Genus Pomovirus Research Articles

Draft Genome Resource for the Potato Powdery Scab Pathogen Spongospora subterranea.

The Plasmodiophorida (Phytomyxea, Rhizaria) are a group of protists that infect plants. Of this group, Spongospora subterranea causes major problems for the potato industry by causing powdery scab and root galling of potatoes and as vector for the Potato mop-top virus (PMTV) (genus Pomovirus, family Virgaviridae). A single tuber isolate (SSUBK13) of this uncultivable protist was used to generate DNA for Illumina sequencing. The data were assembled to a draft genome of 28.08 Mb consisting of 2,340 contigs and an L50 of 280. A total of 10,778 genes were predicted and 93% of the BUSCO genes were detected. The presented genome assembly is only the second genome of a plasmodiophorid. The data will accelerate functional genomics to study poorly understood interaction of plasmodiophorids and their hosts.

Molecular and biological characterisation of two novel pomo-like viruses associated with potato (Solanum tuberosum) fields in Colombia.

Potato is the fourth most important crop worldwide that is used as a staple food, after rice, wheat and maize. The crop can be affected by a large number of pathogens, including fungi, oomycetes, bacteria and viruses. Diseases caused by viruses are among the most important factors contributing to reduced quality and yield of the crop. Potato mop-top virus (genus Pomovirus) induces necrotic flecks in the tuber flesh and skin of potato in temperate countries. Spongospora subterranea is the vector of PMTV. Both the virus and its vector cause disease in potato. In Colombia, PMTV has been detected throughout the country together with a novel pomo-like virus in the centre (Cundinamarca and Boyacá) and south west (Nariño) of the country. We studied the molecular and biological characteristics of this novel virus. Its genome resembles those of members of the genus Pomovirus, and it is closely related to PMTV. It induces mild systemic symptoms in Nicotiana benthamiana (mosaic, branch curling), but no symptoms in N. tabacum, N. debneyi and Chenopodium amaranticolor. The proposed name for the virus is "Colombian potato soil-borne virus" (CPSbV). Additionally, another pomo-like virus was identified in Nariño. This virus induces severe systemic stem declining and mild mosaic in N. benthamiana. The tentative name "soil-borne virus 2" (SbV2) is proposed for this virus. No vectors have been identified for these viruses despite several attempts. This work focused on the characterisation of CPSbV. The risk posed by these viruses if they are introduced into new territories is discussed.

First Report of Potato mop top virus Causing Potato Tuber Necrosis in Colorado and New Mexico.

Potato mop top virus (PMTV) is considered the type member of the genus Pomovirus. PMTV is an important pathogen of potato vectored by the plasmodiophorid Spongospora subterranea f. sp. subterranea (Sss), which causes powdery scab of potato (1). Sss and PMTV are usually associated with cool and humid environments. PMTV-infected potato tubers generally exhibit internal hollow necrotic spots or concentric rings, and the virus is known to cause significant economic losses in Northern Europe, North and South America, and Asia (4). PMTV in the United States was first reported in Maine (2). Potato (Solanum tuberosum L.) tubers cv. FL2048 and cv. Atlantic were sent to our laboratory from fields in Saguache County in Colorado and in San Juan County in New Mexico, respectively, during the spring of 2013. The tubers from both locations had multiple, internal, concentric, necrotic arcs and circles. Internal tissue with necrotic lesion from six symptomatic tubers from each location were crushed in liquid nitrogen followed by ribonucleic acid extraction using a Total RNA Isolation kit (Promega Corp., Madison, WI). These extracts were tested by reverse transcription (RT)-PCR using three different sets of previously published primers for molecular detection of PMTV. The primer set H360/C819 targeting the coat protein (CP) on RNA 3 of PMTV yielded an amplicon (H360-CO and H360-NM) of 460 bp (4). The second set of primers, pmtF4/pmtR4 (5), amplified a 417-bp product (PMTF-CO and PMTF-NM) in RNA 2, and the third set, PMTV-P9/PMTV-M9 (3), designed to amplify the region encoding an 8-KD cysteine-rich protein in RNA 3 of PMTV, yielded a 507-bp amplicon (PMTV9-CO and PMTV9-NM). The amplicons generated from RT-PCR using all three sets were cloned (PGEMT-easy) and sequenced. Since the sequences from symptomatic tuber extracts from each location were identical to their respective primer sets, a consensus sequence from each primer set was submitted to National Center for Biotechnology Information (NCBI) GenBank. Sequences obtained from the H360/C819 primer set (GenBank Accession Nos. KM207013 and KM207014 for H360-CO and H360-NM, respectively) were 100% identical to the corresponding CP regions of PMTV isolates from North Dakota (HM776172). Sequences from the pmtF4/pmtR4 primer set (KM207015 and KM207016 for PMTF-CO and PMTF-NM, respectively) were 100% identical to the corresponding protein in RNA2 of PMTV isolates from North Dakota (GenBank HM776171), and sequences from the PMTV-P9/PMTV-M9 primer set (KM207017 and KM207018 for PMTV9-CO and PMTV9-NM respectively) were 99% identical to the corresponding protein in RNA3 of PMTV isolates (AY187010). The 100-99% homology of the sequences from this study to the corresponding PMTV sequences published in NCBI confirmed the occurrence of symptoms in the tubers from both Colorado and New Mexico due to PMTV. None of the symptomatic tubers tested positive for Tobacco rattle virus, Tomato spotted wilt virus, Alfalfa mosaic virus, Potato leafroll virus, or the necrotic strains of Potato virus Y by RT-PCR. To our knowledge, this is the first report of PMTV in potato in states of Colorado and New Mexico.

