Tobravirus Research Articles

Several viral diseases of Lycopersicon esculentum circulating in Ukraine

This paper describes detection of some typical plant viruses infecting Lycopersicon esculentum Mill. plants in Ukraine. Diagnostics using enzyme-linked immunosorbent assay (ELISA) confirmed presence of antigens of viruses belonging to Tobamovirus (PMMoV, ToMV), Cucumovirus (CMV) and Tobravirus (TRV) genera in sap of tomato plants. When studying viral diseases of tomatoes, monoinfection was shown to be prevalent. Tomato mosaic virus (ToMV) was most common.

Identification of Two Tobacco rattle virus Variants Associated with Line Pattern Disease of Bleeding Heart in Ohio

Tobacco rattle virus (TRV) is a member of the Tobravirus genus and has a broad host range including weeds, vegetables, and ornamentals. Several bleeding heart (Lamprocapnos spectabilis) plants showing a yellow line pattern symptom were negative for the Potyvirus group, Alfalfa mosaic, Arabis mosaic, Cucumber mosaic, Impatiens necrotic spot, Tobacco mosaic, Tobacco ringspot, Tomato ringspot, and Tomato spotted wilt viruses by ELISA. cDNAs synthesized from dsRNA template and immunocaptured virions from symptomatic and asymptomatic bleeding heart amplified clear products with Tobravirus group and TRV specific primers targeted at regions in the 194 kDa open reading frame 1 on RNA 1. Cloned amplicon sequences demonstrated two distinct populations of sequences for both primer pairs and the two populations correlated with symptoms, or lack thereof, on bleeding heart. These results represent the first confirmed report of TRV associated with line pattern disease on bleeding heart in Ohio. Accepted for publication 19 December 2012. Published 25 March 2013.

Identification of Two Tobacco rattle virus Sequence Variants Associated with Virus-like Mottle Symptom on Hosta in Ohio

Two Hosta sp. ‘So Sweet’ plants and one Hosta sieboldii (labeled as ‘Albo-marginata’) plant showing a suspected virus-like leaf mottle symptom tested negative for the Potyvirus group, Hosta virus X, Alfalfa mosaic virus, Arabis mosaic virus, Cucumber mosaic virus, Impatiens necrotic spot virus, Tobacco mosaic virus, Tobacco ringspot virus, Tomato ringspot virus, and Tomato spotted wilt virus by ELISA. DsRNA analysis produced a banding profile suggestive of a viral infection, and dsRNA was used as template to synthesize cDNAs for use with tobravirus group and Tobacco rattle virus (TRV) specific PCR primers. Amplicons were cloned and sequenced, and results showed two distinct populations of sequences: the two So Sweet isolates were ∼99% identical to each other but only ∼92% identical to the Albo-marginata isolate. These results represent the first confirmed report of TRV in Hosta in Ohio, and further demonstrate that there are at least two nucleotide sequence variants of the virus infecting Ohio Hosta. Accepted for publication 21 December 2012. Published 30 March 2013.

Tomato and Tobacco Phytoene Desaturase Gene Silencing by Virus-Induced Gene Silencing (VIGS) Technique

Background and Aims: Virus-Induced Gene Silencing (VIGS) is a virus vector technology that exploits antiviral defense mechanism. By infecting plants with recombinant viruses containing host genes inserted in the viral genome, VIGS achieves the RNA silencing process. The purpose of this study was to investigate the possibility of tomato (Lycopersicon esculentum Mill.) and tobacco (Nicotiana benthamiana) phytoene desaturase (PDS) gene silencing, using VIGS technique by VIGS vector containing tomato PDS. Methods: PDS gene encodes one of the important enzymes in the carotenoid biosynthesis pathway. In this method Tobacco rattle tobravirus (TRV) vectors including pTRV1 ،pTRV2 and pTRV2PDS were used. The pTRV2PDS vector carried inserts derived from tomato PDS gene. Vectors were transformed into Escherichia coli strain DH5α. Agrobacterium tumefaciens strain GV3101 cells were transformed by the vectors containing specific genes. Results: Colony PCR with specific primers proved the presence of the PDS and RNAdependent RNA polymerase genes in selected agrobacterium random colonies. Agrobactrium containing pTRV1 ،pTRV2 and pTRV2PDS was inoculated into induction media culture. Finally, acetosyringone was added to pTRV1 culture and then pTRV1 and pTRV2PDS cultures mixed in a 1:1 ratio. Mixed culture was infiltrated into the test plant seedlings using 1ml needless syringe. Conclusion: Results showed the gene silencing in the form of photobleaching and mosaic occurred in tomato and tobacco plant leaves, respectively. However, plant infiltration with pTRV1 and pTRV2 (without PDS gene as control) mixed culture did not show any photobleaching.

