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Narware J, Chakma J, Singh SP, Prasad DR, Meher J, Singh P, Bhargava P, Sawant SB, Pitambara, Singh JP, Manzar N, Kashyap AS. Nanomaterial-based biosensors: a new frontier in plant pathogen detection and plant disease management. Front Bioeng Biotechnol 2025; 13:1570318. [PMID: 40336552 PMCID: PMC12055542 DOI: 10.3389/fbioe.2025.1570318] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/03/2025] [Accepted: 03/26/2025] [Indexed: 05/09/2025] Open
Abstract
Nanotechnology has significantly advanced the detection of plant diseases by introducing nano-inspired biosensors that offer distinct advantages over traditional diagnostic methods. These biosensors, enhanced with novel nanomaterials, exhibit increased sensitivity, catalytic activity, and faster response times, resulting in improved diagnostic efficiency. The increasing impact of climate-induced stress and emerging plant pathogens have created an urgent demand for real-time monitoring systems in agriculture. Nanobiosensors are revolutionizing plant disease management by enabling on-site detection of pests and weeds, facilitating precise pesticide applications. This article provides a comprehensive overview of the development and application of nanobiosensors in real-time plant disease diagnosis. It highlights key innovations, such as smartphone-integrated nanozyme biosensing and lab-on-a-chip technologies. Special emphasis is placed on the detection of molecular biomarkers, demonstrating the critical role of nanobiosensors in addressing the evolving challenges of plant disease management and agricultural sustainability.
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Affiliation(s)
- Jeetu Narware
- Department of Mycology and Plant Pathology, Institute of Agriculture Sciences, Banaras Hindu University, Varanasi, Uttar Pradesh, India
| | - Jharjhari Chakma
- Department of Mycology and Plant Pathology, Institute of Agriculture Sciences, Banaras Hindu University, Varanasi, Uttar Pradesh, India
| | - Satyendra P. Singh
- Department of Mycology and Plant Pathology, Institute of Agriculture Sciences, Banaras Hindu University, Varanasi, Uttar Pradesh, India
| | - Divya Raj Prasad
- Department of Kayachikitsa, Faculty Ayurveda, Institute of Medical sciences, Banaras Hindu University, Varanasi, Uttar Pradesh, India
| | - Jhumishree Meher
- Department of Mycology and Plant Pathology, Institute of Agriculture Sciences, Banaras Hindu University, Varanasi, Uttar Pradesh, India
| | - Prachi Singh
- Bihar Agricultural University, Bhagalpur, Bihar, India
| | | | | | - Pitambara
- Bihar Agricultural University, Bhagalpur, Bihar, India
| | - Jyoti P. Singh
- ICAR-National Bureau of Agriculturally Important Microorganisms, Mau, Uttar Pradesh, India
| | - Nazia Manzar
- ICAR-National Bureau of Agriculturally Important Microorganisms, Mau, Uttar Pradesh, India
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Bangratz M, Comte A, Billard E, Guigma AK, Gandolfi G, Kassankogno AI, Sérémé D, Poulicard N, Tollenaere C. Deciphering mixed infections by plant RNA virus and reconstructing complete genomes simultaneously present within-host. PLoS One 2025; 20:e0311555. [PMID: 39808677 PMCID: PMC11731864 DOI: 10.1371/journal.pone.0311555] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/25/2024] [Accepted: 09/22/2024] [Indexed: 01/16/2025] Open
Abstract
Local co-circulation of multiple phylogenetic lineages is particularly likely for rapidly evolving pathogens in the current context of globalisation. When different phylogenetic lineages co-occur in the same fields, they may be simultaneously present in the same host plant (i.e. mixed infection), with potentially important consequences for disease outcome. This is the case in Burkina Faso for the rice yellow mottle virus (RYMV), which is endemic to Africa and a major constraint on rice production. We aimed to decipher the distinct RYMV isolates that simultaneously infect a single rice plant and to sequence their genomes. To this end, we tested different sequencing strategies, and we finally combined direct cDNA ONT (Oxford Nanopore Technology) sequencing with the bioinformatics tool RVhaplo. This method was validated by the successful reconstruction of two viral genomes that were less than a hundred nucleotides apart (out of a genome of 4450nt length, i.e. 2-3%), and present in artificial mixes at a ratio of up to a 99/1. We then used this method to subsequently analyze mixed infections from field samples, revealing up to three RYMV isolates within one single rice plant sample from Burkina Faso. In most cases, the complete genome sequences were obtained, which is particularly important for a better estimation of viral diversity and the detection of recombination events. The method described thus allows to identify various haplotypes of RYMV simultaneously infecting a single rice plant, obtaining their full-length sequences, as well as a rough estimate of relative frequencies within the sample. It is efficient, cost-effective, as well as portable, so that it could further be implemented where RYMV is endemic. Prospects include unravelling mixed infections with other RNA viruses that threaten crop production worldwide.
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Affiliation(s)
- Martine Bangratz
- PHIM, Plant Health Institute of Montpellier, Univ. Montpellier, IRD, CIRAD, INRAE, Institute Agro, Montpellier, France
| | - Aurore Comte
- PHIM, Plant Health Institute of Montpellier, Univ. Montpellier, IRD, CIRAD, INRAE, Institute Agro, Montpellier, France
| | - Estelle Billard
- PHIM, Plant Health Institute of Montpellier, Univ. Montpellier, IRD, CIRAD, INRAE, Institute Agro, Montpellier, France
| | - Abdoul Kader Guigma
- INERA, Institut de l’Environnement et de Recherches Agricoles, Laboratoire de Phytopathologie, Bobo-Dioulasso, Burkina Faso
| | - Guillaume Gandolfi
- PHIM, Plant Health Institute of Montpellier, Univ. Montpellier, IRD, CIRAD, INRAE, Institute Agro, Montpellier, France
| | - Abalo Itolou Kassankogno
- INERA, Institut de l’Environnement et de Recherches Agricoles, Laboratoire de Phytopathologie, Bobo-Dioulasso, Burkina Faso
| | - Drissa Sérémé
- INERA, Institut de l’Environnement et de Recherches Agricoles, Laboratoire de Virologie et de Biologie Végétale, Kamboinsé, Burkina Faso
| | - Nils Poulicard
- PHIM, Plant Health Institute of Montpellier, Univ. Montpellier, IRD, CIRAD, INRAE, Institute Agro, Montpellier, France
| | - Charlotte Tollenaere
- PHIM, Plant Health Institute of Montpellier, Univ. Montpellier, IRD, CIRAD, INRAE, Institute Agro, Montpellier, France
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Zhu XT, Sanz-Jimenez P, Ning XT, Tahir Ul Qamar M, Chen LL. Direct RNA sequencing in plants: Practical applications and future perspectives. PLANT COMMUNICATIONS 2024; 5:101064. [PMID: 39155503 PMCID: PMC11589328 DOI: 10.1016/j.xplc.2024.101064] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 05/13/2024] [Revised: 07/17/2024] [Accepted: 08/14/2024] [Indexed: 08/20/2024]
Abstract
The transcriptome serves as a bridge that links genomic variation to phenotypic diversity. A vast number of studies using next-generation RNA sequencing (RNA-seq) over the last 2 decades have emphasized the essential roles of the plant transcriptome in response to developmental and environmental conditions, providing numerous insights into the dynamic changes, evolutionary traces, and elaborate regulation of the plant transcriptome. With substantial improvement in accuracy and throughput, direct RNA sequencing (DRS) has emerged as a new and powerful sequencing platform for precise detection of native and full-length transcripts, overcoming many limitations such as read length and PCR bias that are inherent to short-read RNA-seq. Here, we review recent advances in dissecting the complexity and diversity of plant transcriptomes using DRS as the main technological approach, covering many aspects of RNA metabolism, including novel isoforms, poly(A) tails, and RNA modification, and we propose a comprehensive workflow for processing of plant DRS data. Many challenges to the application of DRS in plants, such as the need for machine learning tools tailored to plant transcriptomes, remain to be overcome, and together we outline future biological questions that can be addressed by DRS, such as allele-specific RNA modification. This technology provides convenient support on which the connection of distinct RNA features is tightly built, sustainably refining our understanding of the biological functions of the plant transcriptome.
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Affiliation(s)
- Xi-Tong Zhu
- State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, College of Life Science and Technology, Guangxi University, Nanning 530004, China.
| | - Pablo Sanz-Jimenez
- National Key Laboratory of Crop Genetic Improvement, College of Informatics, Huazhong Agricultural University, Wuhan 430070, China
| | - Xiao-Tong Ning
- State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, College of Life Science and Technology, Guangxi University, Nanning 530004, China
| | - Muhammad Tahir Ul Qamar
- Integrative Omics and Molecular Modeling Laboratory, Department of Bioinformatics and Biotechnology, Government College University Faisalabad (GCUF), Faisalabad 38000, Pakistan
| | - Ling-Ling Chen
- State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, College of Life Science and Technology, Guangxi University, Nanning 530004, China.
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Nunes-Leite L, Liefting LW, Waite DW, Khan S, Thompson JR. High-Throughput Sequencing Methods for the Detection of Two Strawberry Viruses in Post-Entry Quarantine. Viruses 2024; 16:1550. [PMID: 39459884 PMCID: PMC11512301 DOI: 10.3390/v16101550] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/10/2024] [Revised: 08/19/2024] [Accepted: 09/29/2024] [Indexed: 10/28/2024] Open
Abstract
High-throughput sequencing (HTS) technologies may be a useful tool for testing imported plant germplasm for multiple pathogens present in a sample, offering strain-generic detection not offered by most PCR-based assays. Metatranscriptomics (RNAseq) and tiled amplicon PCR (TA-PCR) were tested as HTS-based techniques to detect viruses present in low titres. Strawberry mottle virus (SMoV), an RNA virus, and strawberry vein banding virus (SVBV), a DNA virus, were selected for comparison of RNAseq and TA-PCR with quantitative PCR assays. RNAseq of plant ribosomal RNA-depleted samples of low viral titre was used to obtain datasets from 3 M to 120 M paired-end (PE) reads. RNAseq demonstrated PCR-like sensitivity, able to detect as few as 10 viral copies/µL when 60 million (M) PE reads were generated. The custom TA-PCR primer panels designed for each virus were successfully used to recover most of the reference genomes for each virus. Single- and multiple-target TA-PCR allowed the detection of viruses in samples with around 10 viral copies/µL with a minimum continuous sequence length recovery of 500 bp. The limit of detection of the HTS-based protocols described here is comparable to that of quantitative PCR assays. This work lays the groundwork for an increased flexibility in HTS detection of plant viruses.
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Affiliation(s)
- Luciano Nunes-Leite
- Plant Health and Environment Laboratory, Ministry for Primary Industries, P.O. Box 2095, Auckland 1140, New Zealand; (D.W.W.); (S.K.)
| | - Lia W. Liefting
- Plant Health and Environment Laboratory, Ministry for Primary Industries, P.O. Box 2095, Auckland 1140, New Zealand; (D.W.W.); (S.K.)
| | | | | | - Jeremy R. Thompson
- Plant Health and Environment Laboratory, Ministry for Primary Industries, P.O. Box 2095, Auckland 1140, New Zealand; (D.W.W.); (S.K.)
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Knobloch S, Salimi F, Buaya A, Ploch S, Thines M. RAPiD: a rapid and accurate plant pathogen identification pipeline for on-site nanopore sequencing. PeerJ 2024; 12:e17893. [PMID: 39346055 PMCID: PMC11438431 DOI: 10.7717/peerj.17893] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/26/2024] [Accepted: 07/19/2024] [Indexed: 10/01/2024] Open
Abstract
Nanopore sequencing technology has enabled the rapid, on-site taxonomic identification of samples from anything and anywhere. However, sequencing errors, inadequate databases, as well as the need for bioinformatic expertise and powerful computing resources, have hampered the widespread use of the technology for pathogen identification in the agricultural sector. Here we present RAPiD, a lightweight and accurate real-time taxonomic profiling pipeline. Compared to other metagenomic profilers, RAPiD had a higher classification precision achieved through the use of a curated, non-redundant database of common agricultural pathogens and extensive quality filtering of alignments. On a fungal, bacterial and mixed mock community RAPiD was the only pipeline to detect all members of the communities. We also present a protocol for in-field sample processing enabling pathogen identification from plant sample to sequence within 3 h using low-cost equipment. With sequencing costs continuing to decrease and more high-quality reference genomes becoming available, nanopore sequencing provides a viable method for rapid and accurate pathogen identification in the field. A web implementation of the RAPiD pipeline for real-time analysis is available at https://agrifuture.senckenberg.de.
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Affiliation(s)
- Stephen Knobloch
- Senckenberg Biodiversity and Climate Research Centre, Senckenberg Society for Nature Research, Frankfurt, Germany
- Department of Food Technology, Fulda University of Applied Sciences, Fulda, Germany
| | - Fatemeh Salimi
- Senckenberg Biodiversity and Climate Research Centre, Senckenberg Society for Nature Research, Frankfurt, Germany
- LOEWE Centre for Translational Biodiversity Genomics, Frankfurt, Germany
| | - Anthony Buaya
- Senckenberg Biodiversity and Climate Research Centre, Senckenberg Society for Nature Research, Frankfurt, Germany
| | - Sebastian Ploch
- Senckenberg Biodiversity and Climate Research Centre, Senckenberg Society for Nature Research, Frankfurt, Germany
| | - Marco Thines
- Senckenberg Biodiversity and Climate Research Centre, Senckenberg Society for Nature Research, Frankfurt, Germany
- LOEWE Centre for Translational Biodiversity Genomics, Frankfurt, Germany
- Department of Biological Sciences, Institute of Ecology, Evolution and Diversity, Goethe University Frankfurt, Frankfurt, Germany
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Carrillo-Lopez LM, Villanueva-Verduzco C, Villanueva-Sánchez E, Fajardo-Franco ML, Aguilar-Tlatelpa M, Ventura-Aguilar RI, Soto-Hernández RM. Nanomaterials for Plant Disease Diagnosis and Treatment: A Review. PLANTS (BASEL, SWITZERLAND) 2024; 13:2634. [PMID: 39339607 PMCID: PMC11434773 DOI: 10.3390/plants13182634] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 04/12/2024] [Revised: 08/06/2024] [Accepted: 09/18/2024] [Indexed: 09/30/2024]
Abstract
Currently, the excessive use of pesticides has generated environmental pollution and harmful effects on human health. The controlled release of active ingredients through the use of nanomaterials (NMs) appears to reduce human exposure and ecosystem alteration. Although the use of NMs can offer an alternative to traditional methods of disease diagnosis and control, it is necessary to review the current approach to the application of these NMs. This review describes the most recent and significant advances in using NMs for diagnosing and treating plant diseases (bacteria, phytopathogenic fungi, viruses, and phytopathogenic nematodes) in cultivated plants. Most studies have focused on reducing, delaying, or eliminating bacteria, fungi, viruses, and nematodes in plants. Both metallic (including metal oxides) and organic nanoparticles (NPs) and composites are widely used in diagnosing and controlling plant diseases due to their biocompatibility and ease of synthesis. Few studies have been carried out with regard to carbon-based NPs due to their toxicity, so future studies should address the development of detection tools, ecological and economic impacts, and human health. The synergistic effect of NMs as fertilizers and pesticides opens new areas of knowledge on the mechanisms of action (plant-pathogen-NMs interaction), the interaction of NMs with nutrients, the effects on plant metabolism, and the traceability of NMs to implement sustainable approaches. More studies are needed involving in vivo models under international regulations to ensure their safety. There is still controversy in the release of NMs into the environment because they could threaten the stability and functioning of biological systems, so research in this area needs to be improved.