First Report of Potato mop-top virus in North Dakota.

Potato mop-top virus (PMTV) is the type member of the genus Pomovirus. PMTV is an important pathogen of potato, causing significant economic losses in Northern Europe, North and South America, and Asia (3). PMTV in the United States was first reported in Maine (2). PMTV is vectored by the plasmodiophoromycete Spongospora subterranea cv. subterranea, which causes powdery scab of potato (1). S. subterranea and PMTV are usually associated with cool and humid environments. In the spring of 2010, six potato tubers of cv. Russet Burbank were received from a commercial potato farm in Grand Forks County in North Dakota. The tubers had multiple, internal, concentric, necrotic arcs and circles. The presence of PMTV in the necrotic lesions was verified by a positive double-antibody sandwich-ELISA (Agden Ltd., Ayr, Scotland). The tuber lesions had an absorbance value (405 nm) at least two times greater than that of the negative control sample, which consisted of a healthy tuber. Total RNA was extracted from lesions of six different tubers that tested positive by ELISA using a Total RNA Isolation kit (Promega Corp. Madison, WI). These extracts were tested for PMTV by reverse transcription (RT)-PCR using two different sets of primers. The primer set H360/C819 targeted the coat protein (CP) of PMTV and yielded an amplicon of 460 bp (4). The amplicons generated from the necrotic lesions were cloned (TOPO Cloning; Invitrogen, Carlsbad, CA) and sequenced. Another set of primers, pmtF4/pmtR4, designed to bind to a region in RNA 2 of PMTV, yielded a 417-bp amplicon that also was cloned and sequenced (3). The sequences from all six tuber lesions were identical for the respective primer sets. A consensus sequence for each primer pair was submitted to GenBank (Accession No. HM776171 for primers pmtF4/pmtR4 and No. HM776172 for primers H360/C819). The sequences obtained from the H360/C819 and pmtF4/pmtR4 amplicons were 99% identical to the corresponding regions of PMTV isolates from Northern Europe (GenBank Accession Nos. AM503629 and AJ277556, respectively). Freeze-dried, necrotic tuber tissue from all six tubers was also tested at a USDA Laboratory in Prosser, WA by RT-PCR with the H360/C819 primer pair (4), confirming the results above. Cloning and sequencing of one of the amplicons revealed 100% similarity to the sequence described above for these primers (GenBank Accession No. HM776172), confirming the presence of PMTV in the symptomatic tubers. None of the symptomatic tubers tested positive for Tobacco rattle virus, Tomato spotted wilt virus, Alfalfa mosaic virus, Potato leafroll virus, or the necrotic strains of Potato virus Y by RT-PCR. To our knowledge this is the first report of PMTV in North Dakota.

