Wulf Menzel

Complete genome sequence of the first endornavirus from the ascocarp of the ectomycorrhizal fungus Tuber aestivum Vittad.

The genus Endornavirus was recently approved by the International Committee on Taxonomy of Viruses (ICTV) [1, 10] to be separated from the family Partitiviridae, and endornaviruses have only been described in plants and fungi [4, 15]. They share common properties such as symptom-free infection of their host, occurrence in lowcopy number in host cells, lack of true virions [7], and an efficient vertical transmission in their hosts [16]. Endornaviruses have unique plasmid-like properties that differ markedly from those of other conventionally encapsidated viruses [5, 12, 16]. The genomes of endornaviruses consist of linear double-stranded RNA (dsRNA) with a characteristic, single open reading frame (ORF) of up to 18 kb in length, often preceded by a site-specific nick at the 5’ end [5, 7, 8, 12, 18]. The predicted protein from this ORF harbours in its N-terminal part characteristic amino acid (aa) sequence motifs for viral RNA helicases (VHel) or viral methyltransferases (VMet), while aa sequence motifs for UDP-glycosyltransferases (UGT) and RNA-dependent RNA polymerases (RdRp) are located at close proximity to the 30 end [7]. The remaining polyprotein is unrelated to any known functional proteins in the database. Currently, the INSDC databases contain complete sequences of seven endornaviruses. Three are plant-associated viruses infecting cultivated and wild rice (Oryza sativa endornavirus [OsEV] and Oryza rufipogon endornavirus [OrEV]) [5, 11], respectively), as well as broad bean (Vicia faba endornavirus [VFV]) [13]. In addition, four fungal isolates are known. One isolate each was reported from the mycelia of the violet root rot fungus (Helicobasidium mompa endornavirus 1-670 [HmEV1-670]) [12] and from an informally designated plant-pathogenic oomycete Phytophtora spp. (Phytophtora endornavirus 1 [PEV1]) [6], while two isolates originate from the pathogenic ascomycete Gremmeniella abietina (Gremmeniella abietina type B RNA virus) [15]. Here, we report the complete nucleotide sequence of an isolate of a putatively novel species in the genus Endornavirus from an ectomycorrhizal fungus, isolated from the hypogeous ascomata of a black truffle (Tuber aestivum Vittad.). Accordingly, we propose the name Tuber aestivum endornavirus (TaEV). The TaEV genome structure is highly similar to that of other Ascomycetes-infecting endornaviruses, such as Gremmeniella abietina type B RNA virus [15], containing characteristic motifs for the DEAD-like helicase (DEXDc), VMet and RdRp.

Complete nucleotide sequence of TaV1, a novel totivirus isolated from a black truffle ascocarp (Tuber aestivum Vittad.)