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Affiliation(s)
- Luis M Carrillo-Lopez
- Consejo Nacional de Humanidades, Ciencias y Tecnologías-Botánica, Colegio de Postgraduados Campus Montecillo, Carretera Mexico-Texcoco Km. 36.5, Texcoco 56230, Mexico
| | - Clemente Villanueva-Verduzco
- Departamento de Fitotecnia, Universidad Autónoma Chapingo, Carretera México-Texcoco Km. 38.5, Chapingo 56230, Estado de México, Mexico
| | - Evert Villanueva-Sánchez
- Consejo Nacional de Humanidades, Ciencias y Tecnologías-Laboratorio Nacional de Investigación y Servicio Agroalimentario y Forestal, Universidad Autónoma Chapingo, Carretera México-Texcoco Km. 38.5, Chapingo 56230, Estado de México, Mexico
| | - Marja L Fajardo-Franco
- Posgrado en Manejo Sustentable de Recursos Naturales, Universidad Intercultural del Estado de Puebla, Calle Principal a Lipuntlahuaca, Huehuetla 73475, Puebla, Mexico
| | - Martín Aguilar-Tlatelpa
- Posgrado en Manejo Sustentable de Recursos Naturales, Universidad Intercultural del Estado de Puebla, Calle Principal a Lipuntlahuaca, Huehuetla 73475, Puebla, Mexico
| | - Rosa I Ventura-Aguilar
- CONAHCYT-Recursos Genéticos y Productividad-Fruticultura, Colegio de Postgraduados, Campus Montecillo, Carretera Mexico-Texcoco Km. 36.5, Texcoco 56230, Mexico
| | - Ramón Marcos Soto-Hernández
- Botánica, Colegio de Postgraduados, Campus Montecillo, Carretera Mexico-Texcoco Km. 36.5, Texcoco 56230, Mexico
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Mohanty P, Singh PK, Lenka B, Adhya TK, Verma SK, Ayreen Z, Patro S, Sarkar B, Mohapatra RK, Mishra S. Biofabricated nanomaterials in sustainable agriculture: insights, challenges and prospects. Biofabrication 2024; 16:042003. [PMID: 38981495 DOI: 10.1088/1758-5090/ad60f7] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/03/2024] [Accepted: 07/09/2024] [Indexed: 07/11/2024]
Abstract
One ever-evolving and ever-demanding critical human endeavour is the provision of food security for the growing world population. This can be done by adopting sustainable agriculture through horizontal (expanding the arable land area) and vertical (intensifying agriculture through sound technological approaches) interventions. Customized formulated nanomaterials have numerous advantages. With their specialized physico-chemical properties, some nanoparticulated materials improve the plant's natural development and stress tolerance and some others are good nanocarriers. Nanocarriers in agriculture often coat chemicals to form composites having utilities with crop productivity enhancement abilities, environmental management (such as ecotoxicity reduction ability) and biomedicines (such as the ability to control and target the release of useful nanoscale drugs). Ag, Fe, Zn, TiO2, ZnO, SiO2and MgO nanoparticles (NPs), often employed in advanced agriculture, are covered here. Some NPs used for various extended purposes in modern farming practices, including disease diagnostics and seed treatment are also covered. Thus, nanotechnology has revolutionized agrotechnology, which holds promise to transform agricultural (ecosystems as a whole to ensure food security in the future. Considering the available literature, this article further probes the emergent regulatory issues governing the synthesis and use of nanomaterials in the agriculture sector. If applied responsibly, nanomaterials could help improve soil health. This article provides an overview of the nanomaterials used in the distribution of biomolecules, to aid in devising a safer and eco-friendly sustainable agriculture strategy. Through this, agri-systems that depend on advanced farming practices might function more effectively and enhance agri-productivity to meet the food demand of the rising world population.
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Affiliation(s)
- Pratikhya Mohanty
- Bioenergy Lab, School of Biotechnology, KIIT Deemed to be University, Campus 11, Bhubaneswar, Odisha 751 024, India
| | - Puneet Kumar Singh
- Bioenergy Lab, School of Biotechnology, KIIT Deemed to be University, Campus 11, Bhubaneswar, Odisha 751 024, India
| | - Basundhara Lenka
- Bioenergy Lab, School of Biotechnology, KIIT Deemed to be University, Campus 11, Bhubaneswar, Odisha 751 024, India
| | - Tapan K Adhya
- School of Biotechnology, KIIT Deemed to be University, Bhubaneswar, Odisha 751 024, India
| | - Suresh K Verma
- School of Biotechnology, KIIT Deemed to be University, Bhubaneswar, Odisha 751 024, India
| | - Zobia Ayreen
- School of Biotechnology, KIIT Deemed to be University, Bhubaneswar, Odisha 751 024, India
| | - Shilpita Patro
- School of Biotechnology, KIIT Deemed to be University, Bhubaneswar, Odisha 751 024, India
| | - Biplab Sarkar
- Indian Institute of Agricultural Biotechnology, ICAR-IIAB, Garhkhantanga, Ranchi, Jharkhand 834 003, India
| | - Ranjan K Mohapatra
- Department of Chemistry, Government College of Engineering, Keonjhar 758 002, Odisha, India
| | - Snehasish Mishra
- Bioenergy Lab, School of Biotechnology, KIIT Deemed to be University, Campus 11, Bhubaneswar, Odisha 751 024, India
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Sherwood P, Nordström I, Woodward S, Bohman B, Cleary M. Detecting Pathogenic Phytophthora Species Using Volatile Organic Compounds. Molecules 2024; 29:1749. [PMID: 38675569 PMCID: PMC11052055 DOI: 10.3390/molecules29081749] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/15/2024] [Revised: 03/30/2024] [Accepted: 04/08/2024] [Indexed: 04/28/2024] Open
Abstract
There are several highly damaging Phytophthora species pathogenic to forest trees, many of which have been spread beyond their native range by the international trade of live plants and infested materials. Such introductions can be reduced through the development of better tools capable of the early, rapid, and high-throughput detection of contaminated plants. This study utilized a volatilomics approach (solid-phase microextraction coupled to gas chromatography-mass spectrometry) to differentiate between several Phytophthora species in culture and discriminate between healthy and Phytophthora-inoculated European beech and pedunculate oak trees. We tentatively identified 14 compounds that could differentiate eight Phytophthora species from each other in vitro. All of the Phytophthora species examined, except Phytophthora cambivora, uniquely produced at least one compound not observed in the other species; however, most detected compounds were shared between multiple species. Phytophthora polonica had the most unique compounds and was the least similar of all the species examined. The inoculated seedlings had qualitatively different volatile profiles and could be distinguished from the healthy controls by the presence of isokaurene, anisole, and a mix of three unknown compounds. This study supports the notion that volatiles are suitable for screening plant material, detecting tree pathogens, and differentiating between healthy and diseased material.
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Affiliation(s)
- Patrick Sherwood
- Southern Swedish Forest Research Centre, Swedish University of Agricultural Sciences, Box 190, 234 22 Lomma, Sweden; (I.N.); (M.C.)
| | - Ida Nordström
- Southern Swedish Forest Research Centre, Swedish University of Agricultural Sciences, Box 190, 234 22 Lomma, Sweden; (I.N.); (M.C.)
| | - Steve Woodward
- Department of Plant and Soil Science, School of Biological Sciences, University of Aberdeen, Cruickshank Building, St. Machar Drive, Aberdeen AB24 3UU, UK;
| | - Björn Bohman
- Department of Plant Protection Biology, Swedish University of Agricultural Sciences, Box 190, 234 22 Lomma, Sweden;
| | - Michelle Cleary
- Southern Swedish Forest Research Centre, Swedish University of Agricultural Sciences, Box 190, 234 22 Lomma, Sweden; (I.N.); (M.C.)
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Kudjordjie EN, Schmidt-Høier AS, Brøndum MB, Johnsen MG, Nicolaisen M, Vestergård M. Early assessment of fungal and oomycete pathogens in greenhouse irrigation water using Oxford nanopore amplicon sequencing. PLoS One 2024; 19:e0300381. [PMID: 38489283 PMCID: PMC10942031 DOI: 10.1371/journal.pone.0300381] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/28/2023] [Accepted: 02/26/2024] [Indexed: 03/17/2024] Open
Abstract
Water-borne plant pathogenic fungi and oomycetes are a major threat in greenhouse production systems. Early detection and quantification of these pathogens would enable us to ascertain both economic and biological thresholds required for a timely treatment, thus improving effective disease management. Here, we used Oxford nanopore MinION amplicon sequencing to analyze microbial communities in irrigation water collected from greenhouses used for growing tomato, cucumber and Aeschynanthus sp. Fungal and oomycete communities were characterized using primers that amplify the full internal transcribed spacer (ITS) region. To assess the sensitivity of the MinION sequencing, we spiked serially diluted mock DNA into the DNA isolated from greenhouse water samples prior to library preparation. Relative abundances of fungal and oomycete reads were distinct in the greenhouse irrigation water samples and in water samples from setups with tomato that was inoculated with Fusarium oxysporum. Sequence reads derived from fungal and oomycete mock communities were proportionate in the respective serial dilution samples, thus confirming the suitability of MinION amplicon sequencing for environmental monitoring. By using spike-ins as standards to test the reliability of quantification using the MinION, we found that the detection of spike-ins was highly affected by the background quantities of fungal or oomycete DNA in the sample. We observed that spike-ins having shorter length (538bp) produced reads across most of our dilutions compared to the longer spikes (>790bp). Moreover, the sequence reads were uneven with respect to dilution series and were least retrievable in the background samples having the highest DNA concentration, suggesting a narrow dynamic range of performance. We suggest continuous benchmarking of the MinION sequencing to improve quantitative metabarcoding efforts for rapid plant disease diagnostic and monitoring in the future.
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Affiliation(s)
- Enoch Narh Kudjordjie
- Department of Agroecology, Faculty of Technical Sciences, Aarhus University, Slagelse, Denmark
| | | | | | | | - Mogens Nicolaisen
- Department of Agroecology, Faculty of Technical Sciences, Aarhus University, Slagelse, Denmark
| | - Mette Vestergård
- Department of Agroecology, Faculty of Technical Sciences, Aarhus University, Slagelse, Denmark
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Trippa D, Scalenghe R, Basso MF, Panno S, Davino S, Morone C, Giovino A, Oufensou S, Luchi N, Yousefi S, Martinelli F. Next-generation methods for early disease detection in crops. PEST MANAGEMENT SCIENCE 2024; 80:245-261. [PMID: 37599270 DOI: 10.1002/ps.7733] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 07/20/2023] [Accepted: 08/21/2023] [Indexed: 08/22/2023]
Abstract
Plant pathogens are commonly identified in the field by the typical disease symptoms that they can cause. The efficient early detection and identification of pathogens are essential procedures to adopt effective management practices that reduce or prevent their spread in order to mitigate the negative impacts of the disease. In this review, the traditional and innovative methods for early detection of the plant pathogens highlighting their major advantages and limitations are presented and discussed. Traditional techniques of diagnosis used for plant pathogen identification are focused typically on the DNA, RNA (when molecular methods), and proteins or peptides (when serological methods) of the pathogens. Serological methods based on mainly enzyme-linked immunosorbent assay (ELISA) are the most common method used for pathogen detection due to their high-throughput potential and low cost. This technique is not particularly reliable and sufficiently sensitive for many pathogens detection during the asymptomatic stage of infection. For non-cultivable pathogens in the laboratory, nucleic acid-based technology is the best choice for consistent pathogen detection or identification. Lateral flow systems are innovative tools that allow fast and accurate results even in field conditions, but they have sensitivity issues to be overcome. PCR assays performed on last-generation portable thermocyclers may provide rapid detection results in situ. The advent of portable instruments can speed pathogen detection, reduce commercial costs, and potentially revolutionize plant pathology. This review provides information on current methodologies and procedures for the effective detection of different plant pathogens. © 2023 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.
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Affiliation(s)
- Daniela Trippa
- Dipartimento di Scienze Agrarie Alimentari e Forestali, Università degli Studi di Palermo, Palermo, Italy
| | - Riccardo Scalenghe
- Dipartimento di Scienze Agrarie Alimentari e Forestali, Università degli Studi di Palermo, Palermo, Italy
| | | | - Stefano Panno
- Dipartimento di Scienze Agrarie Alimentari e Forestali, Università degli Studi di Palermo, Palermo, Italy
| | - Salvatore Davino
- Dipartimento di Scienze Agrarie Alimentari e Forestali, Università degli Studi di Palermo, Palermo, Italy
| | - Chiara Morone
- Regione Piemonte - Phytosanitary Division, Torino, Italy
| | - Antonio Giovino
- Council for Agricultural Research and Economics (CREA)-Research Centre for Plant Protection and Certification (CREA-DC), Palermo, Italy
| | - Safa Oufensou
- Dipartimento di Agraria, Università degli Studi di Sassari, Sassari, Italy
| | - Nicola Luchi
- National Research Council, Institute for Sustainable Plant Protection, (CNR-IPSP), Florence, Italy
| | - Sanaz Yousefi
- Department of Horticultural Science, Bu-Ali Sina University, Hamedan, Iran
| | - Federico Martinelli
- Department of Biology, University of Florence, Florence, Italy
- National Research Council, Institute for Sustainable Plant Protection, (CNR-IPSP), Florence, Italy
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11
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Tarquini G, Maestri S, Ermacora P, Martini M. The Oxford Nanopore MinION as a Versatile Technology for the Diagnosis and Characterization of Emerging Plant Viruses. Methods Mol Biol 2024; 2732:235-249. [PMID: 38060129 DOI: 10.1007/978-1-0716-3515-5_16] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/08/2023]
Abstract
The emergence of novel viral epidemics that could affect major crops represents a serious threat to global food security. The early and accurate identification of the causative viral agent is the most important step for a rapid and effective response to disease outbreaks. Over the last years, the Oxford Nanopore Technologies (ONT) MinION sequencer has been proposed as an effective diagnostic tool for the early detection and identification of emerging viruses in plants, providing many advantages compared with different high-throughput sequencing (HTS) technologies. Here, we provide a step-by-step protocol that we optimized to obtain the virome of "Lamon bean" plants (Phaseolus vulgaris L.), an agricultural product with Protected Geographical Indication (PGI) in North-East of Italy, which is frequently subjected to multiple infections caused by different RNA viruses. The conversion of viral RNA in ds-cDNA enabled the use of Genomic DNA Ligation Sequencing Kit and Native Barcoding DNA Kit, which have been originally developed for DNA sequencing. This allowed the simultaneous diagnosis of both DNA- and RNA-based pathogens, providing a more versatile alternative to the use of direct RNA and/or direct cDNA sequencing kits.
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Affiliation(s)
- Giulia Tarquini
- Department of Agriculture, Food, Environmental and Animal Sciences, University of Udine, Udine, Italy
| | - Simone Maestri
- Center for Genomic Science of IIT@SEMM, Fondazione Istituto Italiano di Tecnologia, Milano, Italy
| | - Paolo Ermacora
- Department of Agriculture, Food, Environmental and Animal Sciences, University of Udine, Udine, Italy
| | - Marta Martini
- Department of Agriculture, Food, Environmental and Animal Sciences, University of Udine, Udine, Italy.
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12
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Theologidis I, Karamitros T, Vichou AE, Kizis D. Nanopore-Sequencing Metabarcoding for Identification of Phytopathogenic and Endophytic Fungi in Olive ( Olea europaea) Twigs. J Fungi (Basel) 2023; 9:1119. [PMID: 37998924 PMCID: PMC10672464 DOI: 10.3390/jof9111119] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/26/2023] [Revised: 11/13/2023] [Accepted: 11/16/2023] [Indexed: 11/25/2023] Open
Abstract
Metabarcoding approaches for the identification of plant disease pathogens and characterization of plant microbial populations constitute a rapidly evolving research field. Fungal plant diseases are of major phytopathological concern; thus, the development of metabarcoding approaches for the detection of phytopathogenic fungi is becoming increasingly imperative in the context of plant disease prognosis. We developed a multiplex metabarcoding method for the identification of fungal phytopathogens and endophytes in olive young shoots, using the MinION sequencing platform (Oxford Nanopore Technologies). Selected fungal-specific primers were used to amplify three different genomic DNA loci (ITS, beta-tubulin, and 28S LSU) originating from olive twigs. A multiplex metabarcoding approach was initially evaluated using healthy olive twigs, and further assessed with naturally infected olive twig samples. Bioinformatic analysis of basecalled reads was carried out using MinKNOW, BLAST+ and R programming, and results were also evaluated using the BugSeq cloud platform. Data analysis highlighted the approaches based on ITS and their combination with beta-tubulin as the most informative ones according to diversity estimations. Subsequent implementation of the method on symptomatic samples identified major olive pathogens and endophytes including genera such as Cladosporium, Didymosphaeria, Paraconiothyrium, Penicillium, Phoma, Verticillium, and others.