Evaluation of the National Plant Germplasm System's Garlic Collection for Seven Viruses

Garlic (Allium sativum L. and A. longicuspis Regel.) germplasm maintained by the USDA-ARS Western Regional Plant Introduction Station (WRPIS) was screened for four viruses commonly infecting garlic [Garlic common latent carlavirus (GCLV), Leek yellow stripe potyvirus (LYSV), Onion yellow dwarf potyvirus (OYDV), and Shallot latent carlavirus (SLV)] plus three others [Iris yellow spot tospovirus (IYSV), Tobacco rattle tobravirus (TRV), and Tomato spotted wilt tospovirus (TSWV)]. Over 200 accessions were rated for the incidence of symptomatic plants during two production seasons, in July 2005 and June 2006. In both years, incidence of symptomatic plants ranged from less than 10% to 100% in individual accessions. Laboratory testing of selected accessions showed that the accessions were predominantly infected with OYDV (60% of infections), followed by GCLV (25%) and LYSV (15%). Accepted for publication 26 June 2008. Published 19 September 2008.

EFFECT OF GREEN MANURE CROPS AND ORGANIC AMENDMENTS ON INCIDENCE OF NEMATODE-BORNE TOBACCO RATTLE VIRUS

Tobacco rattle tobravirus (TRV) may infect several ornamental bulb crops and is transmitted by trichodorid nematodes. Paratrichodorus teres, P. pachydermus and Trichodorus similis are the main vectors in the Netherlands. In field experiments the effects of various pre-crops and organic amendments on the TRV Infection Potential of Soils (TRV-IPS) and on disease level in tulip and gladiolus were studied. Organic matter amendment of soil at a rate of 1% dry weight has been shown to reduce the host finding activity of P. teres under laboratory conditions. In a field containing viruliferous P. teres dahlia, italian ryegrass, white mustard and fodder radish were grown or the soil was kept fallow and the resulting TRV-IPS prior to the bulbous test crops was measured by a soil dilution bait test method. The application of organic matter was tested after dahlia as pre-crop. Household waste compost (GFT compost) was applied as a soil mix or planting furrow treatment at 12 tons dry weight per ha for tulip and gladiolus. Spent mushroom compost (Champost) was added as planting furrow treatment at 17 or 12 tons dw/ha, respectively, for tulip and gladiolus. The percentage of TRV diseased plants was determined at flowering in all pre-crop and organic amendment treatments. Champost in the planting furrow and fodder radish as a preceding crop reduced the percentage infection in tulip under favourable conditions for TRV infection. In gladiolus most organic amendments, fodder radish as pre-crop and keeping the soil fallow reduced the TRV infection rate of the plants during the first growing season, but not of the plants grown from the corms in the next year.

Use of multiplex real‐time PCR (TaqMan) for the detection of potato viruses*

Certain viruses affect the quality of potato tubers for either table use or processing. Visual discrimination of these viruses is problematic because of variable symptoms, but is important if proper controls are to be implemented. Work at the Central Science Laboratory has concentrated on the detection of Potato mop‐top pomovirus (PMTV), Tobacco rattle tobravirus (TRV) (both associated with the disease spraing) and the tuber necrotic strain of Potato Y potyvirus (PVYNTN), the symptoms of which can often be confused with spraing. A nucleic acid‐based approach has been adopted as TRV is often found as naked RNA with no associated coat protein, and accurate discrimination of PVY strains is impossible by serology. The multiplex TaqMan assay developed in this work streamlines the testing, replacing two separate tests currently used (a TRV RT‐PCR and a PMTV enzyme‐linked immunosorbent assay) with a single‐tube assay, which has no post‐PCR manipulations. The assay has been shown to be more sensitive than either of the tests which it replaces, allowing 100‐ and 10000‐fold increases in sensitivity for TRV and PMTV detection respectively. The test reliably detected over 40 different isolates of TRV and PMTV obtained from a wide range of cultivars and locations, including samples where existing tests failed. A PCR‐based method capable of discriminating strains of PVY was also developed.

