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Pawpaw (Asimina triloba) trees exhibiting stunting and foliar mosaic, chlorosis, or distortions were observed in New York. In 2021, leaf samples from two symptomatic trees and a sapling, as well as two asymptomatic trees, were tested for the presence of viruses and viroids by high-throughput sequencing (HTS) using total RNA after ribosomal RNA depletion. HTS sequence information revealed tobacco ringspot virus (TRSV) and tomato ringspot virus (ToRSV) in symptomatic but not in asymptomatic leaves. HTS reads and de novo-assembled contigs covering the genomes of both viruses were obtained, with a higher average read depth for RNA2 than RNA1. The occurrence of TRSV and ToRSV was confirmed in the original leaf samples used for HTS and 12 additional trees and saplings from New York and Maryland in 2022 by RT-PCR combined with Sanger sequencing, and DAS-ELISA. Single infections by TRSV in 11 of 14 trees and dual infections by TRSV and ToRSV in 3 of 14 trees were identified. The nucleotide sequence identity of partial gene fragments of TRSV and ToRSV was high among pawpaw isolates (94.9-100% and 91.8-100%, respectively) and between pawpaw isolates and isolates from other horticultural crops (93.6-100% and 71.3-99.3%, respectively). This study is the first to determine the virome of pawpaw.

This document provides the conclusions of the pest survey card that was prepared in the context of the EFSA mandate on plant pest surveillance (M‐2020‐0114) at the request of the European Commission. The full pest survey card for tobacco ringspot virus (TRSV) is published and available online in the EFSA Plant Pest Survey Cards Gallery at the following link and will be updated whenever new information becomes available: https://efsa.europa.eu/plants/planthealth/monitoring/surveillance/tobacco-ringspot-virus [ABSTRACT FROM AUTHOR]

Aiming at the problem of how to achieve the rapid detection of pathogenic microorganisms, this paper takes tobacco ringspot virus as the detection object, designs the impedance detection and analysis platform of tobacco ringspot virus based on microfluidic impedance method, establishes an equivalent circuit model to analyze the experimental results, and determines the optimal detection frequency of tobacco ringspot virus detection. Based on this frequency, an impedance–concentration regression model was established for the detection of tobacco ringspot virus in a tobacco ringspot virus detection device. Based on this model, a tobacco ringspot virus detection device was designed by using an AD5933 impedance detection chip. A comprehensive test study was carried out on the developed tobacco ringspot virus detection device through various testing methods, which verified the feasibility of the tobacco ringspot virus detection device and provided technical support for the field detection of pathogenic microorganisms. [ABSTRACT FROM AUTHOR]

Aiming at the problem of how to achieve the rapid detection of pathogenic microorganisms, this paper takes tobacco ringspot virus as the detection object, designs the impedance detection and analysis platform of tobacco ringspot virus based on microfluidic impedance method, establishes an equivalent circuit model to analyze the experimental results, and determines the optimal detection frequency of tobacco ringspot virus detection. Based on this frequency, an impedance–concentration regression model was established for the detection of tobacco ringspot virus in a tobacco ringspot virus detection device. Based on this model, a tobacco ringspot virus detection device was designed by using an AD5933 impedance detection chip. A comprehensive test study was carried out on the developed tobacco ringspot virus detection device through various testing methods, which verified the feasibility of the tobacco ringspot virus detection device and provided technical support for the field detection of pathogenic microorganisms.

Pawpaw (Asimina triloba) trees exhibiting stunting and foliar mosaic, chlorosis, or distortions were observed in New York. In 2021, leaf samples from two symptomatic trees and a sapling, as well as two asymptomatic trees, were tested for the presence of viruses and viroids by high-throughput sequencing (HTS) using total RNA after ribosomal RNA depletion. HTS sequence information revealed tobacco ringspot virus (TRSV) and tomato ringspot virus (ToRSV) in symptomatic but not in asymptomatic leaves. HTS reads and de novo-assembled contigs covering the genomes of both viruses were obtained, with a higher average read depth for RNA2 than RNA1. The occurrence of TRSV and ToRSV was confirmed in the original leaf samples used for HTS and 12 additional trees and saplings from New York and Maryland in 2022 by RT-PCR combined with Sanger sequencing, and DAS-ELISA. Single infections by TRSV in 11 of 14 trees and dual infections by TRSV and ToRSV in 3 of 14 trees were identified. The nucleotide sequence identity of partial gene fragments of TRSV and ToRSV was high among pawpaw isolates (94.9–100% and 91.8–100%, respectively) and between pawpaw isolates and isolates from other horticultural crops (93.6–100% and 71.3–99.3%, respectively). This study is the first to determine the virome of pawpaw.

Tobacco ringspot virus (TRSV) is an important plant pathogen that causes severe diseases in tobacco plants. In recent years, this virus has been reported in a number of ornamental plants. However, it has never been reported from Japanese iris, a famous traditional ornamental plant distributed in Asian countries. In this study, the near-complete bipartite genome of a nepovirus (TRSV-Iris) infecting Japanese iris from Taiwan of China was determined. The RNA1 and RNA2 are 7512 and 3899 nucleotides in length, and encodes a polyprotein of 2303 and 1078 amino acids, respectively. Sequence analysis indicates that TRSV-Iris shares more than 80% amino acid sequence identity, the threshold values of the species demarcation for the genus Nepovirus, with known TRSV isolates either in the conserved Pro-Pol or CP regions, respectively. Phylogenetic analysis indicates that TRSV-Iris is a TRSV isolate. [ABSTRACT FROM AUTHOR]

The aim of the study was to select, optimize and characterize RT‐PCR tests for the detection of Tobacco ringspot virus (TRSV) in post‐entry quarantine. Among five different tests, the Poojari et al. (Journal of Virological Methods 235, 112–118) and Jossey & Babadoost (Plant Disease 90, 1361) conventionnal RT‐PCR tests were chosen and optimized for this purpose. Ten TRSV isolates, 17 healthy plants from the genera Vitis, Prunus and Malus, four other Nepoviruses or Secoviridae isolates and serial dilutions of three TRSV isolates were used for the complete characterization of the optimized tests. In the tested conditions, the Poojari and Jossey & Babadoost tests respectively showed 100% and 95% inclusivity, 100% and 100% exclusivity, 100% and 96.4% analytical specificity, 100% and 100% diagnostic specificity, [10−7; 10−1] and [10−8; 10−2] limit of detection dilution factors (analytical sensitivity), 91.1% and 90.0% repeatability, 90.3% and 91.1% reproducibility and 100% and 100% selectivity. Due to its higher inclusivity, the optimized Poojari test is recommended for the detection of TRSV isolates for post‐entry quarantine purposes, but can also be used for global surveys. The optimized Jossey & Babadoost test showed the best analytical sensitivity, suggesting that combining both tests can further enhance detection. [ABSTRACT FROM AUTHOR]

