scholarly journals High-throughput sequencing of Potato virus M from tomato in Slovakia reveals a divergent variant of the virus

2019 ◽  
Vol 55 (No. 3) ◽  
pp. 159-166 ◽  
Author(s):  
Miroslav Glasa ◽  
Katarína Šoltys ◽  
Lukáš Predajňa ◽  
Nina Sihelská ◽  
Jaroslav Budiš ◽  
...  
Keyword(s):  
High Throughput ◽  
Potato Virus ◽  
High Throughput Sequencing ◽  
In Silico Analysis ◽  
Rt Pcr ◽  
Tomato Plants ◽  
Virus Diversity ◽  
Group I ◽  
Potato Virus M ◽  
Intraspecies Diversity

High-throughput sequencing (HTS) analysis of tomato (Solanum lycopersicum) samples revealed the presence of Potato virus M (PVM) in this crop in Slovakia. Full-length genomes of three PVM isolates were obtained using both HTS and Sanger sequencing validation. While two isolates (T40 and T50) were shown to belong to major Group I, a divergent T20 isolate was phylogenetically unrelated to any known PVM variant, potentially representing a new phylogenetic group. Despite a relatively high intraspecies diversity (17.3 ± 0.3%), no evidence of recombination was detected in the dataset of available complete PVM sequences. Conventional screening of tomato plants in Slovakia using ELISA and RT-PCR further confirmed a frequent occurrence of PVM in this host. Developed RT-PCR showed its polyvalence to detect the PVM Group I isolates, however, in silico analysis of primer binding sites indicated its compromised use for Group II isolates. Our results further pinpoint the significance of HTS for unbiased unveiling of virus diversity and a need for continual optimisation of molecular detection tools.

scholarly journals Molecular Characterization of Potato Virus Y (PVY) Using High-Throughput Sequencing: Constraints on Full Genome Reconstructions Imposed by Mixed Infection Involving Recombinant PVY Strains

Plants ◽  
2021 ◽  
Vol 10 (4) ◽  
pp. 753
Author(s):  
Miroslav Glasa ◽  
Richard Hančinský ◽  
Katarína Šoltys ◽  
Lukáš Predajňa ◽  
Jana Tomašechová ◽  
...  
Keyword(s):  
High Throughput ◽  
Potato Virus ◽  
Mixed Infection ◽  
High Throughput Sequencing ◽  
Potato Virus Y ◽  
De Novo ◽  
Sweet Pepper ◽  
Complete Sequence ◽  
Genome Region ◽  
Mapping Parameters

In recent years, high throughput sequencing (HTS) has brought new possibilities to the study of the diversity and complexity of plant viromes. Mixed infection of a single plant with several viruses is frequently observed in such studies. We analyzed the virome of 10 tomato and sweet pepper samples from Slovakia, all showing the presence of potato virus Y (PVY) infection. Most datasets allow the determination of the nearly complete sequence of a single-variant PVY genome, belonging to one of the PVY recombinant strains (N-Wi, NTNa, or NTNb). However, in three to-mato samples (T1, T40, and T62) the presence of N-type and O-type sequences spanning the same genome region was documented, indicative of mixed infections involving different PVY strains variants, hampering the automated assembly of PVY genomes present in the sample. The N- and O-type in silico data were further confirmed by specific RT-PCR assays targeting UTR-P1 and NIa genomic parts. Although full genomes could not be de novo assembled directly in this situation, their deep coverage by relatively long paired reads allowed their manual re-assembly using very stringent mapping parameters. These results highlight the complexity of PVY infection of some host plants and the challenges that can be met when trying to precisely identify the PVY isolates involved in mixed infection.

Comparison of high throughput sequencing to standard protocols for virus detection in berry crops

Plant Disease ◽  
2021 ◽  
Author(s):  
Dan Edward Veloso Villamor ◽  
Karen E Keller ◽  
Robert Martin ◽  
Ioannis Emmanouil Tzanetakis
Keyword(s):  
High Throughput ◽  
High Throughput Sequencing ◽  
Wide Spectrum ◽  
Virus Detection ◽  
Rt Pcr ◽  
Host Interactions ◽  
Growing Seasons ◽  
Berry Crops ◽  
Sampling Times ◽  
Better Than