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Affiliation(s)
- Ioannis Theologidis
- Laboratory of Toxicological Control of Pesticides, Scientific Directorate of Pesticides' Control & Phytopharmacy, Benaki Phytopathological Institute, 8 St. Delta Street, 14561 Athens, Attica, Greece
| | - Timokratis Karamitros
- Bioinformatics and Applied Genomics Unit, Department of Microbiology, Hellenic Pasteur Institute, 127 Vasilissis Sofias Avenue, 11521 Athens, Attica, Greece
| | - Aikaterini-Eleni Vichou
- Laboratory of Mycology, Scientific Directorate of Phytopathology, Benaki Phytopathological Institute, 8 St. Delta Street, 14561 Athens, Attica, Greece
| | - Dimosthenis Kizis
- Laboratory of Mycology, Scientific Directorate of Phytopathology, Benaki Phytopathological Institute, 8 St. Delta Street, 14561 Athens, Attica, Greece
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13
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Manzoor S, Nabi SU, Baranwal VK, Verma MK, Parveen S, Rather TR, Raja WH, Shafi M. Overview on century progress in research on mosaic disease of apple (Malus domestica Borkh) incited by apple mosaic virus/apple necrotic mosaic virus. Virology 2023; 587:109846. [PMID: 37586234 DOI: 10.1016/j.virol.2023.109846] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/25/2023] [Revised: 07/04/2023] [Accepted: 07/18/2023] [Indexed: 08/18/2023]
Abstract
Apple mosaic is widely distributed disease throughout the apple growing regions leading to the major adverse effects both qualitatively and quantitatively. Earlier the apple mosaic virus-ApMV was regarded as the only causal agent of the disease, but recently a novel virus apple necrotic mosaic virus-ApNMV have been reported as the causal pathogen from various apple growing countries. Accurate diagnosis of disease and detection of ApMV and ApNMV are of utmost importance, because without this ability we can neither understand nor control this disease. Both the viruses are mostly controlled through quarantine, isolation, sanitation and certification programs depending on sensitive and specific detection methods available. Here we review the 100-year progress in research on apple mosaic disease, which includes history, yield losses, causal agents, their genome organization, replication, traditional to recent detection methods, transmission, distribution and host range of associated viruses and management of the disease.
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Affiliation(s)
- Subaya Manzoor
- Division of Plant Pathology, FOA-SKUAST-K, Wadura, 193201, India
| | - Sajad Un Nabi
- ICAR-Central Institute of Temperate Horticulture, Srinagar, 191132, India.
| | | | - Mahendra K Verma
- ICAR-Central Institute of Temperate Horticulture, Srinagar, 191132, India
| | - Shugufta Parveen
- ICAR-Central Institute of Temperate Horticulture, Srinagar, 191132, India
| | - Tariq Rasool Rather
- Division of Plant Pathology, FOH-SKUAST-K, Shalimar, Srinagar, 190025, India
| | - Wasim H Raja
- ICAR-Central Institute of Temperate Horticulture, Srinagar, 191132, India
| | - Mansoor Shafi
- Department of Plant Resources and Environment, Jeju National University, Jeju-si, 63243, Republic of Korea
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14
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Gan Z, Zhou Q, Zheng C, Wang J. Challenges and applications of volatile organic compounds monitoring technology in plant disease diagnosis. Biosens Bioelectron 2023; 237:115540. [PMID: 37523812 DOI: 10.1016/j.bios.2023.115540] [Citation(s) in RCA: 17] [Impact Index Per Article: 8.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/17/2023] [Revised: 07/09/2023] [Accepted: 07/17/2023] [Indexed: 08/02/2023]
Abstract
Biotic and abiotic stresses are well known to increase the emission of volatile organic compounds (VOCs) from plants. The analysis of VOCs emissions from plants enables timely diagnostic of plant diseases, which is critical for prompting sustainable agriculture. Previous studies have predominantly focused on the utilization of commercially available devices, such as electronic noses, for diagnosing plant diseases. However, recent advancements in nanomaterials research have significantly contributed to the development of novel VOCs sensors featuring exceptional sensitivity and selectivity. This comprehensive review presents a systematic analysis of VOCs monitoring technologies for plant diseases diagnosis, providing insights into their distinct advantages and limitations. Special emphasis is placed on custom-made VOCs sensors, with detailed discussions on their design, working principles, and detection performance. It is noteworthy that the application of VOCs monitoring technologies in the diagnostic process of plant diseases is still in its emerging stage, and several critical challenges demand attention and improvement. Specifically, the identification of specific stress factors using a single VOC sensor remains a formidable task, while environmental factors like humidity can potentially interfere with sensor readings, leading to inaccuracies. Future advancements should primarily focus on addressing these challenges to enhance the overall efficacy and reliability of VOCs monitoring technologies in the field of plant disease diagnosis.
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Affiliation(s)
- Ziyu Gan
- College of Biosystems Engineering and Food Science, Zhejiang University, 866 Yuhangtang Road, Hangzhou, 310058, China
| | - Qin'an Zhou
- College of Biosystems Engineering and Food Science, Zhejiang University, 866 Yuhangtang Road, Hangzhou, 310058, China
| | - Chengyu Zheng
- College of Biosystems Engineering and Food Science, Zhejiang University, 866 Yuhangtang Road, Hangzhou, 310058, China
| | - Jun Wang
- College of Biosystems Engineering and Food Science, Zhejiang University, 866 Yuhangtang Road, Hangzhou, 310058, China.
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15
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Braley LE, Jewell JB, Figueroa J, Humann JL, Main D, Mora-Romero GA, Moroz N, Woodhall JW, White RA, Tanaka K. Nanopore Sequencing with GraphMap for Comprehensive Pathogen Detection in Potato Field Soil. PLANT DISEASE 2023; 107:2288-2295. [PMID: 36724099 DOI: 10.1094/pdis-01-23-0052-sr] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 06/18/2023]
Abstract
Early detection of causal pathogens is important to prevent crop loss from diseases. However, some diseases, such as soilborne diseases, are difficult to diagnose due to the absence of visible or characteristic symptoms. In the present study, the use of the Oxford Nanopore MinION sequencer as a molecular diagnostic tool was assessed due to its long-read sequencing capabilities and portability. Nucleotide samples (DNA or RNA) from potato field soils were sequenced and analyzed using a locally curated pathogen database, followed by identification via sequence mapping. We performed computational speed tests of three commonly used mapping/annotation tools (BLAST, BWA-BLAST, and BWA-GraphMap) and found BWA-GraphMap to be the fastest tool for local searching against our curated pathogen database. The data collected demonstrate the high potential of Nanopore sequencing as a minimally biased diagnostic tool for comprehensive pathogen detection in soil from potato fields. Our GraphMap-based MinION sequencing method could be useful as a predictive approach for disease management by identifying pathogens present in field soil prior to planting. Although this method still needs further experimentation with a larger sample size for practical use, the data analysis pipeline presented can be applied to other cropping systems and diagnostics for detecting multiple pathogens.
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Affiliation(s)
- Lauren E Braley
- Department of Plant Pathology, Washington State University, Pullman, WA 99164-6430, U.S.A
| | - Jeremy B Jewell
- Department of Plant Pathology, Washington State University, Pullman, WA 99164-6430, U.S.A
| | - Jose Figueroa
- Department of Bioinformatics and Genomics, The University of North Carolina at Charlotte, Charlotte, NC 28223, U.S.A
- Department of Bioinformatics and Genomics, The University of North Carolina at Charlotte, Kannapolis, NC 28081, U.S.A
| | - Jodi L Humann
- Department of Horticulture, Washington State University, Pullman, WA 99164-6414, U.S.A
| | - Dorrie Main
- Department of Horticulture, Washington State University, Pullman, WA 99164-6414, U.S.A
| | - Guadalupe A Mora-Romero
- Unidad de Investigación en Ambiente y Salud, Universidad Autónoma de Occidente, Los Mochis, Sinaloa 81223, México
| | - Natalia Moroz
- Department of Plant Pathology, Washington State University, Pullman, WA 99164-6430, U.S.A
| | - James W Woodhall
- Parma Research and Extension Center, University of Idaho, Parma, ID 83660-6699, U.S.A
| | - Richard Allen White
- Department of Bioinformatics and Genomics, The University of North Carolina at Charlotte, Charlotte, NC 28223, U.S.A
- Department of Bioinformatics and Genomics, The University of North Carolina at Charlotte, Kannapolis, NC 28081, U.S.A
| | - Kiwamu Tanaka
- Department of Plant Pathology, Washington State University, Pullman, WA 99164-6430, U.S.A
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16
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Johnson MA, Vinatzer BA, Li S. Reference-Free Plant Disease Detection Using Machine Learning and Long-Read Metagenomic Sequencing. Appl Environ Microbiol 2023; 89:e0026023. [PMID: 37184398 PMCID: PMC10304783 DOI: 10.1128/aem.00260-23] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/16/2023] [Accepted: 04/14/2023] [Indexed: 05/16/2023] Open
Abstract
Surveillance for early disease detection is crucial to reduce the threat of plant diseases to food security. Metagenomic sequencing and taxonomic classification have recently been used to detect and identify plant pathogens. However, for an emerging pathogen, its genome may not be similar enough to any public genome to permit reference-based tools to identify infected samples. Also, in the case of point-of care diagnosis in the field, database access may be limited. Therefore, here we explore reference-free detection of plant pathogens using metagenomic sequencing and machine learning (ML). We used long-read metagenomes from healthy and infected plants as our model system and constructed k-mer frequency tables to test eight different ML models. The accuracy in classifying individual reads as coming from a healthy or infected metagenome were compared. Of all models, random forest (RF) had the best combination of short run-time and high accuracy (over 0.90) using tomato metagenomes. We further evaluated the RF model with a different tomato sample infected with the same pathogen or a different pathogen and a grapevine sample infected with a grapevine pathogen and achieved similar performances. ML models can thus learn features to successfully perform reference-free detection of plant diseases whereby a model trained with one pathogen-host system can also be used to detect different pathogens on different hosts. Potential and challenges of applying ML to metagenomics in plant disease detection are discussed. IMPORTANCE Climate change may lead to the emergence of novel plant diseases caused by yet unknown pathogens. Surveillance for emerging plant diseases is crucial to reduce their threat to food security. However, conventional genomic based methods require knowledge of existing plant pathogens and cannot be applied to detecting newly emerged pathogens. In this work, we explored reference-free, meta-genomic sequencing-based disease detection using machine learning. By sequencing the genomes of all microbial species extracted from an infected plant sample, we were able to train machine learning models to accurately classify individual sequencing reads as coming from a healthy or an infected plant sample. This method has the potential to be integrated into a generic pipeline for a meta-genomic based plant disease surveillance approach but also has limitations that still need to be overcome.
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Affiliation(s)
- Marcela A. Johnson
- School of Plant and Environmental Sciences, Virginia Tech, Blacksburg, Virginia, USA
- Graduate Program in Genetics, Bioinformatics, and Computational Biology, Virginia Tech, Blacksburg, Virginia, USA
| | - Boris A. Vinatzer
- School of Plant and Environmental Sciences, Virginia Tech, Blacksburg, Virginia, USA
| | - Song Li
- School of Plant and Environmental Sciences, Virginia Tech, Blacksburg, Virginia, USA
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17
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Javaran VJ, Poursalavati A, Lemoyne P, Ste-Croix DT, Moffett P, Fall ML. NanoViromics: long-read sequencing of dsRNA for plant virus and viroid rapid detection. Front Microbiol 2023; 14:1192781. [PMID: 37415816 PMCID: PMC10320856 DOI: 10.3389/fmicb.2023.1192781] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/23/2023] [Accepted: 06/06/2023] [Indexed: 07/08/2023] Open
Abstract
There is a global need for identifying viral pathogens, as well as for providing certified clean plant materials, in order to limit the spread of viral diseases. A key component of management programs for viral-like diseases is having a diagnostic tool that is quick, reliable, inexpensive, and easy to use. We have developed and validated a dsRNA-based nanopore sequencing protocol as a reliable method for detecting viruses and viroids in grapevines. We compared our method, which we term direct-cDNA sequencing from dsRNA (dsRNAcD), to direct RNA sequencing from rRNA-depleted total RNA (rdTotalRNA), and found that it provided more viral reads from infected samples. Indeed, dsRNAcD was able to detect all of the viruses and viroids detected using Illumina MiSeq sequencing (dsRNA-MiSeq). Furthermore, dsRNAcD sequencing was also able to detect low-abundance viruses that rdTotalRNA sequencing failed to detect. Additionally, rdTotalRNA sequencing resulted in a false-positive viroid identification due to the misannotation of a host-driven read. Two taxonomic classification workflows, DIAMOND & MEGAN (DIA & MEG) and Centrifuge & Recentrifuge (Cent & Rec), were also evaluated for quick and accurate read classification. Although the results from both workflows were similar, we identified pros and cons for both workflows. Our study shows that dsRNAcD sequencing and the proposed data analysis workflows are suitable for consistent detection of viruses and viroids, particularly in grapevines where mixed viral infections are common.
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Affiliation(s)
- Vahid J. Javaran
- Saint-Jean-sur-Richelieu Research and Development Centre, Agriculture and Agri-Food Canada, Saint-Jean-sur-Richelieu, QC, Canada
- Centre SÈVE, Département de Biologie, Université de Sherbrooke, Sherbrooke, QC, Canada
| | - Abdonaser Poursalavati
- Saint-Jean-sur-Richelieu Research and Development Centre, Agriculture and Agri-Food Canada, Saint-Jean-sur-Richelieu, QC, Canada
- Centre SÈVE, Département de Biologie, Université de Sherbrooke, Sherbrooke, QC, Canada
| | - Pierre Lemoyne
- Saint-Jean-sur-Richelieu Research and Development Centre, Agriculture and Agri-Food Canada, Saint-Jean-sur-Richelieu, QC, Canada
| | - Dave T. Ste-Croix
- Saint-Jean-sur-Richelieu Research and Development Centre, Agriculture and Agri-Food Canada, Saint-Jean-sur-Richelieu, QC, Canada
- Département de phytologie, Faculté des Sciences de l’Agriculture et de l’Alimentation, Université Laval, Québec, QC, Canada
| | - Peter Moffett
- Centre SÈVE, Département de Biologie, Université de Sherbrooke, Sherbrooke, QC, Canada
| | - Mamadou L. Fall
- Saint-Jean-sur-Richelieu Research and Development Centre, Agriculture and Agri-Food Canada, Saint-Jean-sur-Richelieu, QC, Canada
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18
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Lee HJ, Kim SM, Jeong RD. Analysis of Wheat Virome in Korea Using Illumina and Oxford Nanopore Sequencing Platforms. PLANTS (BASEL, SWITZERLAND) 2023; 12:2374. [PMID: 37375999 DOI: 10.3390/plants12122374] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 05/15/2023] [Revised: 06/14/2023] [Accepted: 06/16/2023] [Indexed: 06/29/2023]
Abstract
Wheat (Triticum aestivum L.) is one of the most important staple crops in the world, along with maize and rice. More than 50 plant viruses are known to infect wheat worldwide. To date, there are no studies on the identification of viruses infecting wheat in Korea. Therefore, we investigated virome in wheat from three different geographical regions where wheat is mainly cultivated in Korea using Oxford Nanopore Technology (ONT) sequencing and Illumina sequencing. Five viral species, including those known to infect wheat, were identified using high-throughput sequencing strategies. Of these, barley virus G (BVG) and Hordeum vulgare endornavirus (HvEV) were consistently present in all libraries. Sugarcane yellow leaf virus (SCYLV) and wheat leaf yellowing-associated virus (WLYaV) were first identified in Korean wheat samples. The viruses identified by ONT and Illumina sequencing were compared using a heatmap. Though the ONT sequencing approach is less sensitive, the analysis results were similar to those of Illumina sequencing in our study. Both platforms served as reliable and powerful tools for detecting and identifying wheat viruses, achieving a balance between practicality and performance. The findings of this study will provide deeper insights into the wheat virosphere and further help improve disease management strategies.