The Panel on Plant Health performed a pest categorisation of Xiphinema californicum (Nematoda: Longidoridae) for the EU. The nematode is a well-defined taxon belonging to a group of morphologically similar species called Xiphinema americanum sensu lato. The nematode was described from the USA and is present in some North and South American countries. The nematode is not present in the EU and is regulated by Council Directive 2000/29/EC, listed in Annex I A I as X. californicum Lamberti and Bleve-Zacheo. It is a polyphagous pest found in soil associated with a number of plant species. As a migratory ectoparasitic species, it punctures the cells of plant roots. X. californicum is in principle able to cause direct damage to plants, but its main damage is caused by vectoring the American nepoviruses: Tobacco ringspot virus (TRSV), Tomato ringspot virus (ToRSV) and Cherry rasp leaf virus (CRLV). Soil is a potential pathway for this nematode for entry into the EU. Moist soil, such as soil attached to plants for planting, increases survival of the nematode. The viruses may persist over prolonged periods inside the nematode and viruliferous nematodes may introduce American nepoviruses. Climatic conditions in the EU are similar to those found in the areas where the pest is currently present. Hosts of the nematode (and of associated viruses) are, e.g. grapes, apples and plums, which are also widely cultivated in the EU. The nematode only moves short distances (around 1 m) but may be spread with soil moving activities. Measures are available to inhibit entry via soil as such. Entry of the nematode with soil attached to plants for planting that are not regulated is possible. X. californicum does satisfy all the criteria that are within the remit of EFSA to assess to be regarded as a Union quarantine pest. [ABSTRACT FROM AUTHOR]

A dot-immunobinding assay (DIBA) was optimized and used successfully for the rapid detection of 15 known viruses [ Alfalfa mosaic virus (AMV), Bean pod mottle virus (BPMV), Bean yellow mosaic virus (BYMV), Cowpea mild mottle virus (CPMMV), Cowpea severe mosaic virus (CPSMV), Cucumber mosaic virus (CMV), Peanut mottle virus (PeMoV), Peanut stunt virus (PSV), Southern bean mosaic virus (SBMV), Soybean dwarf virus (SbDV), Soybean mosaic virus (SMV), Soybean vein necrosis virus (SVNV), Tobacco ringspot virus (TRSV), Tomato ringspot virus (ToRSV), and Tobacco streak virus (TSV)] infecting soybean plants in Oklahoma. More than 1000 leaf samples were collected in approximately 100 commercial soybean fields in 24 counties of Oklahoma, during the 2012–2013 growing seasons. All samples were tested by DIBA using polyclonal antibodies of the above 15 plant viruses. Thirteen viruses were detected, and 8 of them were reported for the first time in soybean crops of Oklahoma. The highest average incidence was recorded for PeMoV (13.5%) followed by SVNV (6.9%), TSV (6.4%), BYMV, (4.5%), and TRSV (3.9%), while the remaining seven viruses were detected in less than 2% of the samples tested. The DIBA was quick, and economical to screen more than 1000 samples against 15 known plant viruses in a very short time. [ABSTRACT FROM AUTHOR]

There is limited information on the distribution of blueberry viruses in the U.S. or around the world other than where the viruses were first discovered and characterized. A survey for blueberry viruses was carried out in the U.S. in 2015–2017. Most blueberry viruses have been characterized to the point that sensitive diagnostic assays have been developed. These assays are based on ELISA or variations of PCR, which were employed here to determine the presence of blueberry viruses in major blueberry production and nursery areas of the U.S. The viruses included in this study were: blueberry fruit drop (BFDaV), blueberry latent (BlLV), blueberry leaf mottle (BLMoV), blueberry mosaic (BlMaV), blueberry red ringspot (BRRV), blueberry scorch (BlScV), blueberry shock (BlShV), blueberry shoestring (BlSSV), blueberry virus A (BVA), peach rosette mosaic (PRMV), tobacco ringspot (TRSV), and tomato ringspot (ToRSV). In the Pacific Northwest BlShV was the most widespread virus, with BlScV and ToRSV detected in a limited number of fields in Oregon and Washington, but BlScV was widespread in British Columbia. In the upper midwest, the nematode-borne (ToRSV, TRSV), aphid-transmitted (BlSSV and BVA) and pollen-borne (BLMoV) viruses were most widespread. In the northeast, TRSV, ToRSV, and BlScV, were detected most frequently. In the southeast, BRRV and BNRBV were the most widespread viruses. BlLV, a cryptic virus with no known symptoms or effect on plant growth or yield was present in all regions. There are other viruses present at low levels in each of the areas, but with the lower incidence they pose minimal threat to nursery systems or fruit production. These results indicate that there are hotspots for individual virus groups that normally coincide with the presence of the vectors. The information presented highlights the high risk viruses for nursery and fruit production each pose a different challenge for control.

We report here the development of tobacco ringspot virus (TRSV)-based vectors for the transient expression of foreign genes and for the analysis of endogenous gene function in plants using virus-induced gene silencing. The jellyfish green fluorescent protein (GFP) gene was inserted between the TRSV movement protein (MP) and coat protein (CP) regions, resulting in high in-frame expression of the RNA2-encoded viral polyprotein. GFP was released from the polyprotein via an N-terminal homologous MP-CP cleavage site and a C-terminal foot-and-mouth disease virus (FMDV) 2 A catalytic peptide in Nicotiana benthamiana . The VIGS target gene was introduced in the sense and antisense orientations into a Sna BI site, which was created by mutating the sequence following the CP stop codon. VIGS of phytoene desaturase ( PDS ) in N. benthamiana, Arabidopsis ecotype Col-0, cucurbits and legumes led to obvious photo-bleaching phenotypes. A significant reduction in PDS mRNA levels in silenced plants was confirmed by semi-quantitative RT-PCR. [ABSTRACT FROM AUTHOR]

