Identification Of Plant Viruses Research Articles

Application of Nanopore-Based Sequencing to Identify Virus Infections in Woody Plants.

Plant viruses threaten the yield and quality of crops. Efficient and affordable pathogen diagnosis is crucial to regulate the trade of plant materials and for disease management and control. Sequencing technology based on Illumina platform is a powerful tool for the identification of plant viruses, but it requires long and expensive protocols, cumbersome equipment, and significant cost per library. Nanopore sequencing technology, developed by Oxford Nanopore Technologies (ONT), is a recent sequencing system very easy to use, suitable for onsite-field detection, and associated with low costs. Coupled with its portability, nanopore technology has great application prospects in the field of quick detection of plant viruses. In this protocol, we expose in detail the application of cDNA-PCR nanopore-based sequencing for the detection of plant viruses.

Plant Viruses and Viral Host Plants Newly Reported in South Korea in 2020–2022

With the development of advanced virus diagnostic technologies, numerous viruses, including novel viruses, have been identified explosively from various biological samples around the world over the last decade. For plant viruses, approximately 376 novel viruses have been reported in the last three years. Information on the occurrence and host ranges of plant viral diseases in a particular country or region is very important for diagnosis, quarantine, and control of the viral diseases. Recently, based on active research on the diagnosis and identification of plant viruses, a significant number of newly occurring viruses and new viral host plants have been reported in South Korea. This review paper provides integrated information on plant viruses and viral host plants newly reported in South Korea between 2020 and 2022 to help diagnose, control, and quarantine plant virus diseases in crop fields.

Rapid Identification of Jasmine Virus H Infecting Ixora coccinea by Nanopore Metatranscriptomics.

The global climate change and international trade have facilitated the movement of plants across borders, increasing the risk of introducing novel plant viruses in new territories. Ixora coccinea exhibited virus-like foliar symptoms, including mosaic and mild mottle. An Oxford Nanopore Technologies-based compact and portable MinION platform was used to identify the causal viral pathogen. The complete genome sequence of jasmine virus H (JaVH; 3867 nt, JaVH-CNU) was determined and found to share 88.4-90.3% nucleotide identity with that of Jasminum sambac JaVH isolate in China. Phylogenetic analysis based on the complete amino acid sequences of RNA-dependent RNA polymerase and coat protein revealed that JaVH-CNU was grouped separately with other JaVH isolates. This is the first report of a natural JaVH infection of >i<I. coccinea. The application of rapid nanopore sequencing for plant virus identification was demonstrated and is expected to provide accurate and rapid diagnosis for virus surveillance.

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

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

PM 4/17 (3) Certification scheme for olive trees and rootstocks

<scp>PM</scp> 4/17 (3) Certification scheme for olive trees and rootstocks

Development of a Novel Tissue Blot Hybridization Chain Reaction for the Identification of Plant Viruses.

Assays for the high throughput screening of crops for virus monitoring need to be quick, easy, and low cost. One method involves using tissue blot immunoassays (TBIA), where plant stems are blotted onto nitrocellulose membrane and screened with available antibodies against a range of viruses. TBIAs are inexpensive but limited by antibody availability and specificity. To circumvent the antibody limitations, we developed the tissue blot hybridization chain reaction (TB-HCR). As with TBIA, plant stems are blotted onto a nitrocellulose membrane, however, TB-HCR involves using nucleic acid probes instead of antibodies. We demonstrated for the first time that TB-HCR can be used for plant viruses by designing and testing probes against species from several virus genera including begomovirus, polerovirus, luteovirus, cucumovirus, and alfamovirus. We also explored different hairpin reporter methods such as biotin/streptavidin-AP and the Alexa Fluor-488 Fluorophore. TB-HCR has applications for low-cost diagnostics for large sample numbers, rapid diagnostic deployment for new viruses, and can be performed as a preliminary triage assay prior to downstream applications.