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Affiliation(s)
- Hyo-Jeong Lee
- Department of Applied Biology, Institute of Environmentally Friendly Agriculture, Chonnam National University, Gwangju 61185, Republic of Korea
| | - Sang-Min Kim
- Crop Foundation Research Division, National Institute of Crop Science, Rural Development Administration, Wanju 55365, Republic of Korea
| | - Rae-Dong Jeong
- Department of Applied Biology, Institute of Environmentally Friendly Agriculture, Chonnam National University, Gwangju 61185, Republic of Korea
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19
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Haveman NJ, Schuerger AC, Yu PL, Brown M, Doebler R, Paul AL, Ferl RJ. Advancing the automation of plant nucleic acid extraction for rapid diagnosis of plant diseases in space. FRONTIERS IN PLANT SCIENCE 2023; 14:1194753. [PMID: 37389293 PMCID: PMC10304293 DOI: 10.3389/fpls.2023.1194753] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Figures] [Subscribe] [Scholar Register] [Received: 03/27/2023] [Accepted: 05/23/2023] [Indexed: 07/01/2023]
Abstract
Human space exploration missions will continue the development of sustainable plant cultivation in what are obviously novel habitat settings. Effective pathology mitigation strategies are needed to cope with plant disease outbreaks in any space-based plant growth system. However, few technologies currently exist for space-based diagnosis of plant pathogens. Therefore, we developed a method of extracting plant nucleic acid that will facilitate the rapid diagnosis of plant diseases for future spaceflight applications. The microHomogenizer™ from Claremont BioSolutions, originally designed for bacterial and animal tissue samples, was evaluated for plant-microbial nucleic acid extractions. The microHomogenizer™ is an appealing device in that it provides automation and containment capabilities that would be required in spaceflight applications. Three different plant pathosystems were used to assess the versatility of the extraction process. Tomato, lettuce, and pepper plants were respectively inoculated with a fungal plant pathogen, an oomycete pathogen, and a plant viral pathogen. The microHomogenizer™, along with the developed protocols, proved to be an effective mechanism for producing DNA from all three pathosystems, in that PCR and sequencing of the resulting samples demonstrated clear DNA-based diagnoses. Thus, this investigation advances the efforts to automate nucleic acid extraction for future plant disease diagnosis in space.
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Affiliation(s)
- Natasha J. Haveman
- NASA Utilization & Life Sciences Office (UB-A), Kennedy Space Center, Merritt Island, FL, United States
| | - Andrew C. Schuerger
- Department of Plant Pathology, University of Florida, Space Life Science Lab, Merritt Island, FL, United States
| | - Pei-Ling Yu
- Department of Plant Pathology, University of Florida, Gainesville, FL, United States
| | - Mark Brown
- Claremont BioSolutions Limited Liability Company (LLC), Upland, CA, United States
| | - Robert Doebler
- Claremont BioSolutions Limited Liability Company (LLC), Upland, CA, United States
| | - Anna-Lisa Paul
- Department of Horticultural Sciences, University of Florida, Gainesville, FL, United States
- Interdisciplinary Center for Biotechnology Research, University of Florida, Gainesville, FL, United States
| | - Robert J. Ferl
- Department of Horticultural Sciences, University of Florida, Gainesville, FL, United States
- University of Florida Office of Research, University of Florida, Gainesville, FL, United States
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20
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Haegeman A, Foucart Y, De Jonghe K, Goedefroit T, Al Rwahnih M, Boonham N, Candresse T, Gaafar YZA, Hurtado-Gonzales OP, Kogej Zwitter Z, Kutnjak D, Lamovšek J, Lefebvre M, Malapi M, Mavrič Pleško I, Önder S, Reynard JS, Salavert Pamblanco F, Schumpp O, Stevens K, Pal C, Tamisier L, Ulubaş Serçe Ç, van Duivenbode I, Waite DW, Hu X, Ziebell H, Massart S. Looking beyond Virus Detection in RNA Sequencing Data: Lessons Learned from a Community-Based Effort to Detect Cellular Plant Pathogens and Pests. PLANTS (BASEL, SWITZERLAND) 2023; 12:2139. [PMID: 37299118 PMCID: PMC10255714 DOI: 10.3390/plants12112139] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 05/10/2023] [Revised: 05/26/2023] [Accepted: 05/27/2023] [Indexed: 06/12/2023]
Abstract
High-throughput sequencing (HTS), more specifically RNA sequencing of plant tissues, has become an indispensable tool for plant virologists to detect and identify plant viruses. During the data analysis step, plant virologists typically compare the obtained sequences to reference virus databases. In this way, they are neglecting sequences without homologies to viruses, which usually represent the majority of sequencing reads. We hypothesized that traces of other pathogens might be detected in this unused sequence data. In the present study, our goal was to investigate whether total RNA-seq data, as generated for plant virus detection, is also suitable for the detection of other plant pathogens and pests. As proof of concept, we first analyzed RNA-seq datasets of plant materials with confirmed infections by cellular pathogens in order to check whether these non-viral pathogens could be easily detected in the data. Next, we set up a community effort to re-analyze existing Illumina RNA-seq datasets used for virus detection to check for the potential presence of non-viral pathogens or pests. In total, 101 datasets from 15 participants derived from 51 different plant species were re-analyzed, of which 37 were selected for subsequent in-depth analyses. In 29 of the 37 selected samples (78%), we found convincing traces of non-viral plant pathogens or pests. The organisms most frequently detected in this way were fungi (15/37 datasets), followed by insects (13/37) and mites (9/37). The presence of some of the detected pathogens was confirmed by independent (q)PCRs analyses. After communicating the results, 6 out of the 15 participants indicated that they were unaware of the possible presence of these pathogens in their sample(s). All participants indicated that they would broaden the scope of their bioinformatic analyses in future studies and thus check for the presence of non-viral pathogens. In conclusion, we show that it is possible to detect non-viral pathogens or pests from total RNA-seq datasets, in this case primarily fungi, insects, and mites. With this study, we hope to raise awareness among plant virologists that their data might be useful for fellow plant pathologists in other disciplines (mycology, entomology, bacteriology) as well.
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Affiliation(s)
- Annelies Haegeman
- Plant Sciences Unit, Flanders Research Institute for Agriculture, Fisheries and Food (ILVO), 9820 Merelbeke, Belgium
| | - Yoika Foucart
- Plant Sciences Unit, Flanders Research Institute for Agriculture, Fisheries and Food (ILVO), 9820 Merelbeke, Belgium
| | - Kris De Jonghe
- Plant Sciences Unit, Flanders Research Institute for Agriculture, Fisheries and Food (ILVO), 9820 Merelbeke, Belgium
| | - Thomas Goedefroit
- Plant Sciences Unit, Flanders Research Institute for Agriculture, Fisheries and Food (ILVO), 9820 Merelbeke, Belgium
| | - Maher Al Rwahnih
- Foundation Plant Services, Department of Plant Pathology, University of California, Davis, CA 95616, USA
| | - Neil Boonham
- School of Natural and Environmental Sciences, Newcastle University, Newcastle Upon Tyne NE1 7RU, UK
| | - Thierry Candresse
- UMR 1332 Biologie du Fruit et Pathologie, Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Université de Bordeaux, 33882 Villenave-d’Ornon, France
| | - Yahya Z. A. Gaafar
- Centre for Plant Health, Canadian Food Inspection Agency, 8801 East Saanich Road, North Saanich, BC V8L 1H3, Canada
| | - Oscar P. Hurtado-Gonzales
- Plant Germplasm Quarantine Program, Animal and Plant Health Inspection Service, United States Department of Agriculture (USDA-APHIS), Beltsville, ML 20705, USA
| | - Zala Kogej Zwitter
- Department of Biotechnology and Systems Biology, National Institute of Biology (NIB), 1000 Ljubljana, Slovenia
- Jožef Stefan International Postgraduate School, 1000 Ljubljana, Slovenia
| | - Denis Kutnjak
- Department of Biotechnology and Systems Biology, National Institute of Biology (NIB), 1000 Ljubljana, Slovenia
| | - Janja Lamovšek
- Plant Protection Department, Agricultural Institute of Slovenia (KIS), 1000 Ljubljana, Slovenia
| | - Marie Lefebvre
- UMR 1332 Biologie du Fruit et Pathologie, Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Université de Bordeaux, 33882 Villenave-d’Ornon, France
| | - Martha Malapi
- Biotechnology Risk Analysis Program, Animal and Plant Health Inspection Service, United States Department of Agriculture (USDA-APHIS), Riverdale, ML 20737, USA
| | - Irena Mavrič Pleško
- Plant Protection Department, Agricultural Institute of Slovenia (KIS), 1000 Ljubljana, Slovenia
| | - Serkan Önder
- Department of Plant Protection, Faculty of Agriculture, Eskişehir Osmangazi University, Odunpazarı, Eskişehir 26160, Turkey
| | | | | | - Olivier Schumpp
- Department of Plant Protection, Agroscope, 1260 Nyon, Switzerland
| | - Kristian Stevens
- Foundation Plant Services, Department of Plant Pathology, University of California, Davis, CA 95616, USA
| | - Chandan Pal
- Zespri International Limited, 400 Maunganui Road, Mount Maunganui 3116, New Zealand
| | - Lucie Tamisier
- Unités GAFL et Pathologie Végétale, Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), 84143 Montfavet, France
| | - Çiğdem Ulubaş Serçe
- Department of Plant Production and Technologies, Faculty of Agricultural Sciences and Technologies, Niğde Ömer Halisdemir University, 51240 Niğde, Turkey
| | - Inge van Duivenbode
- Dutch General Inspection Service for Agricultural Seed and Seed Potatoes (NAK), Randweg 14, 8304 AS Emmeloord, The Netherlands
| | - David W. Waite
- Plant Health and Environment Laboratory, Ministry for Primary Industries, Auckland 1140, New Zealand
| | - Xiaojun Hu
- Plant Germplasm Quarantine Program, Animal and Plant Health Inspection Service, United States Department of Agriculture (USDA-APHIS), Beltsville, ML 20705, USA
| | - Heiko Ziebell
- Institute for Epidemiology and Pathogen Diagnostics, Federal Research Centre for Cultivated Plants, Julius Kühn Institute (JKI), Messeweg 11-12, 38104 Braunschweig, Germany
| | - Sébastien Massart
- Plant Pathology Laboratory, University of Liège, Gembloux Agro-Bio Tech, TERRA, 5030 Gembloux, Belgium
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21
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Chen X, Zhou S, Wang Y, Zheng L, Guan S, Wang D, Wang L, Guan X. Nanopore Single-molecule Analysis of Biomarkers: Providing Possible Clues to Disease Diagnosis. Trends Analyt Chem 2023; 162:117060. [PMID: 38106545 PMCID: PMC10722900 DOI: 10.1016/j.trac.2023.117060] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/19/2023]
Abstract
Biomarker detection has attracted increasing interest in recent years due to the minimally or non-invasive sampling process. Single entity analysis of biomarkers is expected to provide real-time and accurate biological information for early disease diagnosis and prognosis, which is critical to the effective disease treatment and is also important in personalized medicine. As an innovative single entity analysis method, nanopore sensing is a pioneering single-molecule detection technique that is widely used in analytical bioanalytical fields. In this review, we overview the recent progress of nanopore biomarker detection as new approaches to disease diagnosis. In highlighted studies, nanopore was focusing on detecting biomarkers of different categories of communicable and noncommunicable diseases, such as pandemic Covid-19, AIDS, cancers, neurologic diseases, etc. Various sensitive and selective nanopore detecting strategies for different types of biomarkers are summarized. In addition, the challenges, opportunities, and direction for future development of nanopore-based biomarker sensors are also discussed.
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Affiliation(s)
- Xiaohan Chen
- Chongqing Institute of Green and Intelligent Technology, Chinese Academy of Sciences, Chongqing, 400714, China
- Chongqing School, University of Chinese Academy of Science, Chongqing, 400714, China
| | - Shuo Zhou
- Chongqing Institute of Green and Intelligent Technology, Chinese Academy of Sciences, Chongqing, 400714, China
- Chongqing School, University of Chinese Academy of Science, Chongqing, 400714, China
| | - Yunjiao Wang
- Chongqing Institute of Green and Intelligent Technology, Chinese Academy of Sciences, Chongqing, 400714, China
- Chongqing School, University of Chinese Academy of Science, Chongqing, 400714, China
| | - Ling Zheng
- Chongqing Institute of Green and Intelligent Technology, Chinese Academy of Sciences, Chongqing, 400714, China
- Chongqing School, University of Chinese Academy of Science, Chongqing, 400714, China
- School of Pharmacy and Bioengineering, Chongqing University of Technology, Chongqing 400054, China
| | - Sarah Guan
- Hinsdale Central High School, Hinsdale, IL 60521, USA
| | - Deqiang Wang
- Chongqing Institute of Green and Intelligent Technology, Chinese Academy of Sciences, Chongqing, 400714, China
- Chongqing School, University of Chinese Academy of Science, Chongqing, 400714, China
| | - Liang Wang
- Chongqing Institute of Green and Intelligent Technology, Chinese Academy of Sciences, Chongqing, 400714, China
- Chongqing School, University of Chinese Academy of Science, Chongqing, 400714, China
- Chongqing Key Laboratory of Intelligent Medicine Engineering for Hepatopancreatobiliary Diseases, University of Chinese Academy of Sciences, Chongqing 401147, China
| | - Xiyun Guan
- Department of Chemistry, Illinois Institute of Technology, Chicago, IL, 60616, USA
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22
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Abou Kubaa R, Amoia SS, Altamura G, Minafra A, Chiumenti M, Cillo F. Nanopore Technology Applied to Targeted Detection of Tomato Brown Rugose Fruit Virus Allows Sequencing of Related Viruses and the Diagnosis of Mixed Infections. PLANTS (BASEL, SWITZERLAND) 2023; 12:999. [PMID: 36903859 PMCID: PMC10005216 DOI: 10.3390/plants12050999] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Subscribe] [Scholar Register] [Received: 01/20/2023] [Revised: 02/10/2023] [Accepted: 02/17/2023] [Indexed: 06/18/2023]
Abstract
Tomato (Solanum lycopersicum) plants from a commercial glasshouse were identified with symptoms compatible with a tomato brown rugose fruit virus (ToBRFV) infection. Reverse transcription-PCR and quantitative PCR confirmed the presence of ToBRFV. Subsequently, the same RNA sample and a second from tomato plants infected with a similar tobamovirus, tomato mottle mosaic virus (ToMMV), were extracted and processed for high-throughput sequencing with the Oxford Nanopore Technology (ONT). For the targeted detection of ToBRFV, the two libraries were synthesized by using six ToBRFV sequence-specific primers in the reverse transcription step. This innovative target enrichment technology enabled deep coverage sequencing of ToBRFV, with 30% of the total reads mapping to the target virus genome and 57% mapping to the host genome. The same set of primers applied to the ToMMV library generated 5% of the total reads mapping to the latter virus, indicating that sequencing of similar, non-target viral sequences was also allowed. Further, the complete genome of pepino mosaic virus (PepMV) was also sequenced from the ToBRFV library, thus suggesting that, even using multiple sequence-specific primers, a low rate of off-target sequencing can usefully provide additional information on unexpected viral species coinfecting the same samples in an individual assay. These results demonstrate that targeted nanopore sequencing can specifically identify viral agents and has sufficient sensitivity towards non-target organisms to provide evidence of mixed virus infections.
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Affiliation(s)
- Raied Abou Kubaa
- Institute for Sustainable Plant Protection—National Research Council, 70126 Bari, Italy
| | - Serafina Serena Amoia
- Institute for Sustainable Plant Protection—National Research Council, 70126 Bari, Italy
- Department of Soil, Plant and Food Sciences, University of Bari Aldo Moro, 70126 Bari, Italy
| | - Giuseppe Altamura
- Institute for Sustainable Plant Protection—National Research Council, 70126 Bari, Italy
- Research, Experimentation and Education Centre in Agriculture (CRSFA) “Basile Caramia”, Via Cisternino 281, 70010 Locorotondo, Italy
| | - Angelantonio Minafra
- Institute for Sustainable Plant Protection—National Research Council, 70126 Bari, Italy
| | - Michela Chiumenti
- Institute for Sustainable Plant Protection—National Research Council, 70126 Bari, Italy
| | - Fabrizio Cillo
- Institute for Sustainable Plant Protection—National Research Council, 70126 Bari, Italy
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23
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Bahri BA, Parvathaneni RK, Spratling WT, Saxena H, Sapkota S, Raymer PL, Martinez-Espinoza AD. Whole genome sequencing of Clarireedia aff. paspali reveals potential pathogenesis factors in Clarireedia species, causal agents of dollar spot in turfgrass. Front Genet 2023; 13:1033437. [PMID: 36685867 PMCID: PMC9849252 DOI: 10.3389/fgene.2022.1033437] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/31/2022] [Accepted: 11/21/2022] [Indexed: 01/06/2023] Open
Abstract
Dollar spot is one of the most damaging diseases in turfgrass, reducing its quality and playability. Two species, Clarireedia monteithiana and C. jacksonii (formerly Sclerotinia homoeocarpa) have been reported so far in the United States To study the Clarireedia genome, two isolates H2 and H3, sampled from seashore paspalum in Hawaii in 2019 were sequenced via Illumina paired-end sequencing by synthesis technology and PacBio SMRT sequencing. Both isolates were identified as C. aff. paspali, a novel species in the United States Using short and long reads, C. aff. paspali H3 contained 193 contigs with 48.6 Mbp and presented the most completed assembly and annotation among Clarireedia species. Out of the 13,428 protein models from AUGUSTUS, 349 cytoplasmic effectors and 13 apoplastic effectors were identified by EffectorP. To further decipher Clarireedia pathogenicity, C. aff. paspali genomes (H2 and H3), as well as available C. jacksonii (LWC-10 and HRI11), C. monteithiana (DRR09 and RB-19) genomes were screened for fifty-four pathogenesis determinants, previously identified in S. sclerotiorum. Seventeen orthologs of pathogenicity genes have been identified in Clarireedia species involved in oxalic acid production (pac1, nox1), mitogen-activated protein kinase cascade (pka1, smk3, ste12), appressorium formation (caf1, pks13, ams2, rgb1, rhs1) and glycolytic pathway (gpd). Within these genes, 366 species-specific SNPs were recorded between Clarireedia species; twenty-eight were non-synonymous and non-conservative. The predicted protein structure of six of these genes showed superimposition of the models among Clarireedia spp. The genomic variations revealed here could potentially lead to differences in pathogenesis and other physiological functions among Clarireedia species.
