The Fusarium graminearum virus China 9 (FgV-ch9) is a member of the genus Betachrysovirus in the Chrysoviridae family and causes hypovirulence in its host, Fusarium graminearum, the causal agent of Fusarium head blight. Although insights into viral biology of FgV-ch9 have expanded in recent years, questions regarding the function of virus-encoded proteins, cis-acting elements, and virus transmission are yet to be answered. Therefore, we developed a tool for the establishment of an artificial 6th segment of FgV-ch9, which encodes a GFP gene flanked by the non-translated regions of FgV-ch9 segment 1. Subsequently, we have proved successful encapsidation of this artificial segment into virus particles as well as its horizontal transmission. Expression of GFP was further verified via immunoassay and life cell imaging. Thus far, we were able to establish for the first time a mini-replicon system for segmented dsRNA viruses replicating in fungi.

The genomes of most known mycoviruses consist of double stranded RNA (dsRNA) or single stranded RNA (ssRNA). Therefore, for all aspects of mycovirology, the research is highly dependent on the quality and quantity of RNA either by the extraction of genomic dsRNA or dsRNA as a replicating intermediate. A common procedure to extract dsRNA is its binding on a cellulose matrix after a phenol/chloroform purification step. A commercial kit for dsRNA extraction facilitated the researchers´ daily work, but is not available anymore. To extract nucleic acids in a standardized good quality and quantity from small amounts of starting material, we compared commercial kits for gDNA extraction to the kits for RNA extraction using fungal material with a high and a low virus titer. Here we show that viral dsRNA can be extracted using commercial gDNA kits from fungal tissue with a high and a low virus titer in the same quality and quantity as it was done with the discontinued dsRNA extraction kit.

The invasive ascomycete Hymenoscyphus fraxineus is the causative agent for ash dieback on the European species Fraxinus excelsior and Fraxinus angustifolia, and there is concern that it is going to replace the native, closely related and nonpathogenic Hymenoscyphus albidus . Fungal management in forests is limited, and alternative approaches for control are needed. Within the scope of the project “FraxForFuture”, several strategies are being investigated. One idea comprises the use of a viral hyperparasite, which can induce a reduced virulence in the fungal host H. fraxineus in an antagonist-like system. This phenomenon, the reduction of fungal virulence by a viral infection, is known as hypovirulence, and a similar method has already been established to control the Chestnut Blight in Europe. We examined 34 isolates of H. fraxineus for both their virulence and presence of a viral infection. Although a predominant number of isolates were found to be infected with Hymenoscyphus mitovirus 1 (HfMV1), no additional viruses were detected, and our data did not indicate a link to reduced virulence. The search for a viral infection was extended to one isolate of H. albidus in which we found and characterized a novel mycovirus. Based on phylogenetic analysis and sequence properties, it was assigned to the genus Victorivirus in the family of Totiviridae and was tentatively denominated as Hymenoscyphus albidus victorivirus 1. This novel and native mycovirus might be suitable for inducing hypovirulence in H. fraxineus as a biocide .

Two novel dsRNA mycoviruses were found in different isolates of Diplodia fraxini, NW FVA 1581 and NW FVA 1706, which were isolated from a root, associated with stem collar necrosis of Fraxinus excelsior L. Both mycelia are infected by a novel fusagravirus, which was named Diplodia fraxini fusagravirus 1 (DfFV1), and isolate NW FVA 1706 is additionally infected by a novel partitivirus, which was denominated as Diplodia fraxini partitivirus 1 (DfPV1). The one-segmented, bicistronic genome of DfFV1 is composed of about 8,500 bp. Their ORFs are connected by a 1 slippery heptamer sequence and the 3’ terminal ORF is coding for the viral RdRp. The genome of DfPV1 is composed of three, monocistronic dsRNA segments ranging from 1,755 bp (dsRNA 1) over 1,588 bp (dsRNA 2) to 1,233 bp (dsRNA 3). Based on genome organization and phylogenetic positions, DfFV1 was assigned to the proposed family of “Fusagraviridae” and DfPV1 to the genus Gammapartitivirus within the family of Partitiviridae. Ultra-structural analysis showed that polysomal structures were stabilized in the single infection and none of these structures could be isolated in the double infection. It is assumed that DfFV1 has an opportunistic lifestyle, being either protected by ribosomes or by transcapsidation from particles of DfPV1.

