, UTRs formed a potentially stable stem-loop structure (Fig. S6), while all of them members of the family Partitiviridae (Ihrmark, 2001; less inverted complementarity was detected within each of the 5′ UTRs Vainio et al., 2010, 2011a, 2011b). Unlike most fungal viruses that of HetRV6-ab6, HetRV6-ab10 and HetRV6-oc1 (Fig. S7). require a compatible anastomosis contact for lateral transmission, par- The conserved motif search implemented in BlastP revealed a titiviruses of Heterobasidion spp. are able to transfer horizontally be- conserved region resembling the following RdRp subfamilies in the tween somatically incompatible strains and even intersterile fungal HetRV6-ab6 polymerase segment: (i) cd01699, aa residues 289–427, species (Ihrmark, 2001; Vainio et al., 2010, 2011a, 2011b). They are E-value 4.76e−06, (ii) pfam 00680, aa residues 303–427, E-value also capable of spore-mediated dispersal and vegetative spread by 1.71e−07, (iii) PHA00497, aa residues 293–403, E-value 1.55e−05. cell-to-cell contacts (Ihrmark et al., 2002, 2004). The same conserved region occurred also in HetRV6-ab10 and In this study, we describe the first discovery of a novel virus spe- HetRV6-oc1. The conserved RdRp motifs 3–8 of dsRNA viruses of cies designated as Heterobasidion RNA virus 6 (HetRV6), found from lower eukaryotes, as determined by Bruenn (1993), were located in four species of Heterobasidion. The virus was distinct from previously the sequences as shown in Table 1. described viruses infecting Heterobasidion, and distantly related to the Curvularia thermal tolerance virus and the F. graminearum virus Phylogenetic affiliation 4(Márquez et al., 2007; Yu et al., 2009). A population analysis was conducted to reveal whether there is evidence of co-speciation between The three long or complete polymerase sequences (from HetRV6- the viruses and their Heterobasidion hosts at a global scale. We also tested ab6, HetRV6-ab10 and HetRV6-oc1) were used for phylogenetic infer- whether the virus is capable of interspecies transmission by hyphal ence. Based on BlastP analysis, no sequence matches were found to anastomosis. Growth rate tests with isogenic virus-infected and virus- the previously known viruses of Heterobasidion species. The closest free fungal isolates were carried out on agar plates and Norway spruce matches were with the Curvularia thermal tolerance virus, CThTV

Table 1 Location of conserved motifs 3–8 as determined by Bruenn (1993) in HetRV6-ab6 and selected dsRNA viruses. The virus name abbreviations are the same as in Table 2 and Fig. 3.

Virus strain Conserved motifs

3 4 5 6 7 8 (256–262)a (318–327) (379–399) (423–430) (479–483) (505–508)

HetRv6-ab6 TAGRLIF DAAKFDSSLP GSTSGHSYNTLMQSICTLVMI GLGDDQHT QYLGK PFDE CThTV AVGRLIL DARKYDAFLD GTTSGHSHNTLLQSICTLIVG SLGDDNIT QYLGK PFKE FgV4 PKGRLIL DAAKFDSSLD GTTSGHNHNTLIQSICSLVIA TLGDDNLT QYLGK PCEE HaV NKLRTIW DWSRFDKRAY GIPSGLFITQLMDSWYNYVML VQGDDSII EVLSY DLLK HetRV3-ec1 TKIRVIY DWSGFDLRSL SIPSGLFVTQFLDSHYNLIMI VQGDDSLI EVLGY DMTK HaV-P LKVRPVY DYSRFDQLAP GVPSGIFMTQILDSFVNLFIF IQGDDNLV EVLGY DVSK HetRV2-pa1 LKVRPVY DWSRYDQLLP GVPSGIFMTQICDSFCNAFLL IQGDDNVI EVLGY DCAK GaRV-MS1 PKTRLVW DFSSFDTKVP GVPSGSWWTQMVDSVVNYILV VLGDDSAF KLLGT DTNE DdV1 PKTRLVW DFSAFDSKVP GVPSGSWWTQIIDSVVNNILI VLGDDSAF KLLGT STDE BCV3 TKVRGVW DWSSFDSSVT GIPSGSYYTSIVGSVVNRLRI TQGDDSLI TFLGR SLDK PcV KKDRTLL DWADFNEQHS GLYSGWRGTTWINTVLNFCYV HGGDDIDL EFFRN SPTR Sc L-A (L1) GKQRAIY DYDDFNSQHS TLLSGWRLTTFMNTVLNWAYM HNGDDVMI EFLRV YLSR

a Location of the conserved motif in the amino acid sequence of HetRV6-ab6. 368 E.J. Vainio et al. / Virology 422 (2012) 366–376

(39–41% similarity with the two partly overlapping ORFs found in the and representing three different fungal isolates). High or low molecular CThTV genome: RNA1gp1 and RNA1gp2, GenBank IDs YP_001976143 weight additional bands were sometimes observed in the dsRNA pro- and YP_001976144). The F. graminearum virus 4, FgV4 (YP_003288790) files, but they were variable in presence and obviously host related shared 37% protein level sequence similarity with HetRV6-ab6. HetRV6 (>3 kb fragment in Fig. 2a, or ca. 1200 bp fragment in Fig. 2b, with also distantly resembled viruses of the subgroup 1 of Partitiviridae sizes corresponding to host chromosomal DNA or rRNA, respectively). (Ghabrial et al., 2008), as well as plant viruses of the genus Potyvirus (family Potyviridae). However, sequence similarity to both groups was Occurrence of HetRV6 among Heterobasidion isolates low, and only short sequence stretches in the most conserved regions of the polymerase segment (around aa positions 300–400 in HetRV6) The two complete HetRV6 polymerase sequences were used to de- were recognized (Table 2). sign consensus primers (HV6F1A and HV6Re2) that allowed the detec- Based on both Bayesian analysis and NJ clustering, HetRV6-ab6, tion of HetRV6 from a Heterobasidion culture collection by RT-PCR. The HetRV6-ab10 and HetRV6-oc1 formed a clear, well supported taxo- primers successfully amplified partial HetRV6 polymerase fragments of nomical cluster together with the CThTV and the FgV4. The three pro- ca. 700 bp from altogether 35 Heterobasidion isolates. This constitutes tein evolution models (Blosum, Poisson and Equalin) used for Bayesian 12.5% of all the Heterobasidion isolates analyzed (n=279), and 72.9% analysis all revealed the same major taxonomical clusters (Fig. 1). The of all dsRNA-positive isolates (Table 3). The frequency of HetRV6 in Poisson model yielded the highest overall posterior probability support our culture collection was 17.1% for H. parviporum;7.7%forH. annosum; for our data set, while Equalin was least supported (Fig. 1). Also the NJ 17.9% for H. abietinum; 14.3% for H. occidentale; and none for H. insulare analysis yielded the same major clusters, indicating 100% bootstrap s. lat., respectively. Examples of amplification products obtained with support for the taxonomical cluster including strains of HetRV6, FGV4 the consensus primers are shown in Fig. 2c. and CThTV (not shown). Based on both Bayesian and NJ analysis, the One of the Heterobasidion isolates revealed to be double-infected: HetRV6 strains and the affiliated viruses (CThTV and FgV4) grouped isolate 94245 from Poland harbored both HetRV6-an2 and the partiti- clearly outside the four taxonomical subgroups of Partitiviridae virus HetRV1-an3 (GenBank accession JN882010). The partitivirus in- (Ghabrial et al., 2008), and were also distinct from the plant viruses of fection was detected by RT-PCR using consensus primers as described the family Potyviridae. The MAFFT alignment used in the dendrogram in Vainio et al. (2011b), and the virus strain (HetRV1-an3) was 95% construction (including Bruenn's conserved regions 3–6) is shown in similar to HetRV1-an1 (HQ541332; Vainio et al., 2011b). supplementary Fig. S1.

