Occurrence and characterization of viruses infecting Amorphophallus in Yunnan, China.

Viral diseases are becoming an important problem in Amorphophallus production due to the propagation of seed corms and their trade across regions. In this study, combined-High-Throughput Sequencing, RT-PCR, electron microscopy, and mechanical inoculation were used to analyze virus-like infected Amorphophallus samples in Yunnan province to investigate the distribution, molecular characterization, and diversity and evolution of Amorphophallus-infecting viruses including three isolates of dasheen mosaic virus and three orthotospoviruses: mulberry vein banding associated virus (MVBaV), tomato zonate spot virus (TZSV) and impatiens necrotic spot virus (INSV). The results showed that DsMV is the dominant virus infecting Amorphophallus, mixed infections with DsMV and MVBaV to Amorphophallus were quite common in Yunnan province, China. This is the first report on infection of Amorphophallus with MVBaV, TZSV, and impatiens necrotic spot virus (INSV) in China. This work will help to develop an effective integrated management strategy to control the spread of Amorphophallus viral diseases.

Diagnosis and Characterization of Plant Viruses Using HTS to Support Virus Management and Tomato Breeding.

Viral diseases pose a significant threat to tomato crops (Solanum lycopersicum L.), one of the world's most economically important vegetable crops. The limited genetic diversity of cultivated tomatoes contributes to their high susceptibility to viral infections. To address this challenge, tomato breeding programs must harness the genetic resources found in native populations and wild relatives. Breeding efforts may aim to develop broad-spectrum resistance against the virome. To identify the viruses naturally infecting 19 advanced lines, derived from native tomatoes, high-throughput sequencing (HTS) of small RNAs and confirmation with PCR and RT-PCR were used. Single and mixed infections with tomato mosaic virus (ToMV), tomato golden mosaic virus (ToGMoV), and pepper huasteco yellow vein virus (PHYVV) were detected. The complete consensus genomes of three variants of Mexican ToMV isolates were reconstructed, potentially forming a new ToMV clade with a distinct 3' UTR. The absence of reported mutations associated with resistance-breaking to ToMV suggests that the Tm-1, Tm-2, and Tm-22 genes could theoretically be used to confer resistance. However, the high mutation rates and a 63 nucleotide insertion in the 3' UTR, as well as amino acid mutations in the ORFs encoding 126 KDa, 183 KDa, and MP of Mexican ToMV isolates, suggest that it is necessary to evaluate the capacity of these variants to overcome Tm-1, Tm-2, and Tm-22 resistance genes. This evaluation, along with the characterization of advanced lines using molecular markers linked to these resistant genes, will be addressed in future studies as part of the breeding strategy. This study emphasizes the importance of using HTS for accurate identification and characterization of plant viruses that naturally infect tomato germplasm based on the consensus genome sequences. This study provides crucial insights to select appropriate disease management strategies and resistance genes and guide breeding efforts toward the development of virus-resistant tomato varieties.

Co-Infection of Tobacco Rattle and Cycas Necrotic Stunt Viruses in Paeonia lactiflora: Detection Strategies, Potential Origins of Infection, and Implications for Paeonia Germplasm Conservation.

Increasing reports of tobacco rattle virus (TRV) and cycas necrotic stunt virus (CNSV) in herbaceous Paeonia worldwide highlight the importance of conserving the genetic resources of this economically important ornamental and medicinal crop. The unknown origin(s) of infection, differential susceptibility of peony cultivars to these viruses, and elusive disease phenotypes for CNSV in peonies make early detection and management challenging. Here, we report the presence of TRV and CNSV in plants of the University of Michigan living peony collection in the United States and a molecular characterization of their strains. Using sequences of the TRV 194 K RNA polymerase gene, we confirmed TRV infections in seven symptomatic plants (1.07% of all plants in the collection). Using newly developed primers, we recovered sequences of the CNSV RdRp gene and the polyprotein 1 gene region from nine out of twelve samples analyzed, including three from symptomless plants. Four of the nine plants had TRV and CNSV co-infections and showed more severe disease symptoms than plants only infected with TRV. Phylogenetic analyses of isolates from the University of Michigan living peony collection and publicly available isolates point to multiple origins of TRV and CNSV infections in this collection. This is the first report of TRV/CNSV co-infection and of a symptomatic detection of CNSV on cultivated P. lactiflora.

