Detection Of Viroid Research Articles

Improving grapevine virus diagnostics: Comparative analysis of three dsRNA enrichment methods for high-throughput sequencing

The extraction of double stranded (ds) RNA is a common enrichment method for the study, characterization, and detection of RNA viruses. In addition to RNA viruses, viroids, and some DNA viruses, can also be detected from dsRNA enriched extracts which makes it an attractive method for detecting a wide range of viruses when coupled with HTS. Several dsRNA enrichment strategies have been developed. The oldest utilizes the selective binding properties of dsRNA to cellulose. More recent methods are based on the application of anti-dsRNA antibodies and viral proteins with a specific affinity for dsRNA. All three methods have been used together with HTS for plant virus detection and study. To our knowledge, this is the first comparative study of three alternative dsRNA enrichment methods for virus and viroid detection through HTS using virus-infected, and healthy grapevine test plants. Extracts were performed in triplicate using methods based on, the anti-dsRNA antibody mAb rJ2 (Millipore Sigma Canada Ltd, Oakville, ON, Canada), the B2 dsRNA binding protein, and ReliaPrep™ Resin (Promega Corporation, Madison, WI, USA). The results show that the workflows for all three methods are effectively comparable, apart from purification steps related to antibody and binding protein construct. Both the cellulose resin and dsRNA binding protein construct methods provide highly enriched dsRNA extracts suitable for HTS with the B2 method providing a 36× and the ReliaPrep™ Resin a 163× increase in dsRNA enrichment compared to the mAb rJ2 antibody. The overall consistency and cost effectiveness of the ReliaPrep™ cellulose resin-based method and the potentially simpler adaptation to robotics made it the method of choice for future transfer to a semi-automated workflow.

Assessment of the health status of citrus propagation material against hop stunt viroid through biological and molecular diagnosis

Hop stunt viroid (HSVd) is the causal agent of citrus cachexia, which can lead to significant crop losses. As part of a preventive management strategy, it is essential to use healthy plants from a certification program. This study aimed to, healthiness of 17 citrus cultivars was evaluated against HSVd using both biological and molecular diagnostics. For biological diagnosis, Etrog citron (Citrus medica L.) clone Arizona 861 S1 was used as an indicator plant, incorporating negative and positive controls for HSVd infection. For molecular diagnosis, reverse transcription polymerase chain reaction (RT-PCR) technique was employed with specific primers for viroid detection. Results indicated that six months after inoculation, the positive control exhibited symptoms associated with viroid infection, such as necrosis in the petiole and stem, leaf epinasty, and necrosis in the midvein. In contrast, both the negative control and evaluated citrus cultivars showed no symptoms. These findings were supported by molecular analysis, which allowed the amplification of a viroid genome region only in the positive control. Both biological and molecular diagnosis confirmed the absence of HSVd in the citrus varieties assessed. This study will allow the evaluation of the citrus plant collections used in the foundation category as a source of buds for plant multiplication of registered and certified seeds for citrus propagation nurseries in Colombia.

Optimization of Amplicon-Based Sequencing for Large-Scale Diagnostics of Known Pome Viruses and Viroids

The implementation of reliable methods for the simultaneous detection of multiple viruses is challenging in plant viral diagnosis. In the present study, we report the development and effectiveness of an optimized version of a robust multiplexed PCR (HiPlex) followed by simultaneous targeted amplicon sequencing to detect 14 viruses and 5 viroids of pome fruit trees. We used amplicon sequencing data from 1,400 pome fruit tree samples previously diagnosed with the HiPlex v.1.0, which included 290 primer pairs for pome viruses. The analysis enabled the identification of highly efficient and specific primer pairs to be assembled in a new HiPlex (dubbed HiPlex v.2.0). Additionally, newly designed primers were implemented for 30% of targeted viruses and viroids. Overall, 116 primer pairs were assembled into the HiPlex v.2.0, resulting in a reduction of the number of primers and multiplex PCR reactions needed by 60 and 50%, respectively. The new HiPlex also includes two nepoviruses, tomato ringspot virus and tobacco ringspot virus, showing flexibility at augmenting and expanding the number of targeted diagnosed viruses using this approach. This optimized set of primers increased the cost-effectiveness and sensitivity while reducing background amplification. Comparable results for virus and viroid detection were obtained using the HiPlex v.2.0 and individual RT-qPCRs, even at a 10,000-fold dilution, demonstrating similar sensitivity to standard PCR-based diagnostics. This new HiPlex-based amplicon sequencing represents a powerful, sensitive, and low-cost tool for large-scale screening of pome fruit trees for all reported viruses and viroids infecting these crops. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

Viroids of the Mediterranean Basin.