Sequence polymorphism Genomic composition Partial HetRV6 polymerase sequences of 645 bp (215 aa) from 35 The dsRNA pattern of HetRV6 composed of a single band of about Heterobasidion isolates were used for population analyses. Based on 2 kb in length, corresponding to a putative viral RNA polymerase pairwise nucleotide and amino acid similarities, all the HetRV6 se- (Fig. 2). Notably, the genomes of the two most closely related viruses, quences were relatively closely related and could be considered to CThTV and FgV4, are bipartite. In FgV4, one genomic segment of represent strains of a single putative virus species, designated as 2383 bp includes a single ORF coding for a putative RdRp, and a Heterobasidion RNA Virus 6 (HetRV6). The speciﬁc strain names are shorter segment of 1739 bp contains two ORFs with unknown func- abbreviated according to their host species and collection number tion. In CThTV, a genomic segment of 2149 bp contains two overlap- (e.g. ab1, ‘abietinum strain 1’;an1,‘annosum strain 1’;oc1,‘occidentale ping ORFs coding for putative viral polymerases, and a second strain 1’; pa1, ‘parviporum strain 1’). genomic segment of 1886 bp codes for two ORFs with unknown func- At the amino acid level, many HetRV6 strains shared identical se- tion. CThTV also sometimes includes a third, subgenomic segment of quences, and only 12 strains were unique. In three cases, identical less than 1 kb in length. In the case of HetRV6, we detected no indica- protein sequences occurred between viruses of H. abietinum and tion of the presence of additional virus-related fragments of similar H. parviporum (for example between HetRV6-ab14 from Austria and size with the RdRp segment (the analysis included altogether 53 indi- HetRV6-pa1 from Russia). Identical sequences were also found between vidual cloned inserts obtained using two different cloning strategies virus strains from geographically distant regions (HetRV6-ab3, HetRV6-

Table 2 Sequence similarity of HetRV6-ab6 as compared to the closest matching sequences determined by NCBI BlastP.

Virus Family Accession and length Similarity

Fusarium graminearum virus 4 (FgV4) Unassigned YP_003288790 (712 aa) 220/587 (38%) Curvularia thermal tolerance virus RNA1gp1 (CThTV) Unassigned YP_001976143 (371 aa) 136/328 (42%) Curvularia thermal tolerance virus RNA1gp2 (CThTV) Unassigned YP_001976144 (282 aa) 93/233 (40%) Zygosaccharomyces bailii virus Z (ZbV-Z) Totiviridae NP_624325 (643 aa) 58/258 (23%) Maize dwarf mosaic virus (MDMV) Potyviridae ABI75204 (825 aa) 42/143 (30%) Aspergillus ochraceous virus (AoR1) Partitiviridae ABV30675 (539 aa) 28/108 (26%) Tobacco etch virus (TEV) Potyviridae AAA98577 (3054 aa) 38/132 (29%) Sorghum mosaic virus (SrMV) Potyviridae CAC84438 (3071 aa) 42/141 (30%) Sweet potato feathery mottle virus (SPFMV) Potyviridae ACY74485 (1369 aa) 30/118 (26%) Raphanus sativus cryptic virus 2 (RSCV2) Partitiviridae YP_001686783 (477 aa) 61/271 (23%) Sweet potato virus C Potyviridae YP_004046670 (3481 aa) 30/118 (26%) Sugarcane mosaic virus (SCMV) Potyviridae AAT57632 (928 aa) 41/140 (30%) Ophiostoma partitivirus 1 (OPV1) Partitiviridae CAJ31886 (539 aa) 38/167 (23%) Gremmeniella abietina RNA virus MS1 (GaRV-MS1) Partitiviridae NP_659027 (539 aa) 38/157 (25%) Pennisetum mosaic virus Potyviridae YP_249455 (3065 aa) 43/161 (27%) Plum pox virus Potyviridae AAX99419 (3140 aa) 36/119 (31%) Turnip mosaic virus Potyviridae BAC79393 (3164 aa) 34/106 (33%) Pea seed-borne mosaic virus Potyviridae BAA01726 (3206 aa) 31/122 (26%) E.J. Vainio et al. / Virology 422 (2012) 366–376 369

Sc L-A (L1) (Totiviridae) PcV (Chrysoviridae) TEV Potyviridae SCMV MDMV 100/100/100 SrMV SPFMV CThTV Unassigned FgV4 100/100/100 HetRV6-oc1 100/100/100 HetRV6-ab10 100/100/100 HetRV6-ab6 GaRV-MS1 Partitiviridae AoR1 PsV-S Subgroup 1 62/67/53 OPV1 BFPV1 100/100/100 DdV1 BCV3 Subgroup 3 RSCV2 92/55/87 99/89/94 ZbV-Z ACD-PV Subgroup 4 HaV HetRv3-ec1 100/100/100 HmV-V70 HaV-P Subgroup 2 HetRV2-pa1 100/100/100 AhpV-2H 0.4

