Incidence and Molecular Characterization of Tobacco Rattle Virus (TRV) in Azad Jammu and Kashmir-Northeast of Pakistan

Tobacco rattle virus (TRV) poses a significant challenge to potato production, particularly in the hilly and mountainous terrains of Azad Jammu and Kashmir (AJK), Pakistan. To address this issue, the present study aimed to evaluate the resistance of ten potato cultivars, Line A, Kurado, Desiree, Bataina, Fleminco, Line B, Pomola, Ronaldo, Rodalph, and Margrata, against TRV. The study was conducted in two districts, Poonch and Sudhnoti, where the in vivo effects of TRV were assessed based on yield and quality characteristics. The research investigated the impact of TRV on plant growth and yield parameters using double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) and reverse transcription-polymerase chain reaction (RT-PCR) techniques, highlighting the role of soil-borne nematodes as vectors. The findings suggested that none of the tested cultivars exhibited complete resistance to TRV, though Desiree and Fleminco demonstrated moderate insensitivity. These results provided valuable insights into TRV management and emphasized the need for further research on resistant germplasm. The study concluded that TRV and its vector posed significant threats to potato production in AJK, with no tested germplasm exhibiting complete resistance. These findings may aid the potato industry in selecting cultivars that help mitigate the financial burden of tuber necrosis caused by TRV. This research represents the first comprehensive screening of potato germplasm against TRV in AJK, underscoring the severity of the virus as a hazard in hilly regions. Future studies involving broader germplasm screening are recommended to validate and strengthen these findings, ultimately facilitating the development of TRV-resistant potato varieties.

Soil-borne potato viruses are an increasing economic threat to crop yield in the future. Potato mop-top virus (PMTV) and Tobacco rattle virus (TRV) and their vector association in field infection in North-western region of Pakistan and Azad Jammu and Kashmir were surveyed in this study. Incidence and distribution of these soil-borne viruses were estimated by field sample collection from Malakand and Hazara divisions of KPK and selected areas of Azad Kashmir. PMTV incidence was 22% in Malakand (highest of 40% in Swat II and Swat V), 24% in Hazara (highest 50% in Galliyat), and 23% from selected areas of Azad Jammu and Kashmir (AJK) (Highest with 40% in Rawalakot and Trarkhal). TRV incidence was 49% in AJK (100% highest field incidence in Khaigala Rawalakot), 53% in Malakand division (80% highest field incidence in Swat II), and 24% in Hazara division (70% highest field incidence in Abbottabad and Mansehra districts). PMTV, TRV vectors are Spongospora subterranea and Trichodorus, Paratrichodorus spp. were found distributed in all surveyed areas. Eighty percent (80%) fields were found infested with S. subterranean, while Trichodorus and Paratrichodorus were found in 64% in Malakand. In Hazara, 84% of surveyed fields were found infested with S. subterranea while 65% were found to be infested with Trichodorus, Paratrichodorus. In AJK S. subterranea was found distributed in 79% of fields. A significant relationship (P value=0.000) between viruses (PMTV and TRV) and their respective vectors were detected in proportionate. The corresponding R2 (0.70 and 0.82) indicates positive relation between viruses and their vectors. Positive Pearson correlation was found among incidence and severity of virus infection (PMTV and TRV) and vectors (S. subterranea; Trichodorus minor, Paratrichodorus), indicating increased disease severity with vector presence and activity. The study will be a tool in vector virus management to economical potato harvest

ABSTRACTThe study involves tobacco rattle virus (TRV) detection in racemes of two-year beet plants, seedlings, grown from seeds of these plants and seedlings from commercially available sugar beet seeds. Parts of sugar beet roots with a history of TRV were used for growing of two-year floriferous plants. Since plants had symptoms of yellow mosaic on leaves and raceme stipules, we tested each raceme separately with antiserum to TRV and broad beans wilt virus (BBWV) by ELISA. BBWV also causes yellow mosaic on leaves and raceme stipules and is usually combined with TRV infection in beet lately. Of 13 flowering plants, 8 were diagnosed with TRV and 2—with BBWV. TRV virus concentration in racemes was more than 0.3 to more than 0.5 optical units. BBWV showed lower concentration—more than 0.25 optical units in a limited number of plants (only 2), which did not interfere with TRV detection in racemes of flowering beet plants. Artificial vegetative propagation of beet has led to TRV transmission with racemes of two-year beet plants of up to 62%. Seedlings, originating from seeds of two-year beet plant, with extinction value of 0.550 optical units for the sample of its raceme, accounts for 20% of TRV transmission. The results of testing seedlings, grown from commercially available seeds, showed 15% TRV transmission for one batch and 20% for the other. TRV infection of racemes was investigated in samples, collected from a seed-production field. We detected 5% TRV infection in racemes of this field. The progeny of such racemes may transmit TRV to commercial fields, let alone nematode transmission of the virus.