New viruses associated with fungi are discovered frequently and have been reported since the early 1960s in members of numerous fungal taxa [8, 9, 12–14, 16]. Here, we report the complete genome sequence of the first virus infecting an ectomycorrhizal fungus. Mycoviruses, most of which have double-stranded (ds) RNA genomes, are currently not known to have any natural vectors. They are transmitted intracellularly by anastomosis of the hyphae or by heterokaryosis (lateral transmission), or they are spread via spores (serial transmission) [6, 7]. Cytoplasmatic exchange in different periods of the fungal life-cycle allows highly efficient virus transmission via different types of fungal propagules, such as sexually and asexually produced spores. Virus-infected fungi may show symptoms such as abnormal colony morphology or sporocarps [7] but in most cases remain symptomless. In contrast, mycoviruses can be responsible for attenuation and enhancement of fungal virulence itself (hypoand hypervirulence), especially in the well-studied group of phytopathogenic fungi [2, 7, 15]. The commercial importance of several saprophytic and phytopathogenic fungi, such as Cryphonectria parasitica, which causes chestnut blight; Ustilago maydis, which causes corn smut, Botrytis cineria; which causes spoilage of strawberries and grapes; and the commonly important white mushroom, Agaricus bisporus, has consequently focused research on commercially relevant fungi to develop biocontrol agents to prevent massive loss of crop yields [10, 15, 19, 21]. In contrast, the mycorrhizas, which have a profound impact on the colonization of terrestrial life, have so far been screened only rudimentarily for the presence of viruses. Considering the extraordinary number of mycorrhizal fungi, it seems odd that ectomycorrhizal macrofungi, which are present in almost any global forest ecosystem, have received much less attention as a source of viruses than other organisms. Here, we report the entire genome sequence of a putatively new totivirus. The virus reported here was isolated from the ascocarp of the hypogeous ectomycorrhizal fungus Tuber aestivum, which is commonly known as black noble truffle, and the name Tuber aestivum virus 1 (TaV1) is proposed. Accordingly, these findings provide the first evidence for the presence of mycoviruses in ectomycorrhizal fungi belonging to the largest fungal phylum (Ascomycota). However, Bai et al. [1] were the first to isolate a smaller dsRNA of 2.2 kbp from the ectomycorrhizal basidomycete Hebeloma circinans (Cortinariaceae). The genomes of members of the family Totiviridae typically contain two open reading frames (ORFs) that can be translated as a single fusion-protein by a -1 frameshift. The predicted proteins encoded by ORF1 and ORF2 show sequence motifs characteristic of viral coat proteins (CP) and viral RNA-dependent RNA polymerases (RdRp), respectively.

Molecular characterization and taxonomy of grapevine leafroll-associated virus 7

The complete nucleotide sequence of an Albanian isolate of grapevine leafroll-associated virus 7 (GLRaV-7-Alb) was determined. The viral genome consists of 16,404 nucleotides and has nine open reading frames (ORFs) that potentially encode proteins, most of which are typical for members of the family Closteroviridae. Only the 25-kDa (ORF8) and 27-kDa (ORF9) proteins had no apparent similarity to other viral proteins in the sequence databases. The genome structure of GLRaV-7-Alb closely resembles that of little cherry virus 1 and cordyline virus 1. In phylogenetic trees constructed with HSP70h sequences, these three viruses cluster together in a clade next to that comprising members of the genus Crinivirus, to which they are more closely related than to the clostero- and ampeloviruses. The molecular properties of these three viruses differ sufficiently from those of members of the three extant genera of the family Closteroviridae to warrant their classification in a novel genus.

A novel Tuber aestivum (Vittad.) mitovirus.

Double-stranded RNA (dsRNA) viruses have been reported from major phyla of the fungal kingdom [8, 11]. Mycovirus infections have been associated with obvious disease symptoms but often remain symptomless [3, 8, 11]. Fungal viruses are known from eleven families: Barnaviridae, Birnaviridae, Chrysoviridae, Cystoviridae, Hypoviridae, Metaviridae, Narnaviridae, Partitiviridae, Reoviridae, Totiviridae and recently also Endornaviridae [1, 8, 11, 14, 15]. The simplest mycoviruses are assigned to the genera Mitovirus and Narnavirus (family Narnaviridae). Members of both genera lack coat proteins, and their linear genomes are characterized by a single open reading frame (ORF) coding for a viral RNA-dependent RNA polymerase (RdRp). Mitoviruses, in contrast to cytoplasmatic narnaviruses, seem to replicate in strict association with mitochondria [2, 8, 11, 12]. Common characteristic features of all mitochondrial viruses are 50and 30-terminal untranslated regions (UTRs) of variable length. It has been suggested that these terminal residues act as stem-loop structures for RdRp recognition and initiation of replication [3, 4]. Mitoviruses have attracted significant attention, as they trigger fungal hypovirulence and phenotypic changes such as reduced growth, sporulation and pigmentation [3–6, 13, 20]. Most mitoviruses are known from phytopathogenic fungal genera only, such as Ophiostoma, Cryphonectria, and Botrytis, and a few from Gremmeniella, Sclerotinia, Thanatephorus, Thielaviopsis, Helicobasidium and the arbuscular mycorrhizae Glomus [4–6, 11, 19–21]. The development of biological control agents against fungal pathogens using mycoviruses has attracted significant scientific attention, whereas the exploration of virus diversity in non-pathogenic fungi has been widely neglected [15, 16]. In this paper, we report the third known virus infecting the ectomycorrhizal fungus Tuber aestivum Vittad. The genome described here is the largest one known in the genus Mitovirus, and based on its host, the name Tuber aestivum mitovirus (TaMV) is proposed. Genome characteristics are highly similar to those of other known mitoviruses, including a single ORF encoding a viral RdRp as well as typical 50 and 30 UTRs.