Background: Genome-wide association study (GWAS) is a useful tool for detecting and characterizing traits of interest including those associated with disease resistance in soybean. The availability of 50,000 single nucleotide polymorphism (SNP) markers (SoySNP50K iSelect BeadChip; www.soybase.org) on 19,652 soybean and wild soybean plant introductions (PIs) in the USDA Soybean Germplasm Collection allows for fast and robust identification of loci associated with a desired phenotype. By using a genome-wide marker set to predict phenotypic values, genomic prediction for phenotype-unknown but genotype-determined PIs has become possible. The goal of this study was to describe the genetic architecture associated with sensitivity to Tobacco ringspot virus (TRSV) infection in the USDA Soybean Germplasm Collection. Results: TRSV-induced disease sensitivities of the 697 soybean PIs were rated on a one to five scale with plants rated as one exhibiting mild symptoms and plants rated as five displaying terminal bud necrosis (i.e., bud blight). The GWAS identified a single locus on soybean chromosome 2 strongly associated with TRSV sensitivity. Cross-validation showed a correlation of 0.55 (P < 0.01) to TRSV sensitivity without including the most significant SNP marker from the GWAS as a covariate, which was a better estimation compared to the mean separation by using significant SNPs. The genomic estimated breeding values for the remaining 18,955 unscreened soybean PIs in the USDA Soybean Germplasm Collection were obtained using the GAPIT R package. To evaluate the prediction accuracy, an additional 55 soybean accessions were evaluated for sensitivity to TRSV, which resulted in a correlation of 0.67 (P < 0.01) between actual and predicted severities. Conclusion: A single locus responsible for TRSV sensitivity in soybean was identified on chromosome 2. Two leucine-rich repeat receptor-like kinase genes were located near the locus and may control sensitivity of soybean to TRSV infection. Furthermore, a comprehensive genomic prediction for TRSV sensitivity for all accessions in the USDA Soybean Germplasm Collection was completed. [ABSTRACT FROM AUTHOR]

Tobacco ringspot virus (TRSV, genus Nepovirus), causes severe diseases in soybean and tobacco plants. TRSV-induced bud blight disease significantly reduced both the yield and quality of soybeans. The function of the encoded viral gene product involved in TRSV infection was unclear due to the limitation of reverse genetics studies on the viral genome. Here, we represent the successful construction of infectious full-length cDNA clones of TRSV genome (RNA1 and RNA2). The cDNAs of TRSV RNA1 and RNA2 were cloned into the binary vector pPZP211 immediately downstream of a double cauliflower mosaic virus 35S promoter and upstream of the nopaline synthase terminator. Seven days after agrobacterium-mediated co-inoculation of these two constructs, Nicotiana benthamiana plants developed a systemic infection with necrotic ringspot symptoms and weak stunting of the leaves, similar to that induced by natural TRSV. The systemic infection was confirmed by transmission electron microscopy and Western blot analysis. Simultaneously, soybean, tomato, and Arabidopsis ecotype Estland were mechanically inoculated with sap prepared from TRSV-agroinfiltrated N. benthamiana leaves, showing typical symptoms of bud blight, necrotic spots, and lethal systemic necrosis, respectively. The system developed herein will be an appealing way to determine TRSV viral gene functions and study host-TRSV interactions. [ABSTRACT FROM AUTHOR]

Tobacco ringspot virus (TRSV) is a quarantine pathogen in Europe. During an official inspection in November 2011, Impatiens walleriana plants showing symptoms were found in a nursery in the Czech Republic. The causal agent of the disease was detected as Nepovirus group A by RT-PCR using specific primers of the Nepovirus group. Sequence analysis of PCR fragments confirms that the detected virus is TRSV. TRSV detection in these plants was further confirmed by ELISA and one-step RT-PCR using specific primers. The coat protein (CP) gene of the Czech TRSV isolate was sequenced, and the sequence analysis showed high identity of both nucleotide (99.28%) and amino acid (99.96%) levels compared with other known TRSV isolates from GenBank. Two amino acid motifs characteristic of nepoviruses, FDDY (FDAY) and FWGR (FYGR), were found equally at positions 80 and 497 of the TRSV CP genes, respectively, including the sequences described in this study. [ABSTRACT FROM AUTHOR]

Vineyards in the Southeastern New England American Viticultural Area were surveyed for the incidence of seven major viruses: grapevine leafroll-associated viruses (GLRaV-1, GLRaV-2, GLRaV-3, and GLRaV-4), grapevine fanleaf virus (GFLV), tomato ringspot virus (ToRSV), and tobacco ringspot virus (TRSV). Viruses were detected by DAS-ELISA and confirmed by RT-PCR and Sanger sequencing. Multiple viruses were present in 19 out of the 25 vineyards surveyed between 2018 and 2020. GLRaV-3 (27.59%) was the most prevalent virus followed by GLRaV-4 (14.90%), GLRaV-1 (13.52%), GLRaV-2 (11.03%), ToRSV (6.34%), GFLV (5.24%), and TRSV (2.62%). Furthermore, phylogenetic analyses of the viral partial genome sequences acquired in this study revealed that the grapevine viruses present in this area are diverse, indicating that they may have been introduced from different sources. Our findings stress the need for improving the sanitary status of planting materials to avoid the introduction and dissemination of viruses to vineyards in this important wine-producing region of New England.

The complete nucleotide sequences of RNA 1 and RNA 2 of the nepovirus potato black ringspot virus (PBRSV) from two different isolates were determined, as well as partial sequences from two additional isolates. RNA1 is 7,579-7,598 nucleotides long and contains one single open reading frame (ORF), which is translated into a large polyprotein with 2,325 amino acids and a molecular weight of 257 kDa. The complete sequence of RNA2 ranges from 3857 to 3918 nt between the different isolates. It encodes a polyprotein of 1079-1082 amino acids with a molecular weight of 120 kDa. Sequence comparison using the Pro-Pol region and CP showed that all four isolates formed two distinct groups, corresponding to potato and arracacha, that were closely related to each other and also to tobacco ringspot virus (TRSV). Comparing our data to those obtained with other nepoviruses, our results confirm that PBRSV belongs to a distinct species and is a member of subgroup A in the genus Nepovirus based on its RNA2 size, genome organization, and nucleotide sequence. [ABSTRACT FROM AUTHOR]