Identification of the principal viruses infecting tomato crops in Tunisia

Serological tests have been used to detect viruses associated with tomato in 257 samples collected in different regions in Tunisia, Cap‐Bon, Sahel and South during successive seasons. The viruses detected were cucumber mosaic cucumovirus (CMV) and tomato aspermy cucumovirus (TAV), potato Y potyvirus (PVY), tobacco etch potyvirus (TEV) and pepper veinal mottle potyvirus (PVMV), tomato mosaic tobamovirus (ToMV) and tobacco mosaic tobamovirus (TMV), tobacco rattle tobravirus (TRV), alfalfa mosaic alfamovirus (AMV), tomato spotted wilt tospovirus (TSWV), tomato ringspot nepovirus (TomRSV) and potato X potexvirus (PVX). Some were detected in all three regions surveyed, at variable frequencies: TMV, CMV, TEV, PVY, ToMV, AMV, TAV, TSWV and TRV. Others were only detected in two regions (PVMV in Cap‐Bon and Sahel and PVX in Sahel and in the south) or one region (TomRSV in Cap‐Bon). Movement of individual viruses from one region to another may be due to movement of specific vectors, as in the case of the thrips‐transmitted TSWV moving from the south to the north. Some of these viruses were found for the first time in Tunisia.

Nematode Transmission of Tobacco Rattle Virus Serves as a Bottleneck to Clear the Virus Population from Defective Interfering RNAs

DI7 is a defective interfering RNA derived from RNA 2 of tobacco rattle tobravirus (TRV) isolate PpK20. Tobacco was transformed with DI7 cDNA fused to the CaMV 35S promoter. Upon infection of the transgenic plants with TRV isolate PpK20 or the serologically unrelated isolate PaY4, the transgenic DI7 RNA started to accumulate at high levels and strongly interfered with accumulation of wild-type (wt) RNA 2. When DI7 transgenic plants infected with isolate PpK20 were used as source plants in nematode-transmission experiments, the vector Paratrichodorus pachydermus efficiently transmitted virus to healthy bait plants. However, the nematodes transmitted only the wt virus present in the transgenic source plants, whereas virus particles containing the abundant, accumulated DI7 RNA were excluded from transmission. Evidence is presented that wt RNA 2 and DI7 RNA are encapsidated in cis by their encoded CPs, which are known to be functional and nonfunctional in transmission, respectively. This mechanism would result in defective interfering RNAs, which rapidly arise after mechanical transmission of the virus in the laboratory, being eliminated from tobraviruses under natural field conditions. Also this mechanism which acts with nematode transmitted virus isolates contrasts with that of vector-transmission of defective potyviruses and luteoviruses by wt helper viruses.

Anemone - an Additional Perennial Ornamental Host of Tobacco Rattle Virus in the U.S.

A previous report (1) drew attention to the occurrence in the U.S. of tobacco rattle tobravirus (TRV) in several perennial ornamentals that move freely in international trade. Here we report the occurrence of TRV in an additional host plant of this type. The virus was identified in anemone (Anemone × hybrida cv. Honorine Jobert) with leaf symptoms consisting of chlorotic blotches, chlorotic line patterns, and distortion. Characteristic tobravirus-like particles with modal lengths of 50, 80, 130, and 180 nm were observed by electron microscopy in partially purified extracts of symptomatic but not of asymptomatic plants. These particles reacted specifically with antibodies to TRV and pea early-browning (PEBV) tobraviruses in immunoelectron microscopic (IEM) assays, produced typical TRV-induced symptoms in Nicotiana benthamiana and N. clevelandii, in which similar particles were detected by IEM, but, like other TRV isolates, did not infect pea (2). No other viruslike particles were observed in partially purified extracts of symptomatic anemone plants. This is the first report of TRV infection in anemone. While TRV occurs widely in Europe, it had been identified previously in the U.S. only in Oregon, Washington, and California (1,2,3). This virus is an important pathogen of several crops, including potato. Several areas of the Midwest, including Minnesota and Wisconsin, have significant seed-potato industries, and further introduction and possible dissemination of TRV pose potential regulatory and quarantine issues. This example of the movement of an exotic pathogen of potential economic importance into new areas underlines the need for closer monitoring of plant material entering international trade in an era of increasing globalization.

Use of PCR as a tool for diagnosis of tobacco rattle tobravirus in seed potatoes

Diagnosis of tobacco rattle tobravirus (TRV) in potato is difficult because of the existence of non‐encapsidated NM‐type strains, because similar symptoms are caused by potato mop‐top furovirus and physiological internal rust spot and because serological tests are inefficient. The aim of this study was first to compare ELISA and PCR tests. The former was found to be ineffective, so only the PCR technique was in a survey of numerous seed‐potato lots (200‐tuber samples) from different EU countries. A large disparity was found according to geographical origin, with infection levels of German, Dutch and French seed potatoes of 10.3, 8.5 and 0.6%, respectively. There is a real risk that planting tubers with such infection levels will newly contaminate soil. Besides, TRV is not considered in the EU texts on marketing of seed potatoes, so there is no legal recourse if seed‐potato lots are found to be TRV‐infected.