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Affiliation(s)
- Bochra Amina Bahri
- Department of Plant Pathology, University of Georgia, Griffin, GA, United States,Institute of Plant Breeding, Genetics and Genomics, University of Georgia, Griffin, GA, United States,*Correspondence: Bochra Amina Bahri,
| | - Rajiv Krishna Parvathaneni
- Department of Plant Pathology, University of Georgia, Griffin, GA, United States,Institute of Plant Breeding, Genetics and Genomics, University of Georgia, Griffin, GA, United States
| | | | - Harshita Saxena
- Institute of Plant Breeding, Genetics and Genomics, University of Georgia, Griffin, GA, United States
| | - Suraj Sapkota
- Department of Plant Pathology, University of Georgia, Griffin, GA, United States
| | - Paul L. Raymer
- Institute of Plant Breeding, Genetics and Genomics, University of Georgia, Griffin, GA, United States,Department of Crop and Soil Sciences, University of Georgia, Griffin, GA, United States
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24
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Ahmed FK, Alghuthaymi MA, Abd-Elsalam KA, Ravichandran M, Kalia A. Nano-Based Robotic Technologies for Plant Disease Diagnosis. NANOROBOTICS AND NANODIAGNOSTICS IN INTEGRATIVE BIOLOGY AND BIOMEDICINE 2023:327-359. [DOI: 10.1007/978-3-031-16084-4_14] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 09/01/2023]
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25
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Hamim I, Sekine KT, Komatsu K. How do emerging long-read sequencing technologies function in transforming the plant pathology research landscape? PLANT MOLECULAR BIOLOGY 2022; 110:469-484. [PMID: 35962900 DOI: 10.1007/s11103-022-01305-5] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 03/03/2022] [Accepted: 07/26/2022] [Indexed: 06/15/2023]
Abstract
Long-read sequencing technologies are revolutionizing the sequencing and analysis of plant and pathogen genomes and transcriptomes, as well as contributing to emerging areas of interest in plant-pathogen interactions, disease management techniques, and the introduction of new plant varieties or cultivars. Long-read sequencing (LRS) technologies are progressively being implemented to study plants and pathogens of agricultural importance, which have substantial economic effects. The variability and complexity of the genome and transcriptome affect plant growth, development and pathogen responses. Overcoming the limitations of second-generation sequencing, LRS technology has significantly increased the length of a single contiguous read from a few hundred to millions of base pairs. Because of the longer read lengths, new analysis methods and tools have been developed for plant and pathogen genomics and transcriptomics. LRS technologies enable faster, more efficient, and high-throughput ultralong reads, allowing direct sequencing of genomes that would be impossible or difficult to investigate using short-read sequencing approaches. These benefits include genome assembly in repetitive areas, creating more comprehensive and exact genome determinations, assembling full-length transcripts, and detecting DNA and RNA alterations. Furthermore, these technologies allow for the identification of transcriptome diversity, significant structural variation analysis, and direct epigenetic mark detection in plant and pathogen genomic regions. LRS in plant pathology is found efficient for identifying and characterization of effectors in plants as well as known and unknown plant pathogens. In this review, we investigate how these technologies are transforming the landscape of determination and characterization of plant and pathogen genomes and transcriptomes efficiently and accurately. Moreover, we highlight potential areas of interest offered by LRS technologies for future study into plant-pathogen interactions, disease control strategies, and the development of new plant varieties or cultivars.
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Affiliation(s)
- Islam Hamim
- Laboratory of Plant Pathology, Graduate School of Agriculture, Tokyo University of Agriculture and Technology, Fuchu, Japan
- International Research Fellow of Japan Society for the Promotion of Science, Tokyo, Japan
- Department of Plant Pathology, Bangladesh Agricultural University, Mymensingh, Bangladesh
| | - Ken-Taro Sekine
- Faculty of Agriculture, University of the Ryukyus, Okinawa, Japan
| | - Ken Komatsu
- Laboratory of Plant Pathology, Graduate School of Agriculture, Tokyo University of Agriculture and Technology, Fuchu, Japan.
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26
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Bollmann-Giolai A, Malone JG, Arora S. Diversity, detection and exploitation: linking soil fungi and plant disease. Curr Opin Microbiol 2022; 70:102199. [PMID: 36108394 DOI: 10.1016/j.mib.2022.102199] [Citation(s) in RCA: 10] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/21/2022] [Revised: 06/22/2022] [Accepted: 08/03/2022] [Indexed: 01/25/2023]
Abstract
Plant-associated fungi are incredibly diverse, comprising over a million species of mycorrhiza, endophytes, saprophytes and pathogens worldwide. This diverse fungal community is highly important for plant health. Many fungi are effective biocontrol agents that can kill or suppress fungal pathogens, with pathogen biocontrol found for both individual microorganisms and plant-associated fungal consortia. Meanwhile, increased plant community diversity aboveground corresponds to an increase in below-ground fungal community diversity, which contributes in turn to improved rhizosphere soil health and pathogen suppression. In this review, we discuss the role of fungal diversity in soil health and plant disease suppression and the various mechanisms by which mycorrhizal and endophytic fungi combat plant pathogenic fungi. We also discuss the array of diagnostic tools, both well-established and newly developed, which are revolutionising fungal pathogen detection and rhizosphere community analysis.
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Affiliation(s)
- Anita Bollmann-Giolai
- Department of Evolutionary Biology and Environmental Studies, University of Zurich, CH-8006 Zurich, Switzerland
| | - Jacob G Malone
- John Innes Centre, Norwich Research Park, Norwich NR4 7UH, UK; School of Biological Sciences, University of East Anglia, Norwich NR4 7TJ, UK.
| | - Sanu Arora
- John Innes Centre, Norwich Research Park, Norwich NR4 7UH, UK.
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27
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MinION Whole-Genome Sequencing in Resource-Limited Settings: Challenges and Opportunities. CURRENT CLINICAL MICROBIOLOGY REPORTS 2022; 9:52-59. [DOI: 10.1007/s40588-022-00183-1] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 10/04/2022] [Indexed: 11/18/2022]
Abstract
Abstract
Purpose of Review
The introduction of MinION whole-genome sequencing technology greatly increased and simplified complete genome sequencing in various fields of science across the globe. Sequences have been generated from complex organisms to microorganisms and are stored in genome databases that are readily accessible by researchers. Various new software for genome analysis, along with upgrades to older software packages, are being generated. New protocols are also being validated that enable WGS technology to be rapidly and increasingly used for sequencing in field settings.
Recent Findings
MinION WGS technology has been implemented in developed countries due to its advantages: portability, real-time analysis, and lower cost compared to other sequencing technologies. While these same advantages are critical in developing countries, MinION WGS technology is still under-utilized in resource-limited settings.
Summary
In this review, we look at the applications, advantages, challenges, and opportunities of using MinION WGS in resource-limited settings.
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28
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Lee HJ, Cho IS, Jeong RD. Nanopore Metagenomics Sequencing for Rapid Diagnosis and Characterization of Lily Viruses. THE PLANT PATHOLOGY JOURNAL 2022; 38:503-512. [PMID: 36221922 PMCID: PMC9561158 DOI: 10.5423/ppj.oa.06.2022.0084] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 06/27/2022] [Revised: 07/28/2022] [Accepted: 08/12/2022] [Indexed: 05/21/2023]
Abstract
Lilies (Lilium spp.) are one of the most important ornamental flower crops grown in Korea. Most viral diseases in lilies are transmitted by infected bulbs, which cause serious economic losses due to reduced yields. Various diagnostic techniques and high-throughput sequencing methods have been used to detect lily viruses. According to Oxford Nanopore Technologies (ONT), MinION is a compact and portable sequencing device. In this study, three plant viruses, lily mottle, lily symptomless, and plantago asiatica mosaic virus, were detected in lily samples using the ONT platform. As a result of genome assembly of reads obtained through ONT, 100% coverage and 90.3-93.4% identity were obtained. Thus, we show that the ONT platform is a promising tool for the diagnosis and characterization of viruses that infect crops.
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Affiliation(s)
- Hyo-Jeong Lee
- Department of Applied Biology, Institute of Environmentally Friendly Agriculture, Chonnam National University, Gwangju 61185,
Korea
| | - In-Sook Cho
- Horticultural and Herbal Crop Environment Division, National Institute of Horticultural and Herbal Science, Rural Development Administration, Wanju 55365,
Korea
| | - Rae-Dong Jeong
- Department of Applied Biology, Institute of Environmentally Friendly Agriculture, Chonnam National University, Gwangju 61185,
Korea
- Corresponding author. Phone) +82-62-530-2075, FAX) +82-62-530-2069, E-mail)
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29
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Waite DW, Liefting L, Delmiglio C, Chernyavtseva A, Ha HJ, Thompson JR. Development and Validation of a Bioinformatic Workflow for the Rapid Detection of Viruses in Biosecurity. Viruses 2022; 14:v14102163. [PMID: 36298719 PMCID: PMC9610911 DOI: 10.3390/v14102163] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/15/2022] [Accepted: 09/25/2022] [Indexed: 11/05/2022] Open
Abstract
The field of biosecurity has greatly benefited from the widespread adoption of high-throughput sequencing technologies, for its ability to deeply query plant and animal samples for pathogens for which no tests exist. However, the bioinformatics analysis tools designed for rapid analysis of these sequencing datasets are not developed with this application in mind, limiting the ability of diagnosticians to standardise their workflows using published tool kits. We sought to assess previously published bioinformatic tools for their ability to identify plant- and animal-infecting viruses while distinguishing from the host genetic material. We discovered that many of the current generation of virus-detection pipelines are not adequate for this task, being outperformed by more generic classification tools. We created synthetic MinION and HiSeq libraries simulating plant and animal infections of economically important viruses and assessed a series of tools for their suitability for rapid and accurate detection of infection, and further tested the top performing tools against the VIROMOCK Challenge dataset to ensure that our findings were reproducible when compared with international standards. Our work demonstrated that several methods provide sensitive and specific detection of agriculturally important viruses in a timely manner and provides a key piece of ground truthing for method development in this space.
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Affiliation(s)
- David W. Waite
- Plant Health and Environment Laboratory, Ministry for Primary Industries, P.O. Box 2095, Auckland 1140, New Zealand
- Correspondence:
| | - Lia Liefting
- Plant Health and Environment Laboratory, Ministry for Primary Industries, P.O. Box 2095, Auckland 1140, New Zealand
| | - Catia Delmiglio
- Plant Health and Environment Laboratory, Ministry for Primary Industries, P.O. Box 2095, Auckland 1140, New Zealand
| | | | - Hye Jeong Ha
- Animal Health Laboratory, Ministry for Primary Industries, Upper Hutt 5018, New Zealand
| | - Jeremy R. Thompson
- Plant Health and Environment Laboratory, Ministry for Primary Industries, P.O. Box 2095, Auckland 1140, New Zealand
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30
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Volk GM, Bonnart R, de Oliveira ACA, Henk AD. Minimizing the deleterious effects of endophytes in plant shoot tip cryopreservation. APPLICATIONS IN PLANT SCIENCES 2022; 10:e11489. [PMID: 36258787 PMCID: PMC9575093 DOI: 10.1002/aps3.11489] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 12/01/2021] [Revised: 02/23/2022] [Accepted: 03/15/2022] [Indexed: 05/05/2023]
Abstract
Plant cryopreservation technologies are used within gene banks for the long-term preservation of vegetatively propagated collections. Surface-sterilized plant tissues grown in the field, greenhouse/screenhouse, growth chamber, or in vitro are the source of shoot tips subjected to vitrification-based cryopreservation methods. Here, we describe the methods used to minimize microbial contamination during the tissue culture initiation process. We also discuss the occurrence and possible elimination of endophytes after extended in vitro culture and during recovery after liquid nitrogen exposure. We describe two case studies in which bacterial endophytes were observed in Citrus gene bank accessions during recovery after cryopreservation. These were identified using the MinION Oxford Nanopore system and Kirby-Bauer disc diffusion assays to examine the bacterial responses to antibiotic exposure. The methods used in this case study could be applied to identify endophytes to better target antimicrobial treatments of plant tissue collections.
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Affiliation(s)
- Gayle M. Volk
- USDA‐ARS National Laboratory for Genetic Resources Preservation1111 S. Mason St.Fort CollinsColorado80521USA
| | - Remi Bonnart
- USDA‐ARS National Laboratory for Genetic Resources Preservation1111 S. Mason St.Fort CollinsColorado80521USA
| | | | - Adam D. Henk
- USDA‐ARS National Laboratory for Genetic Resources Preservation1111 S. Mason St.Fort CollinsColorado80521USA
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31
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Diouf MB, Festus R, Silva G, Guyader S, Umber M, Seal S, Teycheney PY. Viruses of Yams (Dioscorea spp.): Current Gaps in Knowledge and Future Research Directions to Improve Disease Management. Viruses 2022; 14:v14091884. [PMID: 36146691 PMCID: PMC9501508 DOI: 10.3390/v14091884] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/26/2022] [Revised: 08/19/2022] [Accepted: 08/22/2022] [Indexed: 12/24/2022] Open
Abstract
Viruses are a major constraint for yam production worldwide. They hamper the conservation, movement, and exchange of yam germplasm and are a threat to food security in tropical and subtropical areas of Africa and the Pacific where yam is a staple food and a source of income. However, the biology and impact of yam viruses remains largely unknown. This review summarizes current knowledge on yam viruses and emphasizes gaps that exist in the knowledge of the biology of these viruses, their diagnosis, and their impact on production. It provides essential information to inform the implementation of more effective virus control strategies.
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Affiliation(s)
- Mame Boucar Diouf
- INRAE, UR ASTRO, F-97170 Petit-Bourg, France
- CIRAD, UMR AGAP Institut, F-97130 Capesterre-Belle-Eau, France
- UMR AGAP Institut, University Montpellier, CIRAD, INRAE, Institut Agro, F-97130 Capesterre-Belle-Eau, France
| | - Ruth Festus
- Natural Resources Institute, University of Greenwich, Central Avenue, Chatham Maritime, Kent ME4 4TB, UK
| | - Gonçalo Silva
- Natural Resources Institute, University of Greenwich, Central Avenue, Chatham Maritime, Kent ME4 4TB, UK
| | | | - Marie Umber
- INRAE, UR ASTRO, F-97170 Petit-Bourg, France
| | - Susan Seal
- Natural Resources Institute, University of Greenwich, Central Avenue, Chatham Maritime, Kent ME4 4TB, UK
| | - Pierre Yves Teycheney
- CIRAD, UMR PVBMT, F-97410 Saint Pierre, France
- UMR PVBMT, Université de la Réunion, F-97410 Saint-Pierre, France
- Correspondence: ; Tel.: +33-262-492-819
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32
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Sun K, Liu Y, Zhou X, Yin C, Zhang P, Yang Q, Mao L, Shentu X, Yu X. Nanopore sequencing technology and its application in plant virus diagnostics. Front Microbiol 2022; 13:939666. [PMID: 35958160 PMCID: PMC9358452 DOI: 10.3389/fmicb.2022.939666] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/09/2022] [Accepted: 07/05/2022] [Indexed: 11/13/2022] Open
Abstract
Plant viruses threaten crop yield and quality; thus, efficient and accurate pathogen diagnostics are critical for crop disease management and control. Recent advances in sequencing technology have revolutionized plant virus research. Metagenomics sequencing technology, represented by next-generation sequencing (NGS), has greatly enhanced the development of virus diagnostics research because of its high sensitivity, high throughput and non-sequence dependence. However, NGS-based virus identification protocols are limited by their high cost, labor intensiveness, and bulky equipment. In recent years, Oxford Nanopore Technologies and advances in third-generation sequencing technology have enabled direct, real-time sequencing of long DNA or RNA reads. Oxford Nanopore Technologies exhibit versatility in plant virus detection through their portable sequencers and flexible data analyses, thus are wildly used in plant virus surveillance, identification of new viruses, viral genome assembly, and evolution research. In this review, we discuss the applications of nanopore sequencing in plant virus diagnostics, as well as their limitations.
