Wheat Virus Identification Within Infected Tissue Using Nanopore Sequencing Technology
TL;DR: CDNA from infected wheat tissue was sequenced with single-strand, Oxford Nanopore sequencing technology (ONT), and ONT was able to confirm the presence of WSMV, demonstrating that ONT can more accurately identify causal virus agents and has sufficient resolution to provide evidence of causal variants.
Abstract: 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.
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TL;DR: This is the first report describing the use of Oxford Nanopore’s MinION to detect and genotype potato viruses and 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.
Abstract: 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.
41 citations
Cites methods from "Wheat Virus Identification Within I..."
...For this reason, several other methods for the preparation of cDNA libraries, including random RNA priming and the use of second strand cDNA synthesis kits [66] or using whole-transcriptome amplification kits [70] have also been described....
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TL;DR: This review discusses the recent progress in the use of NGS-based techniques for the detection, diagnosis, and identification of plant viral diseases.
Abstract: 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.
41 citations
Cites background from "Wheat Virus Identification Within I..."
...The result of the study showed that nanopore technology can more accurately identify causal virus agents and has sufficient resolution to provide evidence of causal variants [184]....
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...Wheat streak mosaic virus (WSMV) Wheat plant Wheat streak mosaic virus identified [184]...
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TL;DR: A review of metagenomics studies on weeds and wild plants to show the benefits and limitations of this approach and identify knowledge gaps is presented in this paper, where the authors consider key genomics developments that are likely to benefit the field in the near future.
Abstract: 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.
16 citations
11 Nov 2020
TL;DR: An overview of the turfgrass diagnostics efforts used and prospects for disease detection is provided to provide a faster and accurate disease diagnosis and a reduction in damage and cost of control.
Abstract: 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.
13 citations
TL;DR: The utilities, advantages, applications, bottlenecks of NGS, and CRISPR-Cas in plant virology are discussed.
Abstract: 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.
10 citations
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TL;DR: A novel, pocket-sized nanopore sequencer is used at a field diagnostic laboratory in Liberia during the current Ebola virus outbreak to perform rapid sequencing of RNA/DNA from pathogen samples obtained during disease outbreaks.
Abstract: Rapid sequencing of RNA/DNA from pathogen samples obtained during disease outbreaks provides critical scientific and public health information. However, challenges exist for exporting samples to laboratories or establishing conventional sequencers in remote outbreak regions. We successfully used a novel, pocket-sized nanopore sequencer at a field diagnostic laboratory in Liberia during the current Ebola virus outbreak.
172 citations
TL;DR: Phylogenetic analyses of the complete polyprotein, NIa-Pro, NIb, and coat protein sequences of representative species of six genera and unassigned members of the family Potyviridae suggested that TriMV and SCSMV are sister taxa and share a most recent common ancestor with tritimoviruses or ipomoviruses, and the genus Poacevirus is proposed as the type member.
Abstract: Tatineni, S., Ziems, A. D., Wegulo, S. N., and French, R. 2009. Triticum mosaic virus: A distinct member of the family Potyviridae with an unusually long leader sequence. Phytopathology 99:943-950. The complete genome sequence of Triticum mosaic virus (TriMV), a member in the family Potyviridae, has been determined to be 10,266 nucleotides (nt) excluding the 3′ polyadenylated tail. The genome encodes a large polyprotein of 3,112 amino acids with the “hall-mark proteins” of potyviruses, including a small overlapping gene, PIPO, in the P3 cistron. The genome of TriMV has an unusually long 5′ nontranslated region of 739 nt with 12 translation initiation codons and three small open reading frames, which resemble those of the internal ribosome entry site containing 5′ leader sequences of the members of Picornaviridae. Pairwise comparison of 10 putative mature proteins of TriMV with those of representative members of genera in the family Potyviridae revealed 33 to 44% amino acid identity within the highly conserved NIb protein sequence and 15 to 29% amino acid identity within the least conserved P1 protein, suggesting that TriMV is a distinct member in the family Potyviridae. In contrast, TriMV displayed 47 to 65% amino acid sequence identity with available sequences of mature proteins of Sugarcane streak mosaic virus (SCSMV), an unassigned member of the Potyviridae. Phylogenetic analyses of the complete polyprotein, NIa-Pro, NIb, and coat protein sequences of representative species of six genera and unassigned members of the family Potyviridae suggested that TriMV and SCSMV are sister taxa and share a most recent common ancestor with tritimoviruses or ipomoviruses. These results suggest that TriMV and SCSMV should be classified in a new genus, and we propose the genus Poacevirus in the family Potyviridae, with TriMV as the type member. Additional keyword: wheat. Viruses from several different families infect wheat (Triticum aestivum L.) in the Great Plains and other parts of the United States. These viruses include Agropyron mosaic virus (AgMV), Barley yellow dwarf virus, Soil-borne wheat mosaic virus, Triticum mosaic virus (TriMV), Wheat American striate mosaic virus, Wheat mosaic virus, and Wheat streak mosaic virus (WSMV) (6,20,21). Among these viruses, WSMV is an economically important virus causing significant yield losses in the United States (6). TriMV was recently reported from Kansas, naturally infecting WSMV-resistant wheat cultivars (20); however, the impact of this virus on yield losses in wheat remains to be known. The Potyviridae is the largest family of positive-stranded RNA viruses infecting plants, divided into six genera based on their genetic relatedness, vector transmission, and genome organization (2,5). The genus Potyvirus, with Potato virus Y (PVY) as the type member, contains numerous economically important aphidtransmitted virus species and is the most thoroughly characterized genus among the family Potyviridae. Other genera include Rymovirus, with Ryegrass mosaic virus (RGMV) as the type species, transmitted by Abacarus mites; Tritimovirus, with WSMV as the type member, vectored by wheat curl mites (Aceria tosichella); Ipomovirus, with Sweet potato mild mottle virus (SPMMV) as the type species, transmitted by whiteflies; and Macluravirus, with Maclura mosaic virus (MacMV) as the type species, with characteristic short virus particles transmitted by aphids. These five genera all contain monopartite viruses, whereas the genus Bymovirus, with Barley yellow mosaic virus (BaYMV) as the type member, contains bipartite viruses transmitted by plasmodio
81 citations
TL;DR: ‘Mace’ hard red winter wheat (Triticum aestivum L.) was developed by the USDA-ARS and the Nebraska Agricultural Experiment Station and released in December 2007 for its resistance to Wheat streak mosaic virus (WSMV) and adaptation to rainfed and irrigated wheat production systems in Nebraska and adjacent areas in the northern Great Plains.
Abstract: ‘Mace’ (Reg. No. CV-1027, PI 651043) hard red winter wheat (Triticum aestivum L.) was developed by the USDA-ARS and the Nebraska Agricultural Experiment Station and released in December 2007. Mace was selected from the cross Yuma//PI 372129/3/CO850034/4/4*Yuma/5/(KS91H184/Arlin S//KS91HW29/3/NE89526). Mace primarily was released for its resistance to Wheat streak mosaic virus (WSMV) and adaptation to rainfed and irrigated wheat production systems in Nebraska and adjacent areas in the northern Great Plains. Mace was derived from a head selection made from a heterogeneous, in terms of fi eld resistance to WSMV, F 5 line. Resistance to WSMV is conditioned by the Wsm-1 gene, located on an introgressed chromosome arm from Thinopyrum intermedium (Host) Barkworth & D.R. Dewey [Agropyron intermedium (Horst.) Beauv.] present as a 4DL.4AgS chromosomal translocation. Mace was tested under the experimental designation N02Y5117.
77 citations
TL;DR: A study testing the efficacy of a portable nanopore-based massively parallel sequencing (MPS) technology for use in the detection of diverse plant pathogens in selected samples showed that this methodology is useful for detecting unsuspected viral and bacterial pathogens in plant and insect tissues.
Abstract: Plant pathogens are constantly emerging and spreading into new areas and there are often limited postdiagnosis treatment options for infection, making surveillance key to their control. Here we present results from a study testing the efficacy of a portable nanopore-based massively parallel sequencing (MPS) technology for use in the detection of diverse plant pathogens in selected samples. The Oxford MinION device was coupled with whole transcriptome amplification (WTA) to sequence the metatranscriptome of plant and insect tissues infected with either Candidatus Liberibacter asiaticus or plum pox virus. Results showed that this methodology is useful for detecting unsuspected viral and bacterial pathogens in plant and insect tissues. The percentage of generated reads assigned to plum pox virus was 95% from infected tissue and 3% from the viruliferous insect, Myzus persicae. Diaphorina citri sequencing led to 22% of the reads mapping as Ca. L. asiaticus. Plum pox virus and Ca. L. asiaticus were detected in both tissue and insect samples near the beginning of each sequencing run, demonstrating the capability of this methodology to obtain results rapidly. This approach also proved the capability of this system to determine the major components of the insect vector's microbiome and the specific strain of small-genome, high-titer pathogens.
74 citations
TL;DR: In this article, the MinION sequencing platform consistently revealed the presence of several plant virus species, including Dioscorea bacilliform virus, Yam mild mosaic virus and Yam chlorotic necrosis virus.
Abstract: We here assessed the capability of the MinION sequencing approach to detect and characterize viruses infecting a water yam plant. This sequencing platform consistently revealed the presence of several plant virus species, including Dioscorea bacilliform virus, Yam mild mosaic virus and Yam chlorotic necrosis virus. A potentially novel ampelovirus was also detected by a complimentary Illumina sequencing approach. The full-length genome sequence of yam chlorotic necrosis virus was determined using Sanger sequencing, which enabled determination of the coverage and sequencing accuracy of the MinION technology. Whereas the total mean sequencing error rate of yam chlorotic necrosis virus-related MinION reads was 11.25%, we show that the consensus sequence obtained either by de novo assembly or after mapping the MinION reads on the virus genomic sequence was >99.8% identical with the Sanger-derived reference sequence. From the perspective of potential plant disease diagnostic applications of MinION sequencing, these degrees of sequencing accuracy demonstrate that the MinION approach can be used to both reliably detect and accurately sequence nearly full-length positive-sense single-strand polyadenylated RNA plant virus genomes.
53 citations