Search Results (125)

A high-throughput sequencing-based grapevine metagenomic survey was conducted across all grape-growing Canadian provinces (British Columbia, Ontario, Nova Scotia, and Québec) with the objective of better understanding the grapevine virome composition. In total, 310 composite grapevine samples representing nine Vitis vinifera red; five V. vinifera white; seven American–French red; and five white hybrid cultivars were analyzed. dsRNA, enriched using two different methods, was used as the starting material and source of viral nucleic acids in HTS. The virome status on the distribution and incidence in different regions and grapevine cultivars is addressed. Results from this study revealed the presence of 20 viruses and 3 viroids in the samples tested. Twelve viruses, which are in the regulated viruses list under grapevine certification, were identified in this survey. The major viruses detected in this survey and their incidence rates are GRSPaV (26% to 100%), GLRaV-2 (1% to 18%), GLRaV-3 (15% to 63%), GRVFV (0% to 52%), GRGV (0% to 52%), GPGV (3.3% to 77%), GFkV (1.5% to 31.6%), and GRBV (0% to 19.4%). This survey is the first comprehensive virome study using viral dsRNA and a metagenomics approach on grapevine samples from the British Columbia, Ontario, Nova Scotia, and Quebec provinces in Canada. Results from this survey highlight the grapevine virome distribution across four major grapevine-growing regions and their cultivars. The outcome of this survey underlines the need for strengthening current management options to mitigate the impact of virus spread, and the implementation of a domestic grapevine clean plant program to improve the sanitary status of the grapevine ecosystem. Full article

Viroids are subviral plant pathogens composed of non-coding, small, circular, single-stranded RNAs that parasitize the transcriptional machinery of their host cells. For many years, viroid-induced diseases were mistakenly attributed to viruses due to similarities in symptoms and pathogenic behavior. However, advances in molecular biology over the past sixty years have clearly distinguished viroids from viruses and other pathogens in terms of genetic composition, structural features, and replication mechanisms. Citrus trees in the Mediterranean region appear to have been associated with viroid infections since ancient times. Nevertheless, the use of propagation material harboring asymptomatic viroid infections allowed for continued production of high-quality fruit. This delicate equilibrium was disrupted with the spread of novel citrus pathogens, prompting the adoption of new horticultural practices that emphasized the elimination of citrus pathogens—including viroids—from propagation material. Concurrently, a contrasting approach emerged in the late 1960s: the experimental use of “graft-transmissible dwarfing agents”—later identified as citrus viroids—to control citrus tree size. Our lab initiated work on citrus viroid-induced dwarfing in the early 1980s and continued this line of research for nearly two decades. Eventually, we concluded that it was impractical to simultaneously promote rigorous sanitation protocols while advocating for the use of viroids to induce dwarfing. This review summarizes key biological and molecular aspects of citrus and avocado viroids investigated in our laboratory, including the development of diagnostic techniques and the exploration of viroid-induced dwarfing as a horticultural tool. Full article

Peaches (Prunus persica L. Batsch) and nectarines (Prunus persica L. Batsch var. nectarina [Ait.] Maxim) are economically important stone fruits consumed worldwide, both fresh and processed. Viruses and viroids significantly constrain the cultivation and productivity of peach orchards. Climate change may alter vector populations and lead to shifts in agricultural practices, influencing the spread of these viruses and viroids. Additionally, market globalization further intensifies the pressure on peach crops by facilitating the movement of pathogens, increasing the incidence of virus-induced diseases. In this study, we identified the viral and viroid communities in five peach cultivars from Bulgaria and assessed their impact on symptom development. RNA sequencing of symptomatic leaf samples revealed the presence of common peach viruses, such as plum pox virus and prunus necrotic ringspot virus. Notably, we identified peach latent mosaic viroid and cherry green ring mottle virus in Bulgarian peach orchards for the first time. Furthermore, bioassays of indicator plants, ELISA, and Sanger sequencing were performed for each peach tree to complement the RNA sequencing data. These findings provide valuable insights into the composition of viral and viroid pathogens affecting peaches in Bulgaria and will support the development of targeted strategies for monitoring and managing these pathogens, contributing to the sustainable production of peaches in the region. Full article