Detection, distribution and control of Potato mop‐top virus, a soil‐borne virus, in northern Europe

Potato mop‐top virus (PMTV; genus Pomovirus; family Virgaviridae) is transmitted by the soil‐borne Spongospora subterranea f.sp. subterranea, a protoctist that causes powdery scab on potato. PMTV is distributed widely in the potato growing areas in South and North America, Japan and northwestern Europe. This article reviews the current knowledge on detection, distribution and control of PMTV with focus on the Baltic Sea region. Since the 1980s, PMTV has caused great economic losses to potato production in the Nordic countries (Norway, Sweden, Denmark and Finland), but its occurrence in other countries of the Baltic Sea region remained unknown. To fill this knowledge gap, harmonised sampling and virus detection procedures including bioassays and serological and molecular methods were employed by 21 research institutions to detect PMTV in potato tubers and soil samples in 2005–2008. Potato growing areas were widely contaminated with PMTV in the Nordic countries. Only the main seed potato production area in northern Sweden and the High Grade seed potato production zone in Finland were negative for PMTV. Intensive and systematic surveys in Poland in 2004–2008 found no evidence of PMTV, except a single PMTV‐infected tuber detected in 2008. Surveys in the Baltic countries (Lithuania, Latvia and Estonia) and northwestern Russia (Leningrad province) were negative for PMTV, except infection of minitubers in a screenhouse in Latvia in 2005. Varying percentages of tubers expressing spraing symptoms in Sweden, Norway, Denmark and Poland were infected with Tobacco rattle virus, and bioassays indicated similar results for Russia. Incidence of symptomless infections with PMTV was high in tubers of many potato cultivars. Here, we discuss the contrasting patterns of distribution of PMTV in the Baltic Sea region, factors playing a role in dispersal and establishment of PMTV in new fields and means for controlling PMTV and its spread to new areas. We emphasise the use of the current virus‐specific methods for the detection of PMTV in symptomless potato tubers and the high risks of disseminating PMTV to new fields and areas in viruliferous resting spores of S. subterranea in the soil adhering to seed tubers. PMTV‐resistant potato cultivars will provide the only sustainable means for preventing yield losses in the infested fields and the prospects of resistance breeding are summarised.

Detection of Potato mop-top virus in Potato Tubers and Sprouts: Combinations of RNA2 and RNA3 Variants and Incidence of Symptomless Infections

Potato mop-top virus (PMTV, genus Pomovirus) causes severe quality problems by inducing necrotic arcs (spraing symptoms) in potato tubers. In this study, coat protein (CP) gene and read-through domain of RNA2 and 8K gene and 3' untranslated region of RNA3 were characterized from 37 PMTV isolates detected in tubers from fields in Finland and a screenhouse in Latvia. Two distinguishable types of RNA2 and RNA3 were found, each showing only little genetic variability. Sequencing and restriction fragment length polymorphism analysis of polymerase chain reaction amplicons indicated that the majority of PMTV isolates infecting tubers comprise restrictotypes RNA2-II and RNA3-B. The incidence of PMTV-infected tubers in 2006 (2007) was 55 (60), 33 (39), and 62 (68)% in cvs. Kardal, Saturna, and Nicola, respectively, grown in the same field in 2006 (2007). Incidence of PMTV-infected tubers that were symptomless was 100 (90)% in Kardal and 88 (44)% in Saturna, and also high in cvs. Bintje (95%) and Van Gogh (63%), tested only in 2006, whereas it was only 12 (2)% in Nicola. Hence, reliance on visual inspection of spraing will miss a large proportion of infected tubers and risk spreading PMTV to new fields in seed tubers. No specific combination of the types of RNA2 and RNA3 was associated with spraing-expressing or symptomless tubers. Using recombinant PMTV CP for comparison, the concentrations of PMTV CP in tuber and sprout tissue were estimated to reach 57 mug/g. Sprout sap interfered less with enzyme-linked immunosorbent assay than did tuber sap.

