Tobacco Rattle Virus Isolates Research Articles

First report of tobacco rattle virus infecting Weigela florida in the USA.

Weigela (Weigela florida (Bunge) A. DC., Family: Caprifoliaceae) are woody shrubs native to North China, Korea, and Japan. In the U.S., weigela are commonly used as landscape ornamental plants (McNamara et al. 2010). Two viruses have been reported in weigela: tomato spotted wilt orthotospovirus and impatiens necrotic spot orthotospovirus (Sastry et al. 2019). Ten weigela plants, originating from commercial nurseries in Minnesota, exhibiting chlorosis, chlorotic line patterns, and necrosis (e-Xtra) were submitted for virus diagnostics as potted plants at the University of Minnesota Plant Disease Clinic and the Virology Lab in 2019 and 2020 (five plants each year). Under greenhouse conditions, symptoms progressed from chlorosis to necrosis and even plant death in two of the five plants in 2019. Electron microscopy revealed rod-shaped particles of ≈20 nm in diameter and lengths between 40-200 nm with similar morphology to members of the genus Tobravirus (e-Xtra). Virus-like particles were enriched by ultracentrifugation and total nucleic acids were extracted from partial purifications using a phenol:chloroform extraction method (Lockhart et al. 1997). Tobacco rattle virus (TRV) was identified by cloning and sequencing of the 463bp amplicon obtained with the TRV detection primers described in Robinson, 1992. High-throughput sequencing (HTS) was done to confirm the TRV detection. A cDNA library was prepared from purified viral RNA using the TruSeq Stranded mRNA kit and sequenced on Illumina NovaSeq 6000 platform as 150 bp-paired end reads. A total of 44,316,446 raw data reads were obtained, preprocessed using the BBDuk plugin, and de novo assembled using SPAdes assembler. Viral contigs were identified using the NCBI BLASTX tool. The assembly of TRV RNA1 was 6,842 nt with 20,627,348 reads (47% of total reads) mapped to it and an average coverage per nucleotide at 323,639X. The assembly of TRV RNA2 was 3,033 nt with 22,769,253 reads (52% of total reads) mapped to it and an average coverage per nucleotide at 798,660X. NCBI GenBank accession numbers for the assemblies representing RNA1 and RNA2 are OQ408335 and OQ408336, respectively. NCBI BLASTn analysis showed the highest level of nucleotide identity to TRV genomic RNA segments 1 and 2, with 97% and 99% identity to the TRV isolate RNA1 (GQ903771) and RNA2 (GQ903772), respectively, that originated from Michigan potato. No other viral contigs were detected from the virion nucleic acid extraction by HTS, however this enrichment method doesn't exclude other viruses. In addition to using the detection primers by Robinson 1992, we designed primers based on our HTS data: TRV-WG-DetF3 5'- GACGAAGGAGGCTGTCATTGC-3' and TRV-WG-DetR3 5'-CGGACTATCGTGATGCCCATGC- 3'. RT-PCR amplicons from each of the 10 symptomatic plants were cloned and sequenced. Among these clones, Sanger sequence identities ranged between 96-100% compared to the HTS data and 98-99% to the TRV potato isolate from MI. To our knowledge, this is the first report of TRV infecting the ornamental host W. florida worldwide. TRV is a nematode-transmitted viral pathogen of economic importance, most notably in potatoes (Sastry et al. 2019). In the US, TRV has been reported on several landscape ornamentals, horticultural crops, and native habitats. Further research is needed to investigate the impact of TRV on the ornamental industry and the role of ornamentals as reservoirs for cultivated crops like potatoes.

Phylogenetics of tobacco rattle virus isolates from potato (Solanum tuberosum L.) in the USA: a multi-gene approach to evolutionary lineage.

Tobacco rattle virus (TRV) is an important soil-borne virus of potato that is transmitted by stubby-root nematodes. TRV causes corky ringspot, a tuber disease of economic importance to potato production. Utilizing protein-coding regions of the whole genome and a range of computational tools, the genetic diversity, and population structure of TRV isolates from several potato-growing regions (Colorado, Idaho, Indiana, Minnesota, Nebraska, North Dakota, and Washington State) in the USA were determined. Phylogenetic analyses based on RNA2 nucleotide sequences, the coat protein (CP) and nematode transmission (2b) genes, showed geographical clustering of USA isolates with previously known American isolates, while European isolates grouped in a distinct cluster. This was corroborated by the observed genetic differentiation and infrequent gene flow between American and European isolates. Low genetic diversity was revealed among American isolates compared to European isolates. Phylogenetic clustering based on RNA1 genes (RdRp, RdRp-RT, and 1a) were all largely incongruent to that of 1b gene (virus suppressor of RNA silencing). This genetic incongruence suggested the influence of recombination. Furthermore, the RdRp, RdRp-RT, and 1a genes were predicted to be more conserved and under negative selection, while the 1b gene was less constrained. Different evolutionary lineages between TRV RNA1 and RNA2 genomic segments were revealed.