A comprehensive study comparing virus detection between high throughput sequencing (HTS) and standard protocols in 30 berry selections (12 Fragaria, 10 Vaccinium and 8 Rubus) with known virus profiles was completed. The study examined temporal detection of viruses at four sampling times encompassing two growing seasons. Within the standard protocols, RT-PCR proved better than biological indexing. Detection of known viruses by HTS and RT-PCR nearly mirrored each other. HTS provided superior detection compared to RT-PCR on a wide spectrum of virus variants and discovery of novel viruses. More importantly, in most cases where the two protocols showed parallel virus detection, 11 viruses in 16 berry selections were not consistently detected by both methods at all sampling points. Based on these data we propose a four sampling times/two-year testing requirement for berry and potentially other crops to ensure that no virus remains undetected independent of titer, distribution or other virus/virus or virus/host interactions.

scholarly journals High-Throughput Sequencing Reveals Further Diversity of Little Cherry Virus 1 with Implications for Diagnostics

Viruses ◽  
2018 ◽  
Vol 10 (7) ◽  
pp. 385 ◽  
Author(s):  
Asimina Katsiani ◽  
Varvara Maliogka ◽  
Nikolaos Katis ◽  
Laurence Svanella-Dumas ◽  
Antonio Olmos ◽  
...  
Keyword(s):  
Genetic Diversity ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Sour Cherry ◽  
Prunus Species ◽  
Cherry Tree ◽  
High Genetic Diversity ◽  
Viral Genomes ◽  
Virus Diversity ◽  
First Time

Little cherry virus 1 (LChV1, Velarivirus, Closteroviridae) is a widespread pathogen of sweet or sour cherry and other Prunus species, which exhibits high genetic diversity and lacks a putative efficient transmission vector. Thus far, four distinct phylogenetic clusters of LChV1 have been described, including isolates from different Prunus species. The recent application of high throughput sequencing (HTS) technologies in fruit tree virology has facilitated the acquisition of new viral genomes and the study of virus diversity. In the present work, several new LChV1 isolates from different countries were fully sequenced using different HTS approaches. Our results reveal the presence of further genetic diversity within the LChV1 species. Interestingly, mixed infections of the same sweet cherry tree with different LChV1 variants were identified for the first time. Taken together, the high intra-host and intra-species diversities of LChV1 might affect its pathogenicity and have clear implications for its accurate diagnostics.

scholarly journals High-throughput sequencing identifies distinct fecal and mucosal gut microbiota correlating with different mucosal proteins

2016 ◽  
Author(s):  
Li-na Dong ◽  
Jun-ping Wang ◽  
Ping Liu ◽  
Yun-feng Yang ◽  
Jing Feng ◽  
...  
Keyword(s):  
Gut Microbiota ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Flexible Sigmoidoscopy ◽  
Local Effect ◽  
Chinese People ◽  
Rt Pcr ◽  
Clinical Indices ◽  
16Srrna Gene ◽  
Firmicutes Phylum

The intestinal microbiota is associated with human health. The luminal microbiota (LM) and mucosa-associated microbiota (MAM) are distinct ecosystems with different metabolic and immunological functions. Several studies have examined the correlations between the gut microbiota and clinical indices, but few have investigated the relationships between the microbiota and mucosal proteins. We characterized the intestinal LM and MAM in Chinese people and examined the association between these communities and the expression of mucosal proteins. Fresh fecal samples and distal colonic mucosal biopsies were collected from 32 subjects before (fecal) and during (mucosal) flexible sigmoidoscopy. We used high-throughput sequencing targeting the 16SrRNA gene V3–V4 region to analyze the samples and reverse transcription(RT)–PCR to detect the expression of colonic proteins BDNF, ZO1, TLR2, TLR4, AQP3, and AQP8. Differences in the stool and mucosal microbiota were identified and a correlation network analysis performed. The LM and MAM populations differed significantly. In LM, the microbiota composition correlated significantly positively with host age, and Firmicutes (phylum) correlated positively with body mass index (BMI), but inversely with ZO1.At the genus level, systemic indices, such as age, BMI, and BDNF, correlated predominantly with LM, whereas systemic and local indices, such as TLR2, correlated with both MAM and LM. ZO1 and TLR4 which usually exert a local effect, mainly correlated with MAM. Different bacteria were associated with the expression of different proteins. Our data suggest that The microbial compositions of LM and MAM differed. Different gut bacteria may play different roles by regulating the expression of different proteins.

scholarly journals High-Throughput Sequencing Reveals Cyclamen persicum Mill. as a Natural Host for Fig Mosaic Virus