Background Due to the infection with the invasive ascomycete Hymenoscyphus fraxineus , which has been replacing the closely related and non-pathogenic native Hymenoscyphus albidus , the European ashes, Fraxinus excelsior (also known as the common ash), Fraxinus angustifolia (also known as narrow-leaved ash) and Fraxinus ornus (also known as the manna ash) are at risk. Hymenoscyphus fraxineus is the causative agent of ash dieback of the European ashes, but is non-pathogenic to the native Asian ash Fraxinus mandshurica (also known as the Manchurian ash). Even though the invasion of H. fraxineus is a great threat for ashes in Europe, the fungal biology is still poorly understood. By the use of live cell imaging and targeted gene knock-out, the fungal life cycle and host–pathogen interaction can be studied in more detail. Results Here, we developed a protocol for the preparation of protoplasts from mycelium of H. fraxineus , for their regeneration and for stable transformation with reporter genes and targeted gene knock-out by homologous recombination. We obtained mutants with various levels of reporter gene expression which did not correlate with the number of integrations. In an in vitro infection assay, we demonstrated the suitability of reporter gene overexpression for fungal detection in plant tissue after inoculation. As a proof of principle for targeted gene knock-out, the hygromycin resistance cassette of a reporter gene-expressing mutant was replaced with a geneticin resistance cassette. Conclusions The invasive fungal pathogen H. fraxineus is threatening the European ashes. To develop strategies for pest management, a better understanding of the fungal life cycle and its host interaction is crucial. Here, we provide a protocol for stable transformation of H. fraxineus to obtain fluorescence reporter strains and targeted gene knock-out mutants. This protocol will help future investigations on the biology of this pathogen.

From the ascomycete Aspergillus cibarius strain NW-FVA 2590, which was originally isolated from a root, associated with stem collar necrosis of Fraxinus excelsior L., a novel virus was isolated and characterized. Its genome is encoded on three monocistronic dsRNA segments ranging from 3683 bp (dsRNA 1) over 3093 (dsRNA 2) to 2902 bp (dsRNA 3), which are packed in isometric particles of around 35 nm. While the viral RdRp (P1) is encoded on segment 1, protein sequencing showed that two more structural proteins are present which are translated from dsRNA 2 (P2) and dsRNA 3 (P3) and possibly form the viral capsid. Additionally, P2 and P3 may undergo posttranslational modifications since the detected proteins bands deviated from the calculated sizes. Due to its phylogenetic position, the novel virus was grouped in the family of Chrysoviridae and was tentatively denominated as Aspergillus cibarius chrysovirus 1 (AcCV1). Due to its composition, biological properties and phylogenetic position, distant from the genera Alphachrysovirus and Betachrysovirus , we suggest to position AcCV1 in a proposed genus “ Gammachrysovirus ” .

A novel dsRNA mycovirus named Ilyonectria crassa alternavirus 1 (IcAV1) was found in Ilyonectria crassa isolate NW-FVA 1829. The fungus was isolated from an ash (Fraxinus excelsior L.) necrotic trunk disc infected with Hymenoscyphus fraxineus [(T. Kowalski) Baral, Queloz, Hosoya] causing ash dieback. The complete genome of IcAV1 is composed of three segments, each containing a single ORF on the positive-sense RNA. The extreme 5' UTRs of dsRNA 1 (3604 bp), dsRNA 2 (2547 bp), and dsRNA 3 (2518 bp) share a conserved hexadecamer sequence (5'-GGCTGTGTGTTTAGTT-3') and are capped. The 3' UTRs are polyadenylated. In silico analysis showed that the viral RdRP is encoded on dsRNA 1 and the capsid-protein subunits are encoded on dsRNA 3. Maximum-likelihood analysis of the aa sequence of the viral RdRP showed that IcAV1 clusters with alternaviruses from Fusarium spp., while the type member of the proposed family "Alternaviridae", Alternaria alternata virus 1 (AaV1), formed a clade together with Stemphylium lycopersici mycovirus (SlV). The function of the protein encoded on segment 2 is unknown. Based on its genome organization and its phylogenetic position, IcAV1 is suggested to be a new member of the proposed family "Alternaviridae". This is the first report of a mycovirus infecting I. crassa.