TOBACCO RATTLE VIRUS ISOLATES USING COMPLEX DIAGNOSTICS

The experimental data on the search for isolates of the tobacco rattle virus using complex diagnostics of the research potatoes material is presented in the article. As a result of this work, the indicator-plant N. Debneyi was identified, which carries an isolate of tobacco rattle virus.

Complete nucleotide sequences of two closely related tobacco rattle virus RNA2s for which previously possibly only deletion/recombination mutants had been described

The almost complete nucleotide (nt) sequences lacking only short primer-derived 5′ and 3′ terminal portions were determined for the RNA2s of two closely related tobacco rattle virus (TRV) isolates which were detected in recently infected roots of Nicotiana benthamiana bait plants (isolate HaB) or of field-grown potato plants (isolate HaW), respectively. The sequences of c. 1350 5′ proximal nts in these RNA2s were almost identical to those in four previously described TRV isolates from potatoes in the Netherlands and Poland which had been propagated in tobacco leaves. The RNA2s of the earlier isolates, however, consist of only c. 2000 nts, whereas the HaW and HaB RNA2s comprise more than 4000 nts. In addition to the coat protein gene which is present in the RNA2s of all isolates, the HaW and HaB RNA2s contain two further open reading frames which in the previous isolates were apparently lost either entirely or of which small remnants only were left. The RNA2s of the previously described isolates and of our new ones have different RNA1-related 3′ ends which apparently have been acquired from different supporting RNA1s. After transmission to tobacco leaves HaB RNA2 formed deletion/recombination mutants which – as in the previous isolates – consisted of only c. 2000 nts. They had acquired the 3′ end from their supporting RNA1.

Single-nucleotide polymorphisms and reading frame shifts in RNA2 recombinant regions of tobacco rattle virus isolates Slu24 and Deb57.

Two previously sequenced tobacco rattle virus (TRV) isolates, Slu24 and Deb57, from Polish potato fields have recombinant RNA2 species. The 3'-proximal region of the Slu24 RNA2 is derived from the 3' terminus of its supporting RNA1, while that of the Deb57 RNA2 is derived from the 3' terminus of the unrelated RNA1 from the isolate SYM or PpK20. Gene structure annotation revealed open reading frames encoding truncated 16-kDa proteins in the recombinant regions of the RNA2 of Deb57 and Slu24. Reading frame shifts, single nucleotide substitutions and deletions occurred during recombination, including shifts from a stop codon or replacements of an internal stop codon. In the recombinant region of the Deb57 RNA2, the first reading frameshift event starts from the AUG start codon of the truncated 16-kDa protein. The second frameshift event, caused by a single nucleotide deletion upstream of the stop codon, leads to the splitting of the stop codon into two amino acid codons and the continuation of translation. In addition, a U-to-A substitution changes a potential internal stop codon UAA, which is caused by recombination-related frame shifts, into the codon AAA, encoding lysine. The replacement of this internal stop codon with an amino acid codon prevented the premature translation termination of the truncated 16-kDa protein. These recombination-related reading frame shifts are the driving force underlying the major differences in the translated amino acids, consequently leading to their translation into distinct polypeptides. Conversely, single nucleotide substitutions in the recombinant regions of the RNA2 of Deb57 or Slu24 resulted in only minor changes in the translated amino acids.

The genomic RNA1 and RNA2 sequences of the tobacco rattle virus isolates found in Polish potato fields

Four tobacco rattle virus (TRV) isolates were identified from tobacco bait seedlings planted in soil samples from Polish potato fields. Sequence analysis of the genomic RNA1 of the isolates revealed significant similarity to the isolates Ho and AL recently found in Germany. Multiple sequence alignments of the genomic RNA2 indicated that the two isolates from northern Poland (Deb57 and Slu24) are in a cluster with the isolates PSG and PLB found in the Netherlands. The remaining two isolates, from central Poland (11r21 and Mlo7), are in a distinct group with the unique isolate SYM found in England. The RNA2 sequences of the studied isolates range from 1998 nt to 2739 nt in length, and all carry deletions of the 2b and/or 2c genes. The isolate Mlo7 has an atypical RNA2 structure, having its cp gene located in its central region.

