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Cotton leafroll dwarf virus (CLRDV), transmitted by the cotton aphid (Aphis gossypii Glover), was first confirmed in upland cotton (Gossypium hirsutum L.) in Alabama, U.S.A., in 2017. Subsequent observations of symptomatic plants followed by virus infection confirmation via reverse transcriptase PCR (RT-PCR) were made in neighboring states in 2018. To assess the distribution and incidence of CLRDV and the occurrence of presumed symptoms across the southern cotton belt, a multidisciplinary team established sentinel plot survey sites at 16 experiment stations in 11 states stretching from Texas to Virginia and Tennessee to Florida beginning in 2019. Field trials were conducted over a 3-year period using multiple cotton cultivars that were adjusted annually. Cotton plots were evaluated at each location by a single evaluator to attempt to correlate symptom occurrence across the cotton-growing region with virus incidence in cotton plant tissues using RT-PCR. Symptom incidence, based on visual estimation of plants in each plot with presumed symptoms, differed across the region and ranged from 0 to 75%, with a low average over all locations of 11.4% in 2021 to an average high of 28.0% in 2020. Though symptom incidence suggested the presence of CLRDV, there were instances where apparent symptoms were observed but virus presence was not confirmed by RT-PCR. CLRDV has since been confirmed from all locations, which suggests that it has become endemic in cotton production areas throughout the eastern range of the United States. [Figure: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Aphid-transmitted viruses cause significant losses in pepper production worldwide, negatively affecting yield and quality. The emergence of new aphid-transmitted viruses or development of variants as well as the occurrence in mixed infections make management a challenge. Here, we overview the current status of the distribution, incidence and phylogeny of aphids and the viruses they transmit in pepper in Africa; outline the available genetic resources, including sources of resistance, resistance genes and molecular markers; and discuss the recent advances in understanding the genetic basis of resistance to the predominant African viruses infecting pepper. Pepper veinal mottle virus (PVMV; Potyvirus); Potato virus Y (PVY; Potyvirus), Chili veinal mottle virus (ChiVMV; Potyvirus), Cucumber mosaic virus (CMV; Cucumovirus) and Pepper veins yellow virus (PeVYV; Polerovirus) have been reported to be the most widespread and devastating aphid-transmitted viruses infecting pepper across Africa. Co-infection or mixed infection between aphid-transmitted viruses has been detected and the interrelationship between viruses that co-infect chili peppers is poorly understood. Establishing and evaluating existing and new diversity sets with more genetic diversity is an important component of developing host resistance and implementing integrated management strategies. However, more work needs to be done to characterize the aphid-transmitted viral strains across Africa and understand their phylogeny in order to develop more durable host resistance. In addition, a limited number of QTLs associated with resistance to the aphid-transmitted virus have been reported and QTL data are only available for PVY, ChiVMV and CMV mainly against European and Asian strains, although PVMV is likely the most important aphid-transmitted viral disease in Africa. There is a need to identify germplasm resources with resistance against various aphid-transmitted virus strains, and subsequent pyramiding of the resistance using marker-assisted selection could be an effective strategy. The recent advances in understanding the genetic basis of the resistance to the virus and the new breeding techniques that can be leveraged to accelerate breeding for aphid-transmitted virus in pepper are proposed as strategies to more efficiently develop resistant cultivars. The deployment of multi-genetic resistances in pepper is an effective and desirable method of managing viral-diseases in Africa and limit losses for farmers in a sustainable manner.

In this study, total RNA high-throughput sequencing (HTS) of a single symptomatic Phytolacca americana plant enabled the obtention of a nearly complete genome of two new isolates of turnip yellows virus (TuYV), named TuYV-ITA1 and TuYV-ITA2, and revealed a mixed infection with a new variant of citrus exocortis viroid (CEVd), named CEVd-ITA1. The TuYV-ITA2 isolate diverged from the known virus isolates of TuYV and showed variability in the P0 and P5 readthrough domain. Recombination analysis revealed its recombinant nature between TuYV and an unidentified polerovirus. The putative recombination event was identified in the P5 readthrough domain of the TuYMV-ITA2 isolate. Our results thus represent the first report of TuYV in Italy and some molecular evidence for the possible natural co-existence of TuYV and CEVd in a new natural host for both infectious entities. This study is adding further knowledge about the role of weed plants as virus reservoirs, and thus additional biological and impact studies would be desirable to determine in particular the role of P. americana in the spread of TuYV and if this virus should be considered a new threat for the susceptible Italian crops.

Since the ban of neonicotinoid insecticides in the European Union, sugar beet production is threatened by outbreaks of virus yellows (VY) disease, caused by several aphid-transmitted viruses, including the polerovirus beet mild yellowing virus (BMYV). As the symptoms induced may vary depending on multiple infections and other stresses, there is an urgent need for fast screening tests to evaluate resistance/tolerance traits in sugar beet accessions. To address this issue, we exploited the virus-induced gene silencing (VIGS) system, by introducing a fragment of a Beta vulgaris gene involved in chlorophyll synthesis in the BMYV genome. This recombinant virus was able to generate early clear vein chlorosis symptoms in infected sugar beets, allowing easy and rapid visual discernment of infected plants across five sugar beet lines. The recombinant virus displayed similar infectivity as the wild-type, and the insert remained stable within the viral progeny. We demonstrated that the percentage of VIGS-symptomatic plants was representative of the infection rate of each evaluated line, and depending on the susceptibility of the line to BMYV infection, VIGS symptoms may last over months. Our work provides a polerovirus-based VIGS system adapted to sugar beet crop allowing visual and rapid large-scale screens for resistance or functional genomic studies.

microRNAs (miRNAs) influence many biological processes at the post-transcriptional level. However, the molecular characterization of miRNAs in the Myzus persicae response to Brassica yellows virus (BrYV) stress remains unclear. In this study, we present the results of miRNA profiling in Myzus persicae under two different treatments: treatment one (raised on turnip plants), and treatment two (raised on Arabidopsis thaliana). A total of 72 known and 113 novel mature miRNAs were identified in both non-viruliferous and viruliferous aphids, under treatment one. In treatment two, 72 known and 112 novel mature miRNAs were identified in BrYV-free aphids; meanwhile, 71 known and 115 novel miRNAs were identified in BrYV-carrying aphids. Moreover, eight upregulated and four downregulated miRNAs were identified in viruliferous aphids under treatment two, whereas only two miRNAs were differentially expressed under treatment one. These results indicated the relative BrYV level could influence miRNA expression in aphids. KEGG enrichment analysis showed the predicted genes targeted by differentially expressed miRNAs were primarily involved in Peroxisome, neuroactive ligand–receptor interaction, and metabolism of xenobiotics by cytochrome P450 pathways. Taken together, these findings reveal the effect of BrYV on miRNAs in Myzus persicae and provide key clues for further studies on the molecular mechanisms of BrYV transmission via aphids.

Squash (Cucurbita pepo) fields in Mosul suffered from virus-like symptoms, such as vein clearing, severe mosaic, mottling, chlorosis, necrosis, stunting and fruit discoloration. The symptoms were associated with aphids presence in the infected fields. A metatranscriptomic analysis has been applied to detect the causal agent. The result showed that 559,024 mapped reads were related to Cucurbit aphid-borne yellows virus with full coverage to produce 5,662 bp consensus sequence. The virus sequence was deposited in National Center for Biotechnology Information (NCBI) under accession number OQ865059, and the isolate named Iraq. For the first time in Iraq, the infection of the virus has been reported in squash, highlighting the impact of such virus infection that spread epidemically across the squash and other cucurbits fields.