Replication of in vitro tobravirus recombinants shows that the specificity of template recognition is determined by 5' non-coding but not 3' non-coding sequences.

Natural recombinant tobacco rattle tobravirus (TRV) isolates contain sequences from a different tobravirus, pea early browning virus (PEBV). To characterize the sequence requirements for viable recombinant formation hybrid cDNA clones of RNA2 of PEBV and TRV were assembled. Inclusion of 320 nt from the 5' terminus of PEBV or 335 nt from the 5' terminus of TRV in the hybrid RNAs was sufficient to permit their replication by, respectively, PEBV RNA1 or TRV RNA1 regardless of the origin of the 3' terminal region. However, PEBV RNA1 but not TRV RNA1 was sometimes able to support low level replication of RNA2 containing the heterologous 5' terminal region. In vitro translation of PEBV transcripts containing 5' noncoding region deletions supported the hypothesis that in vivo the PEBV coat protein (CP) is expressed from a subgenomic RNA and that, therefore, in the recombinants the CP subgenomic promoter probably is recognized by the replicase of the heterologous virus.

Characterization and vector relation of a serologically distinct isolate of tobacco rattle tobravirus (TRV) transmitted byTrichodorus similis in northern Greece

A virus isolated from diseased tobacco plants growing in Macedonia, northern Greece, had host range and physico-chemical properties typical of a tobravirus. Although it was serologically unrelated to any of the ten tobravirus isolates tested, it reacted in spot hybridization tests with a probe derived from RNA-1 of tobacco rattle virus (TRV) strain SYM. Therefore, the isolate probably represents a previously undescribed serotype of TRV. Male, female and juvenileTrichodorus similis nematodes recovered from the rhizosphere of the diseased tobacco plants transmitted TRV in each of three laboratory experiments. In two of these experiments 50% and 54%, respectively, of the nematodes transmitted virus toPetunia hybrida bait plants, whereas only 18% transmitted virus toNicotiana tabacum plants in a third test. Ultrathin sections of the feeding apparatus of individual nematodes, which had transmitted virus, were examined by electron microscopy. Virus particles were observed, retained as a monolayer in the apices of the oesophageal lumen and as a group of particles within a matrix in the open part of the lumen.

Occurrence of Tobravirus Infections inPeperomia, Hosta,andPhloxin the Midwestern U.S.

Occurrence of Tobravirus Infections in<i>Peperomia, Hosta,</i>and<i>Phlox</i>in the Midwestern U.S.

Increase in Plasmodesmatal Permeability during Cell-to-Cell Spread of Tobacco Rattle Virus from Individually Inoculated Cells.

A microinjection technique was devised for inoculation of single Nicotiana clevelandii leaf trichome cells with virus particles. By removing inoculated trichomes at various times after microinjection, it was shown that at least 4 hr were required for tobacco rattle virus (TRV; tobravirus group) to move out of primarily inoculated cells. Effects of the early stages of TRV infection on plasmodesmatal permeability were examined by microinjection of fluorochrome-labeled molecules. Fluorescein-labeled insulin A chain (Mr, 2921) and fluorescein-labeled dextran (Mr, 4400) were observed to pass out of individual N. clevelandii trichome cells that had been inoculated with TRV by microinjection 5 hr previously. By contrast, Lucifer Yellow CH-labeled dextran (Mr, 10,000) was restricted to the inoculated cell. None of these fluorescent probes were able to move out of uninoculated cells or out of cells that had been inoculated with TRV only 2 hr previously. The movement of macromolecules through plasmodesmata, therefore, coincided with and probably resulted from cell-to-cell movement of TRV. The results are discussed with reference to the interaction of viruses and plasmodesmata and mechanisms of intercellular virus movement.

The leaky UGA termination codon of tobacco rattle virus RNA is suppressed by tobacco chloroplast and cytoplasmic tRNAs(Trp) with CmCA anticodon.

