In this study, we completed the whole genome sequence of a new tobamovirus isolated from tomato plants grown in greenhouses in Jordan during the spring of 2015. The 6393-nt single-stranded RNA ...(ssRNA) genome encodes four proteins, as do other tobamoviruses: two replication-related proteins of 126 kDa and 183 kDa, a 30-kDa movement protein (MP) and a 17.5-kDa coat protein (CP). Phylogenetic analysis showed that this virus does not group with either the tomato mosaic virus (ToMV) or the tobacco mosaic virus (TMV) clades. Instead, it stems from a branch leading to the TMV clade. Analysis of possible recombination events between this virus and representative isolates of closely related tomato-infecting tobamoviruses showed that at least one region originated by recombination. We provide evidence that we have identified a new tobamovirus, for which we propose the name “tomato brown rugose fruit virus”.
Tomato brown rugose fruit virus (ToBRFV), belonging to the genus
, is a highly virulent emerging virus, causing disease outbreaks and significant crop losses worldwide. The growing number of ToBRFV ...epidemic episodes prompted the investigation of the role of seeds in the dissemination of the virus as an important aspect in the overall disease management. Therefore, the objectives of this study were to determine the localization of ToBRFV within tomato seeds and to evaluate its seed transmission characteristics. Seeds extracted from naturally ToBRFV-infected tomato fruits were tested for the presence of the virus using serological, molecular, and biological assays. Three immunolocalization techniques were used to determine the localization and distribution of ToBRFV within the different tissues and parts of tomato seeds. To evaluate seed transmission of ToBRFV, two grow-out experiments were conducted to assess the rate of both vertical (seeds to progeny seedlings) and possible horizontal transmission (plant to plant) based on serological and molecular assays. Seeds extracted from ToBRFV-infected fruits had a 100% contamination rate. The localization of ToBRFV in tomato seeds is only external on the seed coat (testa). Seed transmission rate from seeds to their seedlings was very low (0.08%), while no transmission was recorded from plants to plants in a small-scale greenhouse experimental setup. In conclusion, ToBRFV is a seedborne virus located externally on tomato seed coat and transmitted mechanically from ToBRFV-contaminated tomato seeds to seedlings, which could initiate a disease foci and eventually drive further dissemination and spread of the disease in a new growing area.
•Virome associated to 48 fungi isolated from the sea cucumber Holothuria polii.•Eight fungal isolates contained a total of 10 viruses assembled in silico.•Seven new putative viral species were ...characterized.•New virus taxa proposed.•Nucleocapsid and Ns1 associated to RdRP of mycobunyavirales.
Holothuria polii is a marine animal with an important ecological and economic impact. In the present study we analysed the presence of mycoviruses associated to fungi that were isolated from different H. polii tissues. Among the 48 fungal isolates analysed we identified 10 viruses in 8 strains belonging to 7 fungal species. Five out of nine viruses have a dsRNA genome: three of them belong to the Partitiviridae family, one belongs to a still undefined clade of bipartite viruses and the last one belongs to the Chrysoviridae family. We also identified two viruses belonging to a recently proposed new mycovirus taxon named polymycovirus. Two viruses belong to the positive single stranded RNA clade: one falls into the new Botourmiaviridae family, specifically in the Magoulivirus genus, and the other one falls into a still undefined clade phylogenetically related to tombusviruses. Finally, we also identified a virus with a negative stranded RNA genome showing similarity to a group of viruses recently proposed as a new family of mycoviruses in the order Bunyavirales. A bioinformatics approach comparing two datasets of contigs containing two closely related mycobunyaviruses allowed us to identify putative nucleocapsids (Nc) and non-structural (Ns) associated proteins.
The GenBank/eMBL/DDBJ accession numbers of the sequences reported in this paper are: PRJNA432529, MG913290, MG913291, MG887747, MG887748, MG887749, MG887750, MG887751, MG887752, MG887753, MG887754, MG887755, MG887756, MG887757, MG887758, MG887759, MG887760, MG887761, MG887762, MG887763, MG887764, MG887765, MG887766, MG887767, MH271211, MN163273, MN163274.