Pepper (Capsicum spp.) is an important solanaceous cash crop in Benin; however, productivity is limited due to several key constraints, especially diseases caused by viruses. We sought to understand farmers’ perceptions of viral diseases, management strategies deployed, and to identify the virus population affecting pepper production in Benin. To assess farmers’ perceptions and management of viral diseases, a survey was carried out in four agroecological zones of Benin. A total of 144 pepper farmers were interviewed using the snowball method. A total of 52 pepper leaf samples with virus-like symptoms were collected and diagnosed by reverse-transcriptase polymerase chain reaction (RT-PCR) or PCR. Pepper production systems varied across agroecological zones (P ≤ 0.001) with a predominance of farms practicing monoculture (82%). The majority of farmers (89%) indicated that pests and diseases were the main constraints to increased production. Cucumber mosaic virus (92% of the total samples), Pepper vein yellow virus (52%), and Pepper veinal mottle virus (50%) were the major viruses detected in pepper fields in Benin. There were both single (29%) and mixed (71%) infections of the viruses, suggesting that mixed infections are common for pepper in Benin, confounding efforts to reduce virus infections. Nearly 100% of the farmers surveyed were not aware of these viral diseases. They also could not directly relate symptoms of virus infection to the presence of aphids, whiteflies, or thrips. Farmers relied primarily on synthetic insecticides (93%) to control virus vectors. Interestingly, some farmers applied commercial (12%) and homemade (17%) biopesticides, with neem-based preparations being the most widely used. A total of 15% of farmers used companion cropping with maize, mint or basil and 43% of farmers used crop rotation as a cultural management practice to control viral disease and vector pressure in pepper fields. The implications of this work include the importance of training farmers and extension agents on diagnosis of viruses and their vectors causing viral diseases. This study provides baseline information for the development of host-resistant cultivars and deployment of integrated pest management strategies for pepper in Benin to reduce farmer losses.

Mixed infections of a plant infecting polerovirus, umbravirus, and/or tombusvirus-like associated RNAs (tlaRNAs) produce unique virus disease complexes that exemplify “helper-dependence” interactions, a type of viral synergism that occurs when a “dependent” virus that lacks genes encoding for certain protein products necessary for it to complete its infection cycle can utilize complementary proteins encoded by a co-infecting “helper” virus. While much research has focused on polerovirus-umbravirus or polerovirus-tlaRNA interactions, only recently have umbravirus-tlaRNA interactions begun to be explored. To expand on the limited understanding of umbravirus-tlaRNA interactions in such disease complexes, we established various co-infection pairings of the polerovirus turnip yellows virus (TuYV), the umbravirus carrot mottle virus (CMoV), and three different tlaRNAs—carrot red leaf virus aRNAs (CRLVaRNAs) gamma and sigma, and the TuYVaRNA ST9—in the model plant Nicotiana benthamiana, then investigated the effects of these different co-infections on tlaRNA systemic movement within the host, and on virus accumulation, and aphid and mechanical transmission of each of these viruses. We found that CMoV alone could support systemic movement of each of the tlaRNAs, making this the second report to demonstrate such an interaction between an umbravirus and tlaRNAs. We also report for the first time that CMoV could also impart mechanical transmissibility to the tlaRNAs sigma and ST9, and that co-infections of either of these tlaRNAs with both TuYV and CMoV increased the efficiency with which TuYV could be mechanically co-transmitted with CMoV.

The P0 proteins encoded by Cotton leafroll dwarf virus (CLRDV) serve as viral suppressors of RNA silencing (VSR). CLRDV P0 proteins share a VSR-associated F-box-like motif. This study investigated the role of specific amino acid (aa) residues within the F-box-like motif, elucidating their implications for VSR potency, pathogenicity, and intracellular localization. Analysis of various single aa substitution mutants within the F-box-like motif to understand their effect on P0 proteins’ VSR potency revealed that specific mutations significantly affect P0’s ability to suppress RNA silencing, emphasizing the crucial role of the F-box-like motif. Subcellular localization examinations indicated that the P0 proteins associate with the nucleus and endoplasmic reticulum, which may be linked to their VSR function. Also, the induction of hypersensitive response (HR)-like symptoms and the accumulation of reactive oxygen species by P0 proteins was observed, suggesting their role as pathogenicity factors. These results indicated that specific mutations significantly affect the functions of multifaceted P0 proteins, highlighting the F-box-like motif’s crucial role. Our study highlights the importance of further molecular investigations to elucidate how different CLRDV strains manifest various disease symptoms and severity. This is crucial considering the global economic importance of cotton and the potential emergence of more threatening CLRDV isolates.

Abstract Background Poleroviruses, such as turnip yellows virus (TuYV), are plant viruses strictly transmitted by aphids in a persistent and circulative manner. Acquisition of either virus particles or plant material altered by virus infection is expected to induce gene expression deregulation in aphids which may ultimately alter their behavior. Results By conducting an RNA-Seq analysis on viruliferous aphids fed either on TuYV-infected plants or on an artificial medium containing purified virus particles, we identified several hundreds of genes deregulated in Myzus persicae, despite non-replication of the virus in the vector. Only a few genes linked to receptor activities and/or vesicular transport were common between the two modes of acquisition with, however, a low level of deregulation. Behavioral studies on aphids after virus acquisition showed that M. persicae locomotion behavior was affected by feeding on TuYV-infected plants, but not by feeding on the artificial medium containing the purified virus particles. Consistent with this, genes potentially involved in aphid behavior were deregulated in aphids fed on infected plants, but not on the artificial medium. Conclusions These data show that TuYV particles acquisition alone is associated with a moderate deregulation of a few genes, while higher gene deregulation is associated with aphid ingestion of phloem from TuYV-infected plants. Our data are also in favor of a major role of infected plant components on aphid behavior.

The cocoa crop (Theobroma cacao L.) is known to be a host for several badnaviruses, some of which cause severe disease, while others are asymptomatic. Recently, the first preliminary evidence has been published concerning the occurrence of a polerovirus in cacao. We report here the first near-complete genome sequence of cacao polerovirus (CaPV) by combining bioinformatic searches of cacao transcript databases, with cloning from the infected germplasm. The reported novel genome has all the genome features known for poleroviruses from other species. Pairwise identity analyses of RNA-dependent RNA polymerase and coat protein indicates < 60% similarity of CaPV with any reported poleroviruses; hence, we propose that the polerovirus isolate reported in this study is a novel polerovirus. The genome sequence information was also used to develop a multiplex RT-PCR assay, which was applied to screen a selected range of germplasms and to identify several infected clones. Although there is no evidence that this virus causes any severe disease, this new information, together with a robust diagnostic assay, are of strategic importance in developing protocols for the safe international transfer of cacao germplasms.

Potato leafroll virus (PLRV) is a widespread and one of the most damaging viral pathogens causing significant quantitative and qualitative losses in potato worldwide. The current knowledge of the geographical distribution, standing genetic diversity and the evolutionary patterns existing among global PLRV populations is limited. Here, we employed several bioinformatics tools and comprehensively analyzed the diversity, genomic variability, and the dynamics of key evolutionary factors governing the global spread of this viral pathogen. To date, a total of 84 full-genomic sequences of PLRV isolates have been reported from 22 countries with most genomes documented from Kenya. Among all PLRV-encoded major proteins, RTD and P0 displayed the highest level of nucleotide variability. The highest percentage of mutations were associated with RTD (38.81%) and P1 (31.66%) in the coding sequences. We detected a total of 10 significantly supported recombination events while the most frequently detected ones were associated with PLRV genome sequences reported from Kenya. Notably, the distribution patterns of recombination breakpoints across different genomic regions of PLRV isolates remained variable. Further analysis revealed that with exception of a few positively selected codons, a major part of the PLRV genome is evolving under strong purifying selection. Protein disorder prediction analysis revealed that CP-RTD had the highest percentage (48%) of disordered amino acids and the majority (27%) of disordered residues were positioned at the C-terminus. These findings will extend our current knowledge of the PLRV geographical prevalence, genetic diversity, and evolutionary factors that are presumably shaping the global spread and successful adaptation of PLRV as a destructive potato pathogen to geographically isolated regions of the world.