Exploring the viral landscape of saffron through metatranscriptomic analysis.

Saffron (Crocus sativus L.), a historically significant crop valued for its nutraceutical properties, has been poorly explored from a phytosanitary perspective. This study conducted a thorough examination of viruses affecting saffron samples from Spanish cultivars, using high-throughput sequencing alongside a systematic survey of transcriptomic datasets from Crocus sativus at the Sequence Read Archive. Our analysis unveiled a broad diversity and abundance, identifying 17 viruses across the 52 analyzed libraries, some of which were highly prevalent. This includes known saffron-infecting viruses and previously unreported ones. In addition, we discovered 7 novel viruses from the Alphaflexiviridae, Betaflexiviridae, Potyviridae, Solemoviridae, and Geminiviridae families, with some present in libraries from various locations. These findings indicate that the saffron-associated virome is more complex than previously reported, emphasizing the potential of phytosanitary analysis to enhance saffron productivity.

Development of TaqMan RT-qPCR for the detection of regulated citrus viruses and viroids in Aotearoa New Zealand.

The major citrus species include several economically important fruits, such as orange, mandarin, lemon, limes, grapefruit and pomelos. Since the 1980 s, total production and consumption of citrus has grown strongly with the current annual worldwide production at over 105 million tonnes. New Zealand's citrus exports, for instance, had an estimated worth of NZ$ 11.6 million (approx. US$ 7 million) in 2020. Citrus plants are prone to viral diseases, which can lead to substantial economic losses. In New Zealand, the citrus Import Health Standard (IHS) has identified 22 viruses and viroids that are subject to regulation and requires citrus nursery stock to be free of these pathogens. As such, there is a need for reliable, sensitive, and rapid detection methods to screen for these viruses and viroids during post entry quarantine. In this study, we developed TaqMan RT-qPCR assays for the detection of nine of these regulated viruses and viroids, namely citrus leaf rugose virus (CiLRV), citrus leprosis virus C (CiLV-C), citrus leprosis virus C2 (CiLV-C2), citrus leprosis virus N (CiLV-N), citrus psorosis virus (CPsV), citrus yellow mosaic virus (CYMV), citrus bent leaf viroid (CBLVd), citrus viroid V (CVd-V), and citrus viroid VI (CVd-VI). These assays have been validated and found to be highly sensitive, specific, and reliable. The implementation of these assays will facilitate the safe importation of citrus nursery stock, thus safeguarding the country's horticultural and economic interests.

Molecular Methods for the Simultaneous Detection of Tomato Fruit Blotch Virus and Identification of Tomato Russet Mite, a New Potential Virus-Vector System Threatening Solanaceous Crops Worldwide.

Tomato fruit blotch virus (ToFBV) (Blunervirus solani, family Kitaviridae) was firstly identified in Italy in 2018 in tomato plants that showed the uneven, blotchy ripening and dimpling of fruits. Subsequent High-Throughput Sequencing (HTS) analysis allowed ToFBV to be identified in samples collected in Australia, Brazil, and several European countries, and its presence in tomato crops was dated back to 2012. In 2023, the virus was found to be associated with two outbreaks in Italy and Belgium, and it was included in the EPPO Alert list as a potential new threat for tomato fruit production. Many epidemiologic features of ToFBV need to be still clarified, including transmission. Aculops lycopersici Massee (Acariformes: Eriophyoidea), the tomato russet mite (TRM), is a likely candidate vector, since high population densities were found in most of the ToFBV-infected tomato cultivations worldwide. Real-time RT-PCR tests for ToFBV detection and TRM identification were developed, also as a duplex assay. The optimized tests were then transferred to an RT-ddPCR assay and validated according to the EPPO Standard PM 7/98 (5). Such sensitive, reliable, and validated tests provide an important diagnostic tool in view of the probable threat posed by this virus-vector system to solanaceous crops worldwide and can contribute to epidemiological studies by simplifying the efficiency of research. To our knowledge, these are the first molecular methods developed for the simultaneous detection and identification of ToFBV and TRM.

