Leafroll Virus Research Articles

Diagnostic and historical surveys of sweet cherry (Prunus avium) virus and virus-like diseases in Oregon.

There are over 35 known virus and virus-like diseases of sweet cherry (Prunus avium), some with potential to cause severe economic impact by reducing vegetative growth, vigor, and/or fruit quality. Oregon is the second-ranked state for sweet cherry production in the United States. Statewide surveys were conducted in Oregon sweet cherry orchards for virus and virus-like diversity and distribution. Orchards in key production regions with suspected virus disease symptoms were sampled. Virus-specific ELISA, isothermal amplification, or qPCR were used to test for the presence of common and/or economically important sweet cherry pathogens, including: cherry leaf roll virus (CLRV), little cherry virus 2 (LChV2), prune dwarf virus (PDV), prunus necrotic ringspot virus (PNRSV), tomato ringspot virus (ToRSV), and Candidatus phytoplasma pruni (CaPP). Cherry leaf roll virus, a new virus of sweet cherry in Oregon, was found associated with enation and dieback symptoms in The Dalles. Some viruses were found in new regions, which included: Hood River (PDV, PNRSV, ToRSV) and the Umpqua Valley (PDV, PNRSV). A subsequent survey was conducted in the Mid-Columbia production region for the presence of little cherry symptoms associated with Little Cherry and X-Diseases. All symptomatic samples from The Dalles and Mosier, OR, or Dallesport, WA, tested positive for CaPP, but not LChV2. These findings provide a foundation for the current understanding and management of virus and virus-like diseases of sweet cherry in Oregon and context for further studies into these pathogens and their vectors.

Molecular Determination of Some Important Viruses Causing Infection in Potato Fields in Turkey

Potato is one of the most important agricultural crops worldwide and known to be susceptible to more than 40 viruses in nature. In this research, 298 leaf samples collected from potato fields in Afyon, Nevşehir and Bolu provinces in the previous study were used to determine viruses affecting potato production in the region. The leaves of potato plants showing virus symptoms were subjected to RT-PCR using virus-specific primers, in order to detect the presence of Potato leafroll virus (PLRV), Potato virus A (PVA), Potato virus X (PVX) and Potato virus M (PVM). As a result of the study,one or more viruses were detected in 46 (15.43%) of the 298 leaf samples tested. A total of 43 samples infected with PLRV (14.42%) and 3 sample infected with PVX (1.%). It was determined that the most common virus in plant samples collected from Afyon, Nevşehir and Bolu Central regions was PLRV followed by PVX. PVA and PVM were not detected in the samples. The sequences of some positive isolates of PLRV were obtained and used along with the isolates registered in the GenBank to reveal phylogenetic relationships among them. PLRV isolates from Turkey were classified in Group 2 along with isolates from Serbia, Canada, and Pakistan based on phylogenetic analyses.

A new simple and effective method for PLRV infection to screen for virus resistance in potato

Effective screening of plant germplasm collections for resistance to plant viruses requires that there is a rapid and efficient system in place to challenge individual plants with the virus. Potato leafroll virus (PLRV), a commercially important pathogen of potato, is able naturally to infect only the phloem-associated tissue of plants and is delivered to this tissue by feeding aphids. Mechanical (non-vector-mediated) infection by PLRV does not occur thus screening for PLRV resistance is currently laborious and time consuming. We constructed an infectious cDNA clone of a new (Hutton) isolate of PLRV in the binary vector pDIVA and transformed it into Agrobacterium tumefaciens strain LBA4404. Infiltration of this culture into leaves of Nicotiana benthamiana, a highly susceptible model plant, produced a systemic infection with PLRV, although this approach was not successful for potato. However, a very efficient and reproducible systemic infection of potato was achieved when we submerged cut stems of the plant into the agrobacterium cell suspension and then transplanted the stems into compost to grow roots and new apical leaves. Using a standardised protocol developed for this new PLRV inoculation method we have confirmed the previously described resistance to the virus in the JHI breeding line G8107(1) and identified 62 plant accessions from the Commonwealth Potato Collection in which no PLRV infection was detected.

Development of Reverse Transcription Recombinase Polymerase Amplification (RT-RPA): A Methodology for Quick Diagnosis of Potato Leafroll Viral Disease in Potato.