A novel dsRNA mycovirus was found in Fusarium solani (F. solani) strain NW-FVA 2572. The fungus was originally isolated from a root, associated with stem collar necrosis of Fraxinus excelsior L. The viral genome is composed of four segments, which range from around 3.5 kbp to 1.7 kbp (RNA 1: 3522 bp; RNA 2: 2633 bp; RNA 3: 2403 bp; RNA 4: 1721 bp). The segments share a conserved and capped 5 '-terminus and their 3 '-termini are polyadenylated. Protein sequencing showed that the viral RdRP is encoded on segment 1. The virus clusters together with Aspergillus mycovirus 341 (AsV341), Aspergillus heteromorphus alternavirus 1 (AheAV1), Aspergillus foetidus virus-fast (AfV-F) and Cordyceps chanhua alternavirus 1 (CcAV1). As highest value, the RdRP showed 61.50% identical amino acids with P1 of the AfV-F. The capsid protein is encoded on segment 3, the proteins encoded on RNA 2 and RNA 4 are of unknown function. Segment 4 harbors large UTRs (186 nts at the 5 '-terminus and 311 nts at the 3 '-terminus). Based on its genome organization and phylogenetic position, the virus is suggested to be a new member of the proposed family Alternaviridae and was therefore named Fusarium solani alternavirus 1 (FsAV1). This is the first report of an Alternavirus infecting a fungus of the F. solani species complex (FSSC).

A novel dsRNA virus named “Thelonectria quadrivirus 1” (TQV1) was found in a member of the genus Thelonectria (Ascomycota), isolated from a root associated with stem collar necrosis of Fraxinus excelsior L. The complete genome of TQV1 is composed of four segments, each containing a single ORF on the positive sense RNA. The sequence of the 5´ (5´-(C/T)ACGAAAAA-3´) and 3´termini (5´AT(T/G)AGCAATG(T/C)GC(G/A)CG-3’) of dsRNA 1 (4876 bp), dsRNA 2 (4312 bp), dsRNA 3 (4158 bp), and dsRNA 4 (3933 bp) are conserved. Based on its genome organization and phylogenetic position, TQV1 is suggested to be a new member of the family Quadriviridae. This is the first report of a mycovirus infecting a member of the genus Thelonectria.

While the capsid of viruses in the Alphachrysovirus genus is built of subunits of a single coat protein, the capsid of viruses grouped in the Betachrysovirus genus may consist of subunits of two different proteins. For four of these betachrysoviruses, the detected molecular weights of the putative coat proteins differ from the sizes deduced from the nucleic acid sequence. The origin of these modifications remained unclear and it was hypothesized that the coat proteins undergo unspecific degradation. In our study, we show that these modifications are based on processing steps performed by unknown factors present in extracts of several eukaryotic organisms. Furthermore, we show that the C-terminal domain of P3 is fully degraded after capsid processing and particle assembly.