There has been substantial progress in the Mediterranean countries regarding research on viroids. Twenty-nine viroid species, all belonging to Pospiviroidae and Avsunviroidae genera, have been detected in the Mediterranean Basin. Not only have detection methods, such as reverse transcription-quantitative polymerase chain reaction and next-generation sequencing, been used for viroid detection, along with molecular hybridization techniques allowing for rapid detection, identification, and characterization of known and novel viroids in these countries, but eradication measures have also been taken that allowed for the efficient elimination of certain viroids in a number of Mediterranean countries. The eradication measures were followed as recommended by the European and Mediterranean Plant Protection Organization, which is known by its abbreviation, EPPO. The Mediterranean Region has been a niche for viroids since ancient times due to the warm climate and the socio-cultural conditions that facilitate viroid transmission among different host plant species.

Novel RNA genosensor based on highly stable gold nanoparticles decorated phosphorene nanohybrid with graphene for highly sensitive and low-cost electrochemical detection of coconut cadang-cadang viroid.

Coconut cadang-cadang viroid (CCCVd) is an infectious single-stranded RNA (ssRNA) pathogen, which leads directly to the death of a large number of coconut palm trees and heavy economic loss to coconut farmers. Herein, a novel electrochemical impedance RNA genosensor is presented based on highly stable gold nanoparticles (AuNPs) decorated phosphorene (BP) nanohybrid with graphene (Gr) for highly sensitive, low-cost, and label-free detection of CCCVd. BP-AuNPs are environmentally friendly prepared by ultrasonic-assisted liquid-phase exfoliation of black phosphorus, accompanying direct reduction of chloroauric acid. Gr/BP-AuNPs are facilely prepared by the in situ growth of AuNPs onto the BP surface and its nanohybrid with Gr to improve environmental stability of BP. Gr/BP-AuNP-based RNA genosensor is fabricated by immobilizing the thiol-functionalized single-stranded DNA (ssDNA) oligonucleotide probe onto the surface of Gr/BP-AuNP-modified glassy carbon electrode via gold-thiol interactions, which served as an electrochemical genosensing platform for the label-free impedance detection of CCCVd by hybridization between the functionalized ssDNA probe and the complementary CCCVd ssRNA sequence in a wide linear range from 1.0 × 10-11 to 1.0 × 10-7M with a low limit of detection of 2.8 × 10-12M. This work supplies an experimental support and theoretical direction for the fabrication of RNA biosensors based on graphene-like materials and potential application for a specific diagnosis of plant RNA viral disease in Arecaceae planting industry.

New Insights into Hop Latent Viroid Detection, Infectivity, Host Range, and Transmission.

Hop latent viroid (HLVd), a subviral pathogen from the family Pospiviroidae, is a major threat to the global cannabis industry and is the causative agent for "dudding disease". Infected plants can often be asymptomatic for a period of growth and then develop symptoms such as malformed and yellowing leaves, as well as stunted growth. During flowering, HLVd-infected plants show reduced levels of valuable metabolites. This study was undertaken to expand our basic knowledge of HLVd infectivity, transmission, and host range. HLVd-specific primers were used for RT-PCR detection in plant samples and were able to detect HLVd in as little as 5 picograms of total RNA. A survey of hemp samples obtained from a diseased production system proved sole infection of HLVd (72%) with no coexistence of hop stunt viroid. HLVd was infectious through successive passage assays using a crude sap or total RNA extract derived from infected hemp. HLVd was also highly transmissible through hemp seeds at rates of 58 to 80%. Host range assays revealed new hosts for HLVd: tomato, cucumber, chrysanthemum, Nicotiana benthamiana, and Arabidopsis thaliana (Col-0). Sequence analysis of 77 isolates revealed only 3 parsimony-informative sites, while 10 sites were detected among all HLVd isolates available in the GenBank. The phylogenetic relationship among HLVd isolates allowed for inferring two major clades based on the genetic distance. Our findings facilitate further studies on host-viroid interaction and viroid management.