Potatoes are developed vegetatively from tubers, and therefore potato virus transmission is always a possibility. The potato leafroll virus (PLRV) is a highly devastating virus of the genus Polerovirus and family Luteoviridae and is regarded as the second-most destructive virus after Potato virus Y. Multiple species of aphids are responsible for the persistent and non-propagating transmission of PLRV. Due to intrinsic tuber damage (net necrosis), the yield and quality are drastically diminished. PLRV is mostly found in phloem cells and in extremely low amounts. Therefore, we have attempted to detect PLRV in both potato tuber and leaves using a highly sensitive, reliable and cheap method of one-step reverse transcription-recombinase polymerase amplification (RT-RPA). In this study, an isothermal amplification and detection approach was used for efficient results. Out of the three tested primer sets, one efficiently amplified a 153-bp product based on the coat protein gene. In the present study, there was no cross-reactivity with other potato viruses and the optimal amplification reaction time was thirty minutes. The products of RT-RPA were amplified at a temperature between 38 and 42 °C using a simple heating block/water bath. The present developed protocol of one-step RT-RPA was reported to be highly sensitive for both leaves and tuber tissues equally in comparison to the conventional reverse transcription-polymerase chain reaction (RT-PCR) method. By using template RNA extracted employing a cellular disc paper-based extraction procedure, the method was not only simplified but it detected the virus as effectively as purified total RNA. The simplified one-step RT-RPA test was proven to be successful by detecting PLRV in 129 samples of various potato cultivars (each consisting of leaves and tubers). According to our knowledge, this is the first report of a one-step RT-RPA performed using simple RNA extracted from cellular disc paper that is equally sensitive and specific for detecting PLRV in potatoes. In terms of versatility, durability and the freedom of a highly purified RNA template, the one-step RT-RPA assay exceeds the RT-PCR assay, making it an effective alternative for the certification of planting materials, breeding for virus resistance and disease monitoring.

Identification of grapevine virus G and grapevine virus H in Sardinia, Italy

Grapevine virus G (GVG) and grapevine virus H (GVH) (genus Vitivirus) are recently discovered viruses. Analysis of 38 samples from Sardinian grapevine cultivars for the presence of GVG and GVH was carried out using RT-PCR. All samples were also tested for grapevine Pinot gris virus (GPGV) using RT-PCR, and for grapevine leafroll virus -1, -2 and -3, grapevine virus A (GVA) and B (GVB), arabis mosaic virus (ArMV), grapevine fanleaf virus (GFLV) and grapevine fleck virus (GFkV) using multiplex RT-PCR. GVG was confirmed in four vines, and GVH was detected in only one sample. In phylogenetic analyses of the coat protein (CP) region, the Sardinian GVG isolates clustered separately from isolates from Croatia and New Zealand. The Sardinian GVH isolate clustered with most sequences from other countries, but with greater affinity to isolates from California (USA) for the CP region, whereas it clustered with isolates from Croatia in the RNA-dependent RNA polymerase (RdRp) region. In addition to GVG and GVH, many samples were coinfected with GVA, viruses from the leafroll complex, and GPGV. This is the first record of GVG and GVH occurring in Italy.

Effectiveness of Botanical Pesticides from Wastes of Virginia Tobacco Stems in Reducing Populations of Leaf-Sucking Pest and Occurrence of Virus Disease Symptoms on Potato Plants

The potato plant (Solanum tuberosum L.) is a tuber-producing plant which is rich in carbohydrates, protein and minerals, and can be used as a staple food, and potatoes have great potential as one of the horticultural crops that can support world food diversification. One of the constraining factors in increasing potato productivity is leaf-sucking pests including aphids (Aphids spp.,), whitefly (Bemisia tabaci), and thrips (Thrips Palmi Karny), which besides damaging plants directly, can act as vectors for viruses that cause potato plant diseases such as Potato virus Y (PVY) and Potato leaf roll virus (PLRV). These two viruses can cause quite severe damage and through the health of the seeds they produce, they can reduce potato productivity by up to 70-80%. This study aims to determine the effect of several concentrations of botanical pesticides from Virginia tobacco stem waste on the emergence of viral disease symptoms in potato plants. The experiment was carried out in Sembalun Bumbung Village, East Lombok Regency (Indonesia), which was arranged using a Randomized Block Design consisting of 6 treatments namely control, abamectin, and Virginia tobacco stem waste botanical pesticides with concentrations of 2, 4, 6 and 8 ml/L. The results showed that the botanical pesticides from Virginia tobacco stem waste were effective in reducing leaf-sucking pest populations and suppressing the intensity of leaf-sucking pest attacks and were able to suppress the emergence of viral disease symptoms, with a concentration of 8 ml/L botanical pesticides more effective than other treatments in controlling populations. and attacks by potato leaf-sucking pests suspected as virus vectors.