Molecular Detection of Tobacco rattle virus in Bleeding Heart [Dicentra spectabilis (L.) Lem.] Growing in Alaska

Tobacco rattle virus (TRV) was detected in bleeding heart from South Central Alaskan home gardens in 2010-11. TRV M-type and NM-type isolates were confirmed from these symptomatic bleeding heart plants by reverse transcription (RT)-PCR polymerase chain reaction, protein, serological, and virus transmission assays. RNA1 was sequenced from one of the bleeding heart M-type isolates, and the nucleotide identity ranged from 91% to 94% when compared with six TRV isolates from potato, spinach, and alstroemeria. This is the first detection of TRV from D. spectabilis in Alaska. It is also the first time that M- and NM-type isolates have been distinguished from bleeding heart plants. The significance of these findings is that even though TRV infected plants containing NM-type isolates probably will not be spread to other plants by its specific nematode vector; vegetative propagated roots from TRV infected plants of either type of isolates will continue to be a source of diseased plants to home gardeners. Accepted for publication 18 December 2012. Published 27 February 2013.

RNA2 of TRV SYM breaks the rules for tobravirus genome structure

Currently, all of the RNA2 molecules described for all of the more than thirty sequenced isolates of the three tobraviruses, Tobacco rattle virus (TRV), Pea early-browning virus (PEBV) and Pepper ringspot virus (PepRSV), have the virus coat protein (CP) gene located in the 5′ proximal position. However, sequencing of the RNA2 of the SYM isolate of TRV revealed that this isolate has a unique genome structure in which the virus CP gene is located in the central region of RNA2 downstream of three completely novel open reading frames (ORFN1, ORFN2 and ORFN3). An infectious clone of SYM RNA2 was constructed and mutations were introduced separately into each of the novel genes to interrupt their translation. However, none of the mutations resulted in any noticeable change in the ability of TRV RNA1 or RNA2 to replicate and move systemically in the leaves or roots of infected plants. In addition, individual expression of the novel ORFs either from a Potato virus X (PVX) vector or from a binary plasmid in Agrobacterium tumefaciens did not reveal any potential function.

Complete genomic sequence of a Tobacco rattle virus isolate from Michigan-grown potatoes

Tobacco rattle virus (TRV) causes stem mottle on potato leaves and necrotic arcs and rings in potato tubers, known as corky ringspot disease. Recently, TRV was reported in Michigan potato tubers cv. FL1879 exhibiting corky ringspot disease. Sequence analysis of the RNA-1-encoded 16-kDa gene of the Michigan isolate, designated MI-1, revealed homology to TRV isolates from Florida and Washington. Here, we report the complete genomic sequence of RNA-1 (6,791 nt) and RNA-2 (3,685 nt) of TRV MI-1. RNA-1 is predicted to contain four open reading frames, and the genome structure and phylogenetic analyses of the RNA-1 nucleotide sequence revealed significant homologies to the known sequences of other TRV-1 isolates. The relationships based on the full-length nucleotide sequence were different from than those based on the 16-kDa gene encoded on genomic RNA-1 and reflect sequence variation within a 20-25-aa residue region of the 16-kDa protein. MI-1 RNA-2 is predicted to contain three ORFs, encoding the coat protein (CP), a 37.6-kDa protein (ORF 2b), and a 33.6-kDa protein (ORF 2c). In addition, it contains a region of similarity to the 3' terminus of RNA-1, including a truncated portion of the 16-kDa cistron. Phylogenetic analysis of RNA-2, based on a comparison of nucleotide sequences with other members of the genus Tobravirus, indicates that TRV MI-1 and other North American isolates cluster as a distinct group. TRV M1-1 is only the second North American isolate for which there is a complete sequence of the genome, and it is distinct from the North American isolate TRV ORY. The relationship of the TRV MI-1 isolate to other tobravirus isolates is discussed.

First Report of Tobacco rattle virus in Sedum in Minnesota.