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.

Tobacco rattle virus (TRV), genus Tobravirus, is able to infect a broad range of plant species, possesses worldwide distribution and naturally infects a very large number of cultivated as well as ornamental plants. Members of the plant ectoparasitic nematode genera Trichodorus and Paratrichodorus transmit TRV in a semi-persistent way in a non-replicative process, where virus particles are transferred to the host plant through vector feeding on root epidermal and root hair cells. Our investigations concentrated on ultrastructural and anatomical effects of TRV strain PSG infection in situ following introduction into potato and tobacco tissues by Trichodorus primitivus. Our anatomical observations indicated necrotic changes of rhizodermis and cell wall deformations in primary cortex parenchyma and external phloem layers as a consequence of the interactions between Trichodorus–TRV–host plants. Ultrastructural analyses revealed TRV particles in rhizodermis, cortex and vascular roots of potato and tobacco tissues. These results indicate that TRV strain PSG was transferred into roots and also transported from cell to cell in all root tissues. Complete TRV PSG particles of two lengths were documented in companion cells, phloem fibre and parenchyma, as well as for the first time in immature and mature xylem tracheary elements and xylem parenchyma. Our findings suggested that TRV was systemically transported from the place of direct transfer by the vector to above-ground plant organs (especially leaves). The presence of TRV PSG particles in mesophyll and vascular leaf tissues confirmed our thesis. Moreover, we concluded that TRV systemic movement occurred not only in the phloem, but especially in the xylem, because virus particles were more frequently observed in xylem parenchyma and xylem tracheary elements.

Plant virus-based gene-silencing vectors have been extensively and successfully used to elucidate functional genomics in plants. However, only limited virus-induced gene-silencing (VIGS) vectors can be used in both monocot and dicot plants. Here, we established a dual gene-silencing vector system based on Bamboo mosaic virus (BaMV) and its satellite RNA (satBaMV). Both BaMV and satBaMV vectors could effectively silence endogenous genes in Nicotiana benthamiana and Brachypodium distachyon. The satBaMV vector could also silence the green fluorescent protein (GFP) transgene in GFP transgenic N.benthamiana. GFP transgenic plants co-agro-inoculated with BaMV and satBaMV vectors carrying sulphur and GFP genes, respectively, could simultaneously silence both genes. Moreover, the silenced plants could still survive with the silencing of genes essential for plant development such as heat-shock protein 90 (Hsp90) and Hsp70. In addition, the satBaMV- but not BaMV-based vector could enhance gene-silencing efficiency in newly emerging leaves of N.benthamiana deficient in RNA-dependant RNA polymerase 6. The dual gene-silencing vector system of BaMV and satBaMV provides a novel tool for comparative functional studies in monocot and dicot plants.

Soil disinfection to prevent transmission of tobacco rattle virus to potatoes and flower bulbs

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.

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.

SUMMARY Electron microscopy of ultrathin sections indicated that nucleoprotein particles of raspberry ringspot (RRV) and tobacco rattle (strain CAM; TRV-CAM) viruses occurred in mixed aggregates in cells of doubly infected Nicotiana benthamiana leaves, and in protoplasts doubly infected by inoculation. RRV particles were attached to the sides and ends of TRV-CAM particles that were mostly bound to mitochondria. RRV particles attached to TRV-CAM particles linking mitochondria in clusters are considered to be the structures represented by the aggregates of RRV particle antigen found previously by fluorescent antibody staining in doubly infected protoplasts but not in those infected with RRV alone.

SUMMARY The addition of actinomycin D (25 µg/ml) or cordycepin (1 mm) to protoplast cultures immediately after inoculation with particles of tobacco mosaic, tobacco rattle, tobacco ringspot or potato leafroll viruses resulted in a decrease in the proportion of protoplasts becoming infected, as judged by staining with fluorescent antibody to virus particles. A delay of a few hours between the inoculation and the addition of either inhibitor largely or completely eliminated this effect. In contrast, infection was unaffected by the addition of actinomycin D when the protoplasts were inoculated with RNA preparations from tobacco mosaic or tobacco rattle viruses.