Infections of fungi by mycoviruses are often symptomless but sometimes also fatal, as they perturb sporulation, growth, and, if applicable, virulence of the fungal host. Hypovirulence-inducing mycoviruses, therefore, represent a powerful means to defeat fungal epidemics on crop plants. Infection with Fusarium graminearum virus China 9 (FgV-ch9), a double-stranded RNA (dsRNA) chrysovirus-like mycovirus, debilitates Fusarium graminearum, the causal agent of fusarium head blight. In search for potential symptom alleviation or aggravation factors in F. graminearum, we consecutively infected a custom-made F. graminearum mutant collection with FgV-ch9 and found a mutant with constantly elevated expression of a gene coding for a putative mRNA-binding protein that did not show any disease symptoms despite harboring large amounts of virus. Deletion of this gene, named virus response 1 (vr1), resulted in phenotypes identical to those observed in the virus-infected wild type with respect to growth, reproduction, and virulence. Similarly, the viral structural protein coded on segment 3 (P3) caused virus infection-like symptoms when expressed in the wild type but not in the vr1 overexpression mutant. Gene expression analysis revealed a drastic downregulation of vr1 in the presence of virus and in mutants expressing P3. We conclude that symptom development and severity correlate with gene expression levels of vr1 This was confirmed by comparative transcriptome analysis, showing a large transcriptional overlap between the virus-infected wild type, the vr1 deletion mutant, and the P3-expressing mutant. Hence, vr1 represents a fundamental host factor for the expression of virus-related symptoms and helps us understand the underlying mechanism of hypovirulence.IMPORTANCE Virus infections of phytopathogenic fungi occasionally impair growth, reproduction, and virulence, a phenomenon referred to as hypovirulence. Hypovirulence-inducing mycoviruses, therefore, represent a powerful means to defeat fungal epidemics on crop plants. However, the poor understanding of the molecular basis of hypovirulence induction limits their application. Using the devastating fungal pathogen on cereal crops, Fusarium graminearum, we identified an mRNA binding protein (named virus response 1, vr1) which is involved in symptom expression. Downregulation of vr1 in the virus-infected fungus and vr1 deletion evoke virus infection-like symptoms, while constitutive expression overrules the cytopathic effects of the virus infection. Intriguingly, the presence of a specific viral structural protein is sufficient to trigger the fungal response, i.e., vr1 downregulation, and symptom development similar to virus infection. The advancements in understanding fungal infection and response may aid biological pest control approaches using mycoviruses or viral proteins to prevent future Fusarium epidemics.

The hypovirulence-inducing Fusarium graminearum virus China 9 (FgV-ch9) was described recently and is closely related to the Fusarium graminearum mycovirus-2 (FgV2). Both viruses share common properties of viruses belonging to the family Chrysoviridae. Re-sequencing of FgV-ch9 revealed duplications of the 3′ non-coding regions of segments 2 and 3. Both duplications are arranged in a head-to-tail array, are attached to the complete terminus, and do not affect the encoded gene. An internal duplication was found in segment 5. This duplication resulted in an increase in the size of the encoded protein. In silico analysis showed similar duplications in segments 2 and 3 of FgV2.