First report of Beet soil-borne virus on sugar beet in China

Beet soil-borne virus (BSBV) and Beet virus Q (BVQ) are two members of the genus Pomovirus that are transmitted by Polymyxa betae. BVQ and BSBV are commonly found in fields where Beet necrotic yellow vein virus (BNYVV), the causal agent of rhizomania disease, is present (Henry & Jones, 1986; Koenig et al., 1998; Meunier et al., 2003). During the growing season of 2006, 26 roots of sugar beet exhibiting moderate to severe symptoms of rhizomania were collected from three fields located in Inner Mongolia and Xinjiang Provinces for a survey to determine whether BSBV and BVQ were present in China. Viral RNA was extracted from partially purified viruses of beet roots as described by Han et al. (2000) and tested by RT-PCR using three specific primer sets (BSBV: 5′-ATG GTT GAT CCG CGG TAT GAA G-3′ and 5′-CTA TTC AAC CCA GCG CAA ACC AAC-3′; BVQ: 5′-ATG GTT GAT CCA AGA TAT GAG C-3′ and 5′-CTA TGA GCC GGT CCA CTT CAA TCC-3′; and BNYVV: 5′-GCG GAT CCA TGT CGA GTG AA G GTA GA-3′ and 5′-CGA AGC TTG CTA TTG TCC GGG TGG-3′, based on Accession Nos NC003518, NC003511 and S71490 respectively) and designed to amplify coat protein genes for BSBV, BVQ and BNYVV respectively. Fifteen of the 26 beet samples gave positive reactions for BSBV, 21 for BNYVV and 14 for both BSBV and BNYVV. BVQ was not detected in any sugar beet sample tested. Subsequently, three RT-PCR products amplified in sugar beet samples collected from different locations with the primers specific to BSBV were cloned and sequenced (GenBank Accession Nos EF113300, EF210553 and EF210552). Sequence analysis revealed that these submitted sequences shared nucleotide identity of 98–99% with the German BSBV isolate (Accession No. NC003518). The partially purified viruses from infected sugar beet root were used for mechanical inoculation of Chenopodium quinoa and C. amaranticolor, and local lesions were observed on inoculated leaves. Furthermore, BSBV was also confirmed by RT-PCR (as described above) in the local lesions that appeared approximately 5 days after mechanical inoculation. Combined, the RT-PCR detection, nucleotide sequences and symptoms on inoculated host plants confirm the presence and first occurrence of BSBV in Inner Mongolia and Xinjiang. This work was partially supported by the National Natural Science Foundation of China (30471136 and 30671359).

First Report of Beet virus Q on Sugar Beet in Poland.

The objective of this work was to determine whether Beet virus Q (BVQ), a member of the genus Pomovirus, is present in Poland. BVQ, like Beet necrotic yellow vein virus (BNYVV), is transmitted by Polymyxa betae Keskin. Earlier, BVQ was described as the Wierthe serotype of Beet soilborne virus (BSBV). Now, on the basis of its genomic properties (2), BVQ is recognized as a distinct virus species. BVQ is often found in fields where BSBV and BNYVV are present (4). During the fall of 2005, five plants of a cultivar susceptible to rhizomania (cv. Alyssa) and five resistant to rhizomania (cv. Henrietta) were collected from a field in the Wielkopolska Region of Poland, where BSBV and BNYVV had been previously identified, and tested for BVQ (1). All samples were analyzed by a double antibody sandwich-enzyme linked immunosorbent assay (DAS-ELISA) with antiserum against BNYVV (Bio-Rad, Hercules, CA). Rhizomania was identified only in sugar beet samples of the susceptible variety. The same samples were then tested using a triple antibody sandwich (TAS)-ELISA with commercial antisera against BSBV/BVQ (As-0576.2) and BSBV (As-0576.1) (DSMZ, Braunschweig, Germany). Nine sugar beet plants gave positive reactions with antiserum against BSBV/BVQ and negative reactions with antiserum specific to BSBV. Total RNA extracted from roots of 10 beet samples was then tested using a multiplex reverse transcription-polymerase chain reaction (mRT-PCR) and specific primers designed to amplify a fragment of the RNA2 for BNYVV and BVQ (3). The primers specifically amplified fragments of 545 bp and 291 bp of the BNYVV and BVQ, respectively. BNYVV was detected in all five samples from susceptible sugar beet plants. The presence of BVQ was confirmed in nine of the sugar beer plants, and the RT-PCR products were sequenced. Sequence analysis of the 206-nt amplicon sequence of the Polish isolate of BVQ (GenBank Accession No. DQ309444) indicated 97% nucleotide and 94% amino acid sequence identity with the previously published sequence of BVQ (GenBank Accession No. AJ223596) (2). To my knowledge, this is the first report of the natural occurrence of BVQ on sugar beet in Poland. In Europe, it has been previously reported in Belgium, Bulgaria, France, Germany, Hungary, Italy, the Netherlands, Spain, and Sweden (3,4).