Abstract
The obligatory biotrophic oomycetes Plasmopara viticola is the causal agent of downy mildew, a destructive disease of grapevine worldwide. So far, chemical fungicides are widely employed to ...limit this pathogen, but their adverse effects are stimulating the quest for environmentally friendly alternative approaches. Here, we report on the search for mycoviruses that might be later developed as biocontrol agents for this pathogen. Symptomatic leaves were collected from various regions in Spain and Italy and mycelia associated to leaf lesions was harvested. Total RNA extractions were depleted of rRNA and metatranscriptomes were generated using a high-throughput sequencing approach. The virome associated to leaf lesions was then characterized through a bioinformatic pipeline relying on blast searches against current viral databases. Here, we present an inventory of 283 new RNA viruses: 222 positive strand RNA viruses, 29 negative strand RNA viruses, 27 double-stranded RNA viruses and 5 ORFan virus RdRP segments, which could not be reliably assigned to any existing group in the Riboviria. In addition to ORFan viruses, we found other surprising new evolutionary trajectories in this wide inventory of viruses. The most represented viruses in our collection are those in phylum Lenarviricota, and, among them, a group of mycovirus segments distantly related to narnaviruses, but characterized by a polymerase palm domain lacking subdomain C, with the putative GDD catalytic triad. We also provided evidence of a strict association between two RNA segments that form a new mycovirus clade of positive strand RNA in the phylum Kitrinoviricota, order Martellivirales. In the phylum Negarnaviricota, we report for the first time in the order Mononegavirales a clade of viruses that is ambisense, a feature that so far was present only in the order Bunyavirales. Furthermore, in the same phylum we detected the widespread occurrence and abundant accumulation in our libraries of a distinct mycovirus clade distantly related to the Muvirales and Goujanvirales orders, which so far include only viruses infecting invertebrates. Possible new oomycetes-specific virus clades are also described in the phylum Duplornaviricota. These data greatly expand the evolutionary history of mycoviruses adding new layers of diversity to the realm Riboviria.
Tomato brown rugose fruit virus
(ToBRFV), belonging to the genus
Tobamovirus
, is a highly-virulent emerging virus species, causing disease outbreaks and significant crop losses worldwide. No ...effective measure has been identified to control ToBRFV infection. Therefore, the objectives of this study were to investigate the effectiveness of three seed disinfection treatments on the elimination of ToBRFV in tomato seeds and their effect on seed quality. Seeds extracted from ToBRFV-infected fruits were treated with 2% hydrochloric acid (HCl) for 30 min, 10% trisodium phosphate (TSP) for 3 h, dry heat at 72 °C for 72 h, or their combinations; then, the seeds were tested for the presence of the virus using serological, molecular, and biological assays. Parallel treatments of seeds in distilled water (hydropriming) or untreated seeds were included for comparison. The quality of the treated seeds was also evaluated in terms of seed standard germination, mean time to germination (MTG), and seedling growth. Seeds extracted from ToBRFV-infected fruits had a 100% contamination rate. Treatment of ToBRFV-contaminated seeds with 2% HCl for 30 min or 10% TSP for 3 h resulted in a 100% disinfection rate, while heat treatment at 72 °C for 72 h or hydropriming treatments resulted in a 0% disinfection rate. Furthermore, seed treatment with HCl or hydropriming for 3 h significantly improved seed germination percentage, reduced MTG (faster speed of germination), and increased seedling length. In conclusion, seed treatment with 2% HCl for 30 min not only resulted in a 100% disinfection rate of seeds from the ToBRFV but also improved seed quality of tomato compared to untreated seeds.
Cryphonectria parasitica, the causal agent of chestnut blight is controlled in Europe through natural spread of Cryphonectria hypovirus 1 (CHV1), a mycovirus able to induce hypovirulence to the host. ...In recent years C. parasitica was reported infecting Azerbaijani population of chestnut, but the presence of CHV1 still needs to be confirmed. Aim of this work was to investigate fifty-five C. parasitica isolates collected in Azerbaijan to describe the associated viruses. Our work found i) the first negative-sense ssRNA virus known to infect C. parasitica naturally for which we propose the name Cryphonectria parasitica sclerotimonavirus 1 (CpSV1) and ii) an RNA sequence showing peculiar features suggesting a viral nature for which we propose the name Cryphonectria parasitica ambivirus 1 (CpaV1). The discovery of CpaV1 expands our knowledge of the RNA virosphere suggesting the existence of a new lineage that cannot presently be reliably associated to the monophyletic Riboviria.
•First characterization through high-throughput sequence approach of the virome associated to Cryphonectria parasitica.•First virome characterization of Cryphonectria parasitica isolates from Azerbaijan.•Description of the first negative strand virus infecting Cryphonectria parasitica.•Description of a new class of RNA viruses with ambisense genome organization (ambivirus) infecting Cryphonectria parasitica.
•First report of mycoviruses isolated from fungi from marine environment.•Survey of mycoviruses using multiple approaches for both DNA and RNA genomes.•RNAseq analysis is superior to sRNA for de novo ...assembly of mycoviruses.•Twelve new virus species were characterized molecularly.•Expression of a viral RNA from an endogenized cDNA segment.