Hop latent viroid (HLVd), a 256-nucleotide RNA strand with complementary base-pairing and internal stem loop structures, forms circular or rod-shaped molecules within diseased plants. RT-PCR/RT-qPCR was used to assess HLVd transmission, spread and longevity. The viroid was detected in asymptomatic stock plants and in rooted vegetative cuttings, as well as in recirculated nutrient solution sampled from propagation tables and nozzles. Plant-to-plant spread through root infection in hydroponic cultivation was demonstrated. The viroid survived for 7 days and 4 weeks, respectively, in crushed leaf extracts (sap) or dried leaves/roots at room temperature. Following stem inoculation with infectious sap, HLVd was detected in root tissues within 2–3 weeks and in the foliage within 4–6 weeks. Plants grown under a 12:12 h photoperiod to induce inflorescence development showed more rapid spread of HLVd compared to 24 h lighting. The viroid was subsequently detected in inflorescence tissues, in trichome glands, in dried cannabis flowers and in crude resinous oil extracts. Anthers and pollen from infected male plants and seeds from infected female plants contained HLVd, giving rise to up to 100% infected seedlings. Artificially inoculated tomato and tobacco plants supported viroid replication in roots and leaves. Infected cannabis leaf and root tissues treated with UV-C for 3–5 min or temperatures of 70–90 °C for 30 min contained amplifiable HLVd-RNA. Infectious plant extract treated with 5–10% bleach (0.825% NaOCl) or 1000 ppm hypochlorous acid yielded no RT-PCR bands, suggesting the RNA was degraded. Meristem tip culture from HLVd-infected plants yielded a high frequency of pathogen-free plants, depending on the genotype. Full article

Viroids represent obligate plant pathogens composed exclusively of non-protein coding small single-stranded RNAs that cause high economic losses worldwide. A field survey was carried out to assess the incidence of the peach latent mosaic viroid (PLMVd) in southeastern Kazakhstan, the region of the country where fruit trees are mainly grown. Of 246 stone fruit trees, 20 (8.13%) were infected with the PLMVd. The incidence of the PLMVd in the peach (19.23%; 15/78) was significantly higher than that in the apricot (6.76%; 5/74; p = 0.0234). Eight of the detected viroids were cloned and used for full-genome sequencing. The nucleotide sequence similarity of the selected isolates found in Kazakhstan was 83.9–100%. A phylogenetic analysis indicated three clusters for the Kazakhstani isolates of the PLMVd. Three groups of Kazakhstani viroids differed in their predicted secondary structure. During the survey, the PLMVd was detected and genetically characterized for the first time in Kazakhstan. The obtained results indicate the need to develop state control measures for the PLMVd, including regular monitoring surveys. We identified several SNPs of the PLMVd that had not been previously described. The results may be useful in optimizing diagnostic approaches for detecting stone fruit viroids and preventing their spread through propagation material. Full article

Background:Citrus Exocortis Viroid (CEVd) is a non-coding RNA pathogen capable of infecting a wide range of plant species, despite its lack of protein-coding ability. Viroid infections induce significant alterations in various physiological and biochemical processes, particularly impacting plant metabolism. This study shows the metabolic changes upon viroid infection in tomato plants (Solanum lycopersicum var. ‘MoneyMaker’) exhibiting altered levels of salicylic acid (SA), a key signal molecule involved in the plant defence against this pathogen. Methods: Transgenic RNAi_S5H lines, which have the salicylic acid 5-hydroxylase gene silenced to promote SA accumulation, and NahG lines, which overexpress a salicylate hydroxylase to degrade SA into catechol and prevent its accumulation, were used to establish different SA levels in plants, resulting in varying degrees of resistance to viroid infection. The analysis was performed by using gas chromatography–mass spectrometry (GC-MS) to explore the role of volatile organic compounds (VOCs) in plant immunity against this pathogen. Results: Our results revealed distinct volatile profiles associated with plant immunity, where RNAi_S5H-resistant plants showed significantly enhanced production of monoterpenoids upon viroid infection. Moreover, viroid-susceptible NahG plants emitted a broad range of VOCs, whilst viroid-tolerant RNAi_S5H plants exhibited less variation in VOC emission. Conclusions: This study demonstrates that SA levels significantly influence metabolic responses and immunity in tomato plants infected by CEVd. The identification of differential emitted VOCs upon CEVd infection could allow the development of biomarkers for disease or strategies for disease control. Full article

Among the long non-coding RNAs that are currently recognized as important regulatory molecules influencing a plethora of processes in eukaryotic cells, circular RNAs (circRNAs) represent a distinct class of RNAs that are predominantly produced by back-splicing of pre-mRNA. The most studied regulatory mechanisms involving circRNAs are acting as miRNA sponges, forming R-loops with genomic DNA, and encoding functional proteins. In addition to circRNAs generated by back-splicing, two types of circRNAs capable of autonomous RNA-RNA replication and systemic transport have been described in plants: viroids, which are infectious RNAs that cause a number of plant diseases, and retrozymes, which are transcripts of retrotransposon genomic loci that are capable of circularization due to ribozymes. Based on a number of common features, viroids and retrozymes are considered to be evolutionarily related. Here, we provide an overview of the biogenesis mechanisms and regulatory functions of non-replicating circRNAs produced by back-splicing and further discuss in detail the currently available data on viroids and retrozymes, focusing on their structural features, replication mechanisms, interaction with cellular components, and transport in plants. In addition, biotechnological approaches involving replication-capable plant circRNAs are discussed, as well as their potential applications in research and agriculture. Full article