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Affiliation(s)
- Kai Sun
- Zhejiang Provincial Key Laboratory of Biometrology and Inspection and Quarantine, College of Life Sciences, China Jiliang University, Hangzhou, China
| | - Yi Liu
- Zhejiang Provincial Key Laboratory of Biometrology and Inspection and Quarantine, College of Life Sciences, China Jiliang University, Hangzhou, China
| | - Xin Zhou
- Ausper Biopharma, Hangzhou, China
| | - Chuanlin Yin
- Zhejiang Provincial Key Laboratory of Biometrology and Inspection and Quarantine, College of Life Sciences, China Jiliang University, Hangzhou, China
| | - Pengjun Zhang
- Zhejiang Provincial Key Laboratory of Biometrology and Inspection and Quarantine, College of Life Sciences, China Jiliang University, Hangzhou, China
| | - Qianqian Yang
- Zhejiang Provincial Key Laboratory of Biometrology and Inspection and Quarantine, College of Life Sciences, China Jiliang University, Hangzhou, China
| | - Lingfeng Mao
- Hangzhou Baiyi Technology Co., Ltd., Hangzhou, China
| | - Xuping Shentu
- Zhejiang Provincial Key Laboratory of Biometrology and Inspection and Quarantine, College of Life Sciences, China Jiliang University, Hangzhou, China
- *Correspondence: Xuping Shentu,
| | - Xiaoping Yu
- Zhejiang Provincial Key Laboratory of Biometrology and Inspection and Quarantine, College of Life Sciences, China Jiliang University, Hangzhou, China
- Xiaoping Yu,
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Pecman A, Adams I, Gutiérrez-Aguirre I, Fox A, Boonham N, Ravnikar M, Kutnjak D. Systematic Comparison of Nanopore and Illumina Sequencing for the Detection of Plant Viruses and Viroids Using Total RNA Sequencing Approach. Front Microbiol 2022; 13:883921. [PMID: 35633678 PMCID: PMC9131090 DOI: 10.3389/fmicb.2022.883921] [Citation(s) in RCA: 9] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/25/2022] [Accepted: 03/31/2022] [Indexed: 11/13/2022] Open
Abstract
High-throughput sequencing (HTS) has become an important tool for plant virus detection and discovery. Nanopore sequencing has been rapidly developing in the recent years and offers new possibilities for fast diagnostic applications of HTS. With this in mind, a study was completed, comparing the most established HTS platform (MiSeq benchtop sequencer-Illumina), with the MinION sequencer (Oxford Nanopore Technologies) for the detection of plant viruses and viroids. Method comparisons were performed on five selected samples, containing two viroids, which were sequenced using nanopore technology for the first time and 11 plant viruses with different genome organizations. For all samples, sequencing libraries for the MiSeq were prepared from ribosomal RNA-depleted total RNA (rRNA-depleted totRNA) and for MinION sequencing, direct RNA sequencing of totRNA was used. Moreover, for one of the samples, which contained five different plant viruses and a viroid, three additional variations of sample preparation for MinION sequencing were also used: direct RNA sequencing of rRNA-depleted totRNA, cDNA-PCR sequencing of totRNA, and cDNA-PCR sequencing of rRNA-depleted totRNA. Whilst direct RNA sequencing of total RNA was the quickest of the tested approaches, it was also the least sensitive: using this approach, we failed to detect only one virus that was present in a sample at an extremely low titer. All other MinION sequencing approaches showed improved performance with outcomes similar to Illumina sequencing, with cDNA-PCR sequencing of rRNA-depleted totRNA showing the best performance amongst tested nanopore MinION sequencing approaches. Moreover, when enough sequencing data were generated, high-quality consensus viral genome sequences could be reconstructed from MinION sequencing data, with high identity to the ones generated from Illumina data. The results of this study implicate that, when an appropriate sample and library preparation are selected, nanopore MinION sequencing could be used for the detection of plant viruses and viroids with similar performance as Illumina sequencing. Taken as a balance of practicality and performance, this suggests that MinION sequencing may be an ideal tool for fast and affordable virus diagnostics.
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Affiliation(s)
- Anja Pecman
- Department of Biotechnology and System Biology, National Institute of Biology, Ljubljana, Slovenia.,Jožef Stefan International Postgraduate School, Ljubljana, Slovenia
| | - Ian Adams
- Fera Science Ltd., York, United Kingdom
| | - Ion Gutiérrez-Aguirre
- Department of Biotechnology and System Biology, National Institute of Biology, Ljubljana, Slovenia
| | | | - Neil Boonham
- Institute for Agri-Food Research and Innovation, Newcastle University, Newcastle upon Tyne, United Kingdom
| | - Maja Ravnikar
- Department of Biotechnology and System Biology, National Institute of Biology, Ljubljana, Slovenia
| | - Denis Kutnjak
- Department of Biotechnology and System Biology, National Institute of Biology, Ljubljana, Slovenia
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Tarquini G, Martini M, Maestri S, Firrao G, Ermacora P. The Virome of ‘Lamon Bean’: Application of MinION Sequencing to Investigate the Virus Population Associated with Symptomatic Beans in the Lamon Area, Italy. PLANTS 2022; 11:plants11060779. [PMID: 35336661 PMCID: PMC8951528 DOI: 10.3390/plants11060779] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 01/31/2022] [Revised: 03/10/2022] [Accepted: 03/12/2022] [Indexed: 11/23/2022]
Abstract
‘Lamon bean’ is a protected geographical indication (PGI) for a product of four varieties of bean (Phaseolus vulgaris L.) grown in a specific area of production, which is located in the Belluno district, Veneto region (N.E. of Italy). In the last decade, the ‘Lamon bean’ has been threatened by severe virus epidemics that have compromised its profitability. In this work, the full virome of seven bean samples showing different foliar symptoms was obtained by MinION sequencing. Evidence that emerged from sequencing was validated through RT-PCR and ELISA in a large number of plants, including different ecotypes of Lamon bean and wild herbaceous hosts that may represent a virus reservoir in the field. Results revealed the presence of bean common mosaic virus (BCMV), cucumber mosaic virus (CMV), peanut stunt virus (PSV), and bean yellow mosaic virus (BYMV), which often occurred as mixed infections. Moreover, both CMV and PSV were reported in association with strain-specific satellite RNAs (satRNAs). In conclusion, this work sheds light on the cause of the severe diseases affecting the ‘Lamon bean’ by exploitation of MinION sequencing.
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Affiliation(s)
- Giulia Tarquini
- Department of Agriculture, Food, Environmental and Animal Sciences, University of Udine, I-33100 Udine, Italy; (G.T.); (M.M.); (G.F.)
| | - Marta Martini
- Department of Agriculture, Food, Environmental and Animal Sciences, University of Udine, I-33100 Udine, Italy; (G.T.); (M.M.); (G.F.)
| | - Simone Maestri
- Department of Biotechnology, University of Verona, I-37134 Verona, Italy;
| | - Giuseppe Firrao
- Department of Agriculture, Food, Environmental and Animal Sciences, University of Udine, I-33100 Udine, Italy; (G.T.); (M.M.); (G.F.)
| | - Paolo Ermacora
- Department of Agriculture, Food, Environmental and Animal Sciences, University of Udine, I-33100 Udine, Italy; (G.T.); (M.M.); (G.F.)
- Correspondence:
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Marcolungo L, Passera A, Maestri S, Segala E, Alfano M, Gaffuri F, Marturano G, Casati P, Bianco PA, Delledonne M. Real-Time On-Site Diagnosis of Quarantine Pathogens in Plant Tissues by Nanopore-Based Sequencing. Pathogens 2022; 11:pathogens11020199. [PMID: 35215142 PMCID: PMC8876587 DOI: 10.3390/pathogens11020199] [Citation(s) in RCA: 14] [Impact Index Per Article: 4.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/30/2021] [Revised: 01/23/2022] [Accepted: 01/29/2022] [Indexed: 12/31/2022] Open
Abstract
Rapid and sensitive assays for the identification of plant pathogens are necessary for the effective management of crop diseases. The main limitation of current diagnostic testing is the inability to combine broad and sensitive pathogen detection with the identification of key strains, pathovars, and subspecies. Such discrimination is necessary for quarantine pathogens, whose management is strictly dependent on genotype identification. To address these needs, we have established and evaluated a novel all-in-one diagnostic assay based on nanopore sequencing for the detection and simultaneous characterization of quarantine pathogens, using Xylella fastidiosa as a case study. The assay proved to be at least as sensitive as standard diagnostic tests and the quantitative results agreed closely with qPCR-based analysis. The same sequencing results also allowed discrimination between subspecies when present either individually or in combination. Pathogen detection and typing were achieved within 13 min of sequencing owing to the use of an internal control that allowed to stop sequencing when sufficient data had accumulated. These advantages, combined with the use of portable equipment, will facilitate the development of next-generation diagnostic assays for the efficient monitoring of other plant pathogens.
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Affiliation(s)
- Luca Marcolungo
- Department of Biotechnology, University of Verona, Strada Le Grazie 15, 37134 Verona, Italy; (L.M.); (S.M.); (E.S.); (M.A.); (G.M.)
| | - Alessandro Passera
- Department of Agricultural and Environmental Sciences—Production, Landscape, Agroenergy, University of Milano, Via Celoria 2, 20133 Milan, Italy; (A.P.); (P.C.); (P.A.B.)
| | - Simone Maestri
- Department of Biotechnology, University of Verona, Strada Le Grazie 15, 37134 Verona, Italy; (L.M.); (S.M.); (E.S.); (M.A.); (G.M.)
| | - Elena Segala
- Department of Biotechnology, University of Verona, Strada Le Grazie 15, 37134 Verona, Italy; (L.M.); (S.M.); (E.S.); (M.A.); (G.M.)
| | - Massimiliano Alfano
- Department of Biotechnology, University of Verona, Strada Le Grazie 15, 37134 Verona, Italy; (L.M.); (S.M.); (E.S.); (M.A.); (G.M.)
| | - Francesca Gaffuri
- Servizio Fitosanitario Regione Lombardia Laboratorio Fitopatologico c/o Fondazione Minoprio, 22100 Minoprio, Italy;
| | - Giovanni Marturano
- Department of Biotechnology, University of Verona, Strada Le Grazie 15, 37134 Verona, Italy; (L.M.); (S.M.); (E.S.); (M.A.); (G.M.)
| | - Paola Casati
- Department of Agricultural and Environmental Sciences—Production, Landscape, Agroenergy, University of Milano, Via Celoria 2, 20133 Milan, Italy; (A.P.); (P.C.); (P.A.B.)
| | - Piero Attilio Bianco
- Department of Agricultural and Environmental Sciences—Production, Landscape, Agroenergy, University of Milano, Via Celoria 2, 20133 Milan, Italy; (A.P.); (P.C.); (P.A.B.)
- Institute for Sustainable Plant Protection, National Research Council (IPSP-CNR), Strada delle Cacce, 73, 10135 Turin, Italy
| | - Massimo Delledonne
- Department of Biotechnology, University of Verona, Strada Le Grazie 15, 37134 Verona, Italy; (L.M.); (S.M.); (E.S.); (M.A.); (G.M.)
- Genartis S.r.l., Via P. Mascagni 98, 37060 Castel D’Azzano, Italy
- Correspondence: ; Tel.: +39-045-802-7962
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Yang S, Johnson MA, Hansen MA, Bush E, Li S, Vinatzer BA. Metagenomic sequencing for detection and identification of the boxwood blight pathogen Calonectria pseudonaviculata. Sci Rep 2022; 12:1399. [PMID: 35082361 PMCID: PMC8791934 DOI: 10.1038/s41598-022-05381-x] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/27/2021] [Accepted: 01/10/2022] [Indexed: 11/16/2022] Open
Abstract
Pathogen detection and identification are key elements in outbreak control of human, animal, and plant diseases. Since many fungal plant pathogens cause similar symptoms, are difficult to distinguish morphologically, and grow slowly in culture, culture-independent, sequence-based diagnostic methods are desirable. Whole genome metagenomic sequencing has emerged as a promising technique because it can potentially detect any pathogen without culturing and without the need for pathogen-specific probes. However, efficient DNA extraction protocols, computational tools, and sequence databases are required. Here we applied metagenomic sequencing with the Oxford Nanopore Technologies MinION to the detection of the fungus Calonectria pseudonaviculata, the causal agent of boxwood (Buxus spp.) blight disease. Two DNA extraction protocols, several DNA purification kits, and various computational tools were tested. All DNA extraction methods and purification kits provided sufficient quantity and quality of DNA. Several bioinformatics tools for taxonomic identification were found suitable to assign sequencing reads to the pathogen with an extremely low false positive rate. Over 9% of total reads were identified as C. pseudonaviculata in a severely diseased sample and identification at strain-level resolution was approached as the number of sequencing reads was increased. We discuss how metagenomic sequencing could be implemented in routine plant disease diagnostics.
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Affiliation(s)
- Shu Yang
- School of Plant and Environmental Sciences, Virginia Tech, Blacksburg, VA, USA
| | - Marcela A Johnson
- School of Plant and Environmental Sciences, Virginia Tech, Blacksburg, VA, USA.,Graduate Program in Genetics, Bioinformatics, and Computational Biology, Virginia Tech, Blacksburg, VA, USA
| | - Mary Ann Hansen
- School of Plant and Environmental Sciences, Virginia Tech, Blacksburg, VA, USA
| | - Elizabeth Bush
- School of Plant and Environmental Sciences, Virginia Tech, Blacksburg, VA, USA
| | - Song Li
- School of Plant and Environmental Sciences, Virginia Tech, Blacksburg, VA, USA
| | - Boris A Vinatzer
- School of Plant and Environmental Sciences, Virginia Tech, Blacksburg, VA, USA.
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Amoia SS, Minafra A, Nicoloso V, Loconsole G, Chiumenti M. A New Jasmine Virus C Isolate Identified by Nanopore Sequencing Is Associated to Yellow Mosaic Symptoms of Jasminum officinale in Italy. PLANTS (BASEL, SWITZERLAND) 2022; 11:plants11030309. [PMID: 35161290 PMCID: PMC8839810 DOI: 10.3390/plants11030309] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 12/27/2021] [Revised: 01/20/2022] [Accepted: 01/20/2022] [Indexed: 05/11/2023]
Abstract
Some plants of Jasminum officinale were selected in a nursery for investigation of sanitary status of candidate mother plants before vegetative propagation. The presence of yellow spots and leaf discoloration symptoms pushed for a generic diagnosis through deep sequencing to discover systemic pathogens. Either dsRNA or total RNA were extracted and used in nanopore and Illumina platform for cDNA-PCR, direct RNA and total RNA rRNA-depleted sequencing. A few single reads obtained by nanopore technology or assembled contigs gave unequivocal annotation for the only presence of a jasmine virus C (JaVC, a putative member of genus Carlavirus) isolate. The full-length genome of this isolate was reconstructed, spanning 8490 nucleotides (nt). This isolate shared 90.9% similarity with coat protein sequences and 84% with the entire ORF1 polyprotein, with the other two available JaVC full genomes, isolated from infections in J. sambac in Taiwan and China. The overall nucleotide identity shared by the newly discovered Italian isolate with the Chinese JaVC full genomes was 76.14% (Taiwan) and 75.60% (Fujian). The application of quick nanopore sequencing for virus discovery was assessed. The identification of the virus in a new ornamental host species, largely used in gardening, creates a concern for the potential virus spread and need of testing for production of clean vegetative material.