PhytoPipe: a phytosanitary pipeline for plant pathogen detection and diagnosis using RNA-seq data

BackgroundDetection of exotic plant pathogens and preventing their entry and establishment are critical for the protection of agricultural systems while securing the global trading of agricultural commodities. High-throughput sequencing (HTS) has been applied successfully for plant pathogen discovery, leading to its current application in routine pathogen detection. However, the analysis of massive amounts of HTS data has become one of the major challenges for the use of HTS more broadly as a rapid diagnostics tool. Several bioinformatics pipelines have been developed to handle HTS data with a focus on plant virus and viroid detection. However, there is a need for an integrative tool that can simultaneously detect a wider range of other plant pathogens in HTS data, such as bacteria (including phytoplasmas), fungi, and oomycetes, and this tool should also be capable of generating a comprehensive report on the phytosanitary status of the diagnosed specimen.ResultsWe have developed an open-source bioinformatics pipeline called PhytoPipe (Phytosanitary Pipeline) to provide the plant pathology diagnostician community with a user-friendly tool that integrates analysis and visualization of HTS RNA-seq data. PhytoPipe includes quality control of reads, read classification, assembly-based annotation, and reference-based mapping. The final product of the analysis is a comprehensive report for easy interpretation of not only viruses and viroids but also bacteria (including phytoplasma), fungi, and oomycetes. PhytoPipe is implemented in Snakemake workflow with Python 3 and bash scripts in a Linux environment. The source code for PhytoPipe is freely available and distributed under a BSD-3 license.ConclusionsPhytoPipe provides an integrative bioinformatics pipeline that can be used for the analysis of HTS RNA-seq data. PhytoPipe is easily installed on a Linux or Mac system and can be conveniently used with a Docker image, which includes all dependent packages and software related to analyses. It is publicly available on GitHub at https://github.com/healthyPlant/PhytoPipe and on Docker Hub at https://hub.docker.com/r/healthyplant/phytopipe.

Validation of High-Throughput Sequencing (HTS) for Routine Detection of Citrus Viruses and Viroids.

The management of plant diseases relies on the accurate identification of pathogens that requires a robust and validated tool in terms of specificity, sensitivity, repeatability, and reproducibility. High-throughput sequencing (HTS) has become the method of choice for virus detection when either a complete viral status of a plant is required in a single assay or if an unknown viral agent is expected. To ensure that the most accurate diagnosis is made from an HTS data analysis, a standardized protocol per pathosystem is required. This chapter presents a detailed protocol for the detection of viruses and viroids infecting citrus using HTS. The protocol describes all the steps from sample processing, nucleic acid extraction, and bioinformatic analyses validated to be an efficient method for detection in this pathosystem. The protocol also includes a section on citrus tristeza virus (CTV) genotype differentiation using HTS data.

Detection and Molecular Characterization of Peach Latent Mosaic Viroid Isolates Infecting Stone Fruit Trees in Poland

Abstract Field surveys were carried out to assess the incidence of peach latent mosaic viroid in nine orchards of stone fruit trees in four growing regions of Poland. Forty of 119 peaches, six of 37 apricots, five of eight nectarines and one of four plums trees tested positive by RT-PCR for this viroid. Most of the infected stone fruit trees did not show any symptoms. Leaf yellow mosaic was only noticed on ‘Maycrest’ peach and ‘Saturn’ nectarine trees, and additional symptoms of cracked fruits with corky sutures and enlarged pits were observed on one ‘Maycrest’ tree. One or two isolates from each orchard and each species of infected tree were selected, and together, 11 isolates of peach latent mosaic viroid were used for genetic analyses. The nucleotide sequence similarity of selected isolates found in Poland was 93.6–100%. They shared 82.0–98.8% similarity with the viroid strains from different hosts and geographical regions. The phylogenetic analysis led to the clustering of all isolates detected during this study in the most numerous represented subgroup II-A, composed of peach latent mosaic viroid strains originating from different geographic regions and host plants. Although Polish isolates were clustered in the same subgroup, some differed in length and nucleotide sequences. It was shown that the genetic variability of isolates found in Poland was unrelated to the regions of their origin. A positive signal was obtained in dot-blot hybridization of all analyzed 11 isolates.