Gene-editing technology, specifically the CRISPR-Cas13a system, has shown promise in breeding plants resistant to RNA viruses. This system targets RNA and, theoretically, can also combat RNA-based viroids. To test this, the CRISPR-Cas13a system was introduced into tomato plants via transient expression and into Nicotiana benthamiana through transgenic methods, using CRISPR RNAs (crRNAs) targeting the conserved regions of both sense and antisense genomes of potato spindle tuber viroid (PSTVd). In tomato plants, the expression of CRISPR-Cas13a and crRNAs substantially reduced PSTVd accumulation and alleviated disease symptoms. In transgenic N. benthamiana plants, the PSTVd levels were lower as compared to wild-type plants. Several effective crRNAs targeting the PSTVd genomic RNA were also identified. These results demonstrate that the CRISPR-Cas13a system can effectively target and combat viroid RNAs, despite their compact structures. Full article

Loquat leaves exhibiting obvious yellowing, blistering, mosaic, leaf upward cupping, crinkle, and leaf narrowing were identified in Panzhihua City, Sichuan Province, China. High-throughput sequencing (HTS) with the ribo-depleted cDNA library was employed to identify the virome in the loquat samples; only tomato mosaic virus (ToMV) and citrus exocortis viroid (CEVd) were identified in the transcriptome data. The complete genome sequence of ToMV and CEVd were obtained from the loquat leaves. The full-length genome of the ToMV-loquat is 6376 nt and comprises four open reading frames (ORFs) encoding 183 kDa protein, RNA-dependent RNA polymerase (RdRp), movement protein (MP), and coat protein (CP), respectively. A pairwise identity analysis showed that the complete sequence of the ToMV-loquat had a nucleotide identity between 98.5 and 99.3% with other ToMV isolates. A phylogenetic analysis indicated that ToMV-loquat was more closely related to ToMV-IFA9 (GenBank No. ON156781). A CEVd sequence with 361 nt in length was amplified based on the HTS contigs, sequence alignment indicated CEVd-loquat had the highest identity with the strain of CEVd-Balad (GenBank No. PP869624), phylogenetic analysis showed that CEVd-loquat was more closely related to CEVd-lettuce (GenBank No. ON993891). This significant discovery marks the first documentation and characterization of ToMV and CEVd infecting loquat plants, shedding light on potential threats to loquat cultivation and providing insights for disease management strategies. Full article

Pospiviroids infect a wide range of plant species, and many pospiviroids can be transmitted to potato and tomato. Pospiviroids continue to be a major production constraint as well as of quarantine concern for the movement of germplasm, and are regulated in several countries/regions. The USDA APHIS issued a federal order requiring all imported tomato and pepper seeds be certified free of six pospiviroids of quarantine significance. The six pospiviroids of quarantine interest include CLVd, PCFVd, PSTVd, TASVd, TCDVd, TPMVd. Currently, those six viroids are detected by real-time RT-PCR. CRISPR/Cas-based genome editing has been increasingly used for virus detection in the past five years. We used a rapid Cas13-based Specific High-sensitivity Enzymatic Reporter unLOCKing (SHERLOCK) platform for pospiviroid detection, determined the limits of detection and specificity of CRISPR-Cas13a assays. This platform combines recombinase polymerase amplification (RPA) with CRISPR and CRISPR-associated (CRISPR-Cas) RNA-guided endoribonuclease that is rapid and does not require expensive equipment, and can be adapted for on-site detection. Full article

The human hepatitis delta virus (HDV) is a satellite RNA virus that depends on hepatitis B virus (HBV) surface proteins (HBsAg) to assemble into infectious virions targeting the same organ (liver) as HBV. Until recently, the evolutionary origin of HDV remained largely unknown. The application of bioinformatics on whole sequence databases lead to discoveries of HDV-like agents (DLA) and shed light on HDV’s evolution, expanding our understanding of HDV biology. DLA were identified in heterogeneous groups of vertebrates and invertebrates, highlighting that the evolution of HDV, represented by eight distinct genotypes, is broader and more complex than previously foreseen. In this study, we focused on the characterization of three mammalian DLA discovered in woodchuck (Marmota monax), white-tailed deer (Odocoileus virginianus), and lesser dog-like bat (Peropteryx macrotis) in terms of replication, cell-type permissiveness, and spreading pathways. We generated replication-competent constructs expressing 1.1-fold over-length antigenomic RNA of each DLA. Replication was initiated by transfecting the cDNAs into human (HuH7, HeLa, HEK293T, A549) and non-human (Vero E6, CHO, PaKi, LMH) cell lines. Upon transfection and replication establishment, none of the DLA expressed a large delta antigen. A cell division-mediated viral amplification assay demonstrated the capability of non-human DLA to replicate and propagate in hepatic and non-hepatic tissues, without the requirement of envelope proteins from a helper virus. Remarkably L-HDAg but not S-HDAg from HDV can artificially mediate envelopment of WoDV and DeDV ribonucleoproteins (RNPs) by HBsAg to form infectious particles, as demonstrated by co-transfection of HuH7 cells with the respective DLA expression constructs and a plasmid encoding HBV envelope proteins. These chimeric viruses are sensitive to HDV entry inhibitors and allow synchronized infections for comparative replication studies. Our results provide a more detailed understanding of the molecular biology, evolution, and virus–host interaction of this unique group of animal viroid-like agents in relation to HDV. Full article