Fig. 1. Bayesian analysis of viral polymerase sequences, including three representative strains of HetRV6, and a selection of associated taxa (including viruses recognized by the BlastP analysis, and viruses infecting Heterobasidion species). The alignment was generated using the MAFFT algorithm and includes the sequence regions between the conserved domains 3–6asdeterminedbyBruenn (1993).AhpV-2H,Atkinsonella hypoxylon partitivirus (NP_604475, type species Partitiviridae); PsV-S, Penicillium stoloniferum-virus S (CAJ01909); BCV3, Beet cryptic virus 3 (AAB27624); PcV, Penicillium chrysogenum virus (YP_392482, type species Chrysovirus), Sc LA (L1), Saccharomyces cerevisiae virus L-A (strain L1, NP_620495, Totiviridae); ACD-PV, Amasya cherry disease (ACD)-associated mycovirus (YP_138537); HmV-V70, Helicobasidium mompa dsRNA mycovirus (BAC23065); HaV, Heterobasidion annosum virus (AAK52739); HaV-P, Heterobasidion annosum P-type partitivirus (AAL79540); HetRV2-pa1, Heterobasidion RNA virus 2 (ADL66905); HetRV3-ec1, Heterobasidion RNA Virus 3 (ACO37245), DdV1, Discula destructiva virus 1 (NP_116716), BFPV1, Botryotinia fuckeliana partitivirus 1 (YP_001686789). The remaining virus name abbreviations are as described in Table 2. Strains of HetRV6 are showninbold,andpartitivirusesofHeterobasidion species are underlined. The sequence of CThTV is a composite sequence including two ORFs. The scale bar indicates 0.4 amino acid substitutions per site, and numbers at branch nodes show percentage posterior probabilities using the substitution model Poisson/Blosum/Equalin. The Partitiviridae subgroups 1–4areasdescribed by Ghabrial et al. (2008). ab4 and HetRV6-ab6 from Turkey were identical with HetRV6-ab9 from in sequence at the protein level, as were four virus strains of H. annosum Austria, HetRV6-pa11 from Finland and HetRV6-pa5 from Italy). The (Finland and Poland), four strains from H. abietinum (Austria and Greece), two North American virus strains of H. occidentale were also identical and two strains from H. parviporum (Finland).

A M 3.0 kb – – HetRV6-oc1

1.0 kb –

BCM 1 2 1 23456M

3.0 kb – – 1.0 kb – 0.7 kb 1.0 kb –

D 76 ORF (RdRp) cd01699 (940 – 1356) 1896

1 2050

Fig. 2. Genomic composition of HetRV6. (A) dsRNA extract from Heterobasidion isolate Het6 analyzed by 3.5% polyacrylamide gel electrophoresis with SYBRGold staining; (B) dsRNAs analyzed by 1% agarose gel electrophoresis (EtBr staining) from isolates 1) 07044, 2) 07057; (C) RT-PCR ampliﬁcation products obtained using the primer pair HV6F1A and HV6Re2 from isolates 1) 04179, 2) 08123, 3) 04193, 4) 07047, 5) 00151, 6) 08134; (D) Genomic organization of the dsRNA encoding the HetRV6 RNA-dependent RNA polymerase (the location of the genomic region similar to the conserved domain cd01699 is indicated in gray). M designates the molecular marker (the GeneRuler 100 bp DNA Ladder Plus, Fermentas GmbH, Germany). 370 E.J. Vainio et al. / Virology 422 (2012) 366–376

Table 3 revealed the same three cases of discrepancy between virus and Heterobasidion isolates containing HetRV6. host taxonomy as described above (for strains HetRV6-an1, HetRV6- Country Host species Isolate Host tree Locality Collectorsa pa5 and HetRV6-pa4). Austria H. abietinum 07044 P. abies Rothwald GU At the amino acid level, many identical sequences were shared be- 07047 P. abies Rothwald GU tween virus strains of H. abietinum and H. parviporum, and the NJ 07052 A. alba Rothwald GU analysis revealed only two highly supported clusters (supplementary 07057 P. abies Rothwald GU Fig. S3). Therefore we selected the nucleotide level analysis for re- 07072 A. alba Rothwald GU 07074 P. abies Rothwald GU solving the evolutionary history of HetRV6. 07077 P. abies Rothwald GU H. parviporum 07069 Unknown Rothwald GU Population structure based on F Estonia H. parviporum 136212 P. abies Vöru KM, MH ST Finland H. annosum 08001 P. abies Karjalohja KK 04057 P. abies Tammisaari KK To analyze population structure, the virus strains were grouped H. parviporum LAP3.1.4 P. abies Lapinjärvi TP into putative subpopulations according to their host species and geo- RT3.45 P. abies Ruotsinkylä TP graphical origin (Table 4a). When grouped according to the fungal 6R121 P. abies Ruotsinkylä TP RKU3.1.37 P. abies Ruotsinkylä TP host species, the virus strains of H. occidentale (USA) clearly separated RK8B P. abies Ruotsinkylä EV, TP from the European strains of H. abietinum, H. annosum and H. parviporum France H. parviporum 00151 Unknown Unknown CD (FST 0.696–0.762). Within Eurasia, the differentiation was considerably Greece H. abietinum 93675 A. cephalonica Parnon Mt PT lower (FST ranged from 0.230 between viruses of H. parviporum and 93381 Fagus sp. Pindos Mt PT H. abietinum to 0.392 between virus strains of H. parviporum and H. annosum 93667 P. nigra Parnon Mt PT Italy H. parviporum 03107 P. abies Trentino KK H. annosum). Poland H. annosum 94245 P. sylvestris Podanin PL A more detailed analysis of smaller geographical and host-related 94253 P. sylvestris Lipka PL groups (Table 4b) suggested a rather moderate level of differentiation Russia H. parviporum 95162 A. sibirica Perm, Preduralye KK between the central European virus strains of H. parviporum and the 95176 A. sibirica Perm KK 00078 A. sibirica Siberia, Altai KK geographically distant Siberian and Russian viruses of H. parviporum, 08123 P. obovata Siberia, Irkutsk KK as well as viruses of H. abietinum from southern Turkey (Akseki) and 08134 P. obovata Siberia, Buryatia KK central Europe (FST values≤0.256). The four virus strains of H. annosum Turkey H. abietinum 04179 A. cilicica Antalya, Akseki TD, AL from Poland and Finland seemed to be more differentiated compared to 04181 A. cilicica Antalya, Akseki TD, AL other populations (F values≥0.475). It must be noted that low levels 04188 A. cilicica Antalya, Akseki TD, AL ST 04193 A. equi-trojani Balikesir, Edremit TD, AL of genetic variation generally lead to higher FST-estimates than high 04075d A. equi-trojani Balikesir, Edremit TD, AL levels of variation (Meirmans, 2006), and therefore the small number USA H. occidentale Het6 A. concolor Oregon DG, EH of virus strains in part of the populations may have led to artiﬁcially Het12 A. concolor Oregon DG, EH high FST values. The nucleotide diversity was only 0.019 among the a CD, C. Delatour; TD, T. Doğmuş-Lehtijärvi; DG, D. Goheen; EH, E. Hansen; MH, M. two H. occidentale viruses, while it was 0.047, 0.048 and 0.067 for virus Hanso; KK, K. Korhonen; PL, P. Lakomy; AL, A. Lehtijärvi; KM, K. Männiste; TP, T. Piri; populations hosted by H. parviporum, H. annosum and H. abietinum, PT, P. Tsopelas; GU, G. Unger; EV, E. Vainio. respectively.