A novel mitovirus from the hypogeous ectomycorrhizal fungus Tuber excavatum

The evolution of mycorrhizal mutualism had a profound impact on establishment of terrestrial life [19]. Fossil records indicate that the earliest land plants, which had no proper roots, were already colonized by hyphal fungi, which formed vesicles and arbuscules strikingly similar to modern vesicular-arbuscular mycorrhizas. Today, over 90% of plants form mycorrhizal associations [19]. Although saprotrophic fungi play a central role in the forest ecosystem by cycling, translocating and sequestrating decomposed material, mycorrhizal fungi render inorganic decomposed litter accessible to their plant hosts. In return for inorganics, they receive organic compounds, in particular from coniferous and deciduous trees [14, 19]. In spite of their ecological importance, ectomycorrhizal fungi have largely been ignored by virologists interested in studying mycoviruses [10, 20–22]. Such viruses infecting a broad range of fungi may show a high degree of specialization to their hosts, since some genera exclusively infect fungi, whereas others associated with certain virus families infect multior unicellular organisms [10]. Mycoviruses have been reported from almost the entire range of fungal phyla [9, 15, 17] and possess single-stranded (ssRNA) genomes (genera Botrexvirus, Mycoflexivirus; families Narnaviridae, Endornaviridae, Hypoviridae) [9, 11, 12] or doublestranded (dsRNA) genomes (families Barnaviridae, Birnaviridae, Chrysoviridae, Cystoviridae, Metaviridae, Partitiviridae, Pseudoviridae, Reoviridae and Totiviridae). Members of the Birnaviridae and Cystoviridae are reported to infect fungal hosts [9, 15, 21], but they are not listed to infect fungi in the current ICTV master species list 2009 (version 9; http://talk.ictvonline.org/files/ictv_documents/m/msl/1231.aspx) [7, 10]. Until recently, the family Narnaviridae contained viruses exclusively found in fungi [1]. Since Narnaviridae contains records recently added to the INSDC database of sequences derived from plant material (e.g., ‘grapevine associated narnavirus-1’; GenBank accession GU108586), an alternative explanation for these findings would be that these sequences were actually derived from endophytic filamentous fungi and yeast associated with the plant tissues, which has also been suggested and partially proven by Al Rwahnih et al. [1]. Moreover, given the fact that true plant-pathogenic viruses of the genus Ourmiavirus have been proposed to belong to the family Narnaviridae challanges the current classification of virus genera and the host specificity of members of the family Narnaviridae [18]. The genera Mitovirus and Narnavirus include the simplest mycoviruses, which lack coat proteins and are characterized by a single open reading frame (ORF) coding for a viral RNA-directed RNA polymerase (RdRp) [2–6, 16]. Mitoviruses are strictly associated with mitochondria and possess characteristic 50and 30-terminal untranslated regions (UTRs) of variable length, which are most likely responsible for replicase recognition [3, 4]. While most mitoviruses are found in plant-pathogenic fungi, such as Ophiostoma, Chrysoporthe or Botrytis, members of a small J. B. Stielow CBS-KNAW Fungal Biodiversity Centre, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands

Complete nucleotide sequence of the type isolate of Cowpea mild mottle virus from Ghana

