High-Throughput Sequencing Reveals New Viroid Species in Opuntia in Mexico.

In the main cactus pear (Opuntia ficus-indica)-producing region in the State of Mexico, fruit production occupies the largest cultivated area with 15,800 ha, while 900 ha are cultivated for edible young Opuntia pads ("nopalitos") which are consumed as vegetables. Two composite samples consisting of cladodes of plants for fruit production (n = 6) and another of "nopalitos" (n = 6) showing virus-like symptoms were collected. Both sample sets were subjected to high-throughput sequencing (HTS) to identify the viruses and viroids. The HTS results were verified using RT-PCR and Sanger sequencing. Subsequently, 86 samples including cladodes from "nopalitos", plants for fruit production, xoconostles, and some wild Opuntia were analyzed via RT-PCR with specific primers for the viruses and viroids previously detected via HTS. Three viruses were discovered [Opuntia virus 2 (OV2), cactus carlavirus 1 (CCV-1), and Opuntia potexvirus A (OPV-A)], along with a previously reported viroid [Opuntia viroid 1 (OVd-1)]. Additionally, two new viroids were identified, provisionally named the Mexican opuntia viroid (MOVd, genus Pospiviroid) and Opuntia viroid 2 (OVd-2, genus Apscaviroid). A phylogenetic analysis, pairwise identity comparison, and conserved structural elements analysis confirmed the classification of these two viroids as new species within the Pospiviroidae family. This is the first report of a pospiviroid and two apscaviroids infecting cactus pears in the world. Overall, this study enhances our understanding of the virome associated with cactus pears in Mexico.

Development of a CRISPR/SHERLOCK-Based Method for Rapid and Sensitive Detection of Selected Pospiviroids.

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.

Hop stunt viroid infection induces heterochromatin reorganization.

Viroids are pathogenic noncoding RNAs that completely rely on their host molecular machinery to accomplish their life cycle. Several interactions between viroids and their host molecular machinery have been identified, including interference with epigenetic mechanisms such as DNA methylation. Despite this, whether viroids influence changes in other epigenetic marks such as histone modifications remained unknown. Epigenetic regulation is particularly important during pathogenesis processes because it might be a key regulator of the dynamism of the defense response. Here we have analyzed the changes taking place in Cucumis sativus (cucumber) facultative and constitutive heterochromatin during hop stunt viroid (HSVd) infection using chromatin immunoprecipitation (ChIP) of the two main heterochromatic marks: H3K9me2 and H3K27me3. We find that HSVd infection is associated with changes in both H3K27me3 and H3K9me2, with a tendency to decrease the levels of repressive epigenetic marks through infection progression. These epigenetic changes are connected to the transcriptional regulation of their expected targets, genes, and transposable elements. Indeed, several genes related to the defense response are targets of both epigenetic marks. Our results highlight another host regulatory mechanism affected by viroid infection, providing further information about the complexity of the multiple layers of interactions between pathogens/viroids and hosts/plants.

Predicting symptom severity in PSTVd-infected tomato plants using the PSTVd genome sequence.

Viroids, one of the smallest known infectious agents, induce symptoms of varying severity, ranging from latent to severe, based on the combination of viroid isolates and host plant species. Because viroids are transmissible between plant species, asymptomatic viroid-infected plants may serve as latent sources of infection for other species that could exhibit severe symptoms, occasionally leading to agricultural and economic losses. Therefore, predicting the symptoms induced by viroids in host plants without biological experiments could remarkably enhance control measures against viroid damage. Here, we developed an algorithm using unsupervised machine learning to predict the severity of disease symptoms caused by viroids (e.g., potato spindle tuber viroid; PSTVd) in host plants (e.g., tomato). This algorithm, mimicking the RNA silencing mechanism thought to be linked to viroid pathogenicity, requires only the genome sequences of the viroids and host plants. It involves three steps: alignment of synthetic short sequences of the viroids to the host plant genome, calculation of the alignment coverage, and clustering of the viroids based on coverage using UMAP and DBSCAN. Validation through inoculation experiments confirmed the effectiveness of the algorithm in predicting the severity of disease symptoms induced by viroids. As the algorithm only requires the genome sequence data, it may be applied to any viroid and plant combination. These findings underscore a correlation between viroid pathogenicity and the genome sequences of viroid isolates and host plants, potentially aiding in the prevention of viroid outbreaks and the breeding of viroid-resistant crops.