First Report of Beet virus Q in Spain.

Beet virus Q (BVQ) is a member of the genus Pomovirus that is transmitted by Polymyxa betae Keskin. Initially described as the Wierthe serotype of Beet soilborne virus (BSBV), BVQ is now considered a distinct virus species based on its genomic properties (1). BVQ is commonly found in fields where BSBV and the causal agent of rhizomania disease, Beet necrotic yellow vein virus (BNYVV), are also present. Simultaneous infection of sugar beet plants with multiple virus species could affect disease symptom expression (4). For this reason, the pathogenicity of BVQ and its role in the epidemiology of rhizomania disease remain a subject of study. During 2004, six soil samples were collected from different sites in the Castilla-La Mancha Region in Spain (Albacete and Ciudad Real provinces) where rhizomania symptoms were observed in BNYVV-tolerant sugar beet cultivars. Soil from the Hainaut Region of Belgium, infected with BNYVV, BSBV, and BVQ and supplied by Prof. C. Bragard (Unité de Phytopathologie, Université Catholique de Louvain, Belgium) was used as a positive control. Sugar beet plants (cv. Asso) were grown in the soil samples for 45 days at 24°C and then root tissue was harvested. All samples were analyzed using enzyme-linked immunosorbent assay (ELISA) with commercial BNYVV antiserum (BIOREBA AG, Reinach, Switzerland) and BSBV/BVQ antisera (IC10 and 6G2) supplied by R. Koenig (Federal Biological Research Centre for Agriculture and Forestry, Braunschweig, Germany). Total RNA extracted from sugar beet roots as previously described (3) was tested using reverse transcription-polymerase chain reaction (RT-PCR). Primers BVQ3F (5'-GTT TTC AAA CTT GCC ATC CT-3') and BVQ3R2 (5'-CCA CAA TGG GCC AAT AGA-3'), which amplify a 690-bp fragment of the triple gene block region of BVQ RNA 3, were designed based on the published sequence (GenBank Accession No. AJ223598). The presence of BSBV and BNYVV was assayed using RT-PCR with previously described primers (2,3). BVQ was detected from plants grown in soil collected from La Roda (Albacete) in Spain and from Hainaut in Belgium. The fragments amplified from Spanish sample with BVQ3F and BVQ3R2 (GenBank Accession No. AY849375) showed 95.9% nucleotide sequence identity with the previously published sequence of BVQ (1). The La Roda BVQ isolate was mechanically transmitted to Chenopodium quinoa from infected sugar beet root tissue. BVQ was detected using RT-PCR in local lesions that appeared approximately 5 days after inoculation and subsequently spread along veins. To our knowledge, this is the first report of BVQ in soil from Spain, although it has been previously reported in Belgium, Bulgaria, France, Germany, Hungary, and the Netherlands (2). BSBV and BNYVV (type A) were detected in all six Spanish samples, as well as in the Belgian soil.

Sequence Divergence of Four Soilborne Sugarbeet-Infecting Viruses

Soilborne viruses are among the most harmful pathogens of sugarbeet (Beta vulgaris L.ssp. vulgaris) but most of them lack information on genetic variability due to paucity of sequence data. Only one isolate of Beet soil borne virus (BSBV; genus Pomovirus), Beet virus Q (BVQ; genus Pomovirus) and Beet soil borne mosaic virus (BSBMV; genus Benyvirus) has been characterised for the coat protein (CP) gene. In this study, the CP gene sequences of three isolates each of BSBV and Beet necrotic yellow vein virus (BNYVV; genus Benyvirus) (France, Germany and USA), two isolates of BVQ (France and Germany), and one isolate of BSBMV (USA) were determined. Phylogenetic analyses including sequences from databanks indicated that the French BNYVV isolate of this study belongs to so-called P-type, the American isolate to A-type and the German isolate to B-type. The CP genes of the three BSBV isolates characterised in this study and the one available from databank were highly identical (98.4-99.0% at nucleotide level; one variable amino acid). The BSBMV isolate studied here differed from the previously characterised isolate for five nucleotides and four amino acids in the CP region. The two BVQ isolates characterised in this study contained three additional nucleotides resulting in an additional amino acid residue (arginine) at CP position 86, as compared to the only isolate available in databank.