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Haveman NJ, Schuerger AC. Diagnosing an Opportunistic Fungal Pathogen on Spaceflight-Grown Plants Using the MinION Sequencing Platform. ASTROBIOLOGY 2022; 22:1-6. [PMID: 34793258 DOI: 10.1089/ast.2021.0049] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 06/13/2023]
Abstract
Sustainable agriculture in microgravity is integral to future long-term human space exploration. To ensure the efficient and sustainable cultivation of plants in space, a contingency plan to monitor plant health and mitigate plant diseases is necessary. Yet, neither methods nor tools currently exist to evaluate the plant microbial interactions or to diagnose potential plant diseases in space-based bioregenerative life support systems. In this study, we show how the MinION sequencing platform can be used to diagnose the opportunistic pathogen Fusarium oxysporum sensu lato, a fungal infection on Zinnia hybrida (zinnia) plants that were grown on the International Space Station (ISS) in 2015-2016. Genomic DNA from the infected plant material (root and leaf tissues) retrieved from the ISS were extracted and sequenced. In addition, pure cultures of Burkholderia contaminans, F. oxysporum sensu lato, and Fusarium sporotrichioides were used as controls to test the specificity of the bioinformatics pipeline developed. The results show that the MinION platform can be used to accurately differentiate between fusaria species and strengthens the case for using the platform as a rapid plant disease diagnostic tool in space.
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Affiliation(s)
- Natasha J Haveman
- Department of Horticultural Sciences, University of Florida, Gainesville, Florida, USA
| | - Andrew C Schuerger
- Department of Plant Pathology, University of Florida, Merritt Island, Florida, USA
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Vazquez-Iglesias I, McGreig S, Pufal H, Robinson R, Clover GRG, Fox A, Boonham N, Adams IP. A novel high-throughput sequencing approach reveals the presence of a new virus infecting Rosa: rosa ilarvirus-1 (RIV-1). J Virol Methods 2021; 300:114417. [PMID: 34902457 DOI: 10.1016/j.jviromet.2021.114417] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/05/2021] [Revised: 12/03/2021] [Accepted: 12/08/2021] [Indexed: 01/11/2023]
Abstract
Roses are one of the most valuable ornamental flowering shrubs grown worldwide. Despite the widespread of rose viruses and their impact on cultivation, they have not been studied in detail in the United Kingdom (UK) since the 1980's. As part of a survey of rose viruses entering the UK, 35 samples were collected at Heathrow Airport (London, UK) and were tested by RT-qPCR for different common rose viruses. Of the 35 samples tested using RT-qPCR for prunus necrotic ringspot virus (PNRSV; genus Ilarvirus), 10 were positive. Confirmatory testing was performed using RT-PCR with both PNRSV-specific and ilarvirus-generic primers, and diverse results were obtained: One sample was exclusively positive when using the ilarvirus-generic primers, and subsequent sequencing of the RT-PCR product revealed homology to other ilarviruses but not PNRSV. Further work to characterise the virus was performed using high throughput sequencing, both the MinION Flongle and Illumina MiSeq. The sequencing confirmed the presence of a new virus within group 2 of the genus Ilarvirus and we propose the name "rosa ilarvirus-1″ (RIV-1). Here, we describe the identification of a novel virus using the low-cost Flongle flow cell and discuss its potential as a front-line diagnostic tool.
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Affiliation(s)
- Ines Vazquez-Iglesias
- Fera Science Ltd., York Biotech Campus, Sand Hutton, York, YO41 1LZ, United Kingdom; School of Natural and Environmental Sciences, Agriculture Building, King's Road, Newcastle upon Tyne, NE1 7RU, United Kingdom.
| | - Sam McGreig
- Fera Science Ltd., York Biotech Campus, Sand Hutton, York, YO41 1LZ, United Kingdom
| | - Hollie Pufal
- Fera Science Ltd., York Biotech Campus, Sand Hutton, York, YO41 1LZ, United Kingdom
| | - Rebekah Robinson
- The Royal Horticultural Society, Wisley, Woking, Surrey, GU23 6QB, United Kingdom
| | - Gerard R G Clover
- The Royal Horticultural Society, Wisley, Woking, Surrey, GU23 6QB, United Kingdom
| | - Adrian Fox
- Fera Science Ltd., York Biotech Campus, Sand Hutton, York, YO41 1LZ, United Kingdom
| | - Neil Boonham
- School of Natural and Environmental Sciences, Agriculture Building, King's Road, Newcastle upon Tyne, NE1 7RU, United Kingdom
| | - Ian P Adams
- Fera Science Ltd., York Biotech Campus, Sand Hutton, York, YO41 1LZ, United Kingdom; Institute of Agri Food Research, Newcastle University, Newcastle upon Tyne, NE1 7RU, United Kingdom
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40
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Mango Endophyte and Epiphyte Microbiome Composition during Fruit Development and Post-Harvest Stages. HORTICULTURAE 2021. [DOI: 10.3390/horticulturae7110495] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/16/2022]
Abstract
The influence of the development stage and post-harvest handling on the microbial composition of mango fruit plays a central role in fruit health. Hence, the composition of fungal and bacterial microbiota on the anthoplane, fructoplane, stems and stem-end pulp of mango during fruit development and post-harvest handling were determined using next-generation sequencing of the internal transcribed spacer and 16S rRNA regions. At full bloom, the inflorescence had the richest fungal and bacterial communities. The young developing fruit exhibited lower fungal richness and diversities in comparison to the intermediate and fully developed fruit stages on the fructoplane. At the post-harvest stage, lower fungal and bacterial diversities were observed following prochloraz treatment both on the fructoplane and stem-end pulp. Ascomycota (52.8%) and Basidiomycota (43.2%) were the most dominant fungal phyla, while Penicillium, Botryosphaeria, Alternaria and Mucor were detected as the known post-harvest decay-causing fungal genera. The Cyanobacteria (35.6%), Firmicutes (26.1%) and Proteobacteria (23.1%) were the most dominant bacterial phyla. Changes in the presence of Bacillus subtilis following post-harvest interventions such as prochloraz suggested a non-target effect of the fungicide. The present study, therefore, provides the primary baseline data on mango fungal and bacterial diversity and composition, which can be foundational in the development of effective disease (stem-end rot) management strategies.
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Current practices and emerging possibilities for reducing the spread of oomycete pathogens in terrestrial and aquatic production systems in the European Union. FUNGAL BIOL REV 2021. [DOI: 10.1016/j.fbr.2021.10.001] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022]
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Javaran VJ, Moffett P, Lemoyne P, Xu D, Adkar-Purushothama CR, Fall ML. Grapevine Virology in the Third-Generation Sequencing Era: From Virus Detection to Viral Epitranscriptomics. PLANTS (BASEL, SWITZERLAND) 2021; 10:plants10112355. [PMID: 34834718 PMCID: PMC8623739 DOI: 10.3390/plants10112355] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 09/16/2021] [Revised: 10/16/2021] [Accepted: 10/29/2021] [Indexed: 05/30/2023]
Abstract
Among all economically important plant species in the world, grapevine (Vitis vinifera L.) is the most cultivated fruit plant. It has a significant impact on the economies of many countries through wine and fresh and dried fruit production. In recent years, the grape and wine industry has been facing outbreaks of known and emerging viral diseases across the world. Although high-throughput sequencing (HTS) has been used extensively in grapevine virology, the application and potential of third-generation sequencing have not been explored in understanding grapevine viruses and their impact on the grapevine. Nanopore sequencing, a third-generation technology, can be used for the direct sequencing of both RNA and DNA with minimal infrastructure. Compared to other HTS methods, the MinION nanopore platform is faster and more cost-effective and allows for long-read sequencing. Due to the size of the MinION device, it can be easily carried for field viral disease surveillance. This review article discusses grapevine viruses, the principle of third-generation sequencing platforms, and the application of nanopore sequencing technology in grapevine virus detection, virus-plant interactions, as well as the characterization of viral RNA modifications.
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Affiliation(s)
- Vahid Jalali Javaran
- Saint-Jean-sur-Richelieu Research and Development Centre, Agriculture and Agri-Food Canada, Saint-Jean-sur-Richelieu, QC J3B 3E6, Canada; (V.J.J.); (P.L.); (D.X.)
- Département de Biologie, Centre SÈVE, Université de Sherbrooke, Sherbrooke, QC J1K 2R1, Canada;
| | - Peter Moffett
- Département de Biologie, Centre SÈVE, Université de Sherbrooke, Sherbrooke, QC J1K 2R1, Canada;
| | - Pierre Lemoyne
- Saint-Jean-sur-Richelieu Research and Development Centre, Agriculture and Agri-Food Canada, Saint-Jean-sur-Richelieu, QC J3B 3E6, Canada; (V.J.J.); (P.L.); (D.X.)
| | - Dong Xu
- Saint-Jean-sur-Richelieu Research and Development Centre, Agriculture and Agri-Food Canada, Saint-Jean-sur-Richelieu, QC J3B 3E6, Canada; (V.J.J.); (P.L.); (D.X.)
| | - Charith Raj Adkar-Purushothama
- Département de Biochimie, Faculté de Médecine des Sciences de la Santé, 3201 rue Jean-Mignault, Sherbrooke, QC J1E 4K8, Canada;
| | - Mamadou Lamine Fall
- Saint-Jean-sur-Richelieu Research and Development Centre, Agriculture and Agri-Food Canada, Saint-Jean-sur-Richelieu, QC J3B 3E6, Canada; (V.J.J.); (P.L.); (D.X.)
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Gogolev YV, Ahmar S, Akpinar BA, Budak H, Kiryushkin AS, Gorshkov VY, Hensel G, Demchenko KN, Kovalchuk I, Mora-Poblete F, Muslu T, Tsers ID, Yadav NS, Korzun V. OMICs, Epigenetics, and Genome Editing Techniques for Food and Nutritional Security. PLANTS (BASEL, SWITZERLAND) 2021; 10:1423. [PMID: 34371624 PMCID: PMC8309286 DOI: 10.3390/plants10071423] [Citation(s) in RCA: 11] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 05/13/2021] [Revised: 06/30/2021] [Accepted: 07/07/2021] [Indexed: 12/22/2022]
Abstract
The incredible success of crop breeding and agricultural innovation in the last century greatly contributed to the Green Revolution, which significantly increased yields and ensures food security, despite the population explosion. However, new challenges such as rapid climate change, deteriorating soil, and the accumulation of pollutants require much faster responses and more effective solutions that cannot be achieved through traditional breeding. Further prospects for increasing the efficiency of agriculture are undoubtedly associated with the inclusion in the breeding strategy of new knowledge obtained using high-throughput technologies and new tools in the future to ensure the design of new plant genomes and predict the desired phenotype. This article provides an overview of the current state of research in these areas, as well as the study of soil and plant microbiomes, and the prospective use of their potential in a new field of microbiome engineering. In terms of genomic and phenomic predictions, we also propose an integrated approach that combines high-density genotyping and high-throughput phenotyping techniques, which can improve the prediction accuracy of quantitative traits in crop species.
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Affiliation(s)
- Yuri V. Gogolev
- Federal Research Center Kazan Scientific Center of Russian Academy of Sciences, Kazan Institute of Biochemistry and Biophysics, 420111 Kazan, Russia;
- Federal Research Center Kazan Scientific Center of Russian Academy of Sciences, Laboratory of Plant Infectious Diseases, 420111 Kazan, Russia;
| | - Sunny Ahmar
- Institute of Biological Sciences, University of Talca, 1 Poniente 1141, Talca 3460000, Chile; (S.A.); (F.M.-P.)
| | | | - Hikmet Budak
- Montana BioAg Inc., Missoula, MT 59802, USA; (B.A.A.); (H.B.)
| | - Alexey S. Kiryushkin
- Laboratory of Cellular and Molecular Mechanisms of Plant Development, Komarov Botanical Institute of the Russian Academy of Sciences, 197376 Saint Petersburg, Russia; (A.S.K.); (K.N.D.)
| | - Vladimir Y. Gorshkov
- Federal Research Center Kazan Scientific Center of Russian Academy of Sciences, Kazan Institute of Biochemistry and Biophysics, 420111 Kazan, Russia;
- Federal Research Center Kazan Scientific Center of Russian Academy of Sciences, Laboratory of Plant Infectious Diseases, 420111 Kazan, Russia;
| | - Goetz Hensel
- Centre for Plant Genome Engineering, Institute of Plant Biochemistry, Heinrich-Heine-University, 40225 Dusseldorf, Germany;
- Centre of the Region Haná for Biotechnological and Agricultural Research, Czech Advanced Technology and Research Institute, Palacký University Olomouc, 78371 Olomouc, Czech Republic
| | - Kirill N. Demchenko
- Laboratory of Cellular and Molecular Mechanisms of Plant Development, Komarov Botanical Institute of the Russian Academy of Sciences, 197376 Saint Petersburg, Russia; (A.S.K.); (K.N.D.)
| | - Igor Kovalchuk
- Department of Biological Sciences, University of Lethbridge, Lethbridge, AB T1K 3M4, Canada; (I.K.); (N.S.Y.)
| | - Freddy Mora-Poblete
- Institute of Biological Sciences, University of Talca, 1 Poniente 1141, Talca 3460000, Chile; (S.A.); (F.M.-P.)
| | - Tugdem Muslu
- Faculty of Engineering and Natural Sciences, Sabanci University, 34956 Istanbul, Turkey;
| | - Ivan D. Tsers
- Federal Research Center Kazan Scientific Center of Russian Academy of Sciences, Laboratory of Plant Infectious Diseases, 420111 Kazan, Russia;
| | - Narendra Singh Yadav
- Department of Biological Sciences, University of Lethbridge, Lethbridge, AB T1K 3M4, Canada; (I.K.); (N.S.Y.)
| | - Viktor Korzun
- Federal Research Center Kazan Scientific Center of Russian Academy of Sciences, Laboratory of Plant Infectious Diseases, 420111 Kazan, Russia;
- KWS SAAT SE & Co. KGaA, Grimsehlstr. 31, 37555 Einbeck, Germany
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Silva G, Tomlinson J, Onkokesung N, Sommer S, Mrisho L, Legg J, Adams IP, Gutierrez-Vazquez Y, Howard TP, Laverick A, Hossain O, Wei Q, Gold KM, Boonham N. Plant pest surveillance: from satellites to molecules. Emerg Top Life Sci 2021; 5:275-287. [PMID: 33720345 PMCID: PMC8166340 DOI: 10.1042/etls20200300] [Citation(s) in RCA: 12] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/18/2020] [Revised: 02/19/2021] [Accepted: 02/22/2021] [Indexed: 11/18/2022]
Abstract
Plant pests and diseases impact both food security and natural ecosystems, and the impact has been accelerated in recent years due to several confounding factors. The globalisation of trade has moved pests out of natural ranges, creating damaging epidemics in new regions. Climate change has extended the range of pests and the pathogens they vector. Resistance to agrochemicals has made pathogens, pests, and weeds more difficult to control. Early detection is critical to achieve effective control, both from a biosecurity as well as an endemic pest perspective. Molecular diagnostics has revolutionised our ability to identify pests and diseases over the past two decades, but more recent technological innovations are enabling us to achieve better pest surveillance. In this review, we will explore the different technologies that are enabling this advancing capability and discuss the drivers that will shape its future deployment.