Viromes of Field-Grown Tomatoes and Peppers in Tennessee Revealed by RNA Sequencing Followed by Bioinformatic Analysis

The prevalent viruses in a population of 106 tomato and 53 pepper field-grown plants in Tennessee was investigated by RNA sequencing and analysis as two separate pools of ribosomal-depleted total RNA. Contigs of potato leafroll virus (PLRV), red clover vein mosaic virus, tomato spotted wilt virus, and a novel rhabdovirus were detected only in tomato and those of bell pepper endornavirus (BPEV) and pepper cryptic virus 2 (PCV-2) only in pepper. Additionally, contigs of five other viruses (solanum nigrum ilarvirus 1, SNIV-1; southern tomato virus, STV; tobacco vein-clearing virus, ToVCV; tomato mosaic virus, ToMV; and tomato ringspot virus, ToRSV) were detected in both tomatoes and peppers. Moreover, contigs were assembled of a potentially novel tobamovirus from both tomatoes and peppers. The presence of PLRV, STV, ToVCV, and the novel tobamovirus sequences in the original tomato samples and those of BPEV, PCV-2, ToMV, and ToRSV in the original pepper samples were confirmed by RT-PCR followed by Sanger sequencing. This study represents the first survey of viromes of field-grown tomatoes and peppers in Tennessee. To the best of our knowledge, SNIV-1, STV, and ToVCV have not been reported previously to be associated with peppers elsewhere in the world.

Potato viruses in Kazakhstan and methods for obtaining virus-free seed material

Potato (Solanum tuberosum L.) is a staple food worldwide, including in Kazakhstan. The potato yield in 2021 in the country was only 20 tonnes per ha, which is a rather low indicator. One of the main reasons for the insufficient yield of potatoes in Kazakhstan is the low quality of seed material, and the main requirement for high-quality seed material is the absence of viral diseases. Viral diseases in crops are a major impediment to sustainable potato production, as they cause large losses in crop quantity and quality. To date, 40 viral potato diseases have been discovered worldwide. Depending on the infection of viral diseases, the yield can be reduced by up to 90% in crop production. In this review, we discuss in detail the current state of potato viral diseases in Kazakhstan and characterise the most common potato viruses in the country, including potato virus M (PVM), potato virus (PVS), potato virus X (PVX), potato virus Y (PVY) and potato leafroll virus (PLRV). To ensure food security of the country and prevent the spread of infection with viral diseases, popular methods (ELISA, RT-PCR and detection of specific types of targets using microarrays) for the early detection of potato plant viruses in seed and field material are considered. For the mass production of virus-free potato plant material, the use of temporary immersion bioreactors is discussed, as well as the use of modern genetic engineering methods to obtain plant varieties resistant to the most common viral diseases.

Prevalence and molecular characterization of important potato viruses in the Tokat province of Turkey.

It is believed that viruses affect potato yield more than any other pathogens worldwide. We report here on a survey of the four most common potato viruses in the Tokat Province of northern Turkey. Leaf samples were collected from potato plants showing signs of viral diseases in five districts of the province. Over 400 leaf samples were tested using RT-PCR with virus-specific primers. Among the one or more viruses detected in 218 (52%) leaf samples, Potato virus Y (PVY) was the most common (47.1%), followed by potato virus S (PVS; 16.7%), potato virus X (PVX; 6.0%) and potato leaf roll virus (PLRV; 5.3%). The most common mixed infections were PVY + PVS (6.9%). A phylogenetic analysis of the gene sequences showed all Turkish PVS isolates to be clustered with the PVSO group, two PVY isolates with the PVYN-WI group and one isolate with the PVYNTN group. Turkish PVX isolates are in the Type X group of the two major PVX isolate groups. The Turkish PLRV isolates were separated into two major groups depending on the results of the phylogenetic analysis, with six cases in Group 1 and one in Group 2. PVY, PVX, PVS and PLRV were detected in potato production areas in Tokat. A phylogenetic comparison of the gene sequences revealed all Turkish isolates to be immigrant members of the world populations of these viruses. Our results emphasize the importance of the strict quarantine control of plant materials entering Turkey.