During 2006 and 2007, a survey on the incidence and distribution of fourteen grapevine viruses was carried out in the Skadar Lake basin, one of the two main grapevine-growing areas of Montenegro. In total 165 samples were collected from four red ('Vranac', 'Kratošija', 'Merlot' and 'Cardinal'), two white ('Chardonnay' and 'Rkaciteli') and a few unknown grapevine varieties in the vicinity of Podgorica and Bar. The phytosanitary status of the collected samples was analysed by DAS- ELISA and the presence of Grapevine fanleaf virus ( GFLV), Grapevine leafroll-associated virus 1 ( GLRaV-1), Grapevine leafroll-associated virus 2 ( GLRaV-2) and Grapevine leafroll-associated virus 3 ( GLRaV-3) was confirmed in some of them. The most frequently found virus in assayed samples was GLRaV-3 (54.5%), followed by GFLV (23%), GLRaV-1 (20%) and GLRaV-2 (0.6%). These serological analyses showed the absence of Grapevine leafroll-associated virus 6 ( GLRaV-6), Grapevine leafroll-associated virus 7 ( GLRaV-7), Raspberry bushy dwarf virus ( RBDV), Strawberry latent ringspot virus ( SLRSV), Tomato ringspot virus (To RSV), Raspberry ringspot virus (Rp RSV), Arabis mosaic virus (Ar MV), Tobacco ringspot virus ( TRSV), Tomato black ring virus ( TBRV) and Cherry leaf roll virus ( CLRV) from all tested samples. [ABSTRACT FROM AUTHOR]

The article offers information on several American nepoviruses that can cause degeneration and decline on grapevines. The nepoviruses include the Blueberry leaf mottle virus (BLMoV), the Peach rosette mosaic virus (PRMV), and the Tobacco ringspot virus (TRSV). Information on the main symptoms, transmission, and detection of the nepoviruses is provided.

Abstract: A virus was isolated from potted plants of an unidentified species of Aeonium, a succulent ornamental very common in Southern Italy, showing chlorotic spots and rings on both leaf surfaces. It was successfully transmitted by sap inoculation to a limited range of hosts, including Nicotiana benthamiana which was used for ultrastructural observations and virus purification. Virus particles are isometric, ca. 30nm in diameter, have a single type of coat protein (CP) subunits 54kDa in size, that encapsidate single-stranded positive-sense RNA species of 7549 (RNA1) and 4010 (RNA2) nucleotides. A third RNA molecule 3472 nts in size entirely derived from RNA2 was also found. The structural organization of both genomic RNAs and the cytopathological features were comparable to those of nepoviruses. In addition, amino acid sequence comparisons of CP and the Pro-Pol region (a sequence containing parts of the proteinase and polymerase) with those of other nepoviruses showed that the Aeonium virus belongs to the subgroup A of the genus Nepovirus and is phylogenetically close to, but serologically distinct from tobacco ringspot virus (TRSV). Based on the species demarcation criteria for the family Secoviridae, the virus under study appears to be a novel member of the genus Nepovirus for which the name of Aeonium ringspot virus (AeRSV) is proposed. [Copyright &y& Elsevier]

Abstract: Protein Kinase R (PKR) inhibits translation initiation following double-stranded RNA (dsRNA) binding and thereby represses viral replication. Human cytomegalovirus (HCMV) encodes two noncanonical dsRNA binding proteins, IRS1 and TRS1, and the expression of at least one of these PKR antagonists is essential for HCMV replication. In this study, we investigated the role of dsRNA binding by TRS1 in PKR inhibition. We found that purified TRS1 binds specifically to dsRNA with an affinity lower than that of PKR. Point mutants in the TRS1 dsRNA binding domain that were deficient in rescuing the replication of vaccinia virus lacking its PKR antagonist E3L were unable to bind to dsRNA but retained the ability bind to PKR. Thus TRS1 binding to dsRNA and to PKR are separable. Overall, our results are most consistent with a model in which TRS1 binds simultaneously to both dsRNA and PKR to inhibit PKR activation. [Copyright &y& Elsevier]

Pawpaw (Asimina triloba (L.) Dunal, Annonaceae) is a fruit tree native to eastern North America, increasingly grown for commercial production in the United States (Callaway, 1992; Layne, 1996), Europe, and Western Asia (Brannan and Coyle, 2021; Lolletti et al., 2021). In 2012, virus-like symptoms were noticed in a 0.3 ha pawpaw orchard at Michigan State University Plant Pathology Research Station; ~30% of the trees presented symptoms which included foliar mosaic, vein yellowing, and necrosis, and were first mistaken for nutrient (magnesium/zinc) deficiency. Trees were treated for magnesium/zinc deficiency but continued to decline in fruit yield and overall vigor, and typically died within 3─4years after symptoms were first observed (Fig. S1). Preliminary testing using Agdia ImmunoStrips for cucumber mosaic virus, impatiens necrotic spot virus, tobacco mosaic virus, tomato spotted wilt virus and the genus Potyvirus were negative. However, icosahedral virus particles were observed by TEM (Fig. S2). To establish virus identity, we deep-sequenced tissue from a symptomatic pawpaw obtained from same site in summer 2021. Virus particles were purified , and virion-associated nucleic acids (VANA) were extracted using the Purelink viral RNA/DNA kit (Invitrogen) (Maclot et al., 2021). Both viral RNA and DNA were subjected to high-throughput sequencing (HTS) on the Illumina NextSeq 500 platform (GIGA, University of Liege, Belgium). A total of 574,274 trimmed reads (150 nt read length) were de novo assembled using Geneious Prime 2022.2.2 software (https://www.geneious.com) and subjected to BLASTn analysis. Two contigs of 7511 bp (average coverage: 1048) and 3924 bp (average coverage: 3012) showed 94% and 95% nt identities with tobacco ringspot virus (TRSV) RNA1 isolate YW (MT042825) and RNA2 isolate OH19 (MT561435) respectively. These two contigs (Accession no. OP589177 and OP589178) covered the complete TRSV genome for each segment. HTS found no other plant-associated viral / virus-like sequences in this symptomatic pawpaw sample. To further confirm TRSV infection, leaf extract from this sample was tested with RT-PCR using primers specific to the RdRp gene of TRSV RNA1 (Forward, 5'-TAACCTCATTGCAGTTGATCCTT-3'; Reverse, 5'-TAATTCAAGCTCAGGTCTCTTCT-3'; 739 bp amplicon) and the coat protein of TRSV RNA2 (Forward, 5'-TCATGCTTAAAGATGCAGATGTG-3'; Reverse, 5'-TATAAAGCTCCGCACTAGAAAACA-3'; 753 bp amplicon). Sanger sequence analysis showed 99.5% and 99.8% nt identity between the amplicons and the HTS contigs (RNA1 and RNA2 respectively) assembled from the pawpaw sample, and the amplicons likewise matched GenBank TRSV sequences (91.7% and 95.6% nt identities respectively with TRSV RNA1 isolate CmTX-H (MN504766) and TRSV RNA2 isolate IA-1-2017 (MT563079)). We further screened for TRSV infection in leaves from four symptomatic and three non-symptomatic pawpaw trees collected from the same site in 2022. RT-PCR revealed positive infection in all four symptomatic samples and one of the three (33%) non-symptomatic samples. Our results confirm the presence of TRSV infection in symptomatic pawpaw trees and emphasize the importance of also monitoring non-symptomatic trees. We confirmed graft transmission with 100% transmission rate observed in 200 trees grafted from a TRSV-infected pawpaw (Shenandoah cultivar), and investigation of other transmission vectors is on going. Because of TRSV's wide host range (Tolin, 2008), its broad transmission profile in other crops (via nematodes, thrips, seeds, sap inoculation, and grafting) (Hill and Whitham, 2014), and the notable decline observed in infected pawpaws from different cultivars (10-35, NC-1, Overleese, Pennsylvania-Golden, Shenandoah, Sunflower, Wabash), TRSV appears to pose a new threat to pawpaw orchards. To the best of our knowledge, this is the first report of TRSV infecting pawpaw in North America and the world.