Sedums (Sedum spp.; Crassulaceae) are perennial landscape plants that are grown widely because they are drought tolerant and winter hardy. Plants of Sedum 'Matrona' showing faint foliar ringspot symptoms were collected at a nursery retail outlet in St. Paul, MN in July 2008 and tested for possible viral infection by transmission electron microscopic (TEM) examination of negatively stained, partially purified leaf tissue extracts (1). The only virus-like particles observed were rigid, rod-shaped particles similar to those of Tobacco rattle virus (TRV) and other tobraviruses. A random sample of 100 measurements showed particles 20 nm in diameter with two modal lengths of 115 nm and 175 nm. These virus-like particles were confirmed to be those of TRV by immunosorbent electron microscopy (1) using antiserum to TRV (ATCC PVAS 75) and by reverse transcription (RT)-PCR using total RNA extracted with the RNeasy Kit (Qiagen, Valencia, CA) and primers that yield a 462-bp amplicon from TRV RNA 1 (4). An amplicon of the expected size was obtained by RT-PCR and its nucleotide sequence (GenBank Accession No. GQ268817) had 95 to 99% identity to published TRV sequences (AAW13192 and AAB48382). Two additional amplicons generated by RT-PCR from separate plants were identical in size and nucleotide sequence to the first. On the basis of virion morphology, serological relatedness, and sequence identity, the virus associated with mild ringspot symptoms in sedum was identified as an isolate of TRV. To our knowledge, this represents the first report of TRV incidence in sedum. Although Arabis mosaic virus is the only other virus reported to occur in sedum (2), we have observed numerous, flexuous filamentous 750 to 800 nm virus-like particles in partially purified extracts of a range of sedums showing mild mosaic and/or vein-clearing symptoms in Minnesota. Similar virus-like particles were not observed by TEM in partially purified extracts from TRV-infected 'Matrona' plants, suggesting that they did not contribute to the symptoms observed. We have reported previously (3) the occurrence of TRV in a variety of widely grown perennial ornamentals that provide potential sources of inoculum for spread of this virus by nematode vectors (Trichodorus and Paratrichodorus spp.) that occur commonly in garden soil, and Sedum is now added to the list of potential TRV reservoir plants.

First Report of Tobacco rattle virus in Spinach in California.

In 2009 in coastal California (Santa Barbara County), commercially grown spinach (Spinacia oleracea) in two nearby fields exhibited symptoms of a previously unrecognized virus-like disease. Symptoms consisted of general chlorosis and bright yellow blotches and spots. Necrotic spots were also associated with the disease. In affected fields, disease occurred in limited, irregularly shaped patches that ranged from one to several meters in diameter. Symptomatic plants were unmarketable and these small patches of spinach were not harvested. With a transmission electron microscope, rigid, rod-shaped particles with a clear central canal were observed from plant sap of the symptomatic spinach. Analysis by a double-antibody sandwich-ELISA assay (Agdia Inc., Elkhart, IN) for Tobacco rattle virus (TRV) showed that the symptomatic plants were positive. Symptomatic spinach from the field was used for mechanical transmission to Chenopodium quinoa, C. murale, C. capitatum, spinach, and sugar beet (Beta vulgaris). All inoculated plants showed chlorotic local lesions and sugar beet showed chlorotic local lesions with rings. To further confirm the presence of TRV, reverse transcription (RT)-PCR was conducted. Total RNA was extracted from the mechanically inoculated symptomatic spinach plants using an RNeasy Plant Kit (Qiagen Inc., Valencia, CA) and used as a template in RT-PCR with forward (5'-TACATCACATCTGCCTGC-3') and reverse (5'-CTTCATTCACACAACCCTTG-3') primers specific to the movement protein gene from the spinach isolate of TRV (GenBank Accession No. AJ007294). Amplicons of the expected size (approximately 562 bp) were obtained. The RT-PCR products were sequenced (GenBank Accession No. GU002156) and compared with TRV sequences in GenBank to confirm the identity of the products. Sequences obtained had 96% nucleotide identity and 97% amino acid identity with TRV sequences available under the GenBank Accession Nos. FJ357571 and AJ007294. On the basis of the data from electron microscopy and serological and molecular analyses, the virus was identified as TRV. Soil samples collected from one of the fields were assayed for nematodes; however, Paratrichodorus or Trichodorus species were not recovered. To our knowledge, this is the first report of TRV in spinach in California. TRV has also been reported in spinach in England (1) and Germany (2).