Systematic mutagenesis of Polerovirus protein P0 reveals distinct and overlapping amino acid functions in Nicotiana glutinosa

The protein P0 serves as the viral suppressor of RNA silencing (VSR) for poleroviruses, but elicits the hypersensitive response (HR) in specific Nicotiana species. We subjected P0 proteins from turnip yellows virus (P0Tu) and potato leafroll virus (P0PL) to serial deletion and performed extensive site-directed mutagenesis of P0Tu. Most deletions of the N-terminus and many substitution mutations disrupted both HR elicitation and VSR activity. Two conserved blocks of amino acid residues were found to be associated with HR. A double lysine to arginine substitution in HR-specific block 1 caused P0Tu to elicit a more robust HR. Conversely, deletion or mutation of block 2 in the C-terminus preserved VSR activity, but impaired HR elicitation, allowing virus escape from Nicotiana glutinosa resistance when expressed in the heterologous potato virus X vector. Our observations suggest that P0 residues responsible for suppressing RNA silencing and eliciting HR have overlapping, but distinct functions.

Polerovirus N-terminal readthrough domain structures reveal molecular strategies for mitigating virus transmission by aphids

Poleroviruses, enamoviruses, and luteoviruses are icosahedral, positive sense RNA viruses that cause economically important diseases in food and fiber crops. They are transmitted by phloem-feeding aphids in a circulative manner that involves the movement across and within insect tissues. The N-terminal portion of the viral readthrough domain (NRTD) has been implicated as a key determinant of aphid transmission in each of these genera. Here, we report crystal structures of the NRTDs from the poleroviruses turnip yellow virus (TuYV) and potato leafroll virus (PLRV) at 1.53-Å and 2.22-Å resolution, respectively. These adopt a two-domain arrangement with a unique interdigitated topology and form highly conserved dimers that are stabilized by a C-terminal peptide that is critical for proper folding. We demonstrate that the PLRV NRTD can act as an inhibitor of virus transmission and identify NRTD mutant variants that are lethal to aphids. Sequence conservation argues that enamovirus and luteovirus NRTDs will follow the same structural blueprint, which affords a biological approach to block the spread of these agricultural pathogens in a generalizable manner.

Complete genome sequence of a novel polerovirus infecting chickpea (Cicer arietinum L.).

The complete genome sequence of a novel polerovirus identified in chickpea (C. arietinum L.) is presented. Its sequence was assembled using small RNA sequencing and assembly (sRSA) and confirmed by RT-PCR, 5' and 3' RACE, and Sanger sequencing. According to the current ICTV sequence demarcation criterion of greater than 10% amino acid (aa) sequence divergence in all gene products when compared to other poleroviruses, the newly identified polerovirus should be classified as a member of a new species, and we propose the name "chickpea leafroll virus" (CpLRV) for this virus. The genome of CpLRV is 5,770 nucleotides (nt) long and is organized into seven open reading frames (ORFs), designated as ORF0, ORF1, ORF2, ORF3a, ORF3, ORF4, and ORF5, which code for putative P0, P1, P1-P2, P3a, P3, P4, and P3-P5 proteins, respectively. The 5' untranslated region (UTR) consists of 27 nt, starting with the conserved sequence 5'-ACAAAA-3', which is typical of poleroviruses, while the 3' UTR consists of 229 nt. Phylogenetic analysis based on the aa sequences of P0, P1, P1-P2, P3, P4, and P3-P5 showed that CpLRV clustered with members of the genus Polerovirus and is closely related to chickpea chlorotic stunt virus (CpCSV) and faba bean polerovirus 1 (FBPV1). Recombination analysis suggested that CpLRV is a recombinant of two unknown viruses that share the highest nucleotide sequence similarity with FBPV1 (76.9% identity) and suakwa aphid-borne yellows virus (SAbYV) (64.8% identity). The putative recombination event was identified in the 5' region of the CpLRV genome, the region that encodes proteins P0, P1, and P1-P2. This is the first report of a polerovirus infecting chickpea in Mexico.