Supplementary material for journal article "Development of a Novel Tissue Blot Hybridization Chain Re-action for the Identification of Plant Viruses"

Supplementary material for journal article "Development of a Novel Tissue Blot Hybridization Chain Re-action for the Identification of Plant Viruses"

Potato viruses of 7 commercial cultivars grown in field Primorsky Krai of Russia

Scientific relevance. Plant viruses cause a significant economic loss to potato production, especially if plants are infected at early growth stages and infections are mixed. Viral diseases reduce both yield and quality of harvested crops. Detection and identification of plant viruses are key important to prevent their spreading and to achieve potential yield predetermined by characteristics of varieties.Research methods. Seven potato varieties, bred in Russia and overseas, were used in the field experiment: Smak, Avgustin, Yantar, Laperla, Labella, Red Lady, Sante, Belmonda. Viral infection rate was measured by the percent of plants with symptoms to the total number of plants. In addition to infection frequency, a disease rate was described after visual estimation. Total RNA was isolated from the collected leaves according to Bekesiova I. et al. 1999 [13]. Qualitative and quantitative estimation of plant viruses in the samples were conducted by single-step real-time RT-PCR with fluorescent detection with the Applied Biosystems QuantStudio 5 and commercial kits “Potato Virus X, Y, M, L, S, A, PSTVd-RT” (Syntol Company) according to the official protocol of the kits.Results. As a result of our research, symptoms of mixed viral infection were described for potato varieties depending on concentrations and proportions of these viruses in a plant. Mixed viral infection in the potato field in Primorsky Krai comprised PVY, PVX, PVA, PVS, PVM, also PLRV and PSTVd.

Next-generation sequencing identification and multiplex RT-PCR detection for viruses infecting cigar and flue-cured tobacco.

Early, precise and simultaneous identification of plant viruses is of great significance for preventing virus spread and reducing losses in agricultural yields. In this study, the identification of plant viruses from symptomatic samples collected from a cigar tobacco planting area in Deyang and a flue-cured tobacco planting area in Luzhou city, Sichuan Province, China, was conducted by deep sequencing of small RNAs (sRNAs) through an Illumina sequencing platform, and plant virus-specific contigs were generated based on virus-derived siRNA sequences. Additionally, sequence alignment and phylogenetic analysis were performed to determine the species or strains of these viruses. A total of 27930450, 21537662 and 28194021 clean reads were generated from three pooled samples, with a total of 105 contigs mapped to the closest plant viruses with lengths ranging from 34 ~ 1720nt. The results indicated that the major viruses were potato virus Y, Chilli veinal mottle virus, tobacco vein banding mosaic virus, tobacco mosaic virus and cucumber mosaic virus. Subsequently, a fast and sensitive multiplex reverse transcription polymerase chain reaction assay was developed for the simultaneous detection of the most frequent RNA viruses infecting cigar and flue-cured tobacco in Sichuan. These results provide a theoretical basis and convenient methods for the rapid detection and control of viruses in cigar- and flue-cured tobacco.

Identification of Plant Viruses Infecting Pear Using RNA Sequencing.

Asian pear (Pyrus pyrifolia) is a widely cultivated and commercially important fruit crop, which is occasionally subject to severe economic losses due to latent viral infections. Thus, the aim of the present study was to examine and provide a comprehensive overview of virus populations infecting a major pear cultivar (‘Singo’) in Korea. From June 2017 to October 2019, leaf samples (n = 110) of pear trees from 35 orchards in five major pear-producing regions were collected and subjected to RNA sequencing. Most virus-associated contigs matched the sequences of known viruses, including apple stem grooving virus (ASGV) and apple stem pitting virus (ASPV). However, some contigs matched the sequences of apple green crinkle-associated virus and cucumber mosaic virus. In addition, three complete or nearly complete genomes were constructed based on transcriptome data and subjected to phylogenetic analyses. Based on the number of virus-associated reads, ASGV and ASPV were identified as the dominant viruses of ‘Singo.’ The present study describes the virome of a major pear cultivar in Korea, and looks into the diversity of viral communities in this cultivar. This study can provide valuable information on the complexity of genetic variability of viruses infecting pear trees.