At the nucleotide level, only two virus strains shared identical se- Evidence of purifying selection quences: HetRV6-ab8 and HetRV6-ab10, both from Austrian isolates of H. abietinum. The two North American viruses of H. occidentale The low level of sequence polymorphism observed at protein level shared 98.1% of nucleotide similarity, while their similarity compared compared to nucleotide level indicated that many mutations were to the Eurasian virus strains was 84.3–87.9% and 90.2–93.0% at nucleo- silent, and suggested that purifying selection might be operating on tide and protein level, respectively. The Eurasian virus strains shared HetRV6. We estimated potential selection pressures using the ratio over 88.5% nucleotide similarity and 94.4% protein similarity compared of substitution rates at non-synonymous and synonymous sites, as- b to each other. suming dN/dS 1anddN/dS >1 as hallmark signatures of purifying and positive selection, respectively (Kryazhimskiy and Plotkin, 2008). The ω Evolutionary history based on NJ analysis and haplotype networks average ratio (dN/dS) was 0.054 among HetRV6 strains, suggesting purifying (negative) selection. Moreover, according to the Z-test of se- The NJ analysis of nucleotide sequences revealed clear separation lection, the probability of rejecting the null hypothesis of neutrality b ﬁ of the North American virus strains of H. occidentale from all Eurasian (dN =dS) in favor of purifying selection (dN dS) was signi cant at the − viruses (Fig. 3). Among the Eurasian virus strains, one highly sup- 5% level for all different sequence pairs (the test statistic dN dS ranged − − ported cluster included four viruses of H. annosum from Finland and from 1.78 to 10.36 between different sequence pairs, data not Poland. Another small cluster included three viruses of H. parviporum shown). from Siberia. Also the Finnish, Estonian, Austrian and western Russian virus strains of H. parviporum showed a tendency to cluster together. Between-species transfer of HetRV6 These observations suggest host-related population differentiation for HetRV6. H. abietinum 04188 (hosting HetRV6-ab6) was used as a donor in However, two virus strains of H. parviporum, HetRV6-pa5 from virus transmission experiments. HetRV6-ab6 was successfully trans- Italy and HetRV6-pa4 from France, appeared to be more closely related ferred into both H. annosum (isolate S49-5) and H. parviporum (RK5A). to virus strains of H. abietinum than to the remaining viruses of H. parvi- Single hyphal tip cultures isolated from the recipient mycelia were porum. Moreover, one highly supported taxonomical cluster included found to contain the introduced virus strain, and were analyzed by gen- four virus strains of H. abietinum from Greece, Austria and Turkey, as otyping with mitochondrial and nuclear markers. In both cases, the well as one Greek virus strain of H. annosum (HetRV6-an1). In these recipient hyphal tip isolates had an identical genotype with the original cases, virus taxonomy seemed to disagree with host taxonomy. dsRNA-free isolates and therefore did not appear to have received The Median Joining haplotype network analysis revealed the same nuclear or mitochondrial genetic material from the donor. Examples major clusters as the NJ analysis (supplementary Fig. S2). Notably, it of genetic ﬁngerprints are shown in Fig. 4. E.J. Vainio et al. / Virology 422 (2012) 366–376 371

85 HetRV6-pa16 (RK8B, H. parviporum, Finland) 71 HetRV6-pa17 (136212, H. parviporum, Estonia) 60 HetRV6-pa10 (RKU3.1.37, H. parviporum, Finland) 62 HetRV6-pa9(LAP3.1.4, H. parviporum, Finland) 34 HetRV6-pa12 (6R.121, H. parviporum, Finland) 50 HetRV6-pa11 (RT3.45, H. parviporum, Finland) 77 HetRV6-pa1 (95162, H. parviporum, Russia Perm) 36 HetRV6-pa2 (95176, H. parviporum, Russia Perm) 92 HetRV6-pa6 (07069, H. parviporum, Austria) HetRV6-pa3 (00078, H. parviporum, Russia Altai) 96 HetRV6-pa7 (08123, H. parviporum, Russia Siberia) 97 96 HetRV6-pa8 (08134,H. parviporum, Russia Siberia) 97 HetRV6-ab6 (04188, H. abietinum, Turkey Akseki) 99 HetRV6-ab5 (04181, H. abietinum, Turkey Akseki) HetRV6-ab4 (04179, H. abietinum, Turkey Akseki) 79 HetRV6-pa5 (03107, H. parviporum, Italy) 97 HetRv6-ab9 (07047, H. abietinum, Austria)

74 21 HetRV6-ab14 (07077, H. abietium, Austria) 26 HetRV6-ab3 (04075d, H. abietinum, Turkey Edremit) 23 HetRv6-ab11 (07057, H. abietinum, Austria) 18 HetRV6-pa4 (00151, H. parviporum, France) 98 HetRV6-ab10 (07052, H. abietinum, Austria) 99 HetRV6-ab8 (07044, H. abietinum, Austria) HetRV6-an4 (04057, H. annosum, Finland)

95 HetRV6-an3 (94253, H. annosum, Poland) 100 HetRV6-an5 (08001, H. annosum, Finland) 49 HetRV6-an2 (94245, H. annosum, Poland) HetRV6-an1 (93667, H. annosum, Greece)

100 HetRv6-ab1 (93381, H. abietinum, Greece) 99 HetRV6-ab7 (04193, H. abietinum, Turkey Edremit) 58 HetRv6-ab12 (07072, H. abietinum, Austria) 62 HetRV6-ab13 (07074, H. abietinum, Austria) 45 HetRv6-ab2 (93675, H. abietinum, Greece) HetRV6-oc1 (Het6, H. occidentale, USA) 100 HetRV6-oc2 (Het12, H. occidentale, USA) 0.02

Fig. 3. A Neighbor Joining dendrogram of HetRV6 nucleotide sequences determined in this study. The sequences consisted of 645 nucleotides and they were aligned by ClustalW. The bootstrap values shown at branch nodes were based on 1000 repetitions. The scale bar indicates the number of base substitutions per site computed using the Maximum Composite Likelihood method.

