Identification of Plant Viruses Infecting Pear Using RNA Sequencing.
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ABSTRACT:
SUBMITTER: Kim NY
PROVIDER: S-EPMC8200581 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:BackgroundPlums are a kind of stone fruit, a category that includes peaches, cherries, apricots, and almonds. In Korea, Japanese plum trees are usually cultivated as they best suit the climate. To date, there have been few studies in Korea on viruses infecting plum trees compared to those infecting peach trees.MethodsTo identify viruses and viroids infecting plum trees, we collected leaf samples from six different plum cultivars and subjected them to RNA-sequencing (RNA-seq). Six different plum transcriptomes were de novo assembled using the Trinity assembler followed by BLAST searching against a viral reference database.ResultsWe identified hop stunt viroid (HSVd) and six viruses, including apple chlorotic leaf spot virus (ACLSV), little cherry virus-1 (LChV-1), peach virus D (PeVD), peach leaf pitting-associated virus (PLPaV), plum bark necrosis stem pitting-associated virus (PBNSPaV), and prunus necrotic ringspot virus (PNRSV), from six plum cultivars by RNA-seq. RT-PCR confirmed the infection of HSVd and three viruses-ACLSV, PBNSPaV, and PNRSV-in plum trees. However, RT-PCR demonstrated that plum trees in this study were not infected by LChV-1, PeVD, or PLPaV. It is likely that the three viruses LChV-1, PeVD, and PLPaV as identified by RNA-seq were contaminants from other peach libraries caused by index misassignment, which suggests that careful confirmation by other methods should be carried out in next-generation sequencing (NGS)-based virus identification. Taken together, we identified a viroid and three viruses infecting plum trees in Korea.
| S-EPMC7395596 | biostudies-literature
Project description:BackgroundThe invasion of plant by viruses cause major damage to plants and reduces crop yield and integrity. Devastating plant virus infection has been experienced at different times all over the world, which are attributed to different events of mutation, re-assortment and recombination occurring in the viruses. Strategies for proper virus management has been mostly limited to eradicating the vectors that spreads the plant viruses. However, development of prompt and effective diagnostic methods are required to monitor emerging and re-emerging diseases that may be symptomatic or asymptomatic in the plant as well as the genetic variation and evolution in the plant viruses. A survey of plant viruses infecting field-grown Tobacco crop was conducted in Anhui Province of China by the deep sequencing of sRNAs.MethodsSurvey of plant viruses infecting Tobacco was carried based on 104 samples collected across the province. Nine different sRNA libraries was prepared and custom-made bioinformatics pipeline coupled with molecular techniques was developed to sequence, assemble and analyze the siRNAs for plant virus discovery. We also carried out phylogenetic and recombination analysis of the identified viruses.ResultsTwenty two isolates from eight different virus species including Cucumber mosaic virus, Potato virus Y, Tobacco mosaic virus, Tobacco vein banding Mosaic virus, Pepper mottle virus, Brassica yellow virus, Chilli venial mottle virus, Broad bean wilt virus 2 were identified in tobacco across the survey area. The near-complete genome sequence of the 22 new isolates were determined and analyzed. The isolates were grouped together with known strains in the phylogenetic tree. Molecular variation in the isolates indicated the conserved coding regions have majorly a nucleotide sequence identity of 80-94 % with previously identified isolates. Various events of recombination were discovered among some of the isolates indicating that two or more viruses or different isolates of one virus infect the same host cell.ConclusionThis study describes the discovery of a consortium of plant viruses infecting Tobacco that are broadly distributed in Anhui province of China. It also demonstrates the effectiveness of NGS in identifying plant viruses without a prior knowledge of the virus and the genetic diversity that enhanced mixed infection.
| S-EPMC5096307 | biostudies-literature
Project description:Viruses have caused much mortality and morbidity to humans and pose a serious threat to global public health. The virome with the potential of human infection is still far from complete. Novel viruses have been discovered at an unprecedented pace as the rapid development of viral metagenomics. However, there is still a lack of methodology for rapidly identifying novel viruses with the potential of human infection. This study built several machine learning models to discriminate human-infecting viruses from other viruses based on the frequency of k-mers in the viral genomic sequences. The k-nearest neighbor (KNN) model can predict the human-infecting viruses with an accuracy of over 90%. The performance of this KNN model built on the short contigs (?1 kb) is comparable to those built on the viral genomes. We used a reported human blood virome to further validate this KNN model with an accuracy of over 80% based on very short raw reads (150 bp). Our work demonstrates a conceptual and generic protocol for the discovery of novel human-infecting viruses in viral metagenomics studies.