ABSTRACT Evidence is accumulating that plant viruses alter host plant traits in ways that modify their insect vectors’ behavior. These alterations often enhance virus transmission, which has led to the hypothesis that these effects are manipulations caused by viral adaptation. However, we lack a mechanistic understanding of the genetic basis of these indirect, plant-mediated effects on vectors, their dependence on the plant host, and their relation to the mode of virus transmission. Transcriptome profiling of Arabidopsis thaliana and Camelina sativa plants infected with turnip yellows virus (TuYV) or cauliflower mosaic virus (CaMV) and infested with the common aphid vector Myzus persicae revealed strong virus- and host-specific differences in gene expression patterns. CaMV infection caused more severe effects on the phenotype of both plant hosts than did TuYV infection, and the severity of symptoms correlated strongly with the proportion of differentially expressed genes, especially photosynthesis genes. Accordingly, CaMV infection modified aphid behavior and fecundity more strongly than did infection with TuYV. Overall, infection with CaMV, relying on the noncirculative transmission mode, tends to have effects on metabolic pathways, with strong potential implications for insect vector-plant host interactions (e.g., photosynthesis, jasmonic acid, ethylene, and glucosinolate biosynthetic processes), while TuYV, using the circulative transmission mode, alters these pathways only weakly. These virus-induced deregulations of genes that are related to plant physiology and defense responses might impact both aphid probing and feeding behavior on infected host plants, with potentially distinct effects on virus transmission. IMPORTANCE Plant viruses change the phenotype of their plant hosts. Some of the changes impact interactions of the plant with insects that feed on the plants and transmit these viruses. These modifications may result in better virus transmission. We examine here the transcriptomes of two plant species infected with two viruses with different transmission modes to work out whether there are plant species-specific and transmission mode-specific transcriptome changes. Our results show that both are the case.

Abstract Pepper vein yellows viruses (PeVYV) are phloem-restricted viruses in the genus Polerovirus, family Luteoviridae. Typical viral symptoms of PeVYV including interveinal yellowing of leaves and upward leaf curling were observed in pod pepper plants (Capsicum frutescens) growing in Wenshan city, Yunnan province, China. The complete genome sequence of a virus from a sample of these plants was determined by next-generation sequencing and RT-PCR. Pod pepper vein yellows virus (PoPeVYV) (MT188667) has a genome of 6015 nucleotides, and the characteristic genome organization of a member of the genus Polerovirus. In the 5′ half of its genome (encoding P0 to P4), PoPeVYV is most similar (93.1% nt identity) to PeVYV-3 (Pepper vein yellows virus 3) (KP326573) but diverges greatly in the 3′-part encoding P5, where it is most similar (91.7% nt identity) to tobacco vein distorting virus (TVDV, EF529624) suggesting a recombinant origin. Recombination analysis predicted a single recombination event affecting nucleotide positions 4126 to 5192 nt, with PeVYV-3 as the major parent but with the region 4126–5192 nt derived from TVDV as the minor parent. A full-length clone of PoPeVYV was constructed and shown to be infectious in C. frutescens by RT-PCR and the presence of icosahedral viral particles.

Yellow dwarf viruses (YDVs) are economically destructive viral diseases of cereal crops, which cause the reduction of yield and quality of grains. Cereal yellow dwarf virus-RPV (CYDV-RPV) is one of the most serious virus species of YDVs. These virus diseases cause epidemics in cereal fields in some periods of the year in Turkey depending on potential reservoir natural hosts that play a significant role in epidemiology. This study was conducted to investigate the presence and prevalence of CYDV-RPV in grasses and volunteer cereal host plants including 33 species from Poaceae, Asteraceae, Juncaceae, Geraniaceae, Cyperaceae, and Rubiaceae families in the Trakya region of Turkey. A total of 584 symptomatic grass and volunteer cereal leaf samples exhibiting yellowing, reddening, irregular necrotic patches and dwarfing symptoms were collected from Trakya and tested by ELISA and RT-PCR methods. The screening tests showed that 55 out of 584 grass samples were infected with CYDV-RPV in grasses from the Poaceae family, while none of the other families had no infection. The incidence of CYDV-RPV was detected at a rate of 9.42%. Transmission experiments using the aphid species Rhopalosiphum padi L. showed that CYDV-RPV was transmitted persistently from symptomatic intact grasses such as Avena sterilis, Lolium perenne and Phleum exratum to barley cv. Barbaros seedlings. PCR products of five Turkish RPV grass isolates were sequenced and compared with eleven known CYDV-RPV isolates in the GenBank⁄ EMBL databases. Compared nucleotide and amino acid sequences of CYDV-RPV isolates showed that the identities ranged from 40.38 − 95.86 % to 14.04 − 93.38%, respectively. In this study, 19 grass species from the Poaceae family and two volunteer cereal host plants were determined as natural reservoir hosts of CYDV-RPV in the cereal growing areas of Turkey.

Habanero (Capsicum chinense Jacq.) is widely grown and consumed in West and Central African countries, and viral diseases represent an important production challenge. Diagnosis of the viral species affecting habanero productivity in Benin is limited, and understanding this will enable more efficient host resistance breeding. During 2019 and 2020, we characterized the incidence and severity of the viral diseases infecting nine promising habanero breeding lines and one commercial hybrid check under open field conditions in Benin. The horticultural performance, including yield and yield component traits of the entries, was determined during the 2 years of the experiment. A randomized complete block design was used with three replications, each with 24 plants. Data were recorded on days to 50% flowering and 50% fruit maturity, yield and on the yield components of fruit weight (g), fruit length (cm), and fruit width (mm), as well as disease incidence and severity. In total, 35 leaf samples were collected for viral diagnosis among habanero breeding lines. We found that Pepper veinal mottle virus (PVMV; Potyvirus) was the overwhelmingly predominant virus in our trials, with an 80% incidence; however, we found frequent coinfection of PVMV with Cucumber mosaic virus (CMV, Cucumovirus), Polerovirus, and, to a lesser extent, Chili veinal mottle virus (ChiVMV; Potyvirus). The mean disease incidence across all entries was 60%. AVPP1932 and PBC 2010 had the lowest disease incidence (35% and 43%, respectively), whereas AVPP1929 had the highest (86%) disease incidence. The F1 hybrid check Afadja had the overall highest yield, with 30 t⋅ha−1, followed by AVPP1932, with 19 t⋅ha−1, both in 2019. There was a negative correlation between disease incidence and total yield (r = −0.44; P < 0.001), supporting previous studies indicating that viral diseases are major production constraints for habanero in West Africa. This study provides insight regarding the need to improve habanero for resistance to aphid-transmitted viruses and develop integrated pest management strategies to limit losses in Benin.