The article discusses the virus disease of the blackberry and raspberry plant species in the genus Rubus of the Rosaceae family. It mentions that the Rubus species are prone to infection by viruses during development, propagation, and fruit production stages. It mentions that the viruses including Tobacco ringspot virus, Blackberry chlorotic ringspot virus, and Beet pseudo-yellows virus most commonly infects the Rubus species.

Bean pod mottle virus (BPMV) and Tobacco ringspot virus (TRSV) are two important viral pathogens causing reduction of seed yield and quality in soybean [Glycine max (L.) Merr.]. Various BPMV and TRSV isolates have been observed, but no host resistance has been reported. The objective of this study was to screen a set of modern soybean cultivars for resistance or tolerance to BPMV and TRSV. A total of 303 cultivars currently grown in the mid-southern United States were screened in the greenhouse for their reactions to BPMV and TRSV. Each cultivar was inoculated with a mild and a virulent isolate of each virus. None of the cultivars tested was resistant to BPMV, but a range of tolerance to this virus was observed among the cultivars. An enzyme-linked immunosorbent assay (ELISA) confirmed the presence of BPMV in the high and low tolerance cultivars. Tolerance to BPMV is associated with less of a reduction in plant height and biomass on infected plants. Cultivars were classified into four groups, high, moderate, low, and very low tolerance, on the basis of their having exhibited ≤20, 21 to 40, 41 to 60, or >60% of a reduction in plant height and ≤25, 26 to 50, 51 to 75, and >75% plant biomass reduction, respectively. None of the cultivars was resistant to TRSV, and all showed bud blight symptoms by 10 d after inoculation. However, 55 cultivars recovered 5 wk after inoculation, with more than 80% of the plants growing out of the bud blight symptoms and forming new leaves. The recovered plants, although systemically infected, clearly showed tolerance to TRSV at a later stage. These results may be helpful to soybean producers for selecting tolerant cultivars to grow in areas where BPMV and/or TRSV may cause problems in production. [ABSTRACT FROM AUTHOR]

Abstract: Soluble silicon (Si) provides protection to plants against a variety of abiotic and biotic stress. However, the effects of Si on viral infections are largely unknown. To investigate the role of Si in viral infections, hydroponic studies were conducted in Nicotiana tabacum with two pathogens: Tobacco ringspot virus (TRSV) and Tobacco mosaic virus (TMV). Plants grown in elevated Si showed a delay in TRSV systemic symptom formation and a reduction in symptomatic leaf area, compared to the non-supplemented controls. TRSV-infected plants showed significantly higher levels of foliar Si compared to mock-inoculated plants. However, the Si effect appeared to be virus-specific, since the element did not alter TMV symptoms nor did infection by this virus alter foliar Si levels. Hence, increased foliar Si levels appear to correlate with Si-modulated protection against viral infection. This is all the more intriguing since N. tabacum is classified as a low Si accumulator. [Copyright &y& Elsevier]

I recount the early influences that directed me toward a career in research and then describe some efforts investigating Cowpea mosaic virus and the satellite RNA of Tobacco ringspot virus. These descriptions have a common theme of surprise, how things often can be not as they are expected to be. Finally, I examine the widely held belief that a plant transgene derived from a distant taxonomic source presents a greater risk than a transgene derived from a closely related plant and contend that this also is a situation in which things may not be as they initially seem. [ABSTRACT FROM AUTHOR]

Hairpin ribozymes occur naturally only in the satellite RNAs of tobacco ringspot virus (TRsV), chicory yellow mottle virus (CYMoV) and arabis mosaic virus (ArMV). The catalytic centre of the predominantly studied sTRsV hairpin ribozyme, and of sArMV is organised around a four-way helical junction. We show here that sCYMoV features a five-way helical junction instead. Mutational analysis indicates that the fifth stem does not influence kinetic parameters of the sCYMoV hairpin ribozyme in vitro reactions, and therefore seems an appendix to that junction in the other ribozymes. We report further that all three ribozymes feature a three-way helical junction outside the catalytic core in stem A, with Watson-Crick complementarity to loop nucleotides in stem B. Kinetic analyses of cleavage and ligation reactions of several variants of the sTRsV and sCYMoV hairpin ribozymes in vitro show that the presence of this junction interferes with their reactions, particularly the ligation. We provide evidence that this is not due to a presumed interaction of the afore-mentioned elements in stems A and B. The evolutionary survival of this cis-inhibiting element seems rather to be caused by the coincidence of its position with that of the hammerhead ribozyme in the other RNA polarity. [ABSTRACT FROM AUTHOR]