| S-EPMC7168554 | biostudies-literature
Project description:Apple stem grooving virus (ASGV; genus Capillovirus) is an economically important virus. It has an approx. 6.5 kb, monopartite, linear, positive-sense, single-stranded RNA genome. The present study includes identification of 24 isolates-13 isolates from apple (Pyrus malus L.) and 11 isolates from pear (Pyrus communis L.)-from different agricultural fields in South Korea. The coat protein (CP) gene of the corresponding 23 isolates were amplified, sequenced, and analyzed. The CP sequences showed phylogenetic separation based on their host species, and not on the geography, indicating host adaptation. Further analysis showed that the ASGV isolated in this study followed host adaptation influenced and preferred by the host codon-usage.
| S-EPMC8223792 | biostudies-literature
Project description:The cosmopolitan fungus Rhizoctonia solani has a wide host range and is the causal agent of numerous crop diseases, leading to significant economic losses. To date, no cultivars showing complete resistance to R. solani have been identified and it is imperative to develop a strategy to control the spread of the disease. Fungal viruses, or mycoviruses, are widespread in all major groups of fungi and next-generation sequencing (NGS) is currently the most efficient approach for their identification. An increasing number of novel mycoviruses are being reported, including double-stranded (ds) RNA, circular single-stranded (ss) DNA, negative sense (-)ssRNA, and positive sense (+)ssRNA viruses. The majority of mycovirus infections are cryptic with no obvious symptoms on the hosts; however, some mycoviruses may alter fungal host pathogenicity resulting in hypervirulence or hypovirulence and are therefore potential biological control agents that could be used to combat fungal diseases. R. solani harbors a range of dsRNA and ssRNA viruses, either belonging to established families, such as Endornaviridae, Tymoviridae, Partitiviridae, and Narnaviridae, or unclassified, and some of them have been associated with hypervirulence or hypovirulence. Here we discuss in depth the molecular features of known viruses infecting R. solani and their potential as biological control agents.
| S-EPMC6950361 | biostudies-literature
Project description:Viruses are major pathogens of agricultural crops. Viral infections often start after the virus enters the outer layer of a tissue, and many successful viruses, after local replication in the infected tissue, are able to spread systemically. Quantitative details of virus dynamics in plants, however, are poorly understood, in part, because of the lack of experimental methods which allow the accurate measurement of the degree of infection in individual plant tissues. Recently, a group of researchers followed the kinetics of infection of individual cells in leaves of Nicotiana tabacum plants using Tobacco etch virus (TEV) expressing either Venus or blue fluorescent protein (BFP). Assuming that viral spread occurs from lower to upper leaves, the authors fitted a simple mathematical model to the frequency of cellular infection by the two viral variants found using flow cytometry. While the original model could accurately describe the kinetics of viral spread locally and systemically, we found that many alternative versions of the model, for example, if viral spread starts at upper leaves and progresses to lower leaves or when virus dissemination is stopped due to an immune response, fit the data with reasonable quality, and yet with different parameter estimates. These results strongly suggest that experimental measurements of the virus infection in individual leaves may not be sufficient to identify the pathways of viral dissemination between different leaves and reasons for viral control. We propose experiments that may allow discrimination between the alternatives. By analyzing the kinetics of coinfection of individual cells by Venus and BFP strains of TEV we found a strong deviation from the random infection model, suggesting cooperation between the two strains when infecting plant cells. Importantly, we showed that many mathematical models on the kinetics of coinfection of cells with two strains could not adequately describe the data, and the best fit model needed to assume (i) different susceptibility of uninfected cells to infection by two viruses locally in the leaf vs. systemically from other leaves, and (ii) decrease in the infection rate depending on the fraction of uninfected cells which could be due to a systemic immune response. Our results thus demonstrate the difficulty in reaching definite conclusions from extensive and yet limited experimental data and provide evidence of potential cooperation between different viral variants infecting individual cells in plants.