Cucumber mosaic virus (CMV) is an economically important pathogen on chili plants in Asia. So far, no durable resistant chili varieties were available to obtain virus resistant plants through a classical breeding program. Therefore, several biotechnological approaches to generate resistant plants via virus induced gene silencing (VIGS) were tested. However, due to different target plant species, different CMV isolates and different experimental testing systems, an evaluation of the most efficient construct is very difficult from the published data. To evaluate a suitable construct for generating CMV resistance in chilli, several constructs using the regions of the coat protein from RNA 3 and the suppressors of gene silencing expressed from RNA 2 on CMV genome were introduced into two different binary vectors (pLH6000 and pBIN19) as single gene or as an inverted repeat. Additionally, a chimeric construct GFP+2bIR was generated in both binary vectors. These constructs were transformed in two different tobacco species (N. benthamiana and N. tabaccum). The resistance of these transformants was evaluated using different CMV isolates in a standardized testing system. Immunity, tolerance and recovery phenotypes were verified by symptom expression and virus detection by tissue print immunoblots. Resistance screening on F1 generation revealed that resistance variation between gene constructs and tobacco plants: for single gene constructs (△CP, △2a+2b and △2a+△2b in which start codons of CP and 2b genes were deleted) in pLH6000, the given resistance efficiency rank was pLH6000-△2a+2b > pLH6000-△2a+△2b > pLH6000-△CP in N. benthamiana, while the given rank was not clear in N. tabaccum; however, the given resistance efficiency rank of single gene constructs in pBIN19 was not clear in both tobacco plants because of a lower resistant efficiency. For 2bIR construct, the resistant efficiency in N. benthamiana was higher than in N. tabaccum, and therefore the given rank was pLH6000-2bIR > pBIN19-2bIR in N. benthamiana but not in N. tabaccum. For CPIR, the resistance variation was not clear in both tobacco plants when challenged with heterologous isolate CMVAN. For GFP+2bIR, resistance efficiency was obviously enhanced in both tobacco species with exception of N. benthamiana in pLH6000-GFP+2bIR, all resistant plants were further verified to be immune to CMV-AN by symptom expression, tissue print immunoblot and back inoculation. In addition, three transgenic N. benthamiana lines from pBIN19-2bIR (one line) and pBIN19-GFP+2bIR (two lines) were further challenged with five different CMV isolates. These three lines exhibited broad-resistance against five different CMV isolates. Taken together, (I) the resistance efficiency in tobacco species was ranged from 0 to 100%, which is independent of vectors and/or plant species. (II) Resistance using RNA 3 fragments is lower than with RNA 2 fragments. (III) A chimeric construct with nontarget DNA as flanking sequence showed higher resistant efficiency even when these lines were challenged with heterologous CMV isolates when compared with 2bIR constructs. However the construct should be optimized by exchanging the GFP with a viral sequence before using it to obtain resistant vegetable against CMV in Asian agriculture. Cucumber mosaic virus (CMV) verursacht in Asien bedeutende okonomische Schaden an Chilli. Bis heute sind keine Chilli-Varietaten bekannt, die eine dauerhafte Resistenz uber klassische Zuchtungsprogramme ermoglichen. Aus diesem Grund wurden verschiedene gentechnologische Ansatze fur eine virusinduzierte Resistenz (virus induced gene silencing, VIGS) getestet. In verschiedenen Publikationen wurden unterschiedliche Wirtspflanzen, unterschiedliche CMV-solate und verschiedene Testsysteme verwendet, deswegen ist eine Evaluierung des besten Konstruktes auf der Basis von publizierten Daten sehr schwierig. Zur Bestimmung eines geeigneten Konstruktes zur Generierung von CMV-resistentem Chilli wurden verschiedene Bereiche des Hullproteins der RNA 3 und des gene silencing suppressors der RNA 2 des CMV-Genoms in zwei unterschiedliche binare Vektoren (pBIN 19 und pLH 6000) als „single gene“ oder als „inverted repeat“ Konstrukt kloniert. Zusatzlich wurde ein chimares Konstrukt (GFP-2b IR) in beide Vektoren kloniert. Beide Konstrukte wurden jeweils in zwei Tabakarten (Nicotiana benthamiana und Nicotiana tabaccum) stabil transformiert. Die Resistenz dieser Isolate wurde mit verschiedenen CMV-Isolaten in einem standardisierten Testsystem evaluiert. Die Phanotypen Immunitat, Toleranz und Erholung wurde anhand von Symptomauspragung und Virusnachweis in Gewebeabdrucken mit Hilfe von serologischer Detektion beobachtet. Die Resistenztestung der F1 Generation zeigte eine Variation der Resistenz abhangig vom Genkonstrukt und der Wirtspflanze: Fur die „single gene“ Konstrukte ΔCP, Δ2a+2b und Δ2a+ Δ2b (in denen das Startkodon von CP bzw. 2b entfernt wurde) im binaren Vektor pLH6000 zeigte die Reihenfolge pLH6000- Δ2a+2b > pLH6000- Δ2a+ Δ2b > pLH6000- ΔCP in N. benthamiana, wahrend in transformierten N. tabaccum die Reihenfolge unklar war. Die Rangfolge in beiden Pflanzenspezies war unklar, wenn mit dem binaren Vektor pBIN19 transformiert wurde, da hier generell eine geringe Resistenz beobachtet wurde. Die Resistenz fur das 2bIR Konstrukt war in N. benthamiana hoher als in N. tabaccum und folglich war die Gute der Resistenz in der Reihenfolge pLH6000-2bIR > pBIN19-2bIR in N. benthamiana aber nicht in N. tabaccum. Fur das Konstrukt CPIR folgte die Resistenz keiner erkennbaren Regel in beiden Wirtspflanzen fur den Fall, dass mit dem heterologen Isolat CMVAN infiziert wurde. Bei Pflanzen, die mit dem Konstrukt GFP+2bIR transformiert waren, war eine signifikant bessere Resistenz in beiden Wirtspflanzen zu beobachten, allerdings mit der Ausnahme von N. benthamiana transformiert mit dem Konstrukt pLH6000-GFP2bIR. Die Abwesenheit von Virus wurde bei als immun bewerteten Pflanzen mit Gewebeabdrucken und serologischer Detektion sowie Biotests bestatigt. Zusatzlich zur Testung mit dem homologen Isolat wurden drei Linien (1 x pBIN19-2bIR und 2 x pBIN19-GFP+2bIR) mit weiteren Isolaten auf Resistenz uberpruft. Alle drei Linien zeigten eine breite Resistenz gegenuber funf verschiedenen CMV-Isolaten. Zusammengefasst ergab sich Folgendes: (I) die Reistenzgute in den beiden transformierten Tabakarten variierte von 0 bis 100 %, unabhangig vom Vektor und Pflanzenart. (II) Die Resistenz, die mit Fragmenten der RNA 3 erhalten wurde, war niedriger als diejenige, die mit Fragmenten der RNA 2 erhalten wurde. (III) Ein chimares Konstrukt mit einer virusunabhangigen DNA als flankierende Sequenz zeigte eine bessere Resistenz als 2bIR-Konstrukte, und zwar sogar dann, wenn mit nicht-homologen Isolaten getestet wurde. Trotzdem sollte dieses chimare Konstrukt optimiert werden, indem das GFP gegen virale Sequenzen ausgetauscht wird, bevor es zum Einsatz zur Erzeugung von CMV-resistentem Gemuse in Asien kommt.