High-throughput sequencing (HTS) has been an important tool for the discovery of plant viruses and their surveillance. In 2015, several virus-like symptoms were observed in passion fruit (PF) plants in Bahia state, Brazil. Using HTS technology, bioinformatics tools, RT-PCR, and Sanger sequencing, we identified the cucurbit aphid-borne yellows virus (CABYV, Polerovirus, Solemoviridae) in co-infection with cowpea aphid-borne mosaic virus (CABMV, Potyvirus, Potyviridae) in PF, in green manure, and spontaneous plants in several localities in Bahia. Complete genomes of CABYV-PF isolates were determined and analyzed with other CABYV isolates available in GenBank that have been identified in various countries. Phylogenetic analysis and pairwise identity comparison with CABYV isolates showed that CABYV-PFs are more closely related to French and Spanish isolates. Overall, analyses of all the CABYV genomes revealed that these could represent ten distinct species, and we thus proposed reclassifying these CABYV as isolates into ten species, tentatively named “Polerovirus curcubitaeprimum” to “Polerovirus curcubitaenonum”, and “Polerovirus melo”. CABYV-PF is a member of “Polerovirus curcubitaeprimum”.

Pepper vein yellows virus 5 (PeVYV-5) belongs to a group of emerging poleroviruses (family Solemoviridae) which pose a risk to pepper cultivation worldwide. Since its first detection in Spain in 2013 and the determination of the complete genome sequence of an isolate in 2018, little is known on the presence, genomic variation and molecular properties of this pathogen. As other members of genus Polerovirus, PeVYV-5 encodes a P0 protein that was predicted to act as viral suppressor of RNA silencing (VSR), one of the major antiviral defense mechanisms in plants. The results of the present work have indicated that PeVYV-5 P0 is a potent VSR, which is able to induce the degradation of Argonaute (AGO) endonucleases, the main effectors of RNA silencing. New viral isolates have been identified in samples collected in 2020–2021 and sequencing of their P0 gene has revealed limited heterogeneity, suggesting that the protein is under negative selection. Analysis of natural and engineered P0 variants has pinpointed distinct protein motifs as critical for the VSR role. Moreover, a positive correlation between the VSR activity of the protein and its capability to promote AGO degradation could be established, supporting that such activity essentially relies on the clearance of core components of the RNA silencing machinery.

Pepper vein yellows virus (PeVYV) is a newly recognized Polerovirus extracted from Chinese pepper. The symptoms of PeVYV-infested pepper plants comprise intervein yellow staining, leaf curl formation and other malformations, and leaf internodal shrinkage, but the roles of the viral proteins remain undetermined. The P0 protein of the genus Polerovirus has established post-transcriptional gene silencing (PTGS) activity. This investigation focused on the PeVYV-encoded P0 protein and assessed its potential virulence capacity, PTGS activity, and tendencies to localize in the nucleus. This study revealed that P0 influenced the pathogenic properties of a specific heterologous potato virus X. In addition, P0 proteins impaired local gene silencing, although they did not regulate generalized gene silencing within Nicotiana benthamiana 16c plants. Furthermore, P0 proteins localized mainly in the nucleus, particularly in the nucleolus. P0 deletion mutagenesis demonstrated that the F-box motif (56–72 amino acids, AAs) of P0 was essential for symptom determination, inhibition of PTGS, and subcellular localization. Mutation analysis of the F-box motif of P0 protein indicated that AA 57 of the P0 protein was a pivotal site in symptom development and that AA 56 of the P0 protein was indispensable for inhibiting PTGS and subcellular localization. The outcomes obtained here suggest that further studies should be conducted on the molecular mechanisms of amino acids of the F-box domain of P0 protein in the interaction of PeVYV with plants.

In Sudan yellowing viruses are key production constraints in pulse crops. Field surveys were carried out to identify luteovirids affecting chickpea crops in the major production regions (Gezira Scheme and River Nile State). A total of 415 chickpea plant samples with yellowing and stunting symptoms were collected during the 2013, 2015 and 2018 growing seasons. Serological results (Tissue-blot immunoassays) showed that Luteoviridae and Chickpea chlorotic dwarf virus (CpCDV, genus Mastrevirus, family Geminiviridae) were the most common viruses, with rare infections with Faba bean necrotic yellows virus (FBNYV, genus Nanovirus, family Nanoviridae). Some samples reacted only with a broad-spectrum luteovirid monoclonal antibody (5G4-MAb), and others showed cross reactions between the specific monoclonal antibodies, suggesting the occurrence of new luteovirid variants. Serological results were confirmed by amplification with reverse transcription-polymerase chain reaction (RT-PCR) and sequencing of the partial coat protein gene. Molecular analyses provided a basic, sufficient and reliable characterization for four viruses affecting chickpea that belong to Polerovirus (family Luteoviridae). These were Cucurbit aphid-borne yellows virus (CABYV), Pepper vein yellows virus (PeVYV), Pepo aphid-borne yellows virus (PABYV) and Cotton leafroll dwarf virus (CLRDV), that shared high similarity with the type sequences. Phylogenetic analyses also revealed high similarity to luteovirid species. This study has established reliable, rapid and sensitive molecular tools for the detection of luteovirid species.

Assays for the high throughput screening of crops for virus monitoring need to be quick, easy, and low cost. One method involves using tissue blot immunoassays (TBIA), where plant stems are blotted onto nitrocellulose membrane and screened with available antibodies against a range of viruses. TBIAs are inexpensive but limited by antibody availability and specificity. To circumvent the antibody limitations, we developed the tissue blot hybridization chain reaction (TB-HCR). As with TBIA, plant stems are blotted onto a nitrocellulose membrane, however, TB-HCR involves using nucleic acid probes instead of antibodies. We demonstrated for the first time that TB-HCR can be used for plant viruses by designing and testing probes against species from several virus genera including begomovirus, polerovirus, luteovirus, cucumovirus, and alfamovirus. We also explored different hairpin reporter methods such as biotin/streptavidin-AP and the Alexa Fluor-488 Fluorophore. TB-HCR has applications for low-cost diagnostics for large sample numbers, rapid diagnostic deployment for new viruses, and can be performed as a preliminary triage assay prior to downstream applications.

Massive outbreaks of virus yellows (VY) and syndrome “basses richesses” (SBR) are thought to be responsible for the major loss of sugar beet yields in 2020 in western cantons of Switzerland. Typical yellowing symptoms were visible during field inspections, and control measures were reportedly ineffective or even absent. Both diseases induce yellowing but have distinct etiologies; while VY is caused by aphid-transmitted RNA viruses, SBR is caused by the cixiid-transmitted γ-proteobacterium Candidatus Arsenophonus phytopathogenicus. To clarify the situation, samples from diseased plants across the country were screened for the causal agents of VY and SBR at the end of the season. Beet yellows virus (BYV) and Beet chlorosis virus (BChV) showed high incidence nationwide, and were frequently found together in SBR-infected fields in the West. Beet mild yellowing virus (BMYV) was detected in two sites in the West, while there was no detection of Beet western yellows virus or Beet mosaic virus. The nucleotide diversity of the detected viruses was then investigated using classic and high-throughput sequencing. For both diseases, outbreaks were analyzed in light of monitoring of the respective vectors, and symptoms were reproduced in greenhouse conditions by means of insect-mediated inoculations. Novel quantification tools were designed for BYV, BChV and Ca. A. phytopathogenicus, leading to the identification of specific tissues tropism for these pathogens.

Tombusvirus-like associated RNAs (tlaRNAs) are positive-sense single-stranded RNAs found in plants co-infected with some viruses of the genus Polerovirus. Pod pepper vein yellows virus (PoPeVYV) was recently reported as a new recombinant polerovirus causing interveinal yellowing, stunting, and leaf rolling in Capsicum frutescens plants at Wenshan city, Yunnan province, China. The complete genome sequence of its associated RNA has now been determined by next-generation sequencing and reverse transcription (RT) polymerase chain reaction (PCR). PoPeVYV-associated RNA (PoPeVYVaRNA) (GenBank Accession No. MW323470) has 2970 nucleotides and is closely related to other group II tlaRNAs, particularly tobacco bushy top disease-associated RNA (TBTDaRNA, GenBank Accession No. EF529625). In infection experiments on Nicotiana benthamiana and C. frutescens plants, synergism between PoPeVYVaRNA and PoPeVYV was demonstrated, leading to severe interveinal yellowing of leaves and stunting of plants. The results provide further information on the genetic and biological properties of the various agents associated with pepper vein yellows disease (PeVYD).