RNA-1 molecules from tobacco rattle virus (TRV) and pea early-browning virus (PEBV), two members of the tobravirus group, have recently been shown to contain internal, in-frame UGA termination codons which are suppressed in vitro. Our results suggest that a UGA stop codon also exists in RNA-1 of pepper ringspot virus (PRV), another tobravirus. UGA suppression may therefore be a universal feature of the expression of tobravirus genomes. We have isolated two natural suppressor tRNAs from uninfected tobacco plants on the basis of their ability to promote readthrough over the leaky UGA codon of TRV RNA-1 in a wheat germ extract depleted of endogenous mRNAs and tRNAs. Their amino acid acceptance and nucleotide sequences identify the two UGA-suppressor tRNAs as chloroplast (chl) and cytoplasmic (cyt) tryptophan-specific tRNAs with the anticodon CmCA. These are the first UGA suppressor tRNAs to be identified in plants. They have several interesting features. (i) Chl tRNA(Trp) suppresses the UGA stop codon more efficiently than cyt tRNA(Trp). (ii) Chl tRNA(Trp) contains an A24:U11 pair in the D-stem as does the mutated Escherichia coli UGA-suppressor tRNA(Trp) which is a more active suppressor than wild-type tRNA(Trp). (iii) The suppressor activity of chl tRNA(Trp) is dependent on the nucleotides surrounding the stop codon because it recognizes UGA in the TRV context but not the UGA in the beta-globin context.

Construction and analysis of infectious transcripts synthesized from full-length cDNA clones of both genomic RNAs of pea early browning virus

Full-length cDNA clones of both RNAs of pea early browning virus have been constructed. Synthetic transcripts derived in vitro from these clones are infectious when inoculated onto plants. Electron microscopy revealed the presence of virions in transcript-inoculated plants, and both purified RNA and virions isolated from such plants could be used to infect other plants. Transcripts of RNA1 alone were able to replicate and spread systemically which is a characteristic of members of the tobravirus group of plant viruses.

Analysis of RNA2 of pea early browning virus strain SP5

The tobravirus group includes two viruses, pea early browning virus (PEBV) and tobacco rattle virus (TRV). As part of a project, aimed at using the coat protein gene for synthesis of PEBV resistant transgenic plants, we have cloned the PEBV RNA. One cDNA clone, 2.4 kb in size, was shown by Northern hybridization analysis to be derived from RNA2. The DNA sequence of the 2.4 kb clone is shown. The sequence contains two open reading frames (ORFs) which could encode proteins consisting of 212 amino acids (ORF212) and 255 amino acids (ORF255)

Cultivation factors influencing spraing in potato‐a statistical analysis

In an attempt to study the effects of cultivation factors on the incidence of spraing in potato, on the presence of tobacco rattle tobravirus (TRV) and potato mop‐top furovirus (PMTV), and on the densities of trichodorid nematodes in field soils, a statistical analysis was conducted based on field records provided by a Swedish potato crisp industry. The factors studied were crop rotation, precipitation (irrigation inclusive), planting time, fertilization, plant‐available P, K and B, soil texture and soil pH. The data were collected for 1979‐1987, representing 50 farms (198 fields) in southern Sweden. A correlation was found between TRV infectivity in the autumn and planting time of the potato crop. Thus, late planting was correlated with lower frequency of TRV. By ANOVA test of 5 years of preceding crops, a possible spraing increase was indicated when numbers of potato or barley crops increased in the crop rotations. A possible spraing decrease was indicated when the share of wheat increased. High incidence of spraing, and high densities of trichodorid nematodes, were both correlated with low values of plant‐available potassium, KAL, in soil.

Intraviral homology and subgenomic RNAs of pepper ringspot virus

The Tobraviruses constitute a group of rod-shaped, bipartite, plus-stranded RNA viruses. We report on homologies between the two viral genomic RNA molecules of pepper ringspot virus (PRV) and on the subgenomic components generated from them during infection. It has previously been shown that the 3′-terminal 459 nucleotides of PRV RNA-1 and RNA-2 are identical. Here it is shown that there is a second homology between RNA-1 and a region of RNA-2, located at least 240 nucleotides downstream from its 5′ terminus. In agreement with strains in another Tobravirus group, we observe three subgenomic components in extracts of plants infected with PRV. Two of these, RNAs-1 a (1.6 kb) and −1 b (0.8 kb), are derived from RNA-1 (the larger genomic RNA). They are probably mRNAs for 29- and 16-kDa nonstructural virus proteins. The smaller genomic RNA, RNA-2, generates the subgenomic component RNA-2a (1.3–1.45 kb) which is probably an efficient capsid protein mRNA. Previously reported subgenomic components of 2.8 and 1.1 kb are shown to be electrophoretic artifacts. RNA-2a, but not 1a or 1b, is present in virion RNA preparations, indicating that it is the only subgenomic species to be encapsidated.