First Report of Tobacco rattle virus Associated with Corky Ringspot in Potatoes Grown in North Dakota

Tobacco rattle virus (TRV) belongs to the genus Tobravirus and causes a stem mottle of potato (Solanum tuberosum) foliage and necrotic arcs and rings in tubers referred to as corky ringspot. This virus is generally transmitted by a number of species of stubby-root nematode. The virus is widespread and has been reported in California, Colorado, Florida, Idaho, Michigan, Oregon, Washington, Minnesota, and Wisconsin (2). In the spring of 2009, we received potato tubers of cv. Russet Burbank with internal necrotic arcs very similar to those caused by TRV from potato storages located in Grand Forks and Dickey counties of North Dakota. Total RNA was extracted from the necrotic lesions of two tubers from each location using the Total RNA Isolation kit (Promega Corp., Madison WI). These extracts were tested for TRV by reverse transcription (RT)-PCR using primers complementary to nucleotides 6555 to 6575 (Primer A) and identical to nucleotides 6113 to 6132 (Primer B) within the 3' terminus of TRV-SYM RNA-1 (GenBank Accession No. X06172) (3). The expected 463-bp amplicons from two separate tuber samples from each county were cloned (TOPO Cloning; Invitrogen, Carlsbad, CA) and sequenced. The sequences obtained from the four clones at both locations were found to be identical to each other and were 99% identical to the corresponding regions of TRV isolates from Michigan and Florida (GenBank Accession Nos. EU315226.1 and AF055912.1, respectively). Since sequences from all four clones were identical, only one of the sequences was submitted to Genbank (Accession No. GQ223114) and thus represents a consensus sequence. The extracts also tested positive in RT-PCR with a second set of primers corresponding to sequences in TRV RNA-2 yielding a 3.8-kbp amplicon (1). No evidence was found by RT-PCR for several other viruses that cause tuber necrosis in potato (Potato mop top virus, Tomato spotted wilt virus, Alfalfa mosaic virus, and tuber necrosis strains of Potato virus Y). The virus was mechanically transmitted by inoculating sap from symptomatic tubers from both counties to tobacco cv. Samsun NN, which showed typical bright yellow patches and spots on leaves 2 weeks postinoculation. TRV was confirmed in tobacco by RT-PCR from total RNA extracted from tobacco leaves with both sets of the aforementioned primers. To our knowledge, this is the first report of TRV in North Dakota and the first report of corky ringspot disease of potato in this state.

First Report of Tobacco rattle virus in Peony in Alaska.

Peonies (Paeonia sp.) are highly valued for their large showy flowers in home gardens and commercially in the cut flower industry. In 2007, scattered peony (Paeonia lactiflora 'Sarah Bernhardt') plants cultivated on small plots at the University of Alaska Experimental Station in Fairbanks displayed distinct leaf ringspot patterns. Symptoms were more severe during the cooler months of the growing season (June and September), with symptom remission in the intervening warmer months. Leaf samples from six symptomatic plants were collected in July and from 20 symptomatic plants in September and assayed for viruses. Leaf samples (1 g) were assayed with a general protocol for plant virus extraction and partial purification with differential centrifugation followed by protein detection on stained sodium dodecyl sulfate-polyacrylamide gel electrophoresis (1). No distinct proteins indicative of viral coat protein(s) were detected. Tomato spotted wilt virus (TSWV) and Tobacco rattle virus (TRV), known pathogens of peony, were then specifically targeted. Total RNA was extracted from each sample with an RNeasy Plant Mini kit (Qiagen Inc., Valencia, CA) and used as the template for reverse transcription (RT)-PCR with random primers. TSWV was not detected by RT-PCR with tospovirus group-specific primers (Agdia, Inc., Elkhart, IN). A nested set of primers designed from the TRV 16-kDa protein gene on RNA1 (4) amplified an ~600-bp fragment from one of the symptomatic plants. This DNA was directly sequenced (GenBank Accession No. FJ357572) and BLAST searches in GenBank revealed as much as 95% nucleotide (nt) identity with TRV accessions J04347 and X03685. Additional primer pairs specific for TRV (2) amplified overlapping fragments with expected sizes of ~818, ~515, and ~290 bp from the 29- and 16-kDa protein genes on the 3'-end of RNA1 that were directly sequenced. Assembly of these sequences in Sequencher 4.8 (Gene Codes Corp., Ann Arbor, MI) resulted in a 1,422-nt sequence (Accession No. FJ357571) and Clustal X analysis (3) showed 93 to 94% nt identity to TRV isolates, -ORY (AF034622), -PpK20 (AF314165), -Pp085 (AJ586803), and -SYM (D00155). Mechanical inoculation of partially purified virions from the confirmed TRV-infected peony plant to Nicotiana benthamiana gave no symptoms to occasional ringspots, faintly curled leaves, and chlorotic blotches on N. tabacum 'Samsun', and local lesions on Chenopodium amaranticolor. TRV infection of these hosts was confirmed by RT-PCR. With electron microscopy, rod-shaped particles similar to TRV with a distinct central canal characteristic of TRV were seen occasionally only from inoculated N. benthamiana. On the basis of the biological and molecular data, we have determined the virus in the peony to be an isolate of TRV, tentatively named TRV-Peony. TRV was confirmed in only one other peony based on a sequenced 290-nt PCR fragment with 95% identity with the sequence from the other TRV-infected peony. Lack of TRV detection in the other symptomatic peonies was possibly due to low viral concentrations and interfering plant substances. Documentation of TRV in peonies is especially important to help avoid distribution of virus-infected vegetative propagation material. To our knowledge, this is the first report of TRV in this host in Alaska, but also of this virus in Alaska.