Molecular characterization of variants of a new ‘rule-breaking’ tobacco rattle virus RNA2 in potatoes

Tobacco rattle virus (TRV) is a plant pathogen belonging to the Group IV positive-sense single-stranded RNA viruses. TRV causes disease in various plants (e.g., potato, tomato and tobacco), for which it was classified as a controlled quarantine virus in Korea. This study aimed to develop specific primer sets for the rapid detection of TRV. Two RT-PCR primer sets were developed for specific detection of TRV. Furthermore, nested primer sets were also developed, which is required for high sensitivity detection in plant quarantine. The RT-PCR and nested PCR products had the following sizes: set 5 (1,096→540 bp), and set 7 (878→756 bp), respectively. In addition, a modified positive-control plasmid was also developed for use as a positive control in TRV quarantine. The diagnostic system for TRV detection was verified using samples from Korean quarantine sites for the last five years (2009-2014). A total of 83 cases were detected among various import crops. This system for detection of TRV will continuously contribute to plant quarantine in the future.

Main conclusionSilencing of an ascorbate oxidase (AO) gene in N. benthamiana enhanced disease severity from cucumber mosaic virus (CMV), showing higher accumulation and expansion of the spreading area of CMV.A Nicotiana benthamiana ascorbate oxidase (NbAO) gene was found to be induced upon cucumber mosaic virus (CMV) infection. Virus-induced gene silencing (VIGS) was employed to elucidate the function of AO in N. benthamiana. The tobacco rattle virus (TRV)-mediated VIGS resulted in an efficient silencing of the NbAO gene, i.e., 97.5% and 78.8% in relative quantification as compared to the control groups (TRV::eGFP- and the mock-inoculated plants), respectively. In addition, AO enzymatic activity decreased in the TRV::NtAO-silenced plants as compared to control. TRV::NtAO-mediated NbAO silencing induced a greater reduction in plant height by 15.2% upon CMV infection. CMV titer at 3 dpi was increased in the systemic leaves of NbAO-silenced plants (a 35-fold change difference as compared to the TRV::eGFP-treated group). Interestingly, CMV and TRV titers vary in different parts of systemically infected N. benthamiana leaves. In TRV::eGFP-treated plants, CMV accumulated only at the top half of the leaf, whereas the bottom half of the leaf was “occupied” by TRV. In contrast, in the NbAO-silenced plants, CMV accumulated in both the top and the bottom half of the leaf, suggesting that the silencing of the NbAO gene resulted in the expansion of the spreading area of CMV. Our data suggest that the AO gene might function as a resistant factor against CMV infection in N. benthamiana.

SUMMARYAt a site in eastern Scotland, nine common species of arable weeds were infected with tobacco rattle virus (TRV), and some of these, notably Viola arvensis and Stellaria media, comprised an overwintering reservoir of the virus. TRV was seed‐borne both in naturally and in experimentally infected V. arvensis (2–10%), and occasionally in other weed species. In the glasshouse at 20 oC a naturally infective population of vector nematodes (Tricho‐dorus spp.) kept in soil free of plants retained its infectivity for 20 wk, although few Trichodorus survived for this period. In the field, the incidence of TRV infection in potato (spraing disease) in plots kept free of weeds for 1–5 years was 3–4 times that in weed‐infested plots but Trichodorus numbers did not differ appreciably between the two treatments. Presumably the virus is retained for long periods in its vectors and these feed on potato more frequently when other hosts are not available. Weeds are probably important in the long term as hosts of both TRV and its vectors, but in the short term weed control seems unlikely to prevent potato spraing because of the long persistence of TRV in vector populations.In the field, Trichodorus accumulated near the interface between topsoil and subsoil, and the incidence of spraing was greatest where the topsoil was shallowest. When cucumber seedlings were exposed to virus‐carrying Trichodorus, TRV reached a greater concentration in roots at 20 oC than at 24 oC, and the virus was not detected in roots at 29 oC. In a sandy soil, TRV was transmitted only when the water content exceeded 15%, and at least 30 % water was needed for maximum transmission. Annual records of rainfall and spraing disease suggest that spraing is most prevalent when the summer is wettest.TRV is not confined to cultivated land. Stabilized sand dunes supporting a pure stand of Ammophila armaria were colonized by Trichodorus pachyder‐mus, but TRV was detected only where the plant community had enlarged to include V. arvensis and other dicotyledons. In such situations, TRV may be introduced in the seed of V. arvensis, and the movement of soil by wind probably contributes to the dispersal of Trichodorus.