The genus Polerovirus contains positive-sense, single-stranded RNA plant viruses that cause significant disease in many agricultural crops, including vegetable legumes. This study aimed to identify and determine the abundance of Polerovirus species present within Tasmanian pea crops and surrounding weeds that may act as virus reservoirs. We further sought to examine the genetic diversity of TuYV, the most commonly occurring polerovirus identified. Pea and weed samples were collected during 2019–2020 between October and January from thirty-four sites across three different regions (far northwest, north, and midlands) of Tasmania and tested by RT-PCR assay, with selected samples subject to next-generation sequencing. Results revealed that the presence of polerovirus infection and the prevalence of TuYV in both weeds and pea crops varied across the three Tasmanian cropping regions, with TuYV infection levels in pea crops ranging between 0 and 27.5% of tested plants. Overall, two species members from each genus, Polerovirus and Potyvirus, one member from each of Luteovirus, Potexvirus, and Carlavirus, and an unclassified virus from the family Partitiviridae were also found as a result of NGS data analysis. Analysis of gene sequences of the P0 and P3 genes of Tasmanian TuYV isolates revealed substantial genetic diversity within the collection, with a few isolates appearing more closely aligned with BrYV isolates. Questions remain around the differentiation of TuYV and BrYV species. Phylogenetic inconsistency in the P0 and P3 ORFs supports the concept that recombination may have played a role in TuYV evolution in Tasmania. Results of the evolutionary analysis showed that the selection pressure was higher in the P0 gene than in the P3 gene, and the majority of the codons for each gene are evolving under purifying selection. Future full genome-based analyses of the genetic variations will expand our understanding of the evolutionary patterns existing among TuYV populations in Tasmania.

Viral diseases are major constraints to tomato, cucumber and mung bean production in most areas where these crops are grown. To identify the viruses on the crops in Tajikistan, a field survey was conducted in 2019. Samples of cucumber, mung bean and tomato with virus-like symptoms were collected and the viruses present were diagnosed by RT-PCR and PCR. Across all the samples, a very high proportion of the samples were infected with viruses from the genera Cucumovirus and Potyvirus. Cucumber mosaic virus (CMV; Cucumovirus) was very common in the collected samples of the three crops. As for Potyvirus, Potato virus Y (PVY) was detected in the collected tomato samples, Zucchini yellow mosaic virus (ZYMV) was identified in the collected cucumber samples, and Bean common mosaic virus (BCMV) was detected in 53% of the mung bean samples. Over 68% of the collected samples were infected with two or more viruses, suggesting that mixed infections are common for the three crops. Due to the results that the most identified viruses for the three crops are transmitted by aphids, the management of aphids is extremely important for the production of tomato, cucumber and mung bean in Tajikistan.

Poleroviruses are positive-sense, single-stranded viruses. In this study, we describe the identification of a novel polerovirus isolated from soybean displaying curled leaves. The complete viral genome sequence was identified using high-throughput sequencing and confirmed using rapid amplification of cDNA ends (RACE), RT-PCR and Sanger sequencing. Its genome organization is typical of the members of genus Polerovirus, containing seven putative open reading frames (ORFs). The full genome is composed of single-stranded RNA of 5822 nucleotides in length, with the highest nucleotide sequence identity (79.07% with 63% coverage) for cowpea polerovirus 2 (CPPV2). Amino acid sequence identities of the protein products between the virus and its relatives are below the threshold determined by the International Committee of Taxonomy of Viruses (ICTV) for species demarcation, and this strongly supports this virus’ status as a novel species, for which the name soybean chlorotic leafroll virus (SbCLRV) is proposed. Recombination analysis identified a recombination event in the ORF5 of the 3’ portion in the genome. Phylogenetic analyses of the genome and encoded protein sequences revealed that the new virus is closely related to phasey bean mild yellows virus, CPPV2 and siratro latent polerovirus. Subsequently, we demonstrated the infectivity of SbCLRV in Nicotiana benthamiana via infectious cDNA clone generation and agroinoculation.

Brassica yellows virus (BrYV), a tentative species in the genus Polerovirus, of the Solemoviridae family, is a phloem-restricted and aphid-transmitted virus with at least three genotypes (A, B, and C). It has been found across mainland China, South Korea, and Japan. BrYV was previously undescribed in Tasmania, and its genetic variability in the state remains unknown. Here, we describe a near-complete genome sequence of BrYV (genotype A) isolated from Raphanus raphanistrum in Tasmania using next-generation sequencing and sanger sequencing of RT-PCR products. BrYV-Tas (GenBank Accession no. OM469309) possesses a genome of 5516 nucleotides (nt) and shares higher sequence identity (about 90%) with other BrYV isolates. Phylogenetic analyses showed variability in the clustering patterns of the individual genes of BrYV-Tas. Recombination analysis revealed beginning and ending breakpoints at nucleotide positions 1922 to 5234 nt, with the BrYV isolate LC428359 and BrYV isolate KY310572 identified as major and minor parents, respectively. Results of the evolutionary analysis showed that the majority of the codons for each gene are evolving under purifying selection, though a few codons were also detected to have positive selection pressure. Taken together, our findings will facilitate an understanding of the evolutionary dynamics and genetic diversity of BrYV.

Reverse transcription PCR (RT-PCR) is a popular method for detecting RNA viruses in plants. RT-PCR is usually performed in a classical two-step procedure: in the first step, cDNA is synthesized by reverse transcriptase (RT), followed by PCR amplification by a thermostable polymerase in a separate tube in the second step. However, one-step kits containing multiple enzymes optimized for RT and PCR amplification in a single tube can also be used. Here, we describe an RT-PCR single-enzyme assay based on an RTX DNA polymerase that has both RT and polymerase activities. The expression plasmid pET_RTX_(exo-) was transferred to various E. coli genotypes that either compensated for codon bias (Rosetta-gami 2) or contained additional chaperones to promote solubility (BL21 (DE3) with plasmids pKJE8 or pTf2). The RTX enzyme was then purified and used for the RT-PCR assay. Several purified plant viruses (TMV, PVX, and PVY) were used to determine the efficiency of the assay compared to a commercial one-step RT-PCR kit. The RT-PCR assay with the RTX enzyme was validated for the detection of viruses from different genera using both total RNA and crude sap from infected plants. The detection endpoint of RTX-PCR for purified TMV was estimated to be approximately 0.01 pg of the whole virus per 25 µL reaction, corresponding to 6 virus particles/µL. Interestingly, the endpoint for detection of TMV from crude sap was also 0.01 pg per reaction in simulated crude plant extracts. The longest RNA fragment that could be amplified in a one-tube arrangement was 2379 bp long. The longest DNA fragment that could be amplified during a 10s extension was 6899 bp long. In total, we were able to detect 13 viruses from 11 genera using RTX-PCR. For each virus, two to three specific fragments were amplified. The RT-PCR assay using the RTX enzyme described here is a very robust, inexpensive, rapid, easy to perform, and sensitive single-enzyme assay for the detection of plant viruses.