First Report of Tobacco rattle virus Causing Corky Ringspot in Potatoes Grown in Minnesota and Wisconsin.

In July 2007, potato tubers cv. Russet Burbank (RB) with necrotic arcs and spots were detected in three fields in Buffalo County, Wisconsin and one field in Benson County, Minnesota. Umatilla Russet (UR) potatoes harvested from the west half of a field in Swift County, MN had similar, but visually distinct necrotic lesions. Portions of one field in Minnesota were abandoned, and the stored potato crop from two fields in Wisconsin was rejected by processors, representing a total crop loss due to tuber necrosis. Tuber symptoms displayed in both cultivars resembled those described for corky ringspot caused by Tobacco rattle virus (TRV) (4). Total RNA was isolated from necrotic tuber tissue crushed in liquid nitrogen and extracted using the Total RNA Isolation Kit (Promega Corp., Madison, WI). These extracts were tested for the presence of TRV by reverse transcription (RT)-PCR using primers complementary to nucleotides 6555 to 6575 and identical to nucleotides 6113 to 6132 within the 3' terminal open reading frame of TRV RNA-1 (3). The expected 463-bp fragments were amplified from RB tubers. Nucleotide sequences from a Wisconsin and Minnesota isolate (GenBank Accession Nos. EU569290 and EU569291, respectively) were 99 to 100% identical to the corresponding region in a published TRV sequence (GenBank Accession No. AF055912). A 396-bp fragment was amplified from UR tubers and sequence data (GenBank Accession No. EU569292) indicated a unique 63 nucleotide sequence was substituted for a 129 nucleotide sequence spanning residues 227 to 357 of the 463-bp amplicon from the RB TRV isolates. Seven fragments were sequenced from different UR tubers and the 396-bp fragment was identical among them. The sequence outside the substituted region had 92% identity to the published TRV sequence. Amplification of the full-length TRV RNA2 using primers 179/180 located in the 5' and 3' untranslated regions (2) was successful for 28 and 0% of the RB and UR samples, respectively, suggesting that the RNA2 is not present in these strains or has undergone significant mutation. TRV-infected sap from both potato cultivars was mechanically transmitted to tobacco cv. Samsun NN and these plants subsequently tested positive for TRV by ELISA using ATCC antiserum PVAS 820. Ninety tubers exhibiting mild to severe symptoms of TRV were planted in the greenhouse. Each tuber was bisected laterally; necrotic tissue was removed from one half of the tuber and tested for the presence of TRV using RT-PCR protocols described above for RNA1. The remaining half was bisected horizontally and both sections were planted. Foliage from each emerged plant was subsequently also tested by RT-PCR for TRV RNA1. All RB tubers from Wisconsin tested positive for TRV, but only 7 of 24 emerged plants tested positive. Only 72% of the UR tubers and 4 of 25 emerged plants tested positive. TRV has been confirmed in California, Colorado, Florida, Idaho, Michigan (1), Oregon, and Washington. To our knowledge, this is the first report of corky ringspot in potato caused by TRV in Minnesota and Wisconsin.

First Report of Corky Ringspot Caused by Tobacco rattle virus on Potatoes (Solanum tuberosum) in Michigan.