Plant Viruses Detection and Diagnosis Based on Polymerase Chain Reaction Techniques: Review

Plant virus diseases result in the loss of billions of dollars annually by limiting plant production quantity and quality in the world. Among different strategies adapted for plant virus disease management, proper diagnosis and detection are the most important and essential strategies for the development of appropriate control measures. The current advanced techniques developed for the detection of plant viruses provided the chance to take practical managemental actions timely. Nowadays one of the most advanced diagnosis methods, polymerase chain reaction (PCR) is used extensively for the detection and identification of plant viruses. PCR is the advanced method that allows the specific amplification and hence detection of target DNA sequences from a mixture of nucleic acid extract in which specific amplification of targeted fragments of a single or a few copies of source DNA material is achieved within a few hours. The PCR method copies each piece of DNA fragments through all the cycles that leading to an exponential doubling of copies over time. Several modifications of PCR methods have been developed to boost the effectiveness of the method in diagnostic settings based on their applications. Reverse transcriptase PCR, immunocapture PCR, Multiplex PCR, co-operational PCR, and real-time PCR are the common and widely used types of PCR variants. Keywords : Detection techniques; PCR; Plant virus DOI: 10.7176/ALST/86-02 Publication date: March 31 st 2021

Small RNA-Omics for Plant Virus Identification, Virome Reconstruction, and Antiviral Defense Characterization.

RNA interference (RNAi)-based antiviral defense generates small interfering RNAs that represent the entire genome sequences of both RNA and DNA viruses as well as viroids and viral satellites. Therefore, deep sequencing and bioinformatics analysis of small RNA population (small RNA-ome) allows not only for universal virus detection and genome reconstruction but also for complete virome reconstruction in mixed infections. Viral infections (like other stress factors) can also perturb the RNAi and gene silencing pathways regulating endogenous gene expression and repressing transposons and host genome-integrated endogenous viral elements which can potentially be released from the genome and contribute to disease. This review describes the application of small RNA-omics for virus detection, virome reconstruction and antiviral defense characterization in cultivated and non-cultivated plants. Reviewing available evidence from a large and ever growing number of studies of naturally or experimentally infected hosts revealed that all families of land plant viruses, their satellites and viroids spawn characteristic small RNAs which can be assembled into contigs of sufficient length for virus, satellite or viroid identification and for exhaustive reconstruction of complex viromes. Moreover, the small RNA size, polarity and hotspot profiles reflect virome interactions with the plant RNAi machinery and allow to distinguish between silent endogenous viral elements and their replicating episomal counterparts. Models for the biogenesis and functions of small interfering RNAs derived from all types of RNA and DNA viruses, satellites and viroids as well as endogenous viral elements are presented and discussed.

Real time portable genome sequencing for global food security

Crop losses due to viral diseases and pests are major constraints on food security and income for millions of households in sub-Saharan Africa (SSA). Such losses can be reduced if plant diseases and pests are correctly diagnosed and identified early. Currently, accurate diagnosis for definitive identification of plant viruses and their vectors in SSA mostly relies on standard PCR and next generation sequencing technologies (NGS). However, it can take up to 6 months before results generated using these approaches are available. The long time taken to detect or identify viruses impedes quick, within-season decision-making necessary for early action, crop protection advice and disease control measures by farmers. This ultimately compounds the magnitude of crop losses and food shortages suffered by farmers. The MinION portable pocket DNA sequencer was used, to our knowledge globally for the first time, to sequence whole plant virus genomes. We used this technology to identify the begomoviruses causing the devastating cassava mosaic virus, which is ravaging smallholder farmers’ crops in sub-Saharan Africa.