Pollen is traded internationally as a source of germplasm and for pollination. Thirty-nine viruses and five viroids are known to be pollen transmitted. We investigated whether reverse transcription-polymerase chain reaction (RT-PCR) could be used to detect viruses reliably in pollen. Four extraction methods yielded nucleic acid in appropriate quantity and quality from Tobacco ringspot virus (TRSV)-infected pollen for RT-PCR amplification. One method, the RNeasy Plant Mini Kit was used subsequently to extract nucleic acid of amplifiable quality from nine plant species, and pollen infected with three ilarvirus and two nepovirus species. A real-time TaqMan™ RT-PCR for the detection of TRSV was reliable and specific using 167 extracts of pollen from plants of Nicotiana glutinosa. The assay was highly sensitive, with extracts testing positive to a 10 dilution, equivalent to a single pollen grain. This demonstrated that RT-PCR methods can detect virus-infected pollen reliably, sensitively and specifically. The possible application of these RT-PCR methods to replace current quarantine procedures without compromising biosecurity is discussed. [ABSTRACT FROM AUTHOR]

Abstract: The complete nucleotide sequence and the genome organization were determined of a putative new member of the family Tymoviridae, tentatively named Olive latent virus 3 (OLV-3), recovered in southern Italy from a symptomless olive tree. The sequenced ssRNA genome comprises 7148 nucleotides excluding the poly(A) tail and contains four open reading frames (ORFs). ORF1 encodes a polyprotein of 221.6kDa in size, containing the conserved signatures of the methyltransferase (MTR), papain-like protease (PRO), helicase (HEL) and RNA-dependent RNA polymerase (RdRp) domains of the replication-associated proteins of positive-strand RNA viruses. ORF2 overlaps completely ORF1 and encodes a putative protein of 43.33kDa showing limited sequence similarity with the putative movement protein of Maize rayado fino virus (MRFV). ORF3 codes for a protein with predicted molecular mass of 28.46kDa, identified as the coat protein (CP), whereas ORF4 overlaps ORF3 and encodes a putative protein of 16kDa with sequence similarity to the p16 and p31 proteins of Citrus sudden death-associated virus (CSDaV) and Grapevine fleck virus (GFkV), respectively. Within the family Tymoviridae, OLV-3 genome has the closest identity level (49–52%) with members of the genus Marafivirus, from which, however, it differs because of the diverse genome organization and the presence of a single type of CP subunits. [ABSTRACT FROM AUTHOR]

Grapevine fanleaf virus (GFLV) and Arabis mosaic virus (ArMV) from the genus Nepovirus, family Secoviridae, cause a severe degeneration of grapevines. GFLV and ArMV have a bipartite RNA genome and are transmitted specifically by the ectoparasitic nematodes Xiphinema index and Xiphinema diversicaudatum, respectively. The transmission specificity of both viruses maps to their respective RNA2-encoded coat protein (CP). To further delineate the GFLV CP determinants of transmission specificity, three-dimensional (3D) homology structure models of virions and CP subunits were constructed based on the crystal structure of Tobacco ringspot virus, the type member of the genus Nepovirus. The 3D models were examined to predict amino acids that are exposed at the external virion surface, highly conserved among GFLV isolates but divergent between GFLV and ArMV. Five short amino acid stretches that matched these topographical and sequence conservation criteria were selected and substituted in single and multiple combinations by their ArMV counterparts in a GFLV RNA2 cDNA clone. Among the 21 chimeric RNA2 molecules engineered, transcripts of only three of them induced systemic plant infection in the presence of GFLV RNA1. Nematode transmission assays of the three viable recombinant viruses showed that swapping a stretch of (i) 11 residues in the αB-_C loop near the icosahedral 3-fold axis abolished transmission by X. index but was insufficient to restore transmission by X. diversicaudatum and (ii) 7 residues in the βB-αB loop did not interfere with transmission by the two Xiphinema species. This study provides new insights into GFLV CP determinants of nematode transmission. [ABSTRACT FROM AUTHOR]

A survey was conducted in 2007 to determine the incidence and distribution of viruses in commercial blackberry crops in Alabama. Symptomatic and asymptomatic plants in eight major blackberry producing counties were tested for Impatiens necrotic spot virus, Raspberry bushy dwarf virus, Tobacco ringspot virus, and Tomato ringspot virus using enzyme-linked immunosorbent assay. Seventy-nine percent of the blackberry samples were infected. Tomato ringspot virus was detected in 45% of the samples, followed by Impatiens necrotic spot virus (35%), Tobacco ringspot virus (31%), and Raspberry bushy dwarf virus (26%). Each virus was detected in each of the eight counties surveyed and in each of the 14 blackberry cultivars evaluated in this study. Seventy-eight percent of the asymptomatic blackberry samples tested positive for virus infection, an indication that symptom expression is not necessarily a reliable diagnostic method for virus infection of blackberries. [ABSTRACT FROM AUTHOR]

A producing strain of an anti-tumor and antiviral enzyme L-lysine-α-oxidase from Trichoderma was cultured using a technological device of G. K. Skryabin Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences (Pushchino). L-lysine-α-oxidase activity in the obtained metabolite concentrate was 5.4 U/ml. We studied the effects of the concentrate of active L-lysine-α-oxidase producer on the highly infectious Tobacco ringspot virus and revealed anti-viral activity of it when enzyme concentration was at least 1.0 U/ml. [ABSTRACT FROM AUTHOR]

Abstract: Tomato ringspot virus (ToRSV), Tobacco ringspot virus (TRSV) and Tobacco rattle virus (TRV) are transmitted to healthy plants by viruliferous nematodes in the soil. A method was developed for extraction of genomic viral RNA from virus particles carried within nematodes and a sensitive nested RT-PCR detection assay. The procedure has been adapted to microscale for handling multiple samples. This assay is effective for detection of ToRSV or TRSV in Xiphinema americanum or TRV in Paratrichodorus allius. With this method, viruses can be detected in nematodes fed on infected plants or from field-collected nematodes where the percentage of viruliferous nematodes is unknown. Soil samples from four red raspberry fields infected with ToRSV were collected in 2003 and 2004. Nematodes isolated from these samples were assayed for ToRSV by RT-PCR and compared to cucumber baiting bioassay for virus transmission from the same soil samples. ToRSV was detected in nematodes throughout the season with similar frequencies by the RT-PCR assay and the transmission bioassay. [Copyright &y& Elsevier]