Viruses ◽  
2018 ◽  
Vol 10 (12) ◽  
pp. 684 ◽  
Author(s):  
Toufic Elbeaino ◽  
Armelle Marais ◽  
Chantal Faure ◽  
Elisa Trioano ◽  
Thierry Candresse ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Sequencing ◽  
Viral Infections ◽  
Small Scale ◽  
Rt Pcr ◽  
Cyclamen Persicum ◽  
Sequence Variations ◽  
Pcr Products ◽  
Complete Sequences ◽  
Fig Mosaic Virus

In a search for viral infections, double-stranded RNA (dsRNA) were recovered from a diseased cyclamen (Cyclamen persicum Mill.) accession (Cic) and analyzed by high-throughput sequencing (HTS) technology. Analysis of the HTS data showed the presence of Fig mosaic emaravirus (FMV) in this accession. The complete sequences of six FMV-Cic RNA genomic segments were determined from the HTS data and using Sanger sequencing. All FMV-Cic RNA segments are similar in size to those of FMV from fig (FMV-Gr10), with the exception of RNA-6 that is one nucleotide longer. The occurrence of FMV in cyclamen was investigated through a small-scale survey, from which four plants (out of 18 tested) were found RT-PCR positive. To study sequence variations of cyclamen isolates of FMV, RT-PCR products generated through the amplification of the partially RNA-dependent RNA polymerase (RdRp, RNA-1), glycoprotein (GP, RNA-2), and nucleocapsid (NCP, RNA-3) genes were explored. The nucleotide sequence identities for cyclamen isolates ranged between 86% and 99% in RNA-1, 93% and 99% in RNA-2, and 98% and 99% in RNA-3, while lower identity levels were observed with the sequences of fig isolates. Based on the phylogenetic tree obtained with a 304-nt fragment of RNA3, all FMV isolates from cyclamens were assigned to a single cluster close to fig isolates from the Mediterranean. FMV was graft-transmitted to healthy cyclamens eliciting symptoms similar to those observed in the Cic accession, thus suggesting a causal role of FMV in the symptoms that prompted the investigation. This is the first report of FMV in a non-fig host, Cyclamen persicum, a finding that may help in the control of the mosaic and mosaic-like diseases of fig and cyclamen, respectively.

scholarly journals Tomato Brown Rugose Fruit Virus Contributes to Enhanced Pepino Mosaic Virus Titers in Tomato Plants

Viruses ◽  
2020 ◽  
Vol 12 (8) ◽  
pp. 879 ◽  
Author(s):  
Chen Klap ◽  
Neta Luria ◽  
Elisheva Smith ◽  
Lior Hadad ◽  
Elena Bakelman ◽  
...  
Keyword(s):  
Mosaic Virus ◽  
High Throughput Sequencing ◽  
Amino Acid Position ◽  
Viral Disease ◽  
Mixed Infections ◽  
Rt Pcr ◽  
Tomato Production ◽  
Pepino Mosaic Virus ◽  
Disease Symptoms ◽  
Tomato Plants

The tobamovirus tomato brown rugose fruit virus (ToBRFV), a major threat to tomato production worldwide, has recently been documented in mixed infections with the potexvirus pepino mosaic virus (PepMV) CH2 strain in traded tomatoes in Israel. A study of greenhouse tomato plants in Israel revealed severe new viral disease symptoms including open unripe fruits and yellow patched leaves. PepMV was only detected in mixed infections with ToBRFV in all 104 tested sites, using serological and molecular analyses. Six PepMV isolates were identified, all had predicted amino acids characteristic of CH2 mild strains excluding an isoleucine at amino acid position 995 of the replicase. High-throughput sequencing of viral RNA extracted from four selected symptomatic plants showed solely the ToBRFV and PepMV, with total aligned read ratios of 40.61% and 11.73%, respectively, indicating prevalence of the viruses. Analyses of interactions between the co-infecting viruses by sequential and mixed viral inoculations of tomato plants, at various temperatures, showed a prominent increase in PepMV titers in ToBRFV pre-inoculated plants and in mixed-infected plants at 18–25 °C, compared to PepMV-single inoculations, as analyzed by Western blot and quantitative RT-PCR tests. These results suggest that Israeli mild PepMV isolate infections, preceded by ToBRFV, could induce symptoms characteristic of PepMV aggressive strains.