Growth rate tests on agar plates and Norway spruce stem pieces RK5A and H. annosum S49-5), indicating that the virus had persisted in the host mycelia during the incubations. The HetRV6-ab6 virus strain caused variable effects when introduced to H. parviporum or H. annosum (Table 5). When tested on agar Discussion plates, the virus did not seem to cause a major impact on the growth of H. parviporum: its growth decreased by up to 6.7% at 6 °C and in- This study describes a novel putative virus species that is taxo- creased up to 5.5% at 15 °C due to virus infection. However, in H. anno- nomically distant from all previously known viruses of Heterobasidion sum, the virus increased the growth of the host fungus up to 17.8% at species. The virus was assigned as HetRV6 (Heterobasidion RNA virus 6), both incubation temperatures. In Norway spruce stem pieces (billets), and it was found to be the most common dsRNA virus inhabiting HetRV6-ab6 did not show a significant effect on the growth rate of H. H. annosum s. lat., comprising 72.9% of all dsRNA-positive fungal isolates parviporum (Table 5). The virus appeared to increase the growth of H. in our culture collection (279 Heterobasidion spp. isolates from diverse annosum by 5.5%, but neither this result was statistically significant. locations). In one case, a single Heterobasidion isolate harbored both After the agar plate and wood billet assays, the introduced virus could HetRV6 and a partitivirus (Heterobasidion RNA virus 1). Notably, we be detected by RT-PCR from both the recipient isolates (H. parviporum did not find HetRV6 from H. insulare s. lat., and therefore this virus species might be specifictoH. annosum s. lat. Table 4a Based on phylogenetic analysis HetRV6 is only distantly related Population differentiation (FST) between HetRV6 populations grouped according to to previously known viruses, but resembled the Curvularia thermal host species. Number of strains in each population is shown in parenthesis. tolerance virus (CThTV) and F. graminearum virus 4 (FgV4) with ap- Population H. occidentale H. parviporum H. annosum H. abietinum proximately 40% protein level sequence similarity. This taxonomical (host species) affiliation was highly supported by both Bayesian and NJ analysis. H. occidentale (2) – Both CThTV and FgV4 have been described relatively recently H. parviporum (14) 0.762 (Márquez et al., 2007; Yu et al., 2009), and they are yet unassigned H. annosum (5) 0.741 0.392 taxonomically. A low level of sequence similarity was also detected H. abietinum (14) 0.696 0.230 0.326 – for certain fungal partitiviruses (family Partitiviridae) and plant viruses 372 E.J. Vainio et al. / Virology 422 (2012) 366–376

Table 4b

Nucleotide diversity and population differentiation (FST) between HetRV6 subpopulations grouped based on geographical distribution and host species.

a Geographical subpopulation Nucleotide FST diversity North America Siberia Perm Akseki Austria Finland and Finland and Central Europe Greece and Estonia Poland Edremit

North America, H. occidentale (2) 0.019 – Siberia (Russia), H. parviporum (3) 0.037 0.805 Perm (Russia), H. parviporum (2) 0.031 0.814 0.359 Akseki (Turkey), H. abietinum (3) 0.019 0.867 0.599 0.618 Austria, H. abietinum (7) 0.064 0.701 0.325 0.336 0.354 Finland and Estonia, H. parviporum (6) 0.026 0.835 0.416 0.185 0.634 0.354 Finland and Poland, H. annosum (4) 0.022 0.839 0.635 0.604 0.746 0.475 0.662 Central Europe, H. parviporum (3) 0.055 0.733 0.256 0.190 0.283 n/cb 0.229 0.479 Greece and Edremit (Turkey), 0.066 0.704 0.465 0.501 0.508 0.094 0.503 0.542 0.293 – H. abietinum and H. annosum (5)

a Virus strains were divided into subpopulations based on geographical location and host species. Single virus haplotypes (unique sequences) from individual countries were grouped among the geographically closest population (Estonian strain with Finnish ones, Italian and French strains with Austrian ones, and the Greek strain from H. annosum among the remaining Greek isolates (from H. abietinum)). Some small populations were also combined as they showed no differentiation compared to each other: strains from H. annosum (Finland and Poland) were grouped together, as were the Greek strains and Turkish strains from Edremit (Aegean coast of Turkey, near Greece). The number of strains in each subpopulation is shown in parenthesis. b n/c=FST equal to zero (no differentiation). of the genus Potyvirus (family Potyviridae). The plant Potyviridae have ‘satellite’ elements (Ghabrial et al., 2008). However, based on phyloge- ssRNA genomes with monopartite genomes of ca. 10 kb, coding for netic analysis, HetRV6 and the affiliated taxa clearly clustered outside ten mature proteins (Adams et al., 2005). Therefore their genomic orga- the four recognized subgroups of Partitiviridae. Therefore, HetRV6, nization differs considerably from HetRV6. On the other hand, the size FgV4 and CThTV might be considered as members of a new, yet unas- of the polymerase gene in HetRV6 resembles that of many partiti- signed virus family. viruses. Partitiviruses have a bipartite genome encoding genes for Presently, the only known mycoviruses that code for a single gene polymerase and capsid proteins, and sometimes contain additional (RdRp) are members of the family Narnaviridae (Ghabrial and Suzuki, 2009). They occur in yeasts and filamentous fungi and have ssRNA genomes residing in the cytoplasm (genus Narnavirus) or mitochon- A M12 3 dria (genus Mitovirus). Narnaviruses characteristically have strong secondary structures at their 5′-end in order to evade host nucleases (Esteban et al., 2008), while many mitoviruses have inverted complementary in their terminal sequences, with the potential of forming secondary ‘panhandle’ structures (Hong et al., 1999). The 3′-terminal UTR of HetRV6 could be folded into a potentially stable stem-loop structure, and putative panhandle structures were also indicated by secondary structure analysis. However, as no sequence similarity was detected between HetRV6 and members of Narnaviridae, they seem to be phylogenetically distant. The population structure of HetRV6 shows an interesting correla- tion to that of the host fungi. The global level of sequence variation in HetRV6 can be considered rather moderate (Van Regenmortel, 2007), suggesting that the different strains represent a monophyletic taxon. However, based on phylogenetic clustering and analysis of population differentiation, the North American viruses of H. occidentale clearly differed from the Eurasian virus strains. This is in accordance with the assumption that different strains of this virus species have co- evolved with their geographically separated host populations, and transmission from other fungal genera has not occurred. In Eurasia, a lower level of differentiation between HetRV6 populations was observed. Most of the differentiation seemed to be host- B 12 3 M related, and some geographical differentiation could also be observed. The analysis of population differentiation tentatively suggested more gene flow between virus populations hosted by the closely related H. abietinum and H. parviporum, but lesser amount of gene flow between viruses of the more distantly related H. annosum and H. parviporum or H. abietinum. Among the central and southern European virus strains we also observed three cases where virus taxonomy did not seem to follow the taxonomy of the host fungi. Thus, two virus strains of H. parviporum and one virus of H. annosum grouped among viruses of Fig. 4. Examples of genetic fingerprint patterns of the Heterobasidion isolates used in H. abietinum. These observations suggest that HetRV6 is capable of in- the virus transmission experiment. (A) M13 minisatellite fingerprints. (B) Mitochondrial terspecies transfer in central Europe where three Heterobasidion host LSU fragments. Lanes: 1) virus-positive H. annosum S49-5 (HetRV6-ab6-infected), 2) H. species occur sympatrically. Similarly, Buck et al. (2003) found that annosum S49-5 (original virus-free recipient isolate), 3) H. abietinum 04188 (donor isolate, HetRV6-ab6-infected), M) the GeneRuler 100 bp DNA Ladder Plus (Fermentas GmbH, Ophiostoma mitovirus strains (OMV5) clustered on a geographical Germany). basis rather than according to the fungal host species, and Liu et al. E.J. Vainio et al. / Virology 422 (2012) 366–376 373

Table 5 The effect of HetRV6-ab6 on the growth rate of H. parviporum and H. annosum on MOS agar plates at two different temperatures, and in stem pieces of Norway spruce.