The aim of this study was to investigate the dependence of the observed cleavage rates (kobs) of a tertiary stabilized hammerhead ribozyme (tsHHRz) and of a minimal hammerhead ribozyme (mHHRz), both derived from tobacco ringspot virus, on the type and concentration of divalent metal ions in order to interpret the functional role of high-affinity ions detected by electron paramagnetic resonance (EPR). To measure the fast cleavage of the cistsHHRz, a new method using chemically synthesized fluorescent-labeled RNAs has been developed. The tsHHRz cleavage rate is up to 20-fold faster than that of the mHHRz under similar conditions. The presence of Mn2ions leads to a 60-fold faster cleavage than in the presence of Mg2ions. The functional role of the high-affinity ion was evaluated using neomycin B inhibition studies. Neomycin B reduces the cleavage activity of both ribozymes but the inhibitory effect on tsHHRz is much weaker than that on the mHHRz. EPR data had shown that neomycin B displaces both low-affinity and high-affinity Mn2ions from the mHHRz, but only low-affinity ions from tsHHRz. Inhibition of the tsHHRz activity may be due to the displacement of weakly bound Me2ions required for the local folding leading to cleavage, whereas both the high-affinity ion required for folding and the weakly bound ions are replaced in the mHHRz. The high-affinity metal ion is required for the stabilization of the global HHRz structure, but is not involved in catalysis or stabilization of the transient state. [ABSTRACT FROM AUTHOR]

Background: Low concentrations of free magnesium in the intracellular environment can present critical limitations for hammerhead ribozymes, especially for those that are designed for intermolecular (trans) cleavage of a host or pathogen RNA. Tertiary stabilizing motifs (TSM's) from natural and artificial ribozymes with a "type I" topology have been exploited to stabilize transcleaving hammerheads. Ribozymes with "type II" or "type III" topologies might seem incompatible with conversion to trans-cleavage designs, because opening the loop at the end of stem 1 or stem 2 to accommodate substrate binding is expected to disrupt the TSM and eliminate tertiary stabilization. Results: Stem 1, together with single-stranded segments capping or internal to this stem, contains both the substrate-binding and tertiary stabilization functions. This stem was made discontinuous within the sTRSV hammerhead ribozyme, thereby separating the two functions into discrete structural segments. The resulting ribozyme, designated "RzC," cleaved its 13 nucleotide target substrate at MgCl2 concentrations as low as 0.2 mM at 25°C and 0.5 mM at 37°C. Under multipleturnover conditions, nearly thirty turnovers were observed at the highest substrate:RzC ribozyme ratios. Similar stabilization was observed for several derivatives of RzC. Catalytic activity was diminished or eliminated at sub-millimolar MgCl2 concentrations for ribozymes with weakened or deleted tertiary interactions. Eadie-Hofstee analysis revealed that the stabilized and non-stabilized ribozymes bind their substrates with equivalent affinities, suggesting that differences in observed activity are not the result of diminished binding. Some of the stabilized and non-stabilized ribozymes appear to fold into a heterogeneous collection of conformers, only a subset of which are catalytically active. Conclusion: Hammerhead ribozymes with the "type III" topology can be converted to a tertiary, trans-cleavage design. Separating the stabilization and substrate recognition functions of stem I increases cleavage activity at physiological concentrations of divalent magnesium while retaining recognition of exogenous targets. Trans-cleaving ribozymes that exploit the tertiary stabilizing motifs of all natural hammerhead topologies can therefore be used in intracellular applications. [ABSTRACT FROM AUTHOR]

Bud blight, caused by Tobacco ringspot virus (TRSV; Genus: Nepovirus; Family: Comoviridae), can significantly reduce the seed yield and seed quality of soybean [Glycine max (L.) Merr.]. The identification of resistance genes and the development of resistant cultivars constitute an effective strategy for preventing yield loss. The objectives of this study were to identify and map quantitative trait loci (QTLs) conditioning resistance to bud blight and validate their genomic location with two populations derived from the cross of 'Young' (resistant) × PI 416937 (susceptible). One population consisted of 116 F[sub 4:7] lines and was used to map restriction fragment length polymorphism (RFLP) markers associated with resistance to bud blight. The lines were grown in one-row plots in a randomized complete block design with two replications. The plots were naturally infected with TRSV. At maturity, soybean plots were visually scored according to the number of plants that exhibited terminal bud death. A major QTL was identified and mapped on linkage group (LG) F by the RFLP marker K644_1. It accounted for 82% of the variation in bud blight score. To verify the genomic location of the major bud blight QTL, a second population of Young × PI 416937 that consisted of 180 F[sub 2:3] lines was evaluated. In this population, simple sequence repeat (SSR) markers on LG F near the putative genomic location of the bud blight QTL were utilized. The major QTL conditioning bud blight resistance was confirmed and found to be closely linked to the Satt510 marker. [ABSTRACT FROM AUTHOR]

Tobacco ringspot virus (TRSV) is a plant quarantine virus in Korea. As such, a TRSV examination is conducted when importing various crops. In this study, RT-PCR and nested PCR systems for TRSV detection in quarantine sites, and the modified-positive control plasmid for proving laboratory contamination and false positive reactions were developed. The developed diagnostic system was used to detect TRSV in the quarantine site. It revealed that from 2012 to August 2014, a total of 12 cases were detected in imported various crops. The system is expected to continue contributing to TRSV detection in plant quarantine. [ABSTRACT FROM AUTHOR]

CYMV-T, CYMV-C and CYMV-RS are three strains of CYMV, a nepovirus isolated from chicory in Southern Italy. They are serologically indistinguishable, but display different electrophore- tic and analytical ultracentrifugation patterns. In fact the RNAs obtained from CYMV-T, when analyzed in 2.4% polyacrylamide cylindrical gels, showed the presence of a 170,000 mol. wt. satellite-like RNA (CYMV-T Sat RNA) and several other RNAs, whereas CYMV-RS gave only the genomic RNAs and CYMV-C, in addition, some of the RNAs present in CYMV-T but not the Sat RNA. When these three strains were assayed on Nicotiana glutinosa L., CYMV-C and CYMV-RS elicited typical necrotic rings, whereas CYMV-T infections were symptomless. The conclusions were drawn that CYMV-T Sat RNA is another low mol. wt. RNA able to display viral disease regulation and that CYMV-T, CYMV-C and CYMV-RS could represent an example of natural oscillation among different stages of RNA satellite emergence. [ABSTRACT FROM AUTHOR]