Potato is the fourth most important agricultural commodity in Michigan and is grown for table stock, chip processing, and seed. Tubers are either processed or fresh packed immediately following harvest or sent to storage. Tubers of potato cv. FL1879 representing two separate fields in Huron County were removed from separate storage facilities in February and March of 2007, and substantial internal necrosis was observed in 1 to 2% of the tubers. Symptoms included arcs similar to those caused by Tobacco rattle virus (TRV). This virus is a member of the genus Tobravirus and is transmitted by a number of species of stubby-root nematodes (Paratrichodorus or Trichodorus spp.). Stubby-root nematodes have been reported previously from Michigan (1). To confirm the presence of TRV, nucleic acid extractions were made from these symptomatic tubers (3). Samples were initially tested for TRV by reverse transcription (RT)-PCR using primers specific to the 16 kDa open reading frame on genomic RNA-1 (2). Samples from both storage facilities were positive. The 463-bp RT-PCR product, amplified with these primers, was sequenced (GenBank Accession No. EU315227) and was 99.6% identical to the corresponding region of two TRV isolates from Florida and Washington (GenBank Accession Nos. AF055912 and EU315226, respectively). In addition, sap from cv. FL1879 tuber samples was used to transmit the virus mechanically to tobacco cv. Samsun NN, which produced typical TRV symptoms following inoculation, and sap extracts from the tobacco leaves also tested positive with antiserum specific to TRV upon subsequent ELISA testing. Corky ringspot can result in substantial losses, with entire potato fields being rejected because of internal tuber damage. Once found, fields must be considered permanently at risk to this disease due to the large host range of both the virus and the nematode vector. This disease has been previously found in the United States in California, Colorado, Florida, Idaho, Washington, Oregon, and it is likely present in Indiana. To our knowledge, this is the first report of corky ringspot and TRV on potato in Michigan.

Resistance of transgenic tobacco plants incorporating the putative 57-kDa polymerase read-through gene of Tobacco rattle virus against rub-inoculated and nematode-transmitted virus

Nicotiana tabacum plants were transformed with the 57-kDa read-through domain of the replicase gene of Tobacco rattle virus (TRV) isolate SYM. From a total of six lines containing the viral transgene, four displayed various levels of resistance to TRV infection. Transgenic plants from line 81G were highly resistant to foliar rub-inoculation with the homologous isolate, or with isolates TRV-PpK20 and TRV-PLB, which are almost identical to TRV-SYM in RNA1 sequence. Moreover, 81G plants were moderately resistant to the serologically and genetically distinct, highly pathogenic isolate TRV-GR. Resistance characteristics of line 81G remained stable over six generations. No unambiguous correlation was established between number of transgene insertion loci and level of resistance. Transgene-specific mRNA was clearly detected in plants from susceptible lines but only at an early developmental stage in resistant plants, indicating the operation of a RNA silencing resistance mechanism. Following challenge using viruliferous vector nematodes carrying TRV-PpK20 or by rub inoculation of roots, 81G plants did not show any symptoms and virus was not detected in leaves. However, virus was detected in roots but without apparent effects on plant growth and often at low concentration. When challenged with nematodes carrying TRV-GR, symptoms in aerial parts of 81G plants were less severe and much delayed compared to non-transgenic plants, although younger plants showed less resistance than older ones. No difference was detected in transgene transcript accumulation between leaves and roots of 81G plants. This is the first work reporting a broad level of pathogen derived resistance against two geographically and genetically distinct TRV isolates transmitted directly by their nematode vectors and provides further insight into the expression of transgenic resistance against naturally transmitted soil-borne viruses.

Tobacco rattle virus 29K Movement Protein Is the Elicitor of Extreme and Hypersensitive-like Resistance in Two Cultivars of Solanum tuberosum

Leaf infection experiments were used to analyze the host responses of Solanum tuberosum cultivars known to be resistant or susceptible to natural, nematode-mediated infection of tubers and necrosis induction ("spraing") by Tobacco rattle virus (TRV) isolate PpK20 (TRV-PpK20). Extreme and hypersensitive-like resistance (ER and HR-like, respectively) as well as spreading veinal necrosis and systemic infection were observed. Agroinfection of leaves with a DsRed-expressing TRV cDNA clone revealed ER to function on the single-cell level, inhibiting virus replication and possessing the potential to initiate a cell death response. HR-like necrosis was characterized by initial virus replication and cell-to-cell movement before the onset of necrosis. Transient agroexpression and Potato virus X (PVX)-mediated expression assays demonstrated that the 29K-PpK20 movement protein (MP) can elicit ER and HR-like cell-death. A TRV isolate, PpO85M, known to overcome the resistance to spraing in plants that are resistant to TRV-PpK20 encoded a variant 29K protein which did not elicit HR in PpK20-HR plants. Our results show that the TRV MP is the elicitor of both ER and HR-like cell-death, that no other TRV-encoded proteins or RNA replication are required for its elicitor activity, and that the host reactions are likely to be controlled by single dominant resistance genes.

First Report of Tobacco rattle virus and Cucumber mosaic virus in Phlox paniculata in France.