The number of reported mycoviruses is increasing exponentially due to the current ability to detect mycoviruses using next-generation sequencing (NGS) approaches, with a large number of viral genomes built in-silico using data from fungal transcriptome projects. We decided to screen a collection of fungi originating from a specific marine environment (associated with the seagrass Posidonia oceanica) for the presence of mycoviruses: our findings reveal a wealth of diversity among these symbionts and this complexity will require further studies to address their specific role in this ecological niche. In specific, we identified twelve new virus species belonging to nine distinct lineages: they are members of megabirnavirus, totivirus, chrysovirus, partitivirus and five still undefined clades. We showed evidence of an endogenized virus ORF, and evidence of accumulation of dsRNA from metaviridae retroviral elements. We applied different techniques for detecting the presence of mycoviruses including (i) dsRNA extraction and cDNA cloning, (ii) small and total RNA sequencing through NGS techniques, (iii) rolling circle amplification (RCA) and total DNA extraction analyses, (iv) virus purifications and electron microscopy. We tried also to critically evaluate the intrinsic value and limitations of each of these techniques. Based on the samples we could compare directly, RNAseq analysis is superior to sRNA for de novo assembly of mycoviruses. To our knowledge this is the first report on the virome of fungi isolated from marine environment.
The GenBank/eMBL/DDBJ accession numbers of the sequences reported in this paper are: KT601099–KT601110; KT601114–KT601120; KT592305; KT950836–KT950841.
Indirect evidence of mitochondrial viruses in plants comes from discovery of genomic fragments integrated into the nuclear and mitochondrial DNA of a number of plant species. Here, we report the ...existence of replicating mitochondrial virus in plants: from transcriptome sequencing (RNA-seq) data of infected
, a plant species commonly used as a test plant in virus host range experiments, among other virus contigs, we could assemble a 2.7-kb contig that had highest similarity to mitoviruses found in plant genomes. Northern blot analyses confirmed the existence of plus- and minus-strand RNA corresponding to the mitovirus genome. No DNA corresponding to the genomic RNA was detected, excluding the endogenization of such virus. We have tested a number of
accessions, and the virus was present in a number of commercial varieties but absent from a large collection of Bolivian and Peruvian accessions. The virus could not be transmitted mechanically or by grafting, but it is transmitted vertically through seeds at a 100% rate. Small RNA analysis of a
line carrying the mitovirus and infected by alfalfa mosaic virus showed that the typical antiviral silencing response active against cytoplasmic viruses (21- to 22-nucleotide nt vsRNA peaks) is not active against CqMV1, since in this specific case the longest accumulating vsRNA length is 16 nt, which is the same as that corresponding to RNA from mitochondrial genes. This is evidence of a distinct viral RNA degradation mechanism active inside mitochondria that also may have an antiviral effect.
This paper reports the first biological characterization of a
plant mitovirus in an important crop,
, providing data supporting that mitoviruses have the typical features of cryptic (persistent) plant viruses. We, for the first time, demonstrate that plant mitoviruses are associated with mitochondria in plants. In contrast to fungal mitoviruses, plant mitoviruses are not substantially affected by the antiviral silencing pathway, and the most abundant mitovirus small RNA length is 16 nt.
So far there is no record of a specific virus able to infect both fungal and plant hosts in nature. However, experimental evidence shows that some plant virus RdRPs are able to perform replication in ...trans of genomic or DI RNAs in the yeast Saccharomyces cerevisiae. Furthermore, tobacco mosaic virus was recently shown to replicate in a filamentous ascomycetous fungus. Thus, at least experimentally, some plant viruses can infect some fungi. Endophytic fungi have been reported from many plants and several of these fungi have been shown to contain viruses. Here we tested if mycoviruses derived from a marine plant endophyte can replicate in plant cells. For this purpose, we used partially purified viral particles from isolate MUT4330 of Penicillium aurantiogriseum var. viridicatum which harbors six virus species, some having dsRNA and some positive-strand ssRNA genomes. These were transfected into three distinct plant protoplast cell systems. Time-course analysis of absolute RNA accumulation provided for the first time evidence that viruses of two species belonging to the Partitiviridae and Totiviridae families, can replicate in plant cells without evidence of host adaptation, i.e, changes in their nucleotide sequence.
Summary
To date, no demonstration of a direct correlation between the presence of mycoviruses and the quantitative or qualitative modulation of mycotoxins has been shown. In our study, we transfected ...a virus‐free ochratoxin A (OTA)‐producing isolate of Aspergillus ochraceus with purified mycoviruses from a different A. ochraceus isolate and from Penicillium aurantiogriseum. Among the mycoviruses tested, only Aspergillus ochraceus virus (AoV), a partitivirus widespread in A. ochraceus, caused a specific interaction that led to an overproduction of OTA, which is regulated by the European Commission and is the second most important contaminant of food and feed commodities. Gene expression analysis failed to reveal a specific viral upregulation of the mRNA of genes considered to play a role in the OTA biosynthetic pathway. Furthermore, AoOTApks1, a polyketide synthase gene considered essential for OTA production, is surprisingly absent in the genome of our OTA‐producing isolate. The possible biological and evolutionary implications of the mycoviral regulation of mycotoxin production are discussed.
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