The Ninth Report of the International Committee on Taxonomy of Viruses (ICTV) reports only a few species whose members replicate in fungi. Most of these mycoviruses are described to replicate in phytopathogenic and commercially cultivated fungi. A few reports describe virus-like symptoms and virus-like particles in non-cultivated basidiocarps such as Boletus edulis, Laccaria spp. and Cantharellus spp. However, viral sequences from non-cultivated Agaricomycotina are not available yet. In this report, I present a partial sequence of a virus found in Clitocybe odora (Bull.:Fr.) P. Kumm var. odora coding for a putative RNA-dependent RNA polymerase (RdRp) and a small 20-kDa ORF that may encode a coat protein (CP). The sequence of the putative RdRp (ORF 1) of C. odora clusters with those of the Tanathephorus cucumeris virus RdRp and the Tuber aestivum mitovirus RdRp. In addition to sequence homology, Tanathephorus cucumeris virus shows a similar codon usage and TA content in the 5′- and 3′ non-translated regions, but it does not encode a putative CP. A viral DNA form proposed for Tanathephorus cucumeris virus was not found in Clitocybe odora. This viral sequence does not fit into any of the existing virus taxa.

TMVOhioV was first described 1969 by [1] because it did break resistance of tomato breeding lines containing Tm-1- and Tm-2 resistance genes. It was obtained 1987 from Wetter (Saarbrücken, Germany) and transferred into the DSMZ-Plant Virus Collection (Braunschweig, Germany). A partial sequence of TMVOhioV, the CP gene, has been reported [11] and its comparison with a TMV type isolates (TMVtype), e.g. EMBL: V01409, revealed 50 point mutations in a total of 477 nucleotides (nts) leading to the replacement of only 7 amino acids (aa). In order to investigate the mutations in the non-translated regions and the number of silent mutation in the three other open reading frames (ORF), we sequenced the complete genome of isolate TMVOhioV and compared it to those of other Tobamoviruses.