High-scale analysis of pathogenicity determinants of Potato mop-top virus

Potato mop-top virus (PMTV) is the type member of the genus Pomovirus characterized by a tripartite, single-stranded, positive-sense RNA genome. However, PMTV differs from the other pomoviruses by having an open reading frame (ORF) for a 8-kDa cysteine-rich protein (CRP) on RNA 3, downstream the triple gene block. Site-directed mutagenesis of infectious cDNA clones of PMTV showed that the CRP ORF is not needed for the systemic infection in Nicotiana benthamiana and several other hosts, but it has an effect on the symptom development of PMTV. CRP of PMTV was not capable of RNA silencing suppression in agroinfiltration tests. However, the expression of CRP using vectors based on Potato virus X (PVX) or Tobacco mosaic virus (TMV) resulted in necrotic symptoms on N. benthamiana and tobacco plants, respectively. Sequencing of PMTV isolates obtained from the field revealed significant sequence variability of the CRP. These data suggest that the functions of CRP may be expressed only in certain hosts or at certain phases of the PMTV infection cycle.

Transient coexpression of individual genes encoded by the triple gene block of potato mop-top virus reveals requirements for TGBp1 trafficking.

TGBp1, TGBp2, and TGBp3, three plant virus movement proteins encoded by the "triple gene block" (TGB), may act in concert to facilitate cell-to-cell transport of viral RNA genomes. Transient expression of Potato mop-top virus (genus Pomovirus) movement proteins was used as a model to reconstruct interactions between TGB proteins. In bombarded epidermal cells of Nicotiana benthamiana, green fluorescent protein (GFP)-TGBp1 was distributed uniformly. However, in the presence of TGBp2 and TGBp3, GFP-TGBp1 was directed to intermediate bodies at the cell periphery, and to cell wall-embedded punctate bodies. Moreover, GFP-TGBp1 migrated into cells immediately adjacent to the bombarded cell. These data suggest that TGBp2 and TGBp3 mediate transport of GFP-TGBp1 to and through plasmodesmata. Mutagenesis of TGBp1 suggested that the NTPase and helicase activities of TGBp1 were not required for its transport to intermediate bodies directed by TGBp2 and TGBp3, but these activities were essential for the protein association with cell wall-embedded punctate bodies and translocation of TGBpl to neighboring cells. The C-terminal region of TGBp1 was critical for trafficking mediated by TGBp2 and TGBp3. Mutation analysis also suggested an involvement of the TGBp2 C-terminal region in interactions with TGBp1.

Alternative hosts for potato mop-top virus, genus Pomovirus and its vectorSpongospora subterranea f.sp. subterranea

Seventeen weed species common in the potato fields in Denmark were grown in a hydroponic system infested with viruliferous zoospores ofSpongospora subterranea f.sp.subterranea carrying potato mop-top virus (PMTV). The plants were examined for infection with PMTV andS.s.s. using DAS-ELISA and based on visible symptoms. Only two weed species were found to be infected with PMTV,Chenopodium album andSolanum nigrum, whereas 13 became infected withS.s.s. C. album was infected with PMTV by mechanical inoculation were the infection became systemically and the leaves showed necrotic spots.S. nigrum became local infected with PMTV by mechanical inoculation. By inoculation with viruliferousS.s.s. the roots ofS. nigrum became infected but in three weeks PMTV had not spread into the top of the plants.Nicotiana benthamiana was used as a control susceptible toS.s.s. and PMTV.

First report of Beet virus Q in Belgium.