| S-EPMC9029262 | biostudies-literature
Project description:Viruses-mediated genome editing in plants is a powerful strategy to develop plant cultivars with important and novel agricultural traits. Mulberry alba is an important economic tree species that has been cultivated in China for more than 5000 years. So far, only a few viruses have been identified from mulberry trees, and their application potential is largely unknown. Therefore, mining more virus resources from the mulberry tree can pave the way for the establishment of useful engineering tools. In this study, eight old mulberry plants were gathered in seven geographic areas for virome analysis. Based on transcriptome analysis, we discovered three viruses associated with mulberries: Citrus leaf blotch virus isolate mulberry alba 2 (CLBV-ML2), Mulberry-associated virga-like virus (MaVLV), and Mulberry-associated narna-like virus (MaNLV). The genome of CLBV-ML2 was completely sequenced and exhibited high homology with Citriviruses, considered to be members of the genus Citrivirus, while the genomes of MaVLV and MaNLV were nearly completed lacking the 5' and 3' termini sequences. We tentatively consider MaVLV to be members of the family Virgaviridae and MaNLV to be members of the genus Narnavirus based on the results of phylogenetic trees. The infection experiments showed that CLBV-ML2 could be detected in the inoculated seedlings of both N. benthamiana and Morus alba, while MaVLV could only be detected in N. benthamiana. All of the infected seedlings did not show obvious symptoms.
| S-EPMC9696721 | biostudies-literature
Project description: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.
| S-EPMC9131090 | biostudies-literature
Project description:We used high-throughput sequencing to identify viruses on tomato samples showing virus-like symptoms. Samples were collected from crops in the Iberian Peninsula. Either total RNA or double-stranded RNA (dsRNA) were used as starting material to build the cDNA libraries. In total, seven virus species were identified, with pepino mosaic virus being the most abundant one. The dsRNA input provided better coverage and read depth but missed one virus species compared with the total RNA input. By performing in silico analyses, we determined a minimum sequencing depth per sample of 0.2 and 1.5 million reads for dsRNA and rRNA-depleted total RNA inputs, respectively, to detect even the less abundant viruses. Primers and TaqMan probes targeting conserved regions in the viral genomes were designed and/or used for virus detection; all viruses were detected by qRT-PCR/RT-PCR in individual samples, with all except one sample showing mixed infections. Three virus species (Olive latent virus 1, Lettuce ring necrosis virus and Tomato fruit blotch virus) are herein reported for the first time in tomato crops in Spain.
| S-EPMC8150983 | biostudies-literature
Project description:Deep sequencing has generated 52 contigs derived from five viruses; Apple chlorotic leaf spot virus (ACLSV), Apple stem grooving virus (ASGV), Apple stem pitting virus (ASPV), Apple green crinkle associated virus (AGCaV), and Apricot latent virus (ApLV) were identified from eight apple samples showing small leaves and/or growth retardation. Nucleotide (nt) sequence identity of the assembled contigs was from 68% to 99% compared to the reference sequences of the five respective viral genomes. Sequences of ASPV and ASGV were the most abundantly represented by the 52 contigs assembled. The presence of the five viruses in the samples was confirmed by RT-PCR using specific primers based on the sequences of each assembled contig. All five viruses were detected in three of the samples, whereas all samples had mixed infections with at least two viruses. The most frequently detected virus was ASPV, followed by ASGV, ApLV, ACLSV, and AGCaV which were withal found in mixed infections in the tested samples. AGCaV was identified in assembled contigs ID 1012480 and 93549, which showed 82% and 78% nt sequence identity with ORF1 of AGCaV isolate Aurora-1. ApLV was identified in three assembled contigs, ID 65587, 1802365, and 116777, which showed 77%, 78%, and 76% nt sequence identity respectively with ORF1 of ApLV isolate LA2. Deep sequencing assay was shown to be a valuable and powerful tool for detection and identification of known and unknown virome in infected apple trees, here identifying ApLV and AGCaV in commercial orchards in Korea for the first time.
| S-EPMC5051563 | biostudies-literature