Neonicotinoids are widely used to protect fields against aphid-borne viral diseases. The recent ban of these chemical compounds in the European Union has strongly impacted rapeseed and sugar beet growing practices. The poor sustainability of other insecticide families and the low efficiency of prophylactic methods to control aphid populations and pathogen introduction strengthen the need to characterize the efficiency of new plant protection products targeting aphids. In this study, the impact of Movento® (Bayer S.A.S., Leverkusen, Germany), a tetrameric acid derivative of spirotetramat, on Myzus persicae and on viral transmission was analyzed under different growing temperatures. The results show (i) the high efficiency of Movento® to protect rapeseed and sugar beet plants against the establishment of aphid colonies, (ii) the impact of temperature on the persistence of the Movento® aphicid properties and (iii) a decrease of approximately 10% of the viral transmission on treated plants. These observations suggest a beneficial effect of Movento® on the sanitary quality of treated crops by directly reducing primary infections and indirectly altering, through aphid mortality, secondary infections on which the spread of disease within field depends. These data constitute important elements for the future development of management strategies to protect crops against aphid-transmitted viruses.

Cotton is the most important fiber crop worldwide. To determine the presence of viruses in cotton plants showing leaf roll and vein yellowing symptoms in Henan Province of China, a small RNA-based deep sequencing approach was performed. Analysis of the de novo-assembled contigs followed by reverse transcription PCR allowed the reconstruction of watermelon mosaic virus and an unknown virus. The genome of the unknown virus was determined to be 5870 nucleotides in length, and has a genomic organization with characteristic features of previously reported poleroviruses. Sequence analysis revealed that the virus was closely related to, but significantly different from, cotton leafroll dwarf virus, a polerovirus of the family Solemoviridae. This virus had less than 90% amino acid sequence identity in the products of both ORF0 and ORF1. According to the polerovirus species demarcation criteria set by the International Committee on Taxonomy of Viruses, this virus should be assigned to a new polerovirus species, for which we propose the name “cotton leaf roll virus”.

Cotton leafroll dwarf virus (CLRDV) was first reported in the United States (US) in 2017 from cotton plants in Alabama (AL) and has become widespread in cotton-growing states of the southern US. To investigate the genomic variability among CLRDV isolates in the US, complete genomes of the virus were obtained from infected cotton plants displaying mild to severe symptoms from AL, Florida, and Texas. Eight CLRDV genomes were determined, ranging in size from 5865 to 5867 bp, and shared highest nucleotide identity with other CLRDV isolates in the US, at 95.9–98.7%. Open reading frame (ORF) 0, encoding the P0 silencing suppressor, was the most variable gene, sharing 88.5–99.6% and 81.2–89.3% amino acid similarity with CLRDV isolates reported in cotton growing states in the US and in Argentina and Brazil in South America, respectively. Based on Bayesian analysis, the complete CLRDV genomes from cotton in the US formed a monophyletic group comprising three relatively divergent sister clades, whereas CLRDV genotypes from South America clustered as closely related sister-groups, separate from US isolates, patterns reminiscent of phylogeographical structuring. The CLRDV isolates exhibited a complex pattern of recombination, with most breakpoints evident in ORFs 2 and 3, and ORF5. Despite extensive nucleotide diversity among all available CLRDV genomes, purifying selection (dN/dS < 1) was implicated as the primary selective force acting on viral protein evolution.

Viral diseases are among the most limiting factors in the production of potato in Colombia and the rest of the world. The best strategy to control plant viruses consists on the use of certified seed tubers, control of arthropod vectors and the use of adequate crop management practices that reduce mechanical transmission and the presence of viral reservoirs like weeds and volun-teer plants. However, the successful implementation of these practices relies on the availability of highly sensitive techniques that allow for the asymptomatic detection of viruses. In this work, we tested the performance of Next-generation sequencing (NGS) and real time RT-PCR (RT-qPCR) on a single volunteer potato plant (cv. Diacol-Capiro) growing naturally in a seed-tuber storage facility in Yarumal (Antioquia). Our NGS results demonstrate a mixed infection with Potato virus Y (PVY) and Potato leafroll virus (PLRV). RT-qPCR was performed in roots, main stolons, crown (root collar) and upper, middle and lower leaves using specific primers for PVY, PLRV, Potato virus S (PVS), Potato virus V (PVV), Potato virus X (PVX) and Potato yellow vein virus (PYVV). Only PVY and PLRV were detected in good agreement with the NGS data. This work demonstrates the use-fulness of both techniques for supporting integrated management of plant viruses in potato, in-cluding virus detection in natural reservoirs such as volunteer plants and weeds.

La frutilla (Fragaria x ananassa Duch.) es propagada vegetativamente por lo cual las enfermedades causadas por patógenos sistémicos constituyen uno de los problemas sanitarios más importantes. Más de 30 enfermedades provocadas por virus, fitoplasmas y otros patógenos sistémicos han sido reportadas en frutilla en el mundo. En esta especie se han detectado numerosos áfidos transmisores de los virus colonizando el cultivo. Las plantas libres de virus son usadas en el control de estas enfermedades, lo cual debe ser complementado con la regulación de las poblaciones de los insectos vectores, que son los responsables de dichas enfermedades. El objetivo de esta revisión fue brindar información actualizada de las especies de virus y pulgones encontrados en el cultivo de frutilla en Argentina, como un aporte para definir acciones fitosanitarias tendientes a minimizar el daño producido por estas amenazas bióticas. Los virus detectados en frutilla en Argentina son Strawberry mild yellow edge virus, Strawberry mottle virus, Strawberry crinkle virus y Strawberry polerovirus 1, describiéndose en este trabajo sus aspectos taxonómicos, epidemiológicos y los vectores reportados. Las especies de áfidos detectadas en frutilla en Argentina son: Aphis forbesi Weed, Aphis gossypii Glover, Chaetosiphon fragaefolii (Cockerell), Chaetosiphon minor (Forbes), Chaetosiphon thomasi Hille Ris Lambers, Macrosiphum euphorbiae (Thomas) y Myzus persicae (Sulzer). Las especies de Chaetosiphon han sido reportadas como responsables de las mayores transmisiones de virus en frutilla.

The cotton leafroll dwarf virus (CLRDV) is one of the most devastating pathogens of cotton. This malady, known as cotton blue disease, is widespread in South America where it causes huge crop losses. Recently the disease has been reported from India. We noticed occurrence of cotton blue disease and chickpea stunt disease in adjoining cotton and chickpea fields and got interested in knowing if these two viral diseases have some association. By genetic studies, we have shown here that CLRDV is very close to chickpea stunt disease associated virus (CpSDaV). We were successful in transmitting the CLRDV from cotton to chickpea. Our studies indicate that CpSDaV and CLRDV in India are possibly two different strains of the same virus. These findings would be helpful in managing these serious diseases by altering the cropping patterns.

Forage peanut (Arachis pintoi) is one of the forage crops best adapted to tropical agroecosystems where it is used as ground cover in urban areas and slopes, in the preservation of soils cultivated with coffee, African oil palm and citrus and as animal feed in combination with gramineous plants. A. pintoi is considered to be highly resistant to plagues and diseases; however, in recent years there has been a marked increase of plants showing symptoms typical of viral infection. In this work, Next Generation Sequencing (NGS) was used to confirm the presence of virus in symptomatic A. pintoi plants collected in urban areas in Medellín (Colombia). Transcriptome analysis revealed the presence of 3,291,089 reads related to viruses in the families Potyviridae, Luteoviridae and Alphaflexiviridae and resulted in the complete genome assembly of Peanut mottle virus (9707 nt), Turnip yellows virus (5578 nt) and two variants of a virus with phylogenetic affinity to the genus Allexivirus. These two variants lack ORF6 present in Allexivirus and probably belong to an uncharacterized genus within the family Alphaflexiviridae. The presence of at least three viruses infecting A. pintoi in Colombia highlights the importance of starting a germplasm clean-up program of the plant material used as seed in this crop.