Development of the colorimetric loop-mediated isothermal amplification technique for rapid and sensitive detection of chrysanthemum stunt viroid in chrysanthemum

Development of the colorimetric loop-mediated isothermal amplification technique for rapid and sensitive detection of chrysanthemum stunt viroid in chrysanthemum

A Novel, Precise and High-Throughput Technology for Viroid Detection in Cannabis (MFDetectTM)

Hop latent viroid (HLVd) is a severe disease of cannabis, causing substantial economic losses in plant yield and crop value for growers worldwide. The best way to control the disease is early detection to limit the spread of the viroid in grow facilities. This study describes MFDetectTM as a rapid, highly sensitive, and high-throughput tool for detecting HLVd in the early stages of plant development. Furthermore, in the largest research study conducted so far for HLVd detection in cannabis, we compared MFDetectTM with quantitative RT-PCR in a time course experiment using different plant tissues, leaves, petioles, and roots at different plant developmental stages to demonstrate both technologies are comparable. Our study found leaf tissue is a suitable plant material for HLVd detection, with the viroid titer increasing in the infected leaf tissue with the age of plants. The study showed that other tissue types, including petiole and roots, were equally sensitive to detection via MFDetectTM. The assay developed in this research allows the screening of thousands of plants in a week. The assay can be scaled easily to provide growers with a quick turnaround and a cost-effective diagnostic tool for screening many plants and tissue types at different stages of development.

NanoViromics: long-read sequencing of dsRNA for plant virus and viroid rapid detection.

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.

Development and Validation of a Duplex RT-qPCR for Detection of Peach Latent Mosaic Viroid and Comparison of Different Nucleic-Acid-Extraction Protocols.

Peach latent mosaic viroid (PLMVd) is an important pathogen that causes disease in peaches. Control of this viroid remains problematic because most PLMVd variants are symptomless, and although there are many detection tests in use, the reliability of PCR-based methods is compromised by the complex, branched secondary RNA structure of the viroid and its genetic diversity. In this study, a duplex RT-qPCR method was developed and validated against two previously published single RT-qPCRs, which were potentially able to detect all known PLMVd variants when used in tandem. In addition, in order to simplify the sample preparation, rapid-extraction protocols based on the use of crude sap or tissue printing were compared with commercially available RNA purification kits. The performance of the new procedure was evaluated in a test performance study involving five participant laboratories. The new method, in combination with rapid-sample-preparation approaches, was demonstrated to be feasible and reliable, with the advantage of detecting all different PLMVd isolates/variants assayed in a single reaction, reducing costs for routine diagnosis.

Automating Citrus Budwood Processing for Downstream Pathogen Detection Through Instrument Engineering.

Graft-transmissible, phloem-limited pathogens of citrus such as viruses, viroids, and bacteria are responsible for devastating epidemics and serious economic losses worldwide. For example, the citrus tristeza virus killed over 100 million citrus trees globally, while "Candidatus Liberibacter asiaticus" has cost Florida $9 billion. The use of pathogen-tested citrus budwood for tree propagation is key for the management of such pathogens. The Citrus Clonal Protection Program (CCPP) at the University of California, Riverside, uses polymerase chain reaction (PCR) assays to test thousands of samples from citrus budwood source trees every year to protect California's citrus and to provide clean propagation units to the National Clean Plant Network. A severe bottleneck in the high-throughput molecular detection of citrus viruses and viroids is the plant tissue processing step. Proper tissue preparation is critical for the extraction of quality nucleic acids and downstream use in PCR assays. Plant tissue chopping, weighing, freeze-drying, grinding, and centrifugation at low temperatures to avoid nucleic acid degradation is time-intensive and labor-intensive and requires expensive and specialized laboratory equipment. This paper presents the validation of a specialized instrument engineered to rapidly process phloem-rich bark tissues from citrus budwood, named the budwood tissue extractor (BTE). The BTE increases sample throughput by 100% compared to current methods. In addition, it decreases labor and the cost of equipment. In this work, the BTE samples had a DNA yield (80.25 ng/µL) that was comparable with the CCPP's hand-chopping protocol (77.84 ng/µL). This instrument and the rapid plant tissue processing protocol can benefit several citrus diagnostic laboratories and programs in California and become a model system for tissue processing for other woody perennial crops worldwide.

Detection of multiple viruses and viroid in apple trees in Brazil and their possible association with decline

In an attempt to provide additional information on the virome in apples in Southern Brazil in face of recurrent events of tree decline, plant samples of regional apple orchards have been submitted to HTS (high-throughput sequencing) analysis. Besides common latent viruses, results showed additionally the presence of apple rubbery wood virus 2 (ARWV 2) and citrus concave gum-associated virus (CCGaV) in orchards with decline history.