Tobacco ringspot virus (TRSV), a nepovirus, induced unusually large, expanding lesions in the primary leaves of lima bean (Phaseolus lunatus cv. Nemagreen). During the course of lesion development, two phases were identified on the basis of ultrastructural observations. In the primary phase, cytological changes followed a pattern that was characteristic of compatible or systemic nepovirus infections. Such changes included the formation of large, membranous inclusion bodies in the cytoplasm of mesophyll parenchyma and the accumulation of phytoferritin in the stroma of chloroplasts. The cells maintained their structural integrity and appeared metabolically active. The secondary phase was degradative in nature. Ultrastructural changes observed during this phase were representative of those that occur in a virus-induced incompatible or hypersensitive (necrotic) reaction. Such changes included rounding of chloroplasts, vesiculation and disintegration of the plasma membrane and tonoplast, degeneration of cell organelles, and generalized plasmolysis. Subsequently, the cell walls collapsed, developing folds and loops around the shrunken cytosol. The collective result of these two ultrastructurally distinctive phases was the production of large necrotic lesions in the inoculated leaf, but no systemic viral spread. [ABSTRACT FROM AUTHOR]

High-throughput sequencing (HTS) is becoming the new norm of diagnostics in plant quarantine settings. HTS can be used to detect, in theory, all pathogens present in any given sample. The technique's success depends on various factors, including methods for sample management/preparation and suitable bioinformatic analysis. The Limit of Detection (LoD) of HTS for plant diagnostic tests can be higher than that of PCR, increasing the risk of false negatives in the case of low titer of the target pathogen. Several solutions have been suggested, particularly for RNA viruses, including rRNA depletion of the host, dsRNA, and siRNA extractions, which increase the relative pathogen titer in a metagenomic sample. However, these solutions are costly and time-consuming. Here we present a faster and cost-effective alternative method with lower HTS-LoD similar to or lower than PCR. The technique is called TArget-SPecific Reverse Transcript (TASPERT) pool. It relies on pathogen-specific reverse primers, targeting all RNA viruses of interest, pooled and used in double-stranded cDNA synthesis. These reverse primers enrich the sample for only pathogens of interest. Evidence on how TASPERT is significantly superior to oligodT, random 6-mer, and 20-mer in generating metagenomic libraries containing the pathogen of interest is presented in this proof of concept. [ABSTRACT FROM AUTHOR]

• Tobacco ringspot virus (TRSV) was frequently detected in consumable melon fruits. • The TRSV melon isolate asymptomatically infected melon fruits. • Numerous isolates of TRSV generated and maintained in melon fields. • The TRSV melon isolate symptomatically infected Nicotiana benthamiana plants. Melon is one of the most popular fruits worldwide and has been bred into various cultivars. RNA-sequencing using healthy melon fruit was performed to determine differences in gene expression among cultivars. Unexpected RNA-seq results revealed that viruses asymptomatically infected fruits at a high frequency (16 of 21 fruits examined were infected) and that viral transcripts highly accumulated in comparison with host transcripts (15 %–75 % of total reads). Their nucleotide sequences and phylogenetic analyses indicated that more than 10 novel isolates of tobacco ringspot virus (TRSV) were found in melon fruits. Asymptomatic infection with TRSV on melon fruits was confirmed by both immunoblot and RT-PCR analyses. Numerous isolates of TRSV generated and maintained in melon fields, and this is likely due to their asymptomatic infections. This TRSV melon isolate infected Nicotiana benthamiana plants with stunting and yellowing symptoms. This is the first report of frequent and asymptomatic infection of TRSV in consumable melon fruits. [ABSTRACT FROM AUTHOR]

There is limited information on the distribution of blueberry viruses in the U.S. or around the world other than where the viruses were first discovered and characterized. A survey for blueberry viruses was carried out in the U.S. in 2015–2017. Most blueberry viruses have been characterized to the point that sensitive diagnostic assays have been developed. These assays are based on ELISA or variations of PCR, which were employed here to determine the presence of blueberry viruses in major blueberry production and nursery areas of the U.S. The viruses included in this study were: blueberry fruit drop (BFDaV), blueberry latent (BlLV), blueberry leaf mottle (BLMoV), blueberry mosaic (BlMaV), blueberry red ringspot (BRRV), blueberry scorch (BlScV), blueberry shock (BlShV), blueberry shoestring (BlSSV), blueberry virus A (BVA), peach rosette mosaic (PRMV), tobacco ringspot (TRSV), and tomato ringspot (ToRSV). In the Pacific Northwest BlShV was the most widespread virus, with BlScV and ToRSV detected in a limited number of fields in Oregon and Washington, but BlScV was widespread in British Columbia. In the upper midwest, the nematode-borne (ToRSV, TRSV), aphid-transmitted (BlSSV and BVA) and pollen-borne (BLMoV) viruses were most widespread. In the northeast, TRSV, ToRSV, and BlScV, were detected most frequently. In the southeast, BRRV and BNRBV were the most widespread viruses. BlLV, a cryptic virus with no known symptoms or effect on plant growth or yield was present in all regions. There are other viruses present at low levels in each of the areas, but with the lower incidence they pose minimal threat to nursery systems or fruit production. These results indicate that there are hotspots for individual virus groups that normally coincide with the presence of the vectors. The information presented highlights the high risk viruses for nursery and fruit production each pose a different challenge for control. [ABSTRACT FROM AUTHOR]

The article presents the first report about Blackberry (Rubus sp.) having the disease tobacco ringspot virus (TRSV). Nurseries in the U. S. have reported the virus infection of blackberry samples from North and South Carolina. They were present with TRSV infection particularly affecting in the leaf tissues of plants growing in Alabama.

The article presents an abstract of the article "Tobacco ringspot virus Found in the Cardboard Cycad (Zamia furfuracea) in Florida." This study has focused on Zamia furfuracea, a popular houseplant and easy to grow outdoors in warm climates. It has numerous chlorotic spots on the leaves that eventually became necrotic.

Reports on the occurrence of Tobacco ringspot virus (TobRSV) in gladiolus in India. Cultivation of gladiolus for its cut flowers; Description of other gladiolus diseases; Cultivars that exhibited flower color-break symptoms; Necrotic local lesions induced after inoculation; Transmission of the disease on contaminated tools used during harvesting of flowers and corms.