Las virosis son uno de los problemas fitosanitarios más limitantes para la producción de papa, siendo los virus Potato leafroll virus (PLRV), Potato virus Y (PVY), Potato virus S (PVS) y Potato ye-llow vein virus (PYVV) los que generan mayores pérdidas económicas en la región andina. Gene-ralmente estos virus son transmitidos por insectos hemípteros y por tubérculos-semilla. En esta investigación se evaluó la presencia y distribución del PLRV en tubérculos de Solanum tuberosum variedad Diacol-Capiro y S. phureja variedad Criolla-Colombia obtenidos en Antioquia, utilizando pruebas serológicas y moleculares. Mediante DAS-ELISA se evaluaron 128 muestras consisten-tes en tres tipos de tejidos (piel, yemas latentes y brotes), mientras que con RT-PCR en tiempo real (RT-qPCR), se utilizó un subgrupo de 12 muestras de cada variedad. Utilizando DAS-ELISA, el PLRV se encontró en el 3,1% y 10,1% de las muestras de Diacol-Capiro y Criolla-Colombia, respectivamente; siendo evidente una mayor detección del virus en la piel de los tubérculos de ésta última variedad. Las pruebas de RT-qPCR detectaron el PLRV en 83,3% y 91,6% de las 12 muestras de Diacol-Capiro y de Criolla-Colombia, respectivamente; con valores de Ciclo umbral (CT) entre 17,72 y 30,59. La naturaleza de los amplicones obtenidos por RT-qPCR fue confirmada por secuenciación como parte de una región codificante de la cápside del PLRV (Identidad=99%-100%). Estos resultados enfatizan en la necesidad de utilizar tubérculo-semilla certificados por su sanidad viral en los cultivos de papa del país y de fortalecer dichos programas a partir del empleo de técnicas de detección altamente sensibles como RT-qPCR.

The phloem-limited poleroviruses infect Arabidopsis thaliana without causing noticeable disease symptoms. In order to facilitate visual infection identification, we developed virus-induced gene silencing (VIGS) vectors derived from Turnip yellows virus (TuYV). Short sequences from the host gene AtCHLI1 required for chlorophyll biosynthesis [42 nucleotides in sense or antisense orientation or as an inverted-repeat (IR), or an 81 nucleotide sense fragment] were inserted into the 3′ non-coding region of the TuYV genome to screen for the most efficient and robust silencing vector. All recombinant viruses produced a clear vein chlorosis phenotype on infected Arabidopsis plants due to the expression inhibition of the AtCHLI1 gene. The introduction of a sense-oriented sequence into TuYV genome resulted in a virus exhibiting a more sustainable chlorosis than the virus containing an IR of the same length. This observation was correlated with a higher stability of the sense sequence insertion in the viral genome. In order to evaluate the impact of the TuYV silencing suppressor P0 in the VIGS mechanism a P0 knock-out mutation was introduced into the recombinant TuYV viruses. They induced a similar but milder vein clearing phenotype due to lower viral accumulation. This indicates that P0 does not hinder the performances of the TuYV silencing effect and confirms that in the viral infection context, P0 has no major impact on the production, propagation and action of the short distance silencing signal in phloem cells. Finally, we showed that TuYV can be used to strongly silence the phloem specific AtRTM1 gene. The TuYV-derived VIGS vectors therefore represent powerful tools to easily detect and monitor TuYV in infected plants and conduct functional analysis of phloem-restricted genes. Moreover this example indicates the potential of poleroviruses for use in functional genomic studies of agronomic plants.

Aphid-transmitted plant viruses are a threat for major crops causing massive economic loss worldwide. Members in the Luteoviridae family are transmitted by aphids in a circulative and non-replicative mode. Virions are acquired by aphids when ingesting sap from infected plants and are transported through the gut and the accessory salivary gland (ASG) cells by a transcytosis mechanism relying on virus-specific receptors largely unknown. Once released into the salivary canal, virions are inoculated to plants, together with saliva, during a subsequent feeding. In this paper, we bring in vivo evidence that the membrane-bound Ephrin receptor (Eph) is a novel aphid protein involved in the transmission of the Turnip yellows virus (TuYV, Polerovirus genus, Luteoviridae family) by Myzus persicae. The minor capsid protein of TuYV, essential for aphid transmission, was able to bind the external domain of Eph in yeast. Feeding M. persicae on in planta- or in vitro-synthesized dsRNA targeting Eph-mRNA (dsRNAEph) did not affect aphid feeding behavior but reduced accumulation of TuYV genomes in the aphid's body. Consequently, TuYV transmission efficiency by the dsRNAEph-treated aphids was reproducibly inhibited and we brought evidence that Eph is likely involved in intestinal uptake of the virion. The inhibition of virus uptake after dsRNAEph acquisition was also observed for two other poleroviruses transmitted by M. persicae, suggesting a broader role of Eph in polerovirus transmission. Finally, dsRNAEph acquisition by aphids did not affect nymph production. These results pave the way toward an ecologically safe alternative of insecticide treatments that are used to lower aphid populations and reduce polerovirus damages.

Serological methods are commonly used methods for detection of viruses. Preparation of pure viral antigens is a crucial step in production of antibodies required for serological studies. In this research the gene encoding coat protein of a Beet western yellows virus (BWYV) isolate from Iran was amplified by PCR and was ligated into a bacterial expression vector (pET26b) to obtain pET-BWYV-CP clone. Escherichia coli BL21 was transformed with pET-BWYV-CP and expression of the recombinant coat protein was induced by IPTG. The expressed recombinant coat proteins were purified and used as an antigen for rabbit immunization. The antiserum was able to detect recombinant coat protein in total protein extracts of induced E. coli BL21 cells in western blot analysis.

To identify the pathogens responsible for leaf yellowing symptoms on wheat samples collected from Jinan, China, we tested for the presence of three known barley/wheat yellow dwarf viruses (BYDV-GAV, -PAV, WYDV-GPV) (most likely pathogens) using RT-PCR. A sample that tested negative for the three viruses was selected for small RNA sequencing. Twenty-five million sequences were generated, among which 5% were of viral origin. A novel polerovirus was discovered and temporarily named wheat leaf yellowing-associated virus (WLYaV). The full genome of WLYaV corresponds to 5,772 nucleotides (nt), with six AUG-initiated open reading frames, one non-AUG-initiated open reading frame, and three untranslated regions, showing typical features of the family Luteoviridae. Sequence comparison and phylogenetic analyses suggested that WLYaV had the closest relationship with sugarcane yellow leaf virus (ScYLV), but the identities of full genomic nucleotides and deduced amino acid sequence of coat protein (CP) were 64.9 and 86.2%, respectively, below the species demarcation thresholds (90%) in the family Luteoviridae. Furthermore, agroinoculation of Nicotiana benthamiana leaves with a cDNA clone of WLYaV caused yellowing symptoms on the plant. Our study adds a new polerovirus that is associated with wheat leaf yellowing disease, which would help to identify and control pathogens of wheat.

During the process of virus acquisition by aphids, plants respond to both the virus and the aphids by mobilizing different metabolic pathways. It is conceivable that the plant metabolic responses to both aggressors may be conducive to virus acquisition. To address this question, we analyze the accumulation of the phloem-limited polerovirus Turnip yellows virus (TuYV), which is strictly transmitted by aphids, and aphid’s life traits in six Arabidopsis thaliana mutants (xth33, ss3-2, nata1, myc234, quad, atr1D, and pad4-1). We observed that mutations affecting the carbohydrate metabolism, the synthesis of a non-protein amino acid and the glucosinolate pathway had an effect on TuYV accumulation. However, the virus titer did not correlate with the virus transmission efficiency. Some mutations in A. thaliana affect the aphid feeding behavior but often only in infected plants. The duration of the phloem sap ingestion phase, together with the time preceding the first sap ingestion, affect the virus transmission rate more than the virus titer did. Our results also show that the aphids reared on infected mutant plants had a reduced biomass regardless of the mutation and the duration of the sap ingestion phase.