Citrus is the natural host of at least eight viroid species, providing a natural platform for studying interactions among viroids. The latter manifests as antagonistic or synergistic phenomena. The antagonistic effect among citrus viroids intuitively leads to reduced symptoms caused by citrus viroids, while the synergistic effect leads to an increase in symptom severity. The interaction phenomenon is complex and interesting, and a deep understanding of the underlying mechanisms induced during this viroid interaction is of great significance for the prevention and control of viroid diseases. This paper summarizes the research progress of citrus viroids in recent years, focusing on the interaction phenomenon and analyzing their interaction mechanisms. It points out the core role of the host RNA silencing mechanism and viroid-derived siRNA (vd-siRNA), and provides suggestions for future research directions. Full article

Viroids represent distinctive infectious agents composed solely of short, single-stranded, circular RNA molecules. In contrast to viruses, viroids do not encode for proteins and lack a protective coat protein. Despite their apparent simplicity, viroids have the capacity to induce diseases in plants. Currently, extensive research is being conducted on the replication cycle of viroids within both the Pospiviroidae and Avsunviroidae families, shedding light on the intricacies of the associated host factors. Utilizing the potato spindle tuber viroid as a model, investigations into the RNA structural motifs involved in viroid trafficking between different cell types have been thorough. Nevertheless, our understanding of the host factors responsible for the intra- and inter-cellular movement of viroids remains highly incomplete. This review consolidates our current knowledge of viroid replication and movement within both families, emphasizing the structural basis required and the identified host factors involved. Additionally, we explore potential host factors that may mediate the intra- and inter-cellular movement of viroids, addressing gaps in our understanding. Moreover, the potential application of viroids and the emergence of novel viroid-like cellular parasites are also discussed. Full article

Tomato leaf curl New Delhi virus (ToLCNDV) is an emerging plant pathogen, fast spreading in Asian and Mediterranean regions, and is considered the most harmful geminivirus of cucurbits in the Mediterranean. ToLCNDV infects several plant and crop species from a range of families, including Solanaceae, Cucurbitaceae, Fabaceae, Malvaceae and Euphorbiaceae. Up to now, protection from ToLCNDV infection has been achieved mainly by RNAi-mediated transgenic resistance, and non-transgenic fast-developing approaches are an urgent need. Plant protection by the delivery of dsRNAs homologous to a pathogen target sequence is an RNA interference-based biotechnological approach that avoids cultivating transgenic plants and has been already shown effective against RNA viruses and viroids. However, the efficacy of this approach against DNA viruses, particularly Geminiviridae family, is still under study. Here, the protection induced by exogenous application of a chimeric dsRNA targeting all the coding regions of the ToLCNDV DNA-A was evaluated in zucchini, an important crop strongly affected by this virus. A reduction in the number of infected plants and a delay in symptoms appearance, associated with a tendency of reduction in the viral titer, was observed in the plants treated with the chimeric dsRNA, indicating that the treatment is effective against geminiviruses but requires further optimization. Limits of RNAi-based vaccinations against geminiviruses and possible causes are discussed. Full article

Theodor (“Ted”) Otto Diener (* 28 February 1921 in Zürich, Switzerland; † 28 March 2023 in Beltsville, MD, USA) pioneered research on viroids while working at the Plant Virology Laboratory, Agricultural Research Service, USDA, in Beltsville. He coined the name viroid and defined viroids’ important features like the infectivity of naked single-stranded RNA without protein-coding capacity. During scientific meetings in the 1970s and 1980s, viroids were often discussed at conferences together with other “subviral pathogens”. This term includes what are now called satellite RNAs and prions. Satellite RNAs depend on a helper virus and have linear or, in the case of virusoids, circular RNA genomes. Prions, proteinaceous infectious particles, are the agents of scrapie, kuru and some other diseases. Many satellite RNAs, like viroids, are non-coding and exert their function by thermodynamically or kinetically controlled folding, while prions are solely host-encoded proteins that cause disease by misfolding, aggregation and transmission of their conformations into infectious prion isoforms. In this memorial, we will recall the work of Ted Diener on subviral pathogens. Full article