Cowpea mild mottle virus (CPMMV) was first reported to cause systemic mottling, chlorotic blotches and leaf malformations in cowpea (Vigna unguiculata L.) in 1973 [5] and to be widespread in the Eastern Region of Ghana, West Africa. Since then, it has been described to naturally infect other leguminous crops such as peanut (Arachis hypogaea L.), soybean (Glycine max L.) and common bean (Phaseolus vulgaris L.) [19]. CPMMV symptoms are thought to vary seasonally. On peanut, it might cause systemic chlorosis, chlorotic rings, leaf rolling or veinal necrosis, and on soybean, it is known to cause chlorosis, vein mosaic, and apical necrosis or other malformations [5, 12, 13, 15]. CPMMV has flexuous filamentous particles (approx. 650 nm in length) resembling those of members of the genus Carlavirus. In contrast to the vast majority of carlaviruses, which are aphid-transmitted, CPMMV is transmitted in a non-persistent manner by the whitefly Bemisia tabaci G. [12, 17] and has also been assigned to the genus Carlavirus [2]. It differs from aphid-borne carlaviruses also in its intracellular occurrence in its host plants [6]. In accordance with other carlaviruses infecting members of the family Fabaceae, such as pea streak virus (PeSV) and red clover vein mosaic virus (RCVMV), CPMMV is thought to be seed-borne in a number of its leguminous hosts, even if there have been contradictory reports [5, 11, 12]. The experimental host range of CPMMV includes plant species from several families, which mainly display chlorotic local lesions or systemic mottle upon inoculation/ infection. Since the initial reports from Africa [5, 20], the virus has been discovered in India [13], Thailand [12] and South America [8, 19]. A distinct isolate responsible for pale chlorosis disease in tomato (Solanum lycopersicum L.) was reported from Israel [3, 7, 14]. Comparisons of a tomato pale chlorosis isolate and an eggplant (Solanum melongena L.) isolate from Jordan that causes a mild leaf mosaic with legume isolates from India and West Africa showed that the isolates from solanaceous hosts are very similar to, but clearly distinct from, the legume isolates [15]. The first published nucleotide sequence information about CPMMV (not available in GenBank) was a 30-terminal 0.9-kb fragment of a West African cowpea isolate collected in the eastern region of Ghana [4]. It was generated using a universal primer for carlaviruses and supported the assignment of CPMMV to the genus Carlavirus. Since then, six 30-terminal partial sequences covering 0.9 kb [9] to 2.5 kb [18] have been submitted to GenBank. In this paper, we report the first complete genome sequence of a CPMMV isolate, which is probably the same (type) isolate as that partially sequenced by Badge et al. [4].

Analysis of the tomato mild mottle virus genome indicates that it is the most divergent member of the genus Ipomovirus (family Potyviridae)

The complete genome of a tomato mild mottle virus (ToMMV) isolate was analysed, and some biological features were characterized. The ssRNA genome of ToMMV from Ethiopia encompasses 9283 nucleotides (excluding the 3′ poly(A) tail) and encodes a polyprotein of 3011 amino acids. Phylogenetic and pairwise comparisons with other members of the family Potyviridae revealed that ToMMV is the most divergent member of the genus Ipomovirus, with a genome organization similar to that of members of the species Sweet potato mild mottle virus, the type species of the genus. In contrast to earlier reports, ToMMV isolates from Yemen and Ethiopia were not transmitted by the aphid Myzus persicae, but they were transmitted very erratically by the whitefly Bemisia tabaci. A comparison of the 3′-proximal sequences of different isolates provided evidence for geographically associated genetic variation.

Characterization of an isometric virus isolated from yam (Dioscorea rotundata) in Nigeria suggests that it belongs to a new species in the genus Aureusvirus.

