Potato virus Y (PVY) is the most economically important virus infecting cultivated potato (Solanum tuberosum L.). Accurate diagnosis is crucial to regulate the trade of tubers and for the sanitary selection of plant material for propagation. However, high genetic diversity of PVY represents a challenge for the detection and classification of isolates. Here, the diversity of Irish PVY isolates from a germplasm collection and commercial sites was investigated using conventional molecular and serological techniques. Recombinant PVY isolates were prevalent, with PVYNTNa being the predominant genotype. In addition, we evaluated Nanopore sequencing to detect and reconstruct the whole genome sequence of four viruses (PVY, PVX, PVS, PLRV) and five PVY genotypes in a subset of eight potato plants. De novo assembly of Nanopore sequencing reads produced single contigs covering greater than 90% of the viral genome and sharing greater than 99.5% identity to the consensus sequences obtained with Illumina sequencing. Interestingly, single near full genome contigs were obtained for different isolates of PVY co-infecting the same plant. Mapping reads to available reference viral genomes enabled us to generate near complete genome sequences sharing greater than 99.90% identity to the Illumina-derived consensus. This is the first report describing the use of Oxford Nanopore's MinION to detect and genotype potato viruses. We reconstructed the genome of PVY and other RNA viruses; indicating the technologies potential for virus detection in potato production systems, and for the study of genetic diversity of highly heterogeneous viruses such as PVY.

https://www.mdpi.com/1999-4915/12/4/478/pdf

Characterization of Potato Virus Y Isolates and Assessment of Nanopore Sequencing to Detect and Genotype Potato Viruses.

Plant viral diseases are the foremost threat to sustainable agriculture, leading to several billion dollars in losses every year. Many viruses infecting several crops have been described in the literature; however, new infectious viruses are emerging frequently through outbreaks. For the effective treatment and prevention of viral diseases, there is great demand for new techniques that can provide accurate identification on the causative agents. With the advancements in biochemical and molecular biology techniques, several diagnostic methods with improved sensitivity and specificity for the detection of prevalent and/or unknown plant viruses are being continuously developed. Currently, serological and nucleic acid methods are the most widely used for plant viral diagnosis. Nucleic acid-based techniques that amplify target DNA/RNA have been evolved with many variants. However, there is growing interest in developing techniques that can be based in real-time and thus facilitate in-field diagnosis. Next-generation sequencing (NGS)-based innovative methods have shown great potential to detect multiple viruses simultaneously; however, such techniques are in the preliminary stages in plant viral disease diagnostics. This review discusses the recent progress in the use of NGS-based techniques for the detection, diagnosis, and identification of plant viral diseases. New portable devices and technologies that could provide real-time analyses in a relatively short period of time are prime important for in-field diagnostics. Current development and application of such tools and techniques along with their potential limitations in plant virology are likewise discussed in detail.

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Current Developments and Challenges in Plant Viral Diagnostics: A Systematic Review

High throughput sequencing (HTS) has revolutionised virus detection and discovery, allowing for the untargeted characterisation of whole viromes. Viral metagenomics studies have demonstrated the ubiquity of virus infection - often in the absence of disease symptoms - and tend to discover many novel viruses, highlighting the small fraction of virus biodiversity described to date. The majority of the studies using high-throughput sequencing to characterise plant viromes have focused on economically important crops, and only a small number of studies have considered weeds and wild plants. Characterising the viromes of wild plants is highly relevant, as these plants can affect disease dynamics in crops, often by acting as viral reservoirs. Moreover, the viruses in unmanaged systems may also have important effects on wild plant populations and communities. Here, we review metagenomic studies on weeds and wild plants to show the benefits and limitations of this approach and identify knowledge gaps. We consider key genomics developments that are likely to benefit the field in the near future. Although only a small number of HTS studies have been performed on weeds and wild plants, these studies have already discovered many novel viruses, demonstrated unexpected trends in virus distributions, and highlighted the potential of metagenomics as an approach.

Metagenomic Studies of Viruses in Weeds and Wild Plants: A Powerful Approach to Characterise Variable Virus Communities.