A reverse transcription loop-mediated isothermal amplification assay for the detection of citrus exocortis viroid in Australian citrus

Citrus exocortis viroid (CEVd), the causal agent of exocortis, is a pathogen that is thought to infect all citrus varieties, although it is asymptomatic in most. Symptoms of exocortis develop on susceptible rootstocks, resulting in stunting and yield reduction. To aid the detection and management of CEVd, a rapid near-field assay was developed using reverse transcription loop-mediated isothermal amplification (RT-LAMP) for the detection of the viroids in nursery and field trees. Over 240 CEVd sequences, including sequence variants from representative Australian isolates that induce mild and severe symptoms, were used in the design of the primers. The RT-LAMP successfully detected CEVd in a 1:1000 dilution (236 pg) of plant total RNA indicating high sensitivity, and also detected the viroid in rapid, crude plant extractions. The assay was highly specific to CEVd, given there was no cross-reactivity with other citrus-infecting pathogens. This new assay provides a simple, robust, specific, and sensitive method to detect CEVd in Australian citrus and to our knowledge, is the first RT-LAMP assay to detect any citrus-infecting viroid.

Разработка ПЦР-тест системы для выявления вироида веретеновидности клубней картофеля

The Potato spindle tuber viroid infects potato plants, causing a serious damage to agriculture by reducing yields. Therefore, there is a need to identify this pathogen. Viroids are RNA molecules and so cannot be detected with the immunological methods and the classical PCR. The aim of this work is to develop a method for detection of the Potato spindle tuber viroid. Primers have been selected for the reverse transcription reaction and the subsequent PCR, the annealing temperatures and the size of the amplified fragment have been calculated. Primers and reaction conditions have been tested on plant material. PCR products of the calculated size have been obtained. Determination of their nucleotide sequence confirmed the identification of the genetic material of the Potato spindle tuber viroid. Thus, this PCR test system can be used for detection of the Potato viroid and become the basis for detection of other
 vegetable crops’ viroids.

High-throughput detection of a large set of viruses and viroids of pome and stone fruit trees by multiplex PCR-based amplicon sequencing.

A comprehensive diagnostic method of known plant viruses and viroids is necessary to provide an accurate phytosanitary status of fruit trees. However, most widely used detection methods have a small limit on either the number of targeted viruses/viroids or the number of samples to be evaluated at a time, hampering the ability to rapidly scale up the test capacity. Here we report that by combining the power of high multiplexing PCR (499 primer pairs) of small amplicons (120-135bp), targeting 27 viruses and 7 viroids of fruit trees, followed by a single high-throughput sequencing (HTS) run, we accurately diagnosed the viruses and viroids on as many as 123 pome and stone fruit tree samples. We compared the accuracy, sensitivity, and reproducibility of this approach and contrast it with other detection methods including HTS of total RNA (RNA-Seq) and individual RT-qPCR for every fruit tree virus or viroid under the study. We argue that this robust and high-throughput cost-effective diagnostic tool will enhance the viral/viroid knowledge of fruit trees while increasing the capacity for large scale diagnostics. This approach can also be adopted for the detection of multiple viruses and viroids in other crops.

PM 7/153 (1) Mechanical inoculation of test plants

PM 7/153 (1) Mechanical inoculation of test plants

Molecular detection and characterization of Potato spindle tuber viroid (PSTVd) infecting Tomato (Solanum lycopersicum L.) in Karnataka State of India.

Potato Spindle Tuber Viroid (PSTVd) is a non-coding, infectious, small, circular RNA known to cause disease in agricultural and horticultural plants. In the present work, an investigation was conducted in the southern districts of Karnataka state to assess the possible pospiviroid infections on tomato plants that are considered natural hosts for viroids. A total of 83 tomato samples showing disease symptoms (virus or viroid-like) along with healthy ones were collected and subjected to viroid detection by conventional Reverse Transcriptase Polymer Chain Reaction (RT-PCR) using universal (Pospi1-RE/Pospi1-FW) and a specific set of primers (3H1/2H1). The study confirmed the presence of PSTVd in one of the samples of tomato collected from Banghatta village of Mandy District, with an expected amplicon of ~ 361bp. The bioassay conducted on tomato plants (cv. Rutgers) proved the association of PSTVd, which was further confirmed by RT-PCR. The amplicons were cloned, sequenced, and the representative sequences were deposited in the NCBI GenBank. The sequence alignment and secondary structure analysis of the isolated viroid with other reference sequences revealed the variations in the pathogenicity, central conserved region, and Terminal right domains. The variations observed between the isolated PSTVd with that of other Indian isolates support that viroid may have been transmitted among the crop plants, possibly through seed or mechanical means.