[Viroids : infectious non coding RNAs]. / Les viroïdes : des ARN infectieux non codants.

Viroids are the smallest non-coding infectious RNAs (between 246 and 401 nucleotides) known to be highly structured and replicate autonomously in the host plants. Although they do not encode any peptides, viroids induce visible symptoms in susceptible host plants. This article provides an overview of their physical and biological properties, the diseases they cause and their significance for the plants. The mechanisms underlying the expression of symptoms in host plants, their detection and various strategies employed for diseases prevention are also developed.

Potato spindle tuber viroid (PSTVd) loop 27 mutants promote cell-to-cell movement and phloem unloading of the wild type: Insights into RNA-based viroid interactions.

Variations in infection progression with concurrent or prior infections by different viruses, viroids, or their strains are evident, but detailed investigations into viroid variant interactions are lacking. We studied potato spindle tuber viroid intermediate strain (PSTVd-I) to explore variant interactions. Two mutants, U177A/A182U (AU, replication- and trafficking-competent) and U178G/U179G (GG, replication-competent but trafficking-defective) on loop 27 increased cell-to-cell movement of wild-type (WT) PSTVd without affecting replication. In mixed infection assays, both mutants accelerated WT phloem unloading, while only AU promoted it in separate leaf assays, suggesting that enhancement of WT infection is not due to systemic signals. The mutants likely enhance WT infection due to their loop-specific functions, as evidenced by the lack of impact on WT infection seen with the distantly located G347U (UU) mutant. This study provides the first comprehensive analysis of viroid variant interactions, highlighting the prolonged phloem unloading process as a significant barrier to systemic spread.

Ambiviricota, a novel ribovirian phylum for viruses with viroid-like properties.

Fungi harbor a vast diversity of mobile genetic elements (MGEs). Recently, novel fungal MGEs, tentatively referred to as 'ambiviruses,' were described. 'Ambiviruses' have single-stranded RNA genomes of about 4-5 kb in length that contain at least two open reading frames (ORFs) in non-overlapping ambisense orientation. Both ORFs are conserved among all currently known 'ambiviruses,' and one of them encodes a distinct viral RNA-directed RNA polymerase (RdRP), the hallmark gene of ribovirian kingdom Orthornavirae. However, 'ambivirus' genomes are circular and predicted to replicate via a rolling-circle mechanism. Their genomes are also predicted to form rod-like structures and contain ribozymes in various combinations in both sense and antisense orientations-features reminiscent of viroids, virusoids, ribozyvirian kolmiovirids, and yet-unclassified MGEs (such as 'epsilonviruses,' 'zetaviruses,' and some 'obelisks'). As a first step toward the formal classification of 'ambiviruses,' the International Committee on Taxonomy of Viruses (ICTV) recently approved the establishment of a novel ribovirian phylum, Ambiviricota, to accommodate an initial set of 20 members with well-annotated genome sequences.

Development of TaqMan RT-qPCR for the detection of regulated citrus viruses and viroids in Aotearoa New Zealand.

The major citrus species include several economically important fruits, such as orange, mandarin, lemon, limes, grapefruit and pomelos. Since the 1980 s, total production and consumption of citrus has grown strongly with the current annual worldwide production at over 105 million tonnes. New Zealand's citrus exports, for instance, had an estimated worth of NZ$ 11.6 million (approx. US$ 7 million) in 2020. Citrus plants are prone to viral diseases, which can lead to substantial economic losses. In New Zealand, the citrus Import Health Standard (IHS) has identified 22 viruses and viroids that are subject to regulation and requires citrus nursery stock to be free of these pathogens. As such, there is a need for reliable, sensitive, and rapid detection methods to screen for these viruses and viroids during post entry quarantine. In this study, we developed TaqMan RT-qPCR assays for the detection of nine of these regulated viruses and viroids, namely citrus leaf rugose virus (CiLRV), citrus leprosis virus C (CiLV-C), citrus leprosis virus C2 (CiLV-C2), citrus leprosis virus N (CiLV-N), citrus psorosis virus (CPsV), citrus yellow mosaic virus (CYMV), citrus bent leaf viroid (CBLVd), citrus viroid V (CVd-V), and citrus viroid VI (CVd-VI). These assays have been validated and found to be highly sensitive, specific, and reliable. The implementation of these assays will facilitate the safe importation of citrus nursery stock, thus safeguarding the country's horticultural and economic interests.

