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Pros and Cons in the Use of Next-Generation Sequencing for...
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Pros and Cons in the Use of Next-Generation Sequencing for Plant Virus Diagnosis

A special issue of Viruses (ISSN 1999-4915). This special issue belongs to the section "Viruses of Plants, Fungi and Protozoa".

Deadline for manuscript submissions: closed (30 June 2021) | Viewed by 40130

Special Issue Editor

Dr. Nuredin Habili

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Guest Editor
Visiting Research Fellow, The University of Adelaide, and Senior Research Scientist with the Australian Wine Research Institute, P.O. Box 197, Glen Osmond, Adelaide 5064, SA, Australia
Interests: grapevine viruses; viroids and phytoplasmas; molecular diagnosis; emerging viruses of the grapevine; elimination of viruses from the grapevine by thermotherapy and chemotherapy; molecular diagnosis of Grapevine Trunk Diseases (GTD)
Special Issues, Collections and Topics in MDPI journals

Special Issue Information

The security of the world’s food supply is of vital importance to everyone. Plant viruses, being a highly contagious pathogen, challenge the security of our food supply. Plant diseases, including viruses, result in the loss of $220 billion in agricultural food crops around the globe. However, viruses, unlike other plant diseases and pests, cannot be eradicated by chemical spraying. Once an individual plant is infected, it will carry that virus throughout its lifespan and can even act as a reservoir for insect vectors to cross-contaminate other plants. Diagnostic tools are useful for the management and control of plant viruses. These tools have evolved over recent decades and are becoming more sensitive and specific for virus detection. Early diagnostic procedures involved mechanical inoculation and grafting of sensitive indicator hosts with infected plants, which took days or even years to produce results. These initial techniques were followed by rapid and specific ELISA and PCR-based methods with various levels of sensitivity. The latest diagnostic procedure is next generation sequencing (NGS), which can detect the “virome” of a single sample without the need for previous sequence information. However, NGS appears to be too expensive and time consuming for routine analyses. The fact that most detected viruses in perennial crops like grapevine are symptomless is a cause for concern among biosecurity staff and growers alike. Here, we are seeking advice from expert colleagues on how to address the issue of the detection of the many viruses causing little or no symptoms in a single host by application of NGS.

Dr. Nuredin Habili
Guest Editor

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Keywords

  • plant viruses
  • virome
  • diagnostics
  • next generation sequencing
  • high throughput sequencing
  • RNA-seq
  • bioinformatics
  • RT-PCR
  • amplicon sequencing
  • quasi-species

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Published Papers (7 papers)