Yellowing vein banding disease has been reported infecting cucurbit plants in Bali since 2014. Similar vein banding symptom on chilli pepper was observed previously, and early diagnosis indicated infection of Polerovirus. The objective of this research was to confirm the presence of Polerovirus infection on cucumber plant showing yellow vein banding symptom in Bali. Reverse transcription polymerase chain reaction – based detection method was conducted using specific primer pairs PeVYV-CP-F-BamH1/ PeVYV-CP-R-Pst1followed by sequencing and nucleotide sequence analysis. Specific DNA fragments of ± 650 bp was successfully amplified from field samples. Nucleotide sequence analysis showed that the sequence has the highest similarity > 95% with Pepper vein yellow virus (PeVYV) infecting chili pepper from Indonesia (Bali, and Rembang), Japan, and Greece.

Beet western yellows virus (BWYV), a species of the genus Polerovirus in the family Luteoviridae, is an agriculturally important virus infecting over 150 plant species in 23 dicotyledonous families worldwide. A survey of BWYV in canola fields in Golestan and Tehran provinces of Iran using indirect triple antibody sandwich enzyme-linked immunosorbent assay (TAS-ELISA) indicated 8.3 % infection. The presence of BWYV was confirmed by amplification of the coat protein (CP) gene of the virus via running a reverse transcription-polymerase chain reaction (RT-PCR) on total RNA extracted from ELISA positive leaf tissues. DNA sequences of the BWYV coat protein (CP) gene of seven Iranian isolates were determined and compared at the nucleotide (nt) and amino acid (aa) levels with those of twelve BWYV isolates from different countries deposited in GenBank. Sequence analysis data showed that the identity of BWYV-CP at nt and aa levels among the Iranian isolates were 93.4 % to 100 % and 93.2 % to 100 %, respectively. The maximum similarity of isolates at nt and aa levels were 97.2 and 96.6 %, which occurred among two Iranian isolates (Ir 8 and Ir 100) and four isolates from France (L39967 and X13063) and England (L39973 and L39970). The recombination analysis among the nineteen isolates including seven Iranian isolates revealed that there was no distinct intra-specific recombination event among BWYV isolates. This is the first report of sequencing and analyzing of the BWYV CP gene of Iranian BWYV isolates.

The Luteoviridae is an agriculturally important family of viruses whose replication and transport are restricted to plant phloem. Their genomes encode for four proteins that regulate viral movement. These include two structural proteins that make up the capsid and two non-structural proteins known as P3a and P17. Little is known about how these proteins interact with each other and the host to coordinate virus movement within and between cells. We used quantitative, affinity purification-mass spectrometry to show that the P3a protein of Potato leafroll virus complexes with virus and that this interaction is partially dependent on P17. Bimolecular complementation assays (BiFC) were used to validate that P3a and P17 self-interact as well as directly interact with each other. Co-localization with fluorescent-based organelle markers demonstrates that P3a directs P17 to the mitochondrial outer membrane while P17 regulates the localization of the P3a-P17 heterodimer to plastids. Residues in the C-terminus of P3a were shown to regulate P3a association with host mitochondria by using mutational analysis and also varying BiFC tag orientation. Collectively, our work reveals that the PLRV movement proteins play a game of intracellular hopscotch along host organelles to transport the virus to the cell periphery.

The requirement for the 17-kDa protein (P17) of Potato leafroll virus (PLRV) in virus movement was investigated in four plant species: potato (Solanum tuberosum), Physalis floridana, Nicotiana benthamiana, and N. clevelandii. Two PLRV P17 mutants were characterized, one that does not translate the P17 and another that expresses a P17 missing the first four amino acids. The P17 mutants were able to replicate and accumulate in agroinoculated leaves of potato and P. floridana, but they were unable to move into vascular tissues and initiate a systemic infection in these plants. In contrast, the P17 mutants were able to spread systemically from inoculated leaves in both Nicotiana spp., although the efficiency of infection was reduced relative to wild-type PLRV. Examination of virus distribution in N. benthamiana plants using tissue immunoblotting techniques revealed that the wild-type PLRV and P17 mutants followed a similar movement pathway out of the inoculated leaves. Virus first moved upward to the apical tissues and then downward. The P17 mutants, however, infected fewer phloem-associated cells, were slower than wild-type PLRV in moving out of the inoculated tissue and into apical tissues, and were unable to infect any mature leaves present on the plant at the time of inoculation.

Transgenic Nicotiana benthamiana expressing the minor coat protein P74 of the phloem-limited Beet western yellows virus (BWYV) exhibited an unusual spatial pattern of post-transcriptional gene silencing (PTGS) when infected with BWYV or related viruses. Following infection, transgenic P74 and its mRNA accumulated to only low levels, 21 to 23 nucleotide RNAs homologous to the transgene appeared, and the transgene DNA underwent methylation. The infecting viral RNA, however, was not subject to significant silencing but multiplied readily and produced P74 in the phloem tissues, although the P74 encoded by the transgene disappeared from the phloem as well as the nonvascular tissues.

The plant viral family Luteoviridae is divided into three genera: Luteovirus, Polerovirus and Enamovirus. Without assistance from another virus, members of the family are confined to the cells of the host plant’s vascular system. The first open reading frame (ORF) of poleroviruses and enamoviruses encodes P0 proteins which act as silencing suppressor proteins (VSRs) against the plant’s viral defense-mediating RNA silencing machinery. Luteoviruses, such as barley yellow dwarf virus-PAV (BYDV-PAV), however, have no P0 to carry out the VSR role, so we investigated whether other proteins or RNAs encoded by BYDV-PAV confer protection against the plant’s silencing machinery. Deep-sequencing of small RNAs from plants infected with BYDV-PAV revealed that the virus is subjected to RNA silencing in the phloem tissues and there was no evidence of protection afforded by a possible decoy effect of the highly abundant subgenomic RNA3. However, analysis of VSR activity among the BYDV-PAV ORFs revealed systemic silencing suppression by the P4 movement protein, and a similar, but weaker, activity by P6. The closely related BYDV-PAS P4, but not the polerovirus potato leafroll virus P4, also displayed systemic VSR activity. Both luteovirus and the polerovirus P4 proteins also showed transient, weak local silencing suppression. This suggests that systemic silencing suppression is the principal mechanism by which the luteoviruses BYDV-PAV and BYDV-PAS minimize the effects of the plant’s anti-viral defense.

During a visit to Bali Island in September 2011, we found chilipepper plants exhibiting yellowing symptoms. The infected plants showed quite similar symptom to those of the recently reported Pepper yellow leaf curl virus from Israel and Japan, but there was no shortening of internodes or leaf rolling. We have amplified part of the genome of a virus associated with this disease using degenerate primers for members of the genus Polerovirus. The reverse transcription-polymerase chain reaction (RT-PCR) from symptom-bearing samples resulted in the amplification of a 650 bp band which is the expected size. RT-PCR from healthy samples did not produce an amplicon. Based on our results, we concluded that yellowing disease on chilipepper observed in Payangan, Gianyar, Bali is associated with a virus belonging to the genus Polerovirus. This is the first report on Polerovirus infection in chilipepper in Indonesia. Some researches for elucidation of the virus species and biological/molecular characters are being conducted. Key word: chilipepper, Indonesia, Polerovirus