Phlox paniculata L., a perennial plant from the family Polemoniaceae, is cultivated as an ornamental in gardens and for cut-flower production. In spring 2003, two types of symptoms were observed in P. paniculata plants grown for cut flowers on a farm in the Var department, France. Some plants showed a mild leaf mosaic while others showed leaf browning and delayed growth. In plants showing mild mosaic, Cucumber mosaic virus (CMV) was detected on the basis of the symptoms exhibited by a range of inoculated plants, the observation of isometric particles (approximately 30 nm) with the electron microscope in crude sap preparations from the infected plants, and the positive reaction in double-antibody sandwich (DAS)-ELISA to polyclonal antibodies raised against CMV (1). In double-immunodiffusion analysis, the five tested isolates were shown to belong to group II of CMV strains. To determine if CMV was responsible for the symptoms observed, one isolate was multiplied in Nicotiana tabacum cv. Xanthi-nc plants after isolation from local lesions on Vigna unguiculata and mechanically inoculated to 12 1-year-old P. paniculata plants. At 3 months post inoculation (mpi), all plants showed mild mosaic and CMV was detected by DAS-ELISA. In sap preparations from P. paniculata plants showing leaf browning symptoms, rod-shaped particles with two distinct sizes of 190 to 210 and 70 to 90 nm long, typical of those associated with tobraviruses, were revealed using electron microscopy. Local lesions typical of Tobacco rattle virus (TRV) were observed after inoculation of N. tabacum cv. Xanthi-nc, Chenopodium amaranticolor, and C. quinoa. Total nucleic acid preparations were prepared from symptomatic plants, and amplicons of the expected size (463 bp) were generated by reverse-transcription (RT)-PCR using primers specific to TRV RNA 1 (4). The nucleotide sequence of one amplicon was 93.6% identical to the sequence of a reference TRV isolate (GenBank Accession No. AJ586803). Twelve 1-year-old P. paniculata plants were mechanically inoculated with an extract of infected tissues from one symptomatic P. paniculata plant. TRV was detected 2 to 6 mpi in apical leaves of all inoculated plants by RT-PCR, although the plants did not express symptoms. Since no other pathogens were detected in the source plants, it is plausible that the lack of symptoms in back-inoculated plants is either due to a long incubation period or an interaction with particular environmental factors such as cold conditions. The survey of approximately 200 plants revealed that approximately 7, 10, and 1% were infected by TRV, CMV, or by both viruses, respectively. CMV and TRV were previously detected in P. paniculata in Latvian SSR and in Lithuania (2,3). These results show that sanitary selection of P. paniculata prior to vegetative propagation should include a screening for TRV and CMV infections.

Yeast two-hybrid study of tobacco rattle virus coat protein and 2b protein interactions

The specificity of the interaction between the coat protein (CP) and 2b nematode-transmission helper protein of two isolates, PpK20 and PaY4, of Tobacco rattle virus (TRV) that differ in their transmission characteristics was investigated. Yeast two-hybrid experiments identified a central domain of the CP that promoted CP:CP interaction but inhibited CP:2b interaction. Deletion of a conserved coiled-coil domain from the 2b protein prevented its interaction with CP, whereas deletion of N- and C-terminal domains of the 2b protein greatly enhanced its interaction with CP. A C-terminal flexible domain of the CP was also shown to be important for interaction with the 2b protein. However, this domain was not sufficient to direct isolate-specific interaction of these proteins either in yeast or via a chimeric TRV in planta. Although these two TRV isolates are both transmitted by a shared vector trichodorid nematode, Paratrichodorus pachydermus, the CP from isolate PpK20 did not interact with the 2b protein from isolate PaY4, and vice versa, suggesting that in the field mixed infections of TRV are unlikely to cause promiscuous transmission by alternative trichodorid nematode species.

Molecular detection and identification of potato isolates of Tobacco rattle virus

Sixteen isolates of Tobacco rattle virus (TRV) obtained from the United States and Canada were characterized in the present study. Nucleotide-sequence analysis of open reading frame 4 (ORF4) in RNA1 of seven test isolates revealed a high degree of genetic diversity. Potato isolates of TRV clustered in several distinct groups, with isolates from the same geographic region falling into different groups. Phylogenetic analysis of TRV isolates based on nucleotide sequences and on restriction fragment length polymorphism (RFLP) produced similar groupings. TRV was detected in various potato tissues of inoculated, field-grown plants, including progeny tubers, leaves, and roots, by reverse transcription – polymerase chain reaction (RT–PCR), using a pair of TRV-specific primers targeting ORF4. TRV RNA was detected in highly diluted total RNA preparations (1 / 15 625) and in composite tuber samples (200–400 tubers). RT–PCR followed by RFLP analysis was shown to be an efficient procedure for specific and sensitive detection and identification of TRV in potato.