scholarly journals High throughput sequencing unravels tomato-pathogen interactions towards a sustainable plant breeding

2021 ◽  
Vol 8 (1) ◽  
Author(s):  
Maria Doroteia Campos ◽  
Maria do Rosário Félix ◽  
Mariana Patanita ◽  
Patrick Materatski ◽  
Carla Varanda
Keyword(s):  
Plant Breeding ◽  
High Throughput ◽  
Plant Pathogen ◽  
Molecular Mechanisms ◽  
High Throughput Sequencing ◽  
Molecular Networks ◽  
Sequencing Data ◽  
Sources Of Resistance ◽  
Tomato Plants ◽  
Wide Range

AbstractTomato (Solanum lycopersicum) is one of the most economically important vegetables throughout the world. It is one of the best studied cultivated dicotyledonous plants, often used as a model system for plant research into classical genetics, cytogenetics, molecular genetics, and molecular biology. Tomato plants are affected by different pathogens such as viruses, viroids, fungi, oomycetes, bacteria, and nematodes, that reduce yield and affect product quality. The study of tomato as a plant-pathogen system helps to accelerate the discovery and understanding of the molecular mechanisms underlying disease resistance and offers the opportunity of improving the yield and quality of their edible products. The use of functional genomics has contributed to this purpose through both traditional and recently developed techniques, that allow the identification of plant key functional genes in susceptible and resistant responses, and the understanding of the molecular basis of compatible interactions during pathogen attack. Next-generation sequencing technologies (NGS), which produce massive quantities of sequencing data, have greatly accelerated research in biological sciences and offer great opportunities to better understand the molecular networks of plant–pathogen interactions. In this review, we summarize important research that used high-throughput RNA-seq technology to obtain transcriptome changes in tomato plants in response to a wide range of pathogens such as viruses, fungi, bacteria, oomycetes, and nematodes. These findings will facilitate genetic engineering efforts to incorporate new sources of resistance in tomato for protection against pathogens and are of major importance for sustainable plant-disease management, namely the ones relying on the plant’s innate immune mechanisms in view of plant breeding.

scholarly journals Detection of Banana Mild Mosaic Virus in Musa In Vitro Plants: High-Throughput Sequencing Presents Higher Diagnostic Sensitivity Than (IC)-RT-PCR and Identifies a New Betaflexiviridae Species

Plants ◽  
2022 ◽  
Vol 11 (2) ◽  
pp. 226
Author(s):  
Marwa Hanafi ◽  
Wei Rong ◽  
Lucie Tamisier ◽  
Chadi Berhal ◽  
Nicolas Roux ◽  
...  
Keyword(s):  
Mosaic Virus ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Diagnostic Sensitivity ◽  
Rapid Screening ◽  
Mild Mosaic ◽  
Rt Pcr ◽  
In Vitro Plants ◽  
Mild Mosaic Virus

: The banana mild mosaic virus (BanMMV) (Betaflexiviridae, Quinvirinae, unassigned species) is a filamentous virus that infects Musa spp. and has a very wide geographical distribution. The current BanMMV indexing process for an accession requires the testing of no less than four plants cultivated in a greenhouse for at least 6 months and causes a significant delay for the distribution of the germplasm. We evaluated the sensitivity of different protocols for BanMMV detection from in vitro plants to accelerate the testing process. We first used corm tissues from 137 in vitro plants and obtained a diagnostic sensitivity (DSE) of only 61% when testing four plants per accession. After thermotherapy was carried out to eliminate BanMMV infection, the meristem was recovered and further grown in vitro. The same protocol was evaluated in parallel on the corm tissue surrounding the meristem, as a rapid screening to evaluate virus therapy success, and was compared to the results obtained following the standard protocol. The obtained results showed 28% false negatives when conducting testing from corm tissues, making this protocol unsuitable in routine processes. Furthermore, RT-PCR and high-throughput sequencing (HTS) tests were applied on tissues from the base (n = 39) and the leaves (n = 36). For RT-PCR, the average DSE per sample reached 65% from either the base or leaves. HTS was applied on 36 samples and yielded 100% diagnostic specificity (DSP) and 100% DSE, whatever the sampled tissue, allowing the identification of a new Betaflexiviridae species infecting Musa. These results suggest that a reliable diagnostic of BanMMV from in vitro plants using RT-PCR or HTS technologies might represent an efficient alternative for testing after greenhouse cultivation.

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