The Spatial Diffusion of Cherry Leaf Roll Virus Revealed by a Bayesian Phylodynamic Analysis.

Cherry leaf roll virus (CLRV) is an important plant pathogen that causes severe and detrimental effects on cherry and other fruit plants. Despite recent progress in plant pathology, molecular biology, and population genetics of CLRV, the spatiotemporal spread of this virus remains poorly studied. In this study, we employed a Bayesian phylodynamics framework to investigate the spatial diffusion patterns of CLRV by analyzing the coat protein gene sequences of 81 viral isolates collected from five different countries. Consistent with the trade of cherry, our Bayesian phylodynamic analyses pointed to viral origins in New Zealand and identified multiple migration pathways between Germany and other countries, suggesting that Germany has played an important role in CLRV transmission. The results of our study will be useful in developing sustainable management strategies to control this pathogen.

Complex and simple translational readthrough signals in pea enation mosaic virus 1 and potato leafroll virus, respectively.

Different essential viral proteins are translated via programmed stop codon readthrough. Pea enation mosaic virus 1 (PEMV1) and potato leafroll virus (PLRV) are related positive-sense RNA plant viruses in the family Solemoviridae, and are type members of the Enamovirus and Polerovirus genera, respectively. Both use translational readthrough to express a C-terminally extended minor capsid protein (CP), termed CP-readthrough domain (CP-RTD), from a viral subgenomic mRNA that is transcribed during infections. Limited incorporation of CP-RTD subunits into virus particles is essential for aphid transmission, however the functional readthrough structures that mediate CP-RTD translation have not yet been defined. Through RNA solution structure probing, RNA secondary structure modeling, site-directed mutagenesis, and functional in vitro and in vivo analyses, we have investigated in detail the readthrough elements and complex structure involved in expression of CP-RTD in PEMV1, and assessed and deduced a comparatively simpler readthrough structure for PLRV. Collectively, this study has (i) generated the first higher-order RNA structural models for readthrough elements in an enamovirus and a polerovirus, (ii) revealed a stark contrast in the complexity of readthrough structures in these two related viruses, (iii) provided compelling experimental evidence for the strict requirement for long-distance RNA-RNA interactions in generating the active readthrough signals, (iv) uncovered what could be considered the most complex readthrough structure reported to date, that for PEMV1, and (v) proposed plausible assembly pathways for the formation of the elaborate PEMV1 and simple PLRV readthrough structures. These findings notably advance our understanding of this essential mode of gene expression in these agriculturally important plant viruses.

Screening of cherry leaf roll virus in Sambucus nigra plants in Ukraine

Goal. Cherry leaf roll virus (CLRV) infects naturally a wide range of herbaceous and woody plants, different species of trees, shrubs, horticultural, ornamental, weed plants and causes significant economic losses in many hosts and countries. CLRV on elderberry species was reported in Europe, USA, Iran and North America. Recently this virus has been detected in sour and sweet cherry fruit orchards in Ukraine. Testing of other hosts than Prunus was performed in Ukraine only recently. The aim of the study was to test the black elderberry plants (Sambucus nigra L.). for the presence of CLRV in the Poltava region and Kyiv regions.
 Methods. Sampling was carried out in the summer–autumn period in 2019—2021 in the territories of the Poltava and Kyiv regions and in Kyiv city. Samples of healthy elderberry plants were also analyzed. Visual diagnostics, enzyme-linked immunosorbent assay in DAS-ELISA modification, total RNA extraction, RT-PCR with primers for a 412 bp fragment of the 3’ untranslated region of the CLRV genome, and statistical data analysis were used in this research. PCR products were separated on an 1.5% agarose gel. Commercial CLRV preparations were used for positive controls in DAS-ELISA.
 Results. Thirty three elderberry samples with symptoms of leaf rolling and mosaics of varying degrees were selected in the territory of the Poltava and Kyiv regions and in Kyiv city in 2019 and 2021 and used in the study. ELISA and RT-PCR results showed that 82% of the tested black elderberry samples were infected by CLRV.
 Conclusions. The presence of CLRV, its harmfulness for elderberry plants, and Sambucus potential to serve as a reservoir for the virus indicate the necessity of testing a wider range of plant species for сherry leaf roll virus in Ukraine.