Molecular phylogeny and secondary structure analysis of hop stunt viroid (HSVd) associated with Mulberry (Morus alba) in India.

Hop stunt viroid (HSVd), a small, single stranded, circular, non-coding infectious RNA known to cause infection in various economically important crop plants. In the present investigation, a study was conducted in the southern part of Karnataka districts of India to detect the possible association of HSVd infection in mulberry plants. A total of 41 mulberry plants showing typical viroid-like symptoms along with asymptomatic samples were collected and screened using conventional Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) using a specific set of HSVd-Fw/ HSVd-Re primers. Out of 41 samples, the study confirmed the presence of HSVd in six samples of mulberry collected from Ramanagara (1 sample), Chikkaballapur (3 samples) and Doddaballapura (2 samples) regions with an expected HSVd amplicon size of ∼ 290-300 nucleotides. The mechanical transmission of HSVd was also confirmed on cucumber (cv. Suyo) seedlings through bioassay, which was reconfirmed by RT-PCR. The amplicons were cloned, sequenced, and the representative nucleotide sequences were deposited in the NCBI GenBank. Subsequently, molecular phylogenetic analysis showed that HSVd mulberry isolates from this study were most closely related to grapevine isolates, indicating a common origin. On the other hand, it was shown to belong to a different group from mulberry isolates so far reported from Iran, Italy, Lebanon, and China. The secondary structure analysis of HSVd mulberry Indian isolates exhibited substitutions in the terminal left, pathogenicity, and variable regions compared to those of the Indian grapevine isolates. As far as this study is concerned, HSVd was detected exclusively in some mulberry plants with viral-like symptoms, but the pathogenesis and symptom expression needs to be further investigated to establish the relationship between HSVd and the disease symptoms in the mulberry plants.

Understanding Citrus Viroid Interactions: Experience and Prospects.

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.

Viroids of the Mediterranean Basin.

There has been substantial progress in the Mediterranean countries regarding research on viroids. Twenty-nine viroid species, all belonging to Pospiviroidae and Avsunviroidae genera, have been detected in the Mediterranean Basin. Not only have detection methods, such as reverse transcription-quantitative polymerase chain reaction and next-generation sequencing, been used for viroid detection, along with molecular hybridization techniques allowing for rapid detection, identification, and characterization of known and novel viroids in these countries, but eradication measures have also been taken that allowed for the efficient elimination of certain viroids in a number of Mediterranean countries. The eradication measures were followed as recommended by the European and Mediterranean Plant Protection Organization, which is known by its abbreviation, EPPO. The Mediterranean Region has been a niche for viroids since ancient times due to the warm climate and the socio-cultural conditions that facilitate viroid transmission among different host plant species.

RNA three-dimensional structure drives the sequence organization of potato spindle tuber viroid quasispecies.

RNA viruses and viroids exist and evolve as quasispecies due to error-prone replication. Quasispecies consist of a few dominant master sequences alongside numerous variants that contribute to genetic diversity. Upon environmental changes, certain variants within quasispecies have the potential to become the dominant sequences, leading to the emergence of novel infectious strains. However, the emergence of new infectious variants remains unpredictable. Using mutant pools prepared by saturation mutagenesis of selected stem and loop regions, our study of potato spindle tuber viroid (PSTVd) demonstrates that mutants forming local three-dimensional (3D) structures similar to the wild type (WT) are more likely to accumulate in PSTVd quasispecies. The selection mechanisms underlying this biased accumulation are likely associated with cell-to-cell movement and long-distance trafficking. Moreover, certain trafficking-defective PSTVd mutants can be spread by functional sister genomes in the quasispecies. Our study reveals that the RNA 3D structure of stems and loops constrains the evolution of viroid quasispecies. Mutants with a structure similar to WT have a higher likelihood of being maintained within the quasispecies and can potentially give rise to novel infectious variants. These findings emphasize the potential of targeting RNA 3D structure as a more robust approach to defend against viroid infections.