A Simple Kit of Plate-Trapped Antigen Enzyme-Linked Immunosorbent Assay for Identification of Plant Viruses

Many methods can be used for arriving in a correct virus disease diagnose, and the serological techniques are the most used methods for plant virus identification. The indirect enzyme-linked immunosorbent assay (Indirect-ELISA) or plate-trapped antigen ELISA (PTA-ELISA) has been useful for detection of viruses in a wide range of situations, especially to test a large number of samples in a relatively short period of time. Immune-biological Companies have developed practical kits for direct ELISA or double antibody sandwich (DAS-ELISA), but neither Company has developed kits for PTA-ELISA. As a single universal antibody-conjugate is used for detection of a wide range of plant viruses, the PTA-ELISA technique is more economical, practical and suitable for virus detection. Considering also the great problem of including infectious plant viruses in DAS-ELISA kits, a simple kit for PTA-ELISA was developed for plant virus identification. Extracts from infected plant tissues were added into the ELISA plate wells, which were sealed with plastics and maintained in the refrigerator and Laboratory conditions for different periods of time. The plates were tested by the regular PTA-ELISA and after 20 months of incubation, the plate showed excellent results when used for detection of six virus species from the genera Comovirus, Cucumovirus, Potyvirus and Sobemovirus in infected plant tissues. The ELISA plate trapped virus together with its specific antiserum could constitute a simple kit, which will permit the exchange of antisera among virologists without transferring infectious viruses from one laboratory to another to be used as control.

Integrated analyses using RNA-Seq data reveal viral genomes, single nucleotide variations, the phylogenetic relationship, and recombination for Apple stem grooving virus.

BackgroundNext-generation sequencing (NGS) provides many possibilities for plant virology research. In this study, we performed integrated analyses using plant transcriptome data for plant virus identification using Apple stem grooving virus (ASGV) as an exemplar virus. We used 15 publicly available transcriptome libraries from three different studies, two mRNA-Seq studies and a small RNA-Seq study.ResultsWe de novo assembled nearly complete genomes of ASGV isolates Fuji and Cuiguan from apple and pear transcriptomes, respectively, and identified single nucleotide variations (SNVs) of ASGV within the transcriptomes. We demonstrated the application of NGS raw data to confirm viral infections in the plant transcriptomes. In addition, we compared the usability of two de novo assemblers, Trinity and Velvet, for virus identification and genome assembly. A phylogenetic tree revealed that ASGV and Citrus tatter leaf virus (CTLV) are the same virus, which was divided into two clades. Recombination analyses identified six recombination events from 21 viral genomes.ConclusionsTaken together, our in silico analyses using NGS data provide a successful application of plant transcriptomes to reveal extensive information associated with viral genome assembly, SNVs, phylogenetic relationships, and genetic recombination.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-2994-6) contains supplementary material, which is available to authorized users.

PM 7/125 (1) ELISA tests for viruses

Specific scopeThis Standard describes the most commonly used ELISA tests in virology and how to perform these tests for detection and/or identification of plant viruses.1Specific approval and amendmentApproved as an EPPO Standard in 2015‐09.

Identification and characterization of a maize-associated mastrevirus in China by deep sequencing small RNA populations.

BackgroundMaize streak Reunion virus (MSRV) is a member of the Mastrevirus genus in the family Geminiviridae. Of the diverse and increasing number of mastrevirus species found so far, only Wheat dwarf virus and Sweetpotato symptomless virus 1 have been discovered in China. Recently, a novel, unbiased approach based on deep sequencing of small interfering RNAs followed by de novo assembly of siRNA, has greatly offered opportunities for plant virus identification.MethodsSamples collected from maize leaves was deep sequencing for virus identification. Subsequently, the assay of PCR, rolling circle amplification and Southern blot were used to confirm the presence of a mastrevirus.ResultsMaize streak Reunion virus Yunnan isolate (MSRV-[China:Yunnan 06:2014], abbreviated to MSRV-YN) was identified from maize collected from Yunnan Province, China, by small RNA deep sequencing. The complete genome of this virus was ascertained as 2,880 nucleotides long by conventional sequencing. A phylogenetic analysis showed it shared 96.3 % nucleotide sequence identity with the isolate of Maize streak Reunion virus from La Reunion Island. To our knowledge, this is the first identification of MSRV in China. Analyses of the viral derived small interfering RNAs (vsiRNAs) profile showed that the most abundant MSRV-YN vsiRNAs were 21, 22 and 24 nt long and biased for A and G at their 5’ terminal residue. There was a slightly higher representation of MSRV-YN siRNAs derived from the virion-sense strand genome than the complementary-sense strand genome. Moreover, MSRV-YN vsiRNAs were not uniformly distributed along the genome, and hotspots were detected in the movement protein and coat protein-coding region.ConclusionsA mastrevirus MSRV-YN collected in Yunnan Province, China, was identified by small RNA deep sequencing. This vsiRNAs profile derived from MSRV-YN was characterized, which might contribute to get an insight into the host RNA silencing defense induced by MSRV-YN, and provide guidelines on designing antiviral strategies using RNAi against MSRV-YN.Electronic supplementary materialThe online version of this article (doi:10.1186/s12985-015-0384-3) contains supplementary material, which is available to authorized users.