Fungal strain 6 °C MOS agar plates 15 °C MOS agar plates Spruce wood (average 11.6 °C) 1st repetition 2nd repetition 1st repetition 2nd repetition

H. parviporum −6.7%a (P>0.05) −3.6% (P>0.05) 3.8% (P>0.05) 5.5% (P>0.05) 0% (P>0.05) RK5A (HetRV6-ab6-infected) 12.01±2.49b 13.30±0.82b 15.56±1.16d 13.92±1.22d 9.16±3.41e RK5A (virus-free) 12.88±1.76b 13.80±0.99b 14.99±1.05d 13.20±1.44d 9.16±3.32e ⁎ ⁎ c ⁎ c ⁎ H. annosum 5.3% (Pb0.01) 17.1% (Pb0.001) 12.4% (Pb0.001) 17.8% (Pb0.001) 5,5% (P>0.05) S49-5 (HetRV6-ab6-infected) 20.30±1.15c 23.10±1.53c 13.33±1.28d 14.25±1.28d 10.61±1.50e S49-5 (virus-free) 19.27±1.16c 19.72±1.21c 11.85±0.98d 12.10±1.59d 10.05±1.05e

a Percentage growth difference between the isogenic virus-infected and virus-free strains. The mean growth was measured at the exponential growth phase at the following time points, determined based on the growth of the fungus after an initial incubation period of 3–13 days. b Mean radial growth (in mm) during 10 days of incubation. c Mean radial growth (in mm) during 14 days of incubation. d Mean radial growth (in mm) during 4 days of incubation. e The mean number of wood disks colonized by the inoculated fungal strain (the thickness of each wood disk was ca. 1.5 cm). ⁎ Statistically signiﬁcant values, independent T-test, Pb0.05.