Knockout of SlDCL2b attenuates the resistance of tomato to potato spindle tuber viroid infection.

RNA interference, or RNA silencing, is an important defence mechanism against viroid infection in plants. Plants encode multiple DICER-LIKE (DCL) proteins that are key components of the RNA silencing pathway. However, the roles of different DCLs in defence responses against viroid infection remain unclear. Here, we determined the function of tomato DCL2b (SlDCL2b) in defence responses against potato spindle tuber viroid (PSTVd) infection using SlDCL2b loss-of-function tomato mutant plants. Compared with wild-type plants, mutant plants were more susceptible to PSTVd infection, developing more severe symptoms earlier and accumulating higher levels of PSTVd RNAs. Moreover, we verified the feedback mechanism for the regulation of SlDCL2b expression by miR6026. Functional blocking of tomato miR6026, by expressing its target mimics, can enhance resistance to PSTVd infection in tomato plants. These findings deepen the current understanding of RNAi-based resistance against viroid infection and provide a potentially new strategy for viroid control.

Viroids, Satellite RNAs and Prions: Folding of Nucleic Acids and Misfolding of Proteins.

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.

Molecular and Biological Characteristics of a Peach Latent Mosaic Viroid PC Isolate in Peach from China: Base Mutations in Hairpin Stems and Implications for Symptomatology.

Peach latent mosaic viroid (PLMVd) infects peach trees in China and induces a conspicuous albino phenotype (peach calico, PC) that is closely associated with variants containing a 12-to-14 nucleotide hairpin insertion capped by a U-rich loop. Initially, PC disease distribution was limited to parts of Italy, and it was first detected in the field in China in 2019. To explore the molecular and biological characteristics of PLMVd PC isolates in peach in China, we conducted a comprehensive analysis of disease phenotype development and investigated the data-associated pathogenicity and in vivo dynamics of the Chinese isolate PC-A2 using slash-inoculation into GF-305 peach seedlings. Inoculated seedlings displayed PC symptoms much earlier following topping treatment, and PLMVd infectivity was further assessed using bioassay and semiquantitative RT-PCR experiments. Evolutionary analysis showed that the PC isolate and its progeny variants clustered into a single phylogroup distinct from reference PC-C40 isolates from Italy and PC-K1 and PC-K2 from South Korea. Some PC-A2 progeny variants from green leaves of PC-expressing seedlings showed unbalanced point mutations in hairpin stems compared with the PC-C40 reference sequence and constituted a new stem insertion type. The results reveal associations between the recessive phenotypes of peach albino symptoms and base variation in hairpin stem insertions relative to the PC-C40/chloroplastic heat shock protein 90 reference sequence.

Application of High-Throughput Sequencing for Comprehensive Virome Profiling in Grapevines Shows Yellows in Iran.

A comprehensive study on the whole spectrum of viruses and viroids in five Iranian grapevine cultivars was carried out using sRNA libraries prepared from phloem tissue. A comparison of two approaches to virus detection from sRNAome data indicated a significant difference in the results and performance of the aligners in viral genome reconstruction. The results showed a complex virome in terms of viral composition, abundance, and richness. Thirteen viruses and viroids were identified in five Iranian grapevine cultivars, among which the grapevine red blotch virus and grapevine satellite virus were detected for the first time in Iranian vineyards. Grapevine leafroll-associated virus 1 (GLRaV1) and grapevine fanleaf virus (GFLV) were highly dominant in the virome. However, their frequency and abundance were somewhat different among grapevine cultivars. The results revealed a mixed infection of GLRaV1/grapevine yellow speckle viroid 1 (GYSVd1) and GFLV/GYSVd1 in grapevines that exhibited yellows and vein banding. We also propose a threshold of 14% of complete reconstruction as an appropriate threshold for detection of grapevine viruses that can be used as indicators for reliable grapevine virome profiling or in quarantine stations and certification programs.

Application of Biotechniques for In Vitro Virus and Viroid Elimination in Pome Fruit