Isothermal Nucleic Acid Amplification-Based Lateral Flow Testing for the Detection of Plant Viruses.

Isothermal nucleic acid amplification-based lateral flow testing (INAA-LFT) has emerged as a robust technique for on-site pathogen detection, providing a visible indication of pathogen nucleic acid amplification that rivals or even surpasses the sensitivity of real-time quantitative PCR. The isothermal nature of INAA-LFT ensures consistent conditions for nucleic acid amplification, establishing it as a crucial technology for rapid on-site pathogen detection. However, despite its considerable promise, the widespread application of isothermal INAA amplification-based lateral flow testing faces several challenges. This review provides an overview of the INAA-LFT procedure, highlighting its advancements in detecting plant viruses. Moreover, the review underscores the imperative of addressing the existing limitations and emphasizes ongoing research efforts dedicated to enhancing the applicability and performance of this technology in the realm of rapid on-site testing.

Development and Application of Attenuated Plant Viruses as Biological Control Agents in Japan.

In 1929, it was reported that yellowing symptoms caused by a tobacco mosaic virus (TMV) yellow mosaic isolate were suppressed in tobacco plants that were systemically infected with a TMV light green isolate. Similar to vaccination, the phenomenon of cross-protection involves a whole plant being infected with an attenuated virus and involves the same or a closely related virus species. Therefore, attenuated viruses function as biological control agents. In Japan, many studies have been performed on cross-protection. For example, the tomato mosaic virus (ToMV)-L11A strain is an attenuated isolate developed by researchers and shows high control efficiency against wild-type ToMV in commercial tomato crops. Recently, an attenuated isolate of zucchini yellow mosaic virus (ZYMV)-2002 was developed and registered as a biological pesticide to control cucumber mosaic disease. In addition, attenuated isolates of pepper mild mottle virus (PMMoV), cucumber mosaic virus (CMV), tobacco mild green mosaic virus (TMGMV), melon yellow spot virus (MYSV), and watermelon mosaic virus (WMV) have been developed in Japan. These attenuated viruses, sometimes called plant vaccines, can be used not only as single vaccines but also as multiple vaccines. In this review, we provide an overview of studies on attenuated plant viruses developed in Japan. We also discuss the application of the attenuated strains, including the production of vaccinated seedlings.

Lavender Harbors More Viruses than Previously Thought: First Report of Raspberry Ringspot Virus and Phlox Virus M in Lavandula × intermedia.

Plants of the genus Lavandula are thought to be rarely infected by viruses. To date, only alfalfa mosaic virus, cucumber mosaic virus, tobacco mosaic virus, and tomato spotted wilt virus have been reported in this host. In this study, we identified for the first time raspberry ringspot virus (RpRSV) and phlox virus M (PhlVM) in lavender using herbaceous indexing, enzyme-linked immunosorbent assay, and high-throughput sequencing. Nearly complete genome sequences for both viruses were determined. Phylogenetic and serological characterizations suggest that the obtained RpRSV isolate is a raspberry strain. A preliminary survey of 166 samples indicated RpRSV was spread only in the lavender cultivar 'Grosso', while PhlVM was detected in multiple lavender cultivars. Although RpRSV raspberry strain may have spread throughout Auckland and nearby areas in New Zealand, it is very likely restricted to the genus Lavandula or even to the cultivar 'Grosso' due to the absence or limited occurrence of the nematode vector. Interestingly, all infected lavender plants, regardless of their infection status (by RpRSV, PhlVM, or both) were asymptomatic. RpRSV is an important virus that infects horticultural crops including grapevine, cherry, berry fruits, and rose. It remains on the list of regulated pests in New Zealand. RpRSV testing is mandatory for imported Fragaria, Prunus, Ribes, Rosa, Rubus, and Vitis nursery stock and seeds for sowing, while this is not required for Lavandula importation. Our study revealed that lavender could play a role not only as a reservoir but also as an uncontrolled import pathway of viruses that pose a threat to New Zealand's primary industries.