(2003) described that Cryphonectria hypovirus 1 (CHV-1) strains 2009). As viruses are not present in every hyphal tip or spore of a were more closely related between different Cryphonectria species virus-infected Heterobasidion isolate (Ihrmark et al., 2002, 2004), we than within a single host species in Japan. preferred tissue isolates instead of single spore isolates for virus screen- Further evidence for interspecies transfer of HetRV6 was obtained by ing. The mycelial cultures had been isolated from wood, sporocarp tis- laboratory experiments: we were able to transmit HetRV6 from sue or multiple spores, and were mostly heterokaryotic (the H. abietinum to H. annosum and H. parviporum by hyphal contacts. Previ- percentage of suspect homokaryons were 2%, 3% and 10% in H. parvi- ously it has been shown, that the H. annosum virus (HaV), Heterobasidion porum, H. annosum and H. abietinum, respectively). The virus-infected RNA virus 2 (HetRV2) and Heterobasidion RNA virus 3 (HetRV3) can be isolates were collected during years 1993–2009, and the fungal isolates transmitted between heterokaryotic, incompatible isolates belonging had been stored at 4 °C on malt extract agar slants. to different species of Heterobasidion (Ihrmark et al., 2002; Vainio et al., 2010, 2011a, 2011b). In many fungal species, interspecies virus trans- Extraction of dsRNA by CF11 chromatography fer is limited by vegetative incompatibility (Ghabrial and Suzuki, 2009). However, infection experiments have shown interspecies virus transfer The presence of dsRNA in the Heterobasidion isolates was tested also within the fungal genera Aspergillus (Coenen et al., 1997), Sclerotinia using the CF11 cellulose affinity chromatography method of Morris (Melzer et al., 2002)andCryphonectria (Liu et al., 2003). and Dodds (1979), as modified by Tuomivirta and Hantula (2003), Although the similarity between HetRV6 and CThTV is relatively and Vainio et al. (2010). Briefly, the protocol included homogeniza- low and they are clearly two distinct species, it is noteworthy that tion of mycelia in lysis buffer, followed by phenol and chloroform the latter virus is the only truly symbiotic mycovirus species de- extractions and specific precipitation of dsRNA using CF11 cellulose scribed thus far. We investigated possible effects of HetRV6 on its fungal powder in 15% ethanol. The dsRNA profiles were analyzed by agarose host by comparing the growth of isogenic virus-infected and virus-free gel electrophoresis. Cellophane-covered MOS agar plates (Müller et isolates on agar plates and wood billets. Overall, HetRV6 seemed to al., 1994) were used for the cultivation of fungal mycelia. be cryptic or slightly mutualistic, and its effects on growth were dependent on incubation temperature and also on the host fungal isolate. Complementary DNA synthesis, cloning and sequencing Similarly, Bryner and Rigling (2011) recently described that hypoviruses mediated temperature-dependent effects on the growth of Three dsRNA-containing Heterobasidion isolates (04188, 07052 C. parasitica. The performance of the fungal host might also be influenced and Het6) were selected for cDNA synthesis using the single primer by uneven distribution of the virus in the host fungal mycelium, as has amplification technique of Lambden et al. (1992) as described earlier been described for viruses of R. necatrix (Yaegashi et al., 2011). (Tuomivirta and Hantula, 2003; Vainio et al., 2011a). Briefly, 10–17 g In conclusion, we have characterized a novel, cryptic or slightly of mycelia from each isolate was harvested and pooled for CF11 chro- mutualistic mycovirus, which is somewhat similar to the Curvularia matography, after which 3′-amino-linked T4 RNA adapters (5′-P-GCA thermal tolerance virus and the F. graminearum virus 4, with whom TTC GAC CCC GGG TT-NH2C3-3′) were ligated to the 3′ ends of the it forms a distinct clade from Partitiviridae and Potyviridae. The virus dsRNA segments in order to enable sequencing of the distal ends of showed some geographical and host-related differentiation, and evi- the viral genome. Reverse transcription was conducted using random dence of horizontal transmission between sympatric Heterobasidion hexamers, the T4 RNA-primer (5′-AAC CCG GGT CGA ATG C-3′) and host populations. RevertAid H minus M-MuLV reverse transcriptase (Fermentas GmbH, Germany) or the Revert Aid Premium reverse transcriptase (Fermentas) Material and methods as recommended by the manufacturer. The resulting cDNAs were used for PCR amplification with the T4 RNA-primer and the High Fidelity Fungal isolates PCR enzyme mix (Fermentas) at two different cycling conditions as described earlier (Vainio et al., 2011a). Amplicons in the range of ca. Altogether 279 Heterobasidion spp. isolates were screened for the 500–2000 bp were purified after agarose gel electrophoresis using presence of dsRNA using CF11 chromatography. The culture collec- the E.Z.N.A gel extraction spin protocol kit (Omega Bio-Tek, Georgia, tion included 82 isolates of H. parviporum, 65 isolates of H. annosum, USA), and cloned into the pCR2.1-TOPO cloning vector as instructed 78 isolates of H. abietinum, 14 isolates of H. occidentale, and 40 isolates in the TOPO TA Cloning Kit (Invitrogen, Carlsbad, California, USA). of H. insulare s. lat. (Heterobasidion australe, Heterobasidion ecrustosum, Isolates 04188 and Het6 were also analyzed using a second cloning Heterobasidion orientale or Heterobasidion linzhiense). The locality data approach. The first strand cDNAs were used as templates for second for virus-positive isolates is listed in Table 3, and detailed collection strand synthesis according to the protocol described in the SuperScript data for virus-free isolates is reported in supplementary Table S1 (the Plasmid System for cDNA Synthesis and Plasmid Cloning kit (Life Tech- Austrian host isolates have been previously described in Unger et al., nologies Inc., Rockville, MD, USA). The protocol includes second strand 374 E.J. Vainio et al. / Virology 422 (2012) 366–376 synthesis using Escherichia coli DNA polymerase I, E. coli DNA ligase and For phylogenetic analysis, an amino acid sequence alignment includ- E. coli RNase H, followed by the ligation of SalI-linkers, and PCR ing sequences of HetRV6-ab6, HetRV6-ab10 and HetRV6-oc1, and a selec- amplification with a complementary primer (Vainio et al., 2010). The tion of affiliated virus species was generated using MAFFT version 6.717b resulting amplicons were gel purified and cloned into the pCR2.1- (Katoh et al., 2002; Geneious Pro 5.3, Biomatters Ltd., New Zealand). TOPO cloning vector as described above. The alignment was edited manually according to the conserved motifs Sequencing of the cloned inserts was conducted with the 4200L-2 of RNA-dependent RNA polymerases (RdRps) as determined by Bruenn NEN Global IR2 System (LI-COR Inc., NE, USA) using the SequiTherm (1993). Due to low level of similarity between the sequences (approx- EXCELII sequencing kit (Epicentre Biotechnologies, WI, USA), or at imately 22.7% overall pairwise identity), only the most conserved se- Macrogen Inc., South Korea (www.macrogen.com) using the Applied quence regions were included (the alignment overlaps Bruenn's Biosystems 96-capillary ABI 3730xl DNA analyzer. Based on primary conserved motifs 3–6 that can be recognized also in the ssRNA plant sequence data, specific primers were designed to allow amplification Potyviridae sequences, supplementary Fig. S1). The resulting alignment of sequence positions with insufficient coverage as well as the 5′- and was used for dendrogram construction by Bayesian clustering using the 3′-proximal sequence ends (supplementary Table S2). As a result, MrBayesPlugin of Geneious Pro 5.3 (Huelsenbeck and Ronquist, 2001) each sequence position was covered by at least three cloned inserts and Neighbor Joining clustering using MEGA (Molecular Evolutionary or two overlapping direct PCR products with identical sequence. Genetics Analysis) software version 5 (Tamura et al., 2007). The yeast L-A virus (Totiviridae) was used as an outgroup. The Bayesian analyses were conducted with the gamma among-site rate variation and Consensus primers, RT-PCR and partial sequence determination 1.1×106 cycles for the MCMC algorithm, sampling 1 tree per 200 cycles, and discarding 105 samples as burn-in. The Blosum matrix was selected Consensus primers were designed based on conservative sequence as the protein evolution model based on lowest Bayesian Information regions in the dsRNAs of H. abietinum 04188 and 07052, potentially Criterion (BIC) score obtained by ProtTest (Abascal et al., 2005). We allowing PCR amplification from these and related viruses. The consen- also conducted Bayesian analyses using the Poisson and Equalin models, sus PCR primers HV6F1A (TTG AAT CAC CTG GAC CGT TT) and HV6Re2 as they are not included in the ProtTest software. The Blosum model is a (CAT CAA CCC ATT ATC CAG GT) were expected to amplify a 714 bp fixed rate matrix with substitution rates based on inferred amino acid fragment of the HetRV6 polymerase gene (Table S2). changes from various databases (Huelsenbeck et al., 2008). The Poisson Reverse transcription (RT) was conducted using total RNA samples model is a fixed amino acid model that assumes equal stationary state prepared with the E.Z.N.A Fungal RNA Miniprep kit (Omega Bio-Tek frequencies and equal substitution rates, while the Equalin model Inc. GA, USA). The RT reaction contained 12 μl of total RNA (denatured allows the stationary state frequencies of all amino acids to be different, in boiling water-bath for 5 min and chilled at −80 °C), 0.2 μgofrandom but assumes the same substitution rate (Ronquist et al., 2005). hexamer primer (Fermentas GmbH), 200 U of RevertAid M-MuLV reverse transcriptase (Fermentas GmbH), and dNTPs and buffer as Population genetics recommended by the manufacturer. The PCRs were conducted in 50 μl reaction volumes, including 2 μl The partial HetRV6 sequences were aligned using the ClustalW of the cDNA product, 25 pmol of each primer (HV6F1A and HV6Re2), alignment of MEGA 5 (Tamura et al., 2007). Alignments were con- 1 U of Dynazyme™ II DNA polymerase (Finnzymes, Finland), and ducted both at nucleotide and amino acid level, and they consisted 10 nmol of dNTPs (10 min at 95 °C and 30–35 cycles of: 30 s at of 645 nucleotide characters or 215 amino acid characters without 95 °C, 45 s at 55 °C, 2 min at 72 °C; a final extension of 7 min at 72 °C). any indels. Dendrograms were constructed using the Neighbor Joining The RT-PCR products were used for partial sequence determina- algorithm implemented in MEGA 5. The nucleotide alignment was tion in both orientations using HV6F1A and HV6Re2 as sequencing analyzed by the Maximum Composite Likelihood method, and the primers. The products of two independent PCR reactions were used amino acid alignment using the Poisson correction method (uniform for sequencing from each virus strain, and a third sequencing reaction rates among sites). The optimal trees were statistically evaluated by was carried out in cases of discrepancy between the two sequences. bootstrap analysis with 1000 replications. In seven cases (fungal isolates 95176, 00151, 03107, 04057, 04075d, Median Joining haplotype networks were constructed for the 07074 and Het6), two independent cDNA samples were prepared HetRV6 nucleotide sequences using the Network 4.6.0.0 Program and used for RT-PCR and sequencing to investigate whether the RT (Fluxus Technology Ltd; fluxus-engineering.com; Bandelt et al., 1999). reaction was prone to errors. As an identical sequence was obtained The analysis was conducted with equal weight of characters (10), and from the two corresponding cDNAs in all cases, the remaining sequences ε-value of zero (only minimum length connections were taken into were determined using multiple PCRs from a single cDNA sample from account). each strain. The sequences determined in this study have been depos- Nucleotide diversity (the average number of nucleotide differ- ited in the Genetic sequence database at the National Center for Bio- ences per site between two sequences) was determined with DnaSP technical Information (NCBI) (GenBank ID: HQ189459–HQ189462, version 5 (Librado and Rozas, 2009). Sequence similarity percentages and HQ189465–HQ189495). were calculated by pairwise sequence distance calculation using The possibility of mixed (double) virus infections was investigated MEGA 5 and the Geneious software. by RT-PCR using a recently described consensus primer pair (SConF1 Genetic differentiation among putative HetRV6 subpopulations and SConRL) that detects three different partitivirus species of Hetero- was estimated by F genetic distances as determined for nucleotide basidion (HetRV1, HetRV4 and HetRV5; Vainio et al., 2011b). ST sequences using the DnaSP 5 program. The FST summary statistic is dependent on the amount of within-population genetic variation, Sequence analysis and phylogenetic inference and ranges from zero (no population structure) to one (completely differentiated populations; Meirmans, 2006). Open reading frames were determined using the NCBI ORF Finder The role of selection was investigated by estimating the average program (http://www.ncbi.nlm.nih.gov/gorf/gorf.html). The deduced number of nonsynonymous substitutions per nonsynonymous site amino acid sequences were analyzed by NCBI Protein Blast to search (dN) and synonymous substitutions per synonymous site (dS) for similar sequences and conserved domains. Putative secondary according to the Nei–Gojobori model implemented in the DnaSP pro- structures were analyzed using the RNAstructure program version gram (overall ratio ω = dN/dS). The codon-based Z-test of selection 5.3 with maximum percentage energy difference of 10 and standard was used for statistical testing of the hypotheses of neutrality free energies at 37 °C in kcal/mol. (dN=dS), purifying selection (dN>dS) and positive selection E.J. Vainio et al. / Virology 422 (2012) 366–376 375