Immobilization-mediated reduction in melting temperatures of DNA–DNA and DNA–RNA hybrids: Immobilized DNA probe hybridization studied by SPR

Abstract The melting temperature of probe-target nucleic acid hybrids plays an important role in sensor performance. This study employed surface plasmon resonance (SPR) techniques to investigate the hybridization of both DNA and RNA targets to their complementary surface-bound DNA oligonucleotide and the corresponding melting temperatures. The target molecule was a 26 nucleotide strand of RNA selected because of its utility in detecting the tobacco mosaic virus, a common plant pathogen with an RNA genome. The melting temperatures of duplexes with immobilized probes were determined by hybridization of the DNA and RNA targets with the bound DNA probe at different temperatures using a custom-built thermostated dual-channel SPR cell. Hybridization conditions and binding efficiencies were compared for both DNA and RNA binding to the gold-coated surface-bound DNA probe. The melting temperature for the DNA–DNA duplex was approximately 15 °C higher than the DNA–RNA in the solid state. The melting temperature in solution was also measured for comparison to the surface values. For both RNA and DNA, the melting transitions were substantially lower (∼20 °C) for solid state binding relative to solution. Particularly for surface immobilized DNA–RNA hybrids, the melting temperature was reduced to room temperature or below. Since most biosensor platforms operate at room temperature, they may consequently exhibit poor sensor performance due to weakened probe-target interactions. This result emphasizes that for optimal sensor performance, the hybrid melting temperature should be considered. The ability to study and optimize the binding of complementary strands of genomic materials to bound DNA probes could facilitate the rapid detection and identification of plant viruses having genomic RNA using various biosensor rapid analytical techniques.

Immunocapture-Multiplex RT-PCR for the Simultaneous Detection and Identification of Plant Viruses and Their Strains: Study Case, Potato Virus Y (PVY).

Immunocapture-reverse transcription-polymerase chain reaction (IC-RT-PCR) is a sensitive, reproducible, and robust method for the detection and identification of RNA viruses. The IC step provides a simple method to isolate virus particles from plant tissue, particularly when inhibitory substances are present, and thus enables subsequent use of RT-PCR amplification for large-scale virus testing and typing. The multiplex format of the PCR is often used for the detection and identification of multiple virus/strain simultaneously to save time, labor, and cost. Potato virus Y (PVY) is one of the most economically important viruses infecting potato worldwide. PVY exists as a complex of at least nine strains and many more unclassified recombinants that vary in their genome structures, phenotypes, and their economic importance. In the current chapter, a detailed protocol of an IC-based, multiplex RT-PCR assay for the detection and identification of various PVY strains is described.

Application of Next-Generation Sequencing Technology for Plant Virus Identification

Next-generation sequencing technology (NGS) has been used as a powerful tool for studies on plant pathogens, especially for plant virus identification. NGS is sequence-independent and culture-independent approach, which can be used for detection of RNA viruses, DNA viruses and viroid simultaneously in a plant sample which contains very low titers. Diagnostic for plant viruses is easy by using this revolutionary technology. In the past few years, NGS has been successfully used for survey and identification of viruses in numerous plant species. In this review, we discuss the application and future perspective of NGS in plant virus identification.