Development of the differential detection reverse transcription loop-mediated isothermal amplification method for Barley yellow mosaic virus or Japanese soil-borne wheat mosaic virus from barley.

A differential detection reverse transcription loop-mediated isothermal amplification (DD-RT-LAMP) method was developed to detect either Barley yellow mosaic virus (BaYMV) or Japanese soil-borne wheat mosaic virus (JSBWMV) simultaneously. Both primer sets, which recognized either BaYMV or JSBWMV genomic RNA, amplified DNA more efficiently at 65°C using an isothermal DNA amplification and fluorescence detection device. Furthermore, these primer sets showed unique annealing curves. The peak annealing temperatures of BaYMV and JSBWMV amplification products using specific primer sets were 86.9°C-87.7°C and 84.5°C-85.0°C, respectively, and were clearly distinguishable during an annealing step following the isothermal amplification, monitored using a fluorescence detection device. In the field samples of barley (Hordeum vulgare L.) tested, BaYMV or JSBWMV were detected by DD-RT-LAMP, and the detection results of DD-RT-LAMP were correspondent with the results of reverse transcription-PCR.

One-Enzyme RTX-PCR for the Detection of RNA Viruses from Multiple Virus Genera and Crop Plants.

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.

Viruses Infecting Greenhood Orchids (Pterostylidinae) in Eastern Australia.

The Australasian biogeographic realm is a major centre of diversity for orchids, with every subfamily of the Orchidaceae represented and high levels of endemism at the species rank. It is hypothesised that there is a commensurate diversity of viruses infecting this group of plants. In this study, we have utilised high-throughput sequencing to survey for viruses infecting greenhood orchids (Pterostylidinae) in New South Wales and the Australian Capital Territory. The main aim of this study was to characterise Pterostylis blotch virus (PtBV), a previously reported but uncharacterised virus that had been tentatively classified in the genus Orthotospovirus. This classification was confirmed by genome sequencing, and phylogenetic analyses suggested that PtBV is representative of a new species that is possibly indigenous to Australia as it does not belong to either the American or Eurasian clades of orthotospoviruses. Apart from PtBV, putative new viruses in the genera Alphaendornavirus, Amalgavirus, Polerovirus and Totivirus were discovered, and complete genome sequences were obtained for each virus. It is concluded that the polerovirus is likely an example of an introduced virus infecting a native plant species in its natural habitat, as this virus is probably vectored by an aphid, and Australia has a depauperate native aphid fauna that does not include any species that are host-adapted to orchids.

Development of a one-step RT-qPCR detection assay for the newly described citrus viroid VII.

An apscaviroid, tentatively named citrus viroid VII (CVd-VII), was recently discovered in citrus in Australia. A diagnostic assay using real-time reverse transcription polymerase chain reaction was developed and validated to detect the viroid in citrus plants. The assay showed a high level of sensitivity, reliably detecting 2000 plasmid copies per reaction, while down to 20 plasmid copies per reaction were occasionally detected. The assay showed high specificity, producing no false positives or cross-reactivity with a range of other citrus graft-transmissible pathogens, including viroids, viruses and bacteria. The real-time assay was also found to be more sensitive than the available end-point reverse transcription polymerase chain reaction assay by a factor of 100,000 and could be a useful tool for the rapid detection of CVd-VII in diagnostic and research environments.

Polymer monoliths for the concentration of viruses from environmental waters: A review.