Beet necrotic yellow vein virus (BNYVV), the causal agent of rhizomania disease on sugar beet, has been reported in Belgium for more than 16 years. Other soilborne viruses belonging to the genus Pomovirus, such as Beet soilborne virus (BSBV) (3) and Beet virus Q (BVQ) (1), are suspected pathogens of sugar beets grown in Belgium. During the 2000 growing season, more than 20 fields showing rhizomania-like and yellowing symptoms on sugar beet leaves were investigated for the presence of BVQ, BNYVV, and BSBV. All samples were checked by enzyme-linked immunosorbent assay (ELISA) using commercial BNYVV (Sanofi Diagnostics Pasteur, Marnes-La-Coquette, France) and BSBV/BVQ (DSMZ, Braunschweig, Germany - AS-0576 polyclonal, AS-0576/2 MAb) antisera. RNA was extracted from sugar beet rootlets using an RNeasy extraction kit (Qiagen, Hilden, Germany), before performing a reverse transcription-polymerase chain reaction (RT-PCR) using primers (5'-GCTGGAGTATATCACCGATGAC-3' and 5'-AAAATC TCGGATAGCATCCAAC-3') designed to specifically amplify a 510-bp region of BVQ RNA-1. The presence of BSBV and BNYVV was also checked by RT-PCR using previously described primers (1,2). The BVQ-derived PCR product was sequenced and proved to be more than 99% identical to the Wierthe BVQ isolate nucleotide sequence. Soil transmission of BVQ was demonstrated through a bioassay using soil dilutions with quartz and sugar beet cv. Cadyx as bait. After 6 weeks, BVQ was detected by RT-PCR in bait plants. The putative vector, Polymyxa betae, was identified by lactophenol-cotton blue staining of the roots followed by microscopic examination. BVQ produces irregularly shaped local lesions that appear ≈5 days after mechanical inoculation and tend to spread along veins. BVQ was detected in six fields located in the Polders Region and Brabant Province of Belgium. BVQ was always found in sugar beet samples coinfected with BNYVV and BSBV. The economic significance of BVQ and its interaction with other viruses is not known.

Complete sequence of RNA 1 and the presence of tRNA-like structures in all RNAs of Potato mop-top virus, genus Pomovirus.

The complete nucleotide sequence (6043 nt) of RNA 1 from Potato mop-top virus (PMTV-Sw), the type member of the genus Pomovirus, was determined. The first (5'-terminal) open reading frame (ORF 1) encodes a predicted protein of 148 kDa. ORF 2 extends through the opal stop codon of ORF 1 producing a predicted readthrough protein of 206 kDa which resembles the RNA-dependent RNA polymerases (RdRp) of other fungal-transmitted viruses. It includes a methyltransferase, a helicase and a GDD RdRp motif, respectively. Phylogenetic analyses of RdRps indicated that PMTV is most closely related to Beet soil-borne virus (genus Pomovirus), Broad bean necrosis virus (genus Pomovirus) and Soil-borne wheat mosaic virus (genus Furovirus), and is more distantly related to the other viruses of the former furovirus group. The 5' and 3' termini of RNA 1 in PMTV contained untranslated regions (UTR) of 114 nt and 489 nt, respectively. The 3'-UTR of RNA 1 contained a tRNA-like structure, which has previously been reported in the 3'-UTR of RNA 2 but not RNA 3. However, in this study, the tRNA-like structure was also found in the 3'-UTR of RNA 3, which confirms its presence in the 3'-UTRs of all three RNAs of PMTV.

Genome properties of beet virus Q, a new furo-like virus from sugarbeet, determined from unpurified virus.

Based solely on the information that beet virus Q (BVQ) contains tubular particles, the entire nucleotide sequence of its tripartite genome was determined from unpurified virus in ca. 40 ml crude sap from locally infected Chenopodium quinoa. A starting sequence for RNA 1 was generated using primers corresponding to highly conserved helicase domains in the respective RNAs of furo-, pomo-, peclu-, hordei- and tobraviruses, and was extended by a walking random-primed cDNA approach. The similarity of the 3' ends of furoviral RNAs allowed starting sequences for BVQ RNAs 2 and 3 to be obtained once the 3' end of RNA 1 was known. BVQ RNA 1 encodes a protein with a methyltransferase-like, a variable and a helicase-like region, and for a readthrough protein which, in addition, contains an RNA-dependent RNA polymerase region. RNA 2 carries the coat protein gene, a coat protein read-through protein gene and two additional ORFs which may have arisen by deletions from an originally larger readthrough domain. RNA 3 carries a triple gene block resembling that of several other rod-shaped viruses. The 5' UTRs of the three RNAs have the potential to form a series of hairpins with C-A and C-C mismatches resembling those found in tymoviral RNAs. The 3' ends can be folded into tRNA-like structures which are preceded by a long hairpin-like structure and an upstream pseudoknot domain. BVQ belongs to the recently proposed genus Pomovirus; it shows evolutionary relationships to furoviruses in sensu stricto, peclu-, hordei-, tobra-, tymo-, tobamo-, carla- and potexviruses.