(dNbdS), as implemented in MEGA 5. The Z-test was conducted in Growth rate tests in Norway spruce wood sequence pairs using the Nei–Gojobori (p-distance) model, and vari- ances of the differences were computed with the bootstrap method Growth rates of the isogenic virus-infected and virus-free isolates and 1000 replicates. of H. parviporum RK5A and H. annosum S49-5 were investigated by inoculating them on stem pieces (billets) of Norway spruce (P. abies). The billets were cut from two trees; their length was 40 cm and diame- Virus infection experiments ter ca. 20 cm. The inoculations were made by placing pre-grown mycelial agar plugs (3 cm×0.5 cm) on the top of the billets. Each billet was H. abietinum 04188, hosting HetRV6-ab6, was used as a virus divided in six sectors and treated with three replicate inoculations of a donor, and a collection of dsRNA-free isolates of H. annosum and virus-infected and three inoculations of a virus-free isolate (RK5A or H. parviporum were used as recipients. The following virus-free iso- S49-5). The experiments were conducted in six billets, thus resulting lates were tested: H. parviporum LAP3.1.1, RK5A, RK6B, RK13A, in eighteen parallel repetitions for each isolate. The billets were incubat- RK15A, RK19A, RKU3.1.26, RKU3.2.34 and 7R15; and H. annosum ed on moist sand outdoors in half-shadow. Incubation time was K41-8, S12-2, K19-4, Y1.93, S49-5 and T60-9. All the recipient isolates 1 month and average air temperature during that time was 11.6 °C. had been collected as tissue cultures from decayed wood by T. Piri, After the experiment, the upper parts of the billets were sliced into E. Vainio or J. Hantula from Lapinjärvi, Läyliäinen or Ruotsinkylä in 1.5 cm thick disks, washed in running tap water, and incubated in plas- southern Finland. The isolates were deemed dsRNA-free with both tic bags at room temperature for 1 week. The presence of Heterobasidion CF11 chromatography and RT-PCR. conidiophores on each disk was investigated under a dissecting micro- The infection experiments were conducted as described before scope. The effect of the virus was interpreted as a relative difference in (Ihrmark et al., 2002; Vainio et al., 2010). Briefly, the dsRNA-containing the growth rate between the virus-infected and virus-free strain. The donor isolate and the virus-free recipient were inoculated on the same statistical significance was assessed by T-test in Microsoft Excel 2007. malt agar plate, and incubated for 3 months at 20 °C. Hyphal samples The persistence of HetRV6-ab6 during the experiment was examined were then taken from the recipient side of the culture and subcultured using random samples taken from the wood disks and screened for on MOS plates. Potential dsRNA transmission into the recipient subcul- the presence of HetRV6 as described above. tures was investigated by RT-PCR with specificprimers(HV6F1Aand HV6Re2). Single hyphal tip cultures were isolated from two recipient Acknowledgments isolates found to be virus-positive (H. parviporum RK5A and H. annosum S49-5) in order to select against any contaminating donor hyphae. Pres- This study was financially supported by the Academy of Finland ence of the virus in the hyphal tip cultures was confirmed by RT-PCR. (Project 122565) and the Finnish Forest Research Institute. We thank To confirm that the hyphal tip isolates were identical in genotype (iso- Ms. Maria de las Nieves Lorenzo Gotor, Ms. Marja-Leena Santanen, genic) with the original recipient isolates, and had not received genetic Ms. Sonja Sarsila, and Ms. Sanna Paakkinen for skillful technical assis- material from the donor, their genotypes were determined using mito- tance. The collectors of the Heterobasidion isolates (Y.-C. Dai, C. Delatour, chondrial markers (ML1 and ML2; White et al., 1990, or Mito5, MLS and D. Goheen, M. Hanso, A. Kanaskie, P. Lakomy, K. Männiste, P. Tsopelas, MLF; Garbelotto et al., 1998), as well as multilocus nuclear genotyping G. Unger) are also gratefully acknowledged. with the M13 minisatellite primer (Stenlid et al., 1994), and the RAMS-primers CGA and GAG (Hantula et al., 1996). The DNA samples Appendix A. Supplementary data for genotype determination were extracted using the E.Z.N.A SP Fungal DNA kit (Omega Bio-Tek Inc., GA, USA), and all PCR reactions were Supplementary data to this article can be found online at doi:10. repeated at least twice. 1016/j.virol.2011.10.032.

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