Yam is a primary agricultural commodity in West African countries, especially Nigeria [http://faostat.fao.org]. Only a few viruses have been described to occur in yam (Dioscorea sp.) [1, 8]. These include Dioscorea bacilliform virus (genus Badnavirus) [3] and the potyviruses yam mosaic virus [2], yam mild mosaic virus [7] and yam chlorotic necrotic mosaic virus [22], which form bacilliform and filamentous virions, respectively. In 1998, a virus with isometric particles, referred to here as ‘‘yam spherical virus’’ (YSV), could be identified in a yam sample from Nigeria. In this paper, we report the complete genome sequence of YSV, a putative new member of the genus Aureusvirus. The genomes of viruses of the genus Aureusvirus (family Tombusviridae) consist of a single uncapped, nonpolyadenylated plus-strand RNA containing four open reading frames (ORFs). ORF1 codes for a highly conserved RNA-dependent RNA polymerase, and ORF2 codes for a coat protein (CP), which is expressed from a subgenomic (sg) RNA of approximately 2 kb. The protein encoded by ORF3 has been identified as a movement protein, and ORF4, nested in ORF3, codes for a silencing suppressor. ORFs 3 and 4 are translated from a second sgRNA (0.8 kb). Both sgRNAs are known to be encapsidated and also appear as dsRNA in infected plants [11]. Besides the type member pothos latent virus (PoLV) [16], only three other distinct members of the genus Aureusvirus (http://www. ictvonline.org/virusTaxonomy.asp?version=2012) have been completely sequenced: cucumber leaf spot virus (CLSV) [11], Johnsongrass chlorotic stripe mosaic virus [9] (JCSMV) and maize white line mosaic virus (MWLMV) [17]. Depending on the species, the RNA size ranges from 4.29 kb to 4.43 kb. The genome sequence of a fifth virus, sesame necrotic mosaic virus (SNMV), has been partially determined, and it also shows characteristics of and sequence identities to aureusvirus genomes [23]. Aureusviruses can be efficiently transmitted mechanically. Natural transmission occurs through soil (PoLV) [10, 18] or the soil-inhabiting fungus Olpidium bornovanus (CLSV) [4]. It has also been shown that CLSV can be efficiently transmitted in soil-less cucumber cultures by circulating irrigation water [15].

Characterization of cucumber vein-clearing virus, a whitefly (Bemisia tabaci G.)-transmitted carlavirus

Many important field crops belong to the plant family Cucurbitaceae, and those are commonly affected by a multitude of fungal and viral diseases. Viruses known to be involved belong to greatly differing genera (Potyvirus, Polerovirus, Cucumovirus, Tospovirus, Geminivirus, Crinivirus, Ipomovirus) [4]. A watermelon (Citrullus lanatus) sample with severe leaf symptoms was collected in 2009 in Tanzania. By electron microscopy, large filamentous, flexuous particles resembling those of potyviruses and shorter, carlavirus-like filamentous particles were observed, suggesting a mixed infection. The putative potyvirus was amplified by RTPCR using group-specific primers published by Chen et al. [3]. Following sequence analysis, Moroccan watermelon mosaic virus (MWMV), known to be efficiently transmitted by aphids, was identified. Two carlaviruses occur naturally in Cucurbitaceae (http://www.ictvonline.org/virusTaxonomy.asp?version= 2009): melon yellowing-associated virus (MYaV), a whitefly transmissible virus [7], and the aphidtransmissible muskmelon vein necrosis virus (MuVNV). MYaV has only been reported from Brazil, and it has not been determined if watermelon is susceptible to the virus. MuVNV was first reported in California (U.S.A.) in 1952 and is probably present in China too. It has a narrow host range, almost restricted to members of the Cucurbitaceae [2]. Here, we report the biological and genome characteristics of a member of a new carlavirus species, for which we suggest the name cucumber vein-clearing virus (CuVCV).

Analysis of the complete genome sequence of euphorbia ringspot virus, an atypical member of the genus Potyvirus

The complete genome sequence of an isolate of euphorbia ringspot virus (EuRSV) was determined by deep sequencing and rapid amplification of cDNA ends (RACE) RT-PCR. It has an RNA genome of 10,154 nucleotides in size, excluding the poly(A) tail, and encodes a polyprotein of 3265 amino acids. Phylogenetic analysis from this study supports the earlier taxonomic assignment to the genus Potyvirus; however, a gene encoding the HAM1h protein, inserted between NIb and CP of the EuRSV genome, which was previously only observed for cassava brown streak virus and Ugandan cassava brown streak virus of the genus Ipomovirus, is an unusual feature of this potyvirus, which otherwise has typical potyvirus genome features.