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Affiliation(s)
- Gonçalo Silva
- Natural Resources Institute, University of Greenwich, Central Avenue, Chatham Maritime, Kent ME4 4TB, U.K
| | - Jenny Tomlinson
- Fera Science Ltd., York Biotech Campus, Sand Hutton, York YO41 1LZ, U.K
| | - Nawaporn Onkokesung
- School of Natural and Environmental Sciences, Agriculture Building, Newcastle University, King's Road, Newcastle upon Tyne NE1 7RU, U.K
| | - Sarah Sommer
- School of Natural and Environmental Sciences, Agriculture Building, Newcastle University, King's Road, Newcastle upon Tyne NE1 7RU, U.K
| | - Latifa Mrisho
- International Institute of Tropical Agriculture, Dar el Salaam, Tanzania
| | - James Legg
- International Institute of Tropical Agriculture, Dar el Salaam, Tanzania
| | - Ian P Adams
- Fera Science Ltd., York Biotech Campus, Sand Hutton, York YO41 1LZ, U.K
| | | | - Thomas P Howard
- School of Natural and Environmental Sciences, Agriculture Building, Newcastle University, King's Road, Newcastle upon Tyne NE1 7RU, U.K
| | - Alex Laverick
- School of Natural and Environmental Sciences, Agriculture Building, Newcastle University, King's Road, Newcastle upon Tyne NE1 7RU, U.K
| | - Oindrila Hossain
- Department of Chemical and Biomolecular Engineering, North Carolina State University, Raleigh, NC 27695, U.S.A
| | - Qingshan Wei
- Department of Chemical and Biomolecular Engineering, North Carolina State University, Raleigh, NC 27695, U.S.A
| | - Kaitlin M Gold
- Plant Pathology and Plant Microbe Biology Section, Cornell University, 15 Castle Creek Drive, Geneva, NY 14456, U.S.A
| | - Neil Boonham
- School of Natural and Environmental Sciences, Agriculture Building, Newcastle University, King's Road, Newcastle upon Tyne NE1 7RU, U.K
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Rivarez MPS, Vučurović A, Mehle N, Ravnikar M, Kutnjak D. Global Advances in Tomato Virome Research: Current Status and the Impact of High-Throughput Sequencing. Front Microbiol 2021; 12:671925. [PMID: 34093492 PMCID: PMC8175903 DOI: 10.3389/fmicb.2021.671925] [Citation(s) in RCA: 33] [Impact Index Per Article: 8.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/24/2021] [Accepted: 04/12/2021] [Indexed: 11/30/2022] Open
Abstract
Viruses cause a big fraction of economically important diseases in major crops, including tomato. In the past decade (2011–2020), many emerging or re-emerging tomato-infecting viruses were reported worldwide. In this period, 45 novel viral species were identified in tomato, 14 of which were discovered using high-throughput sequencing (HTS). In this review, we first discuss the role of HTS in these discoveries and its general impact on tomato virome research. We observed that the rate of tomato virus discovery is accelerating in the past few years due to the use of HTS. However, the extent of the post-discovery characterization of viruses is lagging behind and is greater for economically devastating viruses, such as the recently emerged tomato brown rugose fruit virus. Moreover, many known viruses still cause significant economic damages to tomato production. The review of databases and literature revealed at least 312 virus, satellite virus, or viroid species (in 22 families and 39 genera) associated with tomato, which is likely the highest number recorded for any plant. Among those, here, we summarize the current knowledge on the biology, global distribution, and epidemiology of the most important species. Increasing knowledge on tomato virome and employment of HTS to also study viromes of surrounding wild plants and environmental samples are bringing new insights into the understanding of epidemiology and ecology of tomato-infecting viruses and can, in the future, facilitate virus disease forecasting and prevention of virus disease outbreaks in tomato.
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Affiliation(s)
- Mark Paul Selda Rivarez
- Department of Biotechnology and Systems Biology, National Institute of Biology, Ljubljana, Slovenia.,Jožef Stefan International Postgraduate School, Ljubljana, Slovenia
| | - Ana Vučurović
- Department of Biotechnology and Systems Biology, National Institute of Biology, Ljubljana, Slovenia.,Faculty of Agriculture, University of Belgrade, Belgrade, Serbia
| | - Nataša Mehle
- Department of Biotechnology and Systems Biology, National Institute of Biology, Ljubljana, Slovenia
| | - Maja Ravnikar
- Department of Biotechnology and Systems Biology, National Institute of Biology, Ljubljana, Slovenia.,School for Viticulture and Enology, University of Nova Gorica, Nova Gorica, Slovenia
| | - Denis Kutnjak
- Department of Biotechnology and Systems Biology, National Institute of Biology, Ljubljana, Slovenia
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Phytoplasma diseases of plants: molecular diagnostics and way forward. World J Microbiol Biotechnol 2021; 37:102. [PMID: 34009500 DOI: 10.1007/s11274-021-03061-y] [Citation(s) in RCA: 11] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/20/2021] [Accepted: 04/21/2021] [Indexed: 10/21/2022]
Abstract
Phytoplasmas are obligate phytopathogenic bacteria associated with devastating diseases in hundreds of crops across the world. They have been responsible for huge economic losses in many crop plants for decades now. Isolation and establishment of axenic culture of phytoplasma in complex media is a recent progress in phytoplasma research. Earlier methods for phytoplasma disease detection included symptom profiling, microscopy, serology and dodder transmission studies. With advancement in the field of molecular biology, phytoplasma diagnostics and characterisation witnessed radical improvement. Starting from PCR amplification which often necessities a nested PCR on account of low titre of phytoplasmas, to the closed tube quantitative PCR assays and then the ddPCR, an array of diagnostics have been developed for phytoplasma. The isothermal diagnostic platforms are the latest addition to this and the Loop Mediated Isothermal Amplification (LAMP) assay has been applied for the detection of phytoplasma from several hosts. The futuristic approach in phytoplasma detection will be very likely provided by an integration of nanotechnology and molecular diagnostics. Phytoplasma disease management majorly relies on early detection, vector control, use of disease free planting materials and cultivation of resistant varieties. Hence understanding the molecular mechanism of phytoplasma-host interaction is as important as timely and accurate detection, in the management of phytoplasma diseases. Further, the changing climatic scenario and global warming may lead to an upsurge in the phytoplasma diseases spread and severity across the world, making disease management even more challenging.
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Ben Chehida S, Filloux D, Fernandez E, Moubset O, Hoareau M, Julian C, Blondin L, Lett JM, Roumagnac P, Lefeuvre P. Nanopore Sequencing Is a Credible Alternative to Recover Complete Genomes of Geminiviruses. Microorganisms 2021; 9:903. [PMID: 33922452 PMCID: PMC8147096 DOI: 10.3390/microorganisms9050903] [Citation(s) in RCA: 16] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/24/2021] [Revised: 04/21/2021] [Accepted: 04/21/2021] [Indexed: 01/23/2023] Open
Abstract
Next-generation sequencing (NGS), through the implementation of metagenomic protocols, has led to the discovery of thousands of new viruses in the last decade. Nevertheless, these protocols are still laborious and costly to implement, and the technique has not yet become routine for everyday virus characterization. Within the context of CRESS DNA virus studies, we implemented two alternative long-read NGS protocols, one that is agnostic to the sequence (without a priori knowledge of the viral genome) and the other that use specific primers to target a virus (with a priori). Agnostic and specific long read NGS-based assembled genomes of two capulavirus strains were compared to those obtained using the gold standard technique of Sanger sequencing. Both protocols allowed the detection and accurate full genome characterization of both strains. Globally, the assembled genomes were very similar (99.5-99.7% identity) to the Sanger sequences consensus, but differences in the homopolymeric tracks of these sequences indicated a specific lack of accuracy of the long reads NGS approach that has yet to be improved. Nevertheless, the use of the bench-top sequencer has proven to be a credible alternative in the context of CRESS DNA virus study and could offer a new range of applications not previously accessible.
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Affiliation(s)
- Selim Ben Chehida
- CIRAD, UMR PVBMT, F-97410 St Pierre, La Réunion, France; (S.B.C.); (M.H.); (J.-M.L.)
| | - Denis Filloux
- CIRAD, PHIM, F-34398 Montpellier, France; (D.F.); (E.F.); (O.M.); (C.J.); (L.B.); (P.R.)
- PHIM Plant Health Institute, University Montpellier, CIRAD, INRAE, Institut Agro, IRD, F-34398 Montpellier, France
| | - Emmanuel Fernandez
- CIRAD, PHIM, F-34398 Montpellier, France; (D.F.); (E.F.); (O.M.); (C.J.); (L.B.); (P.R.)
- PHIM Plant Health Institute, University Montpellier, CIRAD, INRAE, Institut Agro, IRD, F-34398 Montpellier, France
| | - Oumaima Moubset
- CIRAD, PHIM, F-34398 Montpellier, France; (D.F.); (E.F.); (O.M.); (C.J.); (L.B.); (P.R.)
- PHIM Plant Health Institute, University Montpellier, CIRAD, INRAE, Institut Agro, IRD, F-34398 Montpellier, France
| | - Murielle Hoareau
- CIRAD, UMR PVBMT, F-97410 St Pierre, La Réunion, France; (S.B.C.); (M.H.); (J.-M.L.)
| | - Charlotte Julian
- CIRAD, PHIM, F-34398 Montpellier, France; (D.F.); (E.F.); (O.M.); (C.J.); (L.B.); (P.R.)
- PHIM Plant Health Institute, University Montpellier, CIRAD, INRAE, Institut Agro, IRD, F-34398 Montpellier, France
| | - Laurence Blondin
- CIRAD, PHIM, F-34398 Montpellier, France; (D.F.); (E.F.); (O.M.); (C.J.); (L.B.); (P.R.)
- PHIM Plant Health Institute, University Montpellier, CIRAD, INRAE, Institut Agro, IRD, F-34398 Montpellier, France
| | - Jean-Michel Lett
- CIRAD, UMR PVBMT, F-97410 St Pierre, La Réunion, France; (S.B.C.); (M.H.); (J.-M.L.)
| | - Philippe Roumagnac
- CIRAD, PHIM, F-34398 Montpellier, France; (D.F.); (E.F.); (O.M.); (C.J.); (L.B.); (P.R.)
- PHIM Plant Health Institute, University Montpellier, CIRAD, INRAE, Institut Agro, IRD, F-34398 Montpellier, France
| | - Pierre Lefeuvre
- CIRAD, UMR PVBMT, F-97410 St Pierre, La Réunion, France; (S.B.C.); (M.H.); (J.-M.L.)
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Kutnjak D, Tamisier L, Adams I, Boonham N, Candresse T, Chiumenti M, De Jonghe K, Kreuze JF, Lefebvre M, Silva G, Malapi-Wight M, Margaria P, Mavrič Pleško I, McGreig S, Miozzi L, Remenant B, Reynard JS, Rollin J, Rott M, Schumpp O, Massart S, Haegeman A. A Primer on the Analysis of High-Throughput Sequencing Data for Detection of Plant Viruses. Microorganisms 2021; 9:841. [PMID: 33920047 PMCID: PMC8071028 DOI: 10.3390/microorganisms9040841] [Citation(s) in RCA: 34] [Impact Index Per Article: 8.5] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/14/2021] [Revised: 04/09/2021] [Accepted: 04/10/2021] [Indexed: 12/12/2022] Open
Abstract
High-throughput sequencing (HTS) technologies have become indispensable tools assisting plant virus diagnostics and research thanks to their ability to detect any plant virus in a sample without prior knowledge. As HTS technologies are heavily relying on bioinformatics analysis of the huge amount of generated sequences, it is of utmost importance that researchers can rely on efficient and reliable bioinformatic tools and can understand the principles, advantages, and disadvantages of the tools used. Here, we present a critical overview of the steps involved in HTS as employed for plant virus detection and virome characterization. We start from sample preparation and nucleic acid extraction as appropriate to the chosen HTS strategy, which is followed by basic data analysis requirements, an extensive overview of the in-depth data processing options, and taxonomic classification of viral sequences detected. By presenting the bioinformatic tools and a detailed overview of the consecutive steps that can be used to implement a well-structured HTS data analysis in an easy and accessible way, this paper is targeted at both beginners and expert scientists engaging in HTS plant virome projects.
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Affiliation(s)
- Denis Kutnjak
- Department of Biotechnology and Systems Biology, National Institute of Biology, Večna pot 111, 1000 Ljubljana, Slovenia
| | - Lucie Tamisier
- Plant Pathology Laboratory, Université de Liège, Gembloux Agro-Bio Tech, TERRA, Passage des Déportés, 2, 5030 Gembloux, Belgium; (L.T.); (J.R.); (S.M.)
| | - Ian Adams
- Fera Science Limited, York YO41 1LZ, UK; (I.A.); (S.M.)
| | - Neil Boonham
- Institute for Agri-Food Research and Innovation, Newcastle University, King’s Rd, Newcastle Upon Tyne NE1 7RU, UK;
| | - Thierry Candresse
- UMR 1332 Biologie du Fruit et Pathologie, INRA, University of Bordeaux, 33140 Villenave d’Ornon, France; (T.C.); (M.L.)
| | - Michela Chiumenti
- Institute for Sustainable Plant Protection, National Research Council, Via Amendola, 122/D, 70126 Bari, Italy;
| | - Kris De Jonghe
- Plant Sciences Unit, Flanders Research Institute for Agriculture, Fisheries and Food, Burg. Van Gansberghelaan 96, 9820 Merelbeke, Belgium; (K.D.J.); (A.H.)
| | - Jan F. Kreuze
- International Potato Center (CIP), Avenida la Molina 1895, La Molina, Lima 15023, Peru;
| | - Marie Lefebvre
- UMR 1332 Biologie du Fruit et Pathologie, INRA, University of Bordeaux, 33140 Villenave d’Ornon, France; (T.C.); (M.L.)
| | - Gonçalo Silva
- Natural Resources Institute, University of Greenwich, Central Avenue, Chatham Maritime, Kent ME4 4TB, UK;
| | - Martha Malapi-Wight
- Biotechnology Risk Analysis Programs, Biotechnology Regulatory Services, Animal and Plant Health Inspection Service, U.S. Department of Agriculture, Riverdale, MD 20737, USA;
| | - Paolo Margaria
- Leibniz Institute-DSMZ, Inhoffenstrasse 7b, 38124 Braunschweig, Germany;
| | - Irena Mavrič Pleško
- Agricultural Institute of Slovenia, Hacquetova Ulica 17, 1000 Ljubljana, Slovenia;
| | - Sam McGreig
- Fera Science Limited, York YO41 1LZ, UK; (I.A.); (S.M.)
| | - Laura Miozzi
- Institute for Sustainable Plant Protection, National Research Council of Italy (IPSP-CNR), Strada delle Cacce 73, 10135 Torino, Italy;
| | - Benoit Remenant
- ANSES Plant Health Laboratory, 7 Rue Jean Dixméras, CEDEX 01, 49044 Angers, France;
| | | | - Johan Rollin
- Plant Pathology Laboratory, Université de Liège, Gembloux Agro-Bio Tech, TERRA, Passage des Déportés, 2, 5030 Gembloux, Belgium; (L.T.); (J.R.); (S.M.)
- DNAVision, 6041 Charleroi, Belgium
| | - Mike Rott
- Sidney Laboratory, Canadian Food Inspection Agency, 8801 East Saanich Rd, North Saanich, BC V8L 1H3, Canada;
| | - Olivier Schumpp
- Agroscope, Route de Duillier 50, 1260 Nyon, Switzerland; (J.-S.R.); (O.S.)
| | - Sébastien Massart
- Plant Pathology Laboratory, Université de Liège, Gembloux Agro-Bio Tech, TERRA, Passage des Déportés, 2, 5030 Gembloux, Belgium; (L.T.); (J.R.); (S.M.)
| | - Annelies Haegeman
- Plant Sciences Unit, Flanders Research Institute for Agriculture, Fisheries and Food, Burg. Van Gansberghelaan 96, 9820 Merelbeke, Belgium; (K.D.J.); (A.H.)
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Current Developments and Challenges in Plant Viral Diagnostics: A Systematic Review. Viruses 2021; 13:v13030412. [PMID: 33807625 PMCID: PMC7999175 DOI: 10.3390/v13030412] [Citation(s) in RCA: 55] [Impact Index Per Article: 13.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/07/2021] [Revised: 02/10/2021] [Accepted: 02/18/2021] [Indexed: 12/24/2022] Open
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.
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Fantastic Downy Mildew Pathogens and How to Find Them: Advances in Detection and Diagnostics. PLANTS 2021; 10:plants10030435. [PMID: 33668762 PMCID: PMC7996204 DOI: 10.3390/plants10030435] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 11/30/2020] [Revised: 02/11/2021] [Accepted: 02/15/2021] [Indexed: 12/26/2022]
Abstract
Downy mildews affect important crops and cause severe losses in production worldwide. Accurate identification and monitoring of these plant pathogens, especially at early stages of the disease, is fundamental in achieving effective disease control. The rapid development of molecular methods for diagnosis has provided more specific, fast, reliable, sensitive, and portable alternatives for plant pathogen detection and quantification than traditional approaches. In this review, we provide information on the use of molecular markers, serological techniques, and nucleic acid amplification technologies for downy mildew diagnosis, highlighting the benefits and disadvantages of the technologies and target selection. We emphasize the importance of incorporating information on pathogen variability in virulence and fungicide resistance for disease management and how the development and application of diagnostic assays based on standard and promising technologies, including high-throughput sequencing and genomics, are revolutionizing the development of species-specific assays suitable for in-field diagnosis. Our review provides an overview of molecular detection technologies and a practical guide for selecting the best approaches for diagnosis.
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