Global genetic diversity and evolutionary patterns among Potato leafroll virus populations.

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.

Approved Genetically Modified Potatoes (Solanum tuberosum) for Improved Stress Resistance and Food Safety.

Potatoes (Solanum tuberosum) are one of the most important crops worldwide. However, its production and nutrient content are endangered by both biotic and abiotic stresses. The main yield losses are caused by pest damage (e.g., Colorado potato beetle and aphids), virus disease (e.g., Potato leafroll virus and Potato viruses Y and X), or oomycete pathogens (like Phytophthora infestans), which also significantly affect the production of antinutrients and toxic metabolites of plants. Therefore, the use of genetic engineering could be an efficient tool, not harmful to the environment, and beneficial to the consumer. In this review, we focus on the main sources of problems in the field of potato production according to approved genetic modifications, their traditional solution and positive impact of gene transfection reducing economic losses, use of insecticides, and improving the nutritional properties of potatoes. We summarize all transgenic events that have been performed on potatoes and have been approved for cultivation and/or direct use or processing as feed or food.

INCIDENCE OF APHID BORNE VIRUSES AND THEIR VECTOR DYNAMICS INFECTING POTATO CROP IN DIFFERENT DISTRICTS OF AZAD JAMMU AND KASHMIR, PAKISTAN

Potato crop production is hampered by several fungal, viral, and bacterial diseases. Among the viral Potato leaf roll virus (PLRV), Potato virus Y (PVY), Potato-mop-top-virus (PMTV), Potato virus S (PVS) and Potato virus X (PVX) are the most important. Potato growing fields were surveyed during 2016 and 2017 to determine the incidence of two aphid-borne viruses, PVY and PLRV and to explore the dynamics of potato aphids in four potato-producing district of Azad Jammu & Kashmir (AJK), Pakistan. During survey 930 leaf samples were collected from various locations of Bagh, Sudhnoti, Poonch and Neelum. Parameters determined included aphid population, symptomology, DAC-ELISA based virus incidence, rate of co-infection and disease severity. Overall relative disease incidence of PLRV and PVY was 48.71% and 53.23% respectively. The highest average aphid’s population per field was 390.27 in Sudhnoti due to the low altitude of the surveyed locations. Three aphid species Myzuspersicae Myzusornatusand, Macrosiphum euphorbiaewere identified in AJK potato fields. Themaximum co-infection was recorded from Poonch. It was found that aphid populations decreased with the increase of altitude. The incidence of PLRV and PVY were high where aphid population was high. Therefore, it is necessary to promote awareness among the farmers on virus and aphid symptoms identification and to recommend use of healthy, uncontaminated certified potato seed.

Simple Webserver-Facilitated Method to Design and Synthesize Artificial miRNA Gene and Its Application in Engineering Viral Resistance.

Plant viruses impose serious threats on crop production. Artificial miRNAs can mediate specific and effective gene silencing in plants and are widely used in plant gene function studies and to engineer plant viral resistance. To facilitate the design of artificial miRNA genes, we developed a webserver, AMIRdesigner, which can be used to design oligos for artificial miRNA synthesis using wild-type and permutated MIR171 and MIR164 backbones. The artificial miRNA genes designed by AMIRdesigner can be easily assembled into miRNA clusters for multiple target sites. To validate the server functionality, we designed four artificial miRNA genes targeting four conserved regions in the potato leafroll virus genome using AMIRdesigner. These genes were synthesized with the server-designed oligos and further assembled into a quadruple miRNA cluster, which was cloned into an overexpression vector and transformed into potato plants. Small RNA Northern blot and virus inoculation analyses showed that a high level of artificial miRNA expression and good viral resistance were achieved in some of the transgenic lines. These results demonstrate the utility of our webserver AMIRdesigner for engineering crop viral resistance.

First report of Cucurbit aphid-borne yellows virus causing yellowing disease on pumpkin (Cucurbita pepo) and cucumber (Cucumis sativus) in Bulgaria.

Cucurbit aphid-borne yellows virus (CABYV) was first reported in France in 1992 but has since been observed worldwide (Lecoq et al. 1992; Choi and Choi 2016; Buzkan et al. 2017; Zindovic et al. 2017; Vidal et al. 2018; Khanal and Ali, 2018). This virus has caused severe losses to different crops especially to the members of Cucurbitaceae and yield losses can reach up to 40-50% if infection occurs at early stages (Lecoq et al. 1992). In July 2017, leaf samples showing virus-like symptoms were collected from five pumpkin (Cucurbita pepo L. var. Clypeata Alefield) and two cucumber (Cucumis sativus L. cv. Azuma matsunari) plants, growing in а field near Sadovo, Bulgaria. Nearly all plants in the field were affected and displayed green or yellow mosaic, interveinal yellowing, blisters, and leaf deformation (Fig. 1). The collected samples were all symptomatic and were subjected to double antibody sandwich (DAS) or triple antibody sandwich (TAS) enzyme-linked immunosorbent assay (ELISA) to determine the viral agent(s). Specific monoclonal antibodies (Leibniz institute DSMZ, Germany) raised against Cucumber leaf spot virus, Cucurbit chlorotic yellows virus, Cucurbit yellow stunting disorder virus, Cucumber mosaic virus (CMV), Melon necrotic spot virus, Beet western yellows virus (BWYV), CABYV, Watermelon mosaic virus (WMV), Zucchini yellow mosaic virus (ZYMV), and Cucumber green mottle mosaic virus, were used. The total number of tested samples was seven (n=5 from pumpkin and n=2 from cucumber). All of them displayed positive signals for CABYV and BWYV, both belonging to genus Polerovirus, family Luteoviridae. In addition, ZYMV and/or WMV were detected in pumpkins while CMV and/or WMV were detected in cucumber samples, respectively. To confirm the presence of CABYV and/or BWYV, total RNA was isolated from all seven samples by TRI Reagent® (Sigma, St. Louis, USA) and converted to cDNA with First Strand cDNA Synthesis Kit, Thermo Scientific™. Reverse transcription (RT)-polyemerase chain reaction (PCR) was performed using two pairs of primers (CABYV1FW: 5'-TTATCAGGGGACTATGTTTA-3' and CABYV14REV: 5'-GAGGGGATTTTAACTGACTG-3', and BWYV1FW: 5'-AGTAAGTCCTCCCCAACTGA-3' and BWYV2REV: 5'-CTACCCACGACCGTATTCAT-3'), specifically designed to detect CABYV and BWYV, respectively. Amplicons with expected sizes of 1,930 bp were obtained only with CABYV primers for all samples while no fragments were amplified with BWYV primers. The obtained products from two samples (pumpkin and cucumber) were purified and sent to Macrogen Inc., South Korea, for direct sequencing in both directions. High quality nucleotide sequences were submitted to GenBank We have evaluated the quality of the sequencing and trimmed those parts that did not comply the needed quality. The obtained smaller fragments Nucleotide sequences were submitted to GenBank with accession numbers MK671010 (656 bp) and MK671014 (712bp). These sequences contained ORFs encoding CABYV P1-P2 fusion proteins as determined by Blastp analysis (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE=Proteins). A phylogenetic tree constructed by the Neighbor-joining method using 18 CABYV accessions and Potato leafroll virus as an outlier (Fig. 2) showed that the closest accessions to MK671010 and MK671014 were NC003688 (France) and EU636992 (China) with respective nucleotide identity of 98% and 99%. In 2019, another outbreak was observed in the same field near Sadovo and in a field near Plovdiv planted with pumpkins. Nearly 30% of the plants showed leaf yellowing typical for Polerovirus infection. Screening of collected samples (n=17) by RT-PCR confirmed CABYV presence in 15 samples. Based on available reports and according to our knowledge this is the first report of CABYV in Bulgaria.