Among the Chili breeding lines from the Asian Vegetable Research Center, two were chosen for the screening of a larger selection of Cucumber mosaic virus (CMV) isolates, mainly from Asian countries. The chili line (VC246) showed a resistance against several CMV-isolates and was compared with chili line VC27a that was susceptible to CMV infection. Among the 28 CMV isolates, five were identified as resistance breaking (AN-like) and non-resistance breaking (P3613-like) for the line VC246, whereas all isolates could establish a systemic infection on VC27a. However, further testing revealed that resistance in VC246 was also dependent on the way of inoculation and the inoculums itself. Graft inoculation could overcome the resistance, and the inoculation with isolated viral RNA resulted in no infection at all on the resistant chili line, independent of the virus isolate. Using a pseudo-recombinant approach, we identified RNA2 of resistance breaking isolates as responsible for systemic infection and confined the area within RNA2 to the 3′ terminal part including the ORF 2b. Sequence alignments of that area revealed eight distinct mutations on amino acid level, which was present either in resistance or non-resistance breaking isolates. A reversion from the P3613-like to the AN-like sequence of two of these mutations induced no effect on Capsicum sp., but induced symptoms on several tobacco species distinct from those induced by the wild-type virus. However, pseudorecombinants, each generated from sets of two different AN-like isolates, which were expected to infect VC246 systemically, did not indicating that probably RNA2 must be in a specific context to have the effect. In this case, a generalized attribution of functions to single amino acid exchanges might be impossible or at least extremely difficult.

A new virus, isolated from a mixed infected Brugmansia sp., was characterized. The viral genome was about 6380 nucleotides (nts) long and showed an organization typically for tobamoviruses with overlapping open reading frames (ORF) of the RNA-dependent RNA polymerase (RdRP) and the movement protein (MP). The coat protein (CP) was in the same size as known for other tobamoviruses. Phylogenetic analyses of the complete genome indicated that the new isolate belongs to the tobamovirus subgroup 1, despite strong serological reactions with Ribgrass mosaic virus (RMV) specific antiserum in a plate trapped antigen ELISA. Sequence comparisons with species of the genus Tobamovirus revealed > 10% overall sequence divergence, which indicates according to the rules of the International Committee on Taxonomy of Viruses (ICTV) that this virus is a new species and we suggest the name Brugmansia mild mottle virus (BMMV) for that isolate. Furthermore, we present a diagnostic reverse transcriptase-polymerase chain reaction (RT-PCR) protocol for the detection of this isolate which uses a drastically reduced cycling time.

Ribgrass mosaic virus (RMV), turnip vein-clearing virus (TVCV) and Youcai mosaic virus (YoMV; formerly designated as oilseed rape mosaic virus; ORMV) belong to the genus Tobamovirus and are arranged in one out of three subgroups because of their common host range, serological cross-reactivity and amino acid composition of their coat proteins. The recently defined species Wasabi mottle virus (WMoV) is closely related to the same subgroup. The distinction of the four species is difficult and the lack of sequence information of a wide range of isolates has led to an unclear nomenclature. To clarify this situation we sequenced the coat protein genes from 18 isolates which were serologically related to members of the species of this cluster. The size of the coat protein was conserved with the exception of one isolate which revealed an N-terminal extension due to the mutation of three stop-codons. Phylogenetic analysis of these CP ORFs resulted in a tree with three clusters each containing at least one of the approved species RMV, TVCV and 1ptYoMV/WMoV in which our isolates were distributed. The tree was congruent and did support the present taxonomic status of species within this subgroup.For practical purpose we developed a subgroup 3 specific primer pair and a species differentiating restriction fragment length polymorphism (RFLP). Sequencing of the genome of Streptocarpus flower break virus (SFBV) which is serologically distantly related to the subgroup 3 viruses revealed a distinct genome organization. Therefore we propose that this virus should be regarded as a member of a species not belonging to any of the subgroups so far established.