Turfgrass is a multibillion-dollar industry severely affected by plant pathogens including fungi, bacteria, viruses, and nematodes. Many of the diseases in turfgrass have similar signs and symptoms, making it difficult to diagnose the specific problem pathogen. Incorrect diagnosis leads to the delay of treatment and excessive use of chemicals. To effectively control these diseases, it is important to have rapid and accurate detection systems in the early stages of infection that harbor relatively low pathogen populations. There are many methods for diagnosing pathogens on turfgrass. Traditional methods include symptoms, morphology, and microscopy identification. These have been followed by nucleic acid detection and onsite detection techniques. Many of these methods allow for rapid diagnosis, some even within the field without much expertise. There are several methods that have great potential, such as high-throughput sequencing and remote sensing. Utilization of these techniques for disease diagnosis allows for faster and accurate disease diagnosis and a reduction in damage and cost of control. Understanding of each of these techniques can allow researchers to select which method is best suited for their pathogen of interest. The objective of this article is to provide an overview of the turfgrass diagnostics efforts used and highlight prospects for disease detection.

Turfgrass Disease Diagnosis: Past, Present, and Future.

In recent years, next-generation sequencing (NGS) and contemporary Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR-associated (Cas) technologies have revolutionized the life sciences and the field of plant virology. Both these technologies offer an unparalleled platform for sequencing and deciphering viral metagenomes promptly. Over the past two decades, NGS technologies have improved enormously and have impacted plant virology. NGS has enabled the detection of plant viruses that were previously undetectable by conventional approaches, such as quarantine and archeological plant samples, and has helped to track the evolutionary footprints of viral pathogens. The CRISPR-Cas-based genome editing (GE) and detection techniques have enabled the development of effective approaches to virus resistance. Different versions of CRISPR-Cas have been employed to successfully confer resistance against diverse plant viruses by directly targeting the virus genome or indirectly editing certain host susceptibility factors. Applications of CRISPR-Cas systems include targeted insertion and/or deletion, site-directed mutagenesis, induction/expression/repression of the gene(s), epigenome re-modeling, and SNPs detection. The CRISPR-Cas toolbox has been equipped with precision GE tools to engineer the target genome with and without double-stranded (ds) breaks or donor templates. This technique has also enabled the generation of transgene-free genetically engineered plants, DNA repair, base substitution, prime editing, detection of small molecules, and biosensing in plant virology. This review discusses the utilities, advantages, applications, bottlenecks of NGS, and CRISPR-Cas in plant virology.

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Next-Generation Sequencing and the CRISPR-Cas Nexus: A Molecular Plant Virology Perspective.

Viral diseases are a limiting factor to wheat production. Viruses are difficult to diagnose in the early stages of disease development and are often confused with nutrient deficiencies or other abiotic problems. Immunological methods are useful to identify viruses, but specific antibodies may not be available or require high virus titer for detection. In 2015 and 2017, wheat plants containing Wheat streak mosaic virus (WSMV) resistance gene, Wsm2, were found to have symptoms characteristic of WSMV. Serologically, WSMV was detected in all four samples. Additionally, High Plains wheat mosaic virus (HPWMoV) was also detected in one of the samples. Barley yellow dwarf virus (BYDV) was not detected, and a detection kit was not readily available for Triticum mosaic virus (TriMV). Initially, cDNA cloning and Sanger sequencing were used to determine the presence of WSMV; however, the process was time-consuming and expensive. Subsequently, cDNA from infected wheat tissue was sequenced with single-strand, Oxford Nanopore sequencing technology (ONT). ONT was able to confirm the presence of WSMV. Additionally, TriMV was found in all of the samples and BYDV in three of the samples. Deep coverage sequencing of full-length, single-strand WSMV revealed variation compared with the WSMV Sidney-81 reference strain and may represent new variants which overcome Wsm2. These results demonstrate that ONT can more accurately identify causal virus agents and has sufficient resolution to provide evidence of causal variants.

https://apsjournals.apsnet.org/doi/pdf/10.1094/PDIS-09-18-1700-RE

Christian Webb

Papers

Wheat Virus Identification Within Infected Tissue Using Nanopore Sequencing Technology