Even at low concentrations in environmental waters, some viruses are highly infective, making them a threat to human health. They are the leading cause of waterborne enteric diseases. In agriculture, plant viruses in irrigation and runoff water threat the crops. The low concentrations pose a challenge to early contamination detection. Thus, concentrating the virus particles into a small volume may be mandatory to achieve reliable detection in molecular techniques. This paper reviews the organic monoliths developments and their applications to concentrate virus particles from waters (waste, surface, tap, sea, and irrigation waters). Free-radical polymerization and polyaddition reactions are the most common strategies to prepare the monoliths currently used for virus concentration. Here, the routes for preparing and functionalizing both methacrylate and epoxy-based monoliths will be shortly described, following a revision of their retention mechanisms and applications in the concentration of enteric and plant viruses in several kinds of waters.

High-Throughput Sequencing of Small RNAs for Diagnostics of Grapevine Viruses and Viroids in Russia.

The use of high-throughput sequencing (HTS) technology has led to significant progress in the identification of many viruses and their genetic variants. In this study, we used the HTS platform to sequence small RNAs (sRNAs) of grapevine to study the virome. Isolation of RNA was performed using symptomatic grapevines collected from commercial vineyards in Krasnodar Krai in 2017-2018. To determine the viromes of vineyards, we used an integrated approach that included a bioinformatic analysis of the results of sRNA HTS and the molecular method RT-PCR, which made it possible to identify 13 viruses and 4 viroids. Grapevine leafroll-associated virus 4 (GLRaV-4), Grapevine Syrah Virus-1 (GSyV-1), Raspberry bushy dwarf virus (RBDV), Australian grapevine viroid (AGVd), and Grapevine yellow speckle viroid 2 (GYSVd-2) were identified for the first time in Russia. Out of 38 samples analyzed, 37 had mixed infections with 4-11 viruses, indicating a high viral load. Analysis of the obtained sequences of fragments of virus genomes made it possible to identify recombination events in GLRaV-1, GLRaV-2, GLRaV-3, GLRaV-4, GVT, GPGV, GRSPaV, GVA, and GFLV. The obtained results indicate a wide spread of the viruses and a high genetic diversity in the vineyards of Krasnodar Krai and emphasize the urgent need to develop and implement long-term strategies for the control of viral grapevine diseases.

Identification of Cacao Mild Mosaic Virus (CaMMV) and Cacao Yellow Vein-Banding Virus (CYVBV) in Cocoa (Theobroma cacao) Germplasm.

Cocoa, Theobroma cacao, is an important tropical perennial crop grown widely in the humid tropics. The exchange of cocoa germplasm between germplasm collections and breeding centres is vital for varietal development. Intermediate quarantine facilities, such as the International Cocoa Quarantine Centre, Reading UK (ICQC-R) play a vital role in ensuring the transfer of germplasm whilst minimising the risk of spreading pests and diseases. Current screening procedures combine visual inspection and molecular techniques, which are effective in detecting Cocoa swollen shoot virus (CSSV), a badnavirus, which causes severe losses but are restricted to West Africa. However, the detection of latent or mild virus infections that produce no visual symptoms has been a challenge. Recently two badnavirus species of cocoa producing mild symptoms, cacao mild mosaic virus (CaMMV) and cacao yellow vein-banding virus (CYVBV), have been sequenced. Here, we report new assays for the detection of these two species, for the first time in non-symptomatic accessions. Evolutionary and bioinformatic analyses of the viruses suggest their most recent source was from Trinidad, though there is historic evidence that these viruses may have their origin in South America and then become widespread globally over the last century. We also report a novel colorimetric Loop-mediated isothermal amplification (LAMP) assay for the detection of CYVBV. This simple and accurate method could be employed in field virus testing.

Characterization of Spanish Olive Virome by High Throughput Sequencing Opens New Insights and Uncertainties.

The use of high throughput sequencing (HTS) for the analysis of Spanish olive trees showing leaf yellowing discoloration, defoliation, and/or decline has provided new insights into the olive viruses present in Spain and has opened discussions about the pros and cons of these technologies for diagnostic purposes. In this study, we report for the first time in Spanish orchards the presence of olive leaf yellowing-associated virus (OLYaV), for which the second full coding sequence has been determined. This virus has also been detected in a putative vector, the psyllid Euphyllura olivina. In addition, the presence in Spain of Olea europaea geminivirus (OEGV), recently reported in Italy, has been confirmed, and the full-length sequence of two isolates was obtained by HTS and Sanger sequencing. These results, as well as the detection of other viral sequences related to olive latent virus 3 (OLV-3) and olive viral satellite RNA, raises questions on the biological significance of the findings, about the requirement of standardization on the interpretation of HTS results, and the necessity of additional tests to confirm the relevance of the HTS detection of viral sequences.