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Research

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19 pages, 1601 KiB  
Article
High-Throughput Sequencing of Small RNAs for Diagnostics of Grapevine Viruses and Viroids in Russia
by Emiliya Navrotskaya, Elena Porotikova, Eugeniya Yurchenko, Zsuzsanna Nagyne Galbacs, Eva Varallyay and Svetlana Vinogradova
Viruses 2021, 13(12), 2432; https://doi.org/10.3390/v13122432 - 3 Dec 2021
Cited by 12 | Viewed by 3171
Abstract
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 [...] Read more.
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. Full article
(This article belongs to the Special Issue Pros and Cons in the Use of Next-Generation Sequencing for Plant Virus Diagnosis)
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12 pages, 2472 KiB  
Article
Characterization of Spanish Olive Virome by High Throughput Sequencing Opens New Insights and Uncertainties
by Ana Belén Ruiz-García, Celia Canales, Félix Morán, Manuel Ruiz-Torres, Magdalena Herrera-Mármol and Antonio Olmos
Viruses 2021, 13(11), 2233; https://doi.org/10.3390/v13112233 - 6 Nov 2021
Cited by 7 | Viewed by 3047
Abstract
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 [...] Read more.
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. Full article
(This article belongs to the Special Issue Pros and Cons in the Use of Next-Generation Sequencing for Plant Virus Diagnosis)
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16 pages, 6767 KiB  
Article
HTS-Based Diagnostics of Sugarcane Viruses: Seasonal Variation and Its Implications for Accurate Detection
by Martha Malapi-Wight, Bishwo Adhikari, Jing Zhou, Leticia Hendrickson, Clarissa J. Maroon-Lango, Clint McFarland, Joseph A. Foster and Oscar P. Hurtado-Gonzales
Viruses 2021, 13(8), 1627; https://doi.org/10.3390/v13081627 - 17 Aug 2021
Cited by 12 | Viewed by 3329
Abstract
Rapid global germplasm trade has increased concern about the spread of plant pathogens and pests across borders that could become established, affecting agriculture and environment systems. Viral pathogens are of particular concern due to their difficulty to control once established. A comprehensive diagnostic [...] Read more.
Rapid global germplasm trade has increased concern about the spread of plant pathogens and pests across borders that could become established, affecting agriculture and environment systems. Viral pathogens are of particular concern due to their difficulty to control once established. A comprehensive diagnostic platform that accurately detects both known and unknown virus species, as well as unreported variants, is playing a pivotal role across plant germplasm quarantine programs. Here we propose the addition of high-throughput sequencing (HTS) from total RNA to the routine quarantine diagnostic workflow of sugarcane viruses. We evaluated the impact of sequencing depth needed for the HTS-based identification of seven regulated sugarcane RNA/DNA viruses across two different growing seasons (spring and fall). Our HTS analysis revealed that viral normalized read counts (RPKM) was up to 23-times higher in spring than in the fall season for six out of the seven viruses. Random read subsampling analyses suggested that the minimum number of reads required for reliable detection of RNA viruses was 0.5 million, with a viral genome coverage of at least 92%. Using an HTS-based total RNA metagenomics approach, we identified all targeted viruses independent of the time of the year, highlighting that higher sequencing depth is needed for the identification of DNA viruses. Full article
(This article belongs to the Special Issue Pros and Cons in the Use of Next-Generation Sequencing for Plant Virus Diagnosis)
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16 pages, 1158 KiB  
Article
Quality Assessment and Validation of High-Throughput Sequencing for Grapevine Virus Diagnostics
by Nourolah Soltani, Kristian A. Stevens, Vicki Klaassen, Min-Sook Hwang, Deborah A. Golino and Maher Al Rwahnih
Viruses 2021, 13(6), 1130; https://doi.org/10.3390/v13061130 - 11 Jun 2021
Cited by 22 | Viewed by 4104
Abstract
Development of High-Throughput Sequencing (HTS), also known as next generation sequencing, revolutionized diagnostic research of plant viruses. HTS outperforms bioassays and molecular diagnostic assays that are used to screen domestic and quarantine grapevine materials in data throughput, cost, scalability, and detection of novel [...] Read more.
Development of High-Throughput Sequencing (HTS), also known as next generation sequencing, revolutionized diagnostic research of plant viruses. HTS outperforms bioassays and molecular diagnostic assays that are used to screen domestic and quarantine grapevine materials in data throughput, cost, scalability, and detection of novel and highly variant virus species. However, before HTS-based assays can be routinely used for plant virus diagnostics, performance specifications need to be developed and assessed. In this study, we selected 18 virus-infected grapevines as a test panel for measuring performance characteristics of an HTS-based diagnostic assay. Total nucleic acid (TNA) was extracted from petioles and dormant canes of individual samples and constructed libraries were run on Illumina NextSeq 500 instrument using a 75-bp single-end read platform. Sensitivity was 98% measured over 264 distinct virus and viroid infections with a false discovery rate (FDR) of approximately 1 in 5 positives. The results also showed that combining a spring petiole test with a fall cane test increased sensitivity to 100% for this TNA HTS assay. To evaluate extraction methodology, these results were compared to parallel dsRNA extractions. In addition, in a more detailed dilution study, the TNA HTS assay described here consistently performed well down to a dilution of 5%. In that range, sensitivity was 98% with a corresponding FDR of approximately 1 in 5. Repeatability and reproducibility were assessed at 99% and 93%, respectively. The protocol, criteria, and performance levels described here may help to standardize HTS for quality assurance and accreditation purposes in plant quarantine or certification programs. Full article
(This article belongs to the Special Issue Pros and Cons in the Use of Next-Generation Sequencing for Plant Virus Diagnosis)
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Review

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15 pages, 327 KiB  
Review
Metagenomic Studies of Viruses in Weeds and Wild Plants: A Powerful Approach to Characterise Variable Virus Communities
by Beata Hasiów-Jaroszewska, Dieke Boezen and Mark P. Zwart
Viruses 2021, 13(10), 1939; https://doi.org/10.3390/v13101939 - 27 Sep 2021
Cited by 27 | Viewed by 4096
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 [...] Read more.
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. Full article
(This article belongs to the Special Issue Pros and Cons in the Use of Next-Generation Sequencing for Plant Virus Diagnosis)
31 pages, 1884 KiB  
Review
Current Developments and Challenges in Plant Viral Diagnostics: A Systematic Review
by Gajanan T. Mehetre, Vincent Vineeth Leo, Garima Singh, Antonina Sorokan, Igor Maksimov, Mukesh Kumar Yadav, Kalidas Upadhyaya, Abeer Hashem, Asma N. Alsaleh, Turki M. Dawoud, Khalid S. Almaary and Bhim Pratap Singh
Viruses 2021, 13(3), 412; https://doi.org/10.3390/v13030412 - 5 Mar 2021
Cited by 80 | Viewed by 12439
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 [...] Read more.
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. Full article
(This article belongs to the Special Issue Pros and Cons in the Use of Next-Generation Sequencing for Plant Virus Diagnosis)
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Other

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17 pages, 1350 KiB  
Protocol
Application of Oxford Nanopore Technology to Plant Virus Detection
by Lia W. Liefting, David W. Waite and Jeremy R. Thompson
Viruses 2021, 13(8), 1424; https://doi.org/10.3390/v13081424 - 22 Jul 2021
Cited by 41 | Viewed by 7099
Abstract
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 [...] Read more.
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. Full article
(This article belongs to the Special Issue Pros and Cons in the Use of Next-Generation Sequencing for Plant Virus Diagnosis)
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