New Resources for the Specific and Sensitive Detection of the Emerging Tomato Brown Rugose Fruit Virus.

Plant viruses can evolve towards new pathogenic entities that may eventually cause outbreaks and become epidemics or even pandemics. Seven years ago, tomato brown rugose fruit virus (ToBRFV) emerged, overcoming the genetic resistance that had been employed for more than sixty years against tobamoviruses in tomato. Since then, ToBRFV has spread worldwide, producing significant losses in tomato crops. While new resistances are deployed, the only means of control is the implementation of effective prevention and eradication strategies. For this purpose, in this work, we have designed, assessed, and compared an array of tests for the specific and sensitive detection of the ToBRFV in leaf samples. First, two monoclonal antibodies were generated against a singular peptide of the ToBRFV coat protein; antibodies were utilized to devise a double-antibody-sandwich enzyme-linked immunosorbent assay (DAS-ELISA) test that sensitively detects this virus and has no cross-reactivity with other related tobamoviruses. Second, a real-time quantitative PCR (RT-qPCR) test targeting the RNA-dependent replicase open reading frame (ORF) was designed, and its performance and specificity validated in comparison with the CaTa28 and CSP1325 tests recommended by plant protection authorities in Europe. Third, in line with the tendency to use field-deployable diagnostic techniques, we developed and tested two sets of loop-mediated isothermal amplification (LAMP) primers to double-check the detection of the movement protein ORF of ToBRFV, and one set that works as an internal control. Finally, we compared all of these methods by employing a collection of samples with different ToBRFV loads to evaluate the overall performance of each test.

Direct Metatranscriptomic Survey of the Sunflower Microbiome and Virome.

Sunflowers (Helianthus annuus L.) are susceptible to multiple diseases in field production. In this study, we collected diseased sunflower leaves in fields located in South Dakota, USA, for virome investigation. The leaves showed visible symptoms on the foliage, indicating phomopsis and rust infections. To identify the viruses potentially associated with the disease diagnosed, symptomatic leaves were obtained from diseased plants. Total RNA was extracted corresponding to each disease diagnosed to generate libraries for paired-end high throughput sequencing. Short sequencing reads were assembled de novo and the contigs with similarities to viruses were identified by aligning against a custom protein database. We report the discovery of two novel mitoviruses, four novel partitiviruses, one novel victorivirus, and nine novel totiviruses based on similarities to RNA-dependent RNA polymerases and capsid proteins. Contigs similar to bean yellow mosaic virus and Sclerotinia sclerotiorum hypovirulence-associated DNA virus were also detected. To the best of our knowledge, this is the first report of direct metatranscriptomics discovery of viruses associated with fungal infections of sunflowers bypassing culturing. These newly discovered viruses represent a natural genetic resource from which we can further develop potential biopesticide to control sunflower diseases.

Application of Oxford Nanopore Technology to Plant Virus Detection.

The adoption of Oxford Nanopore Technologies (ONT) sequencing as a tool in plant virology has been relatively slow despite its promise in more recent years to yield large quantities of long nucleotide sequences in real time without the need for prior amplification. The portability of the MinION and Flongle platforms combined with lowering costs and continued improvements in read accuracy make ONT an attractive method for both low- and high-scale virus diagnostics. Here, we provide a detailed step-by-step protocol using the ONT Flongle platform that we have developed for the routine application on a range of symptomatic post-entry quarantine and domestic surveillance plant samples. The aim of this methods paper is to highlight ONT's feasibility as a valuable component to the diagnostician's toolkit and to hopefully stimulate other laboratories towards the eventual goal of integrating high-throughput sequencing technologies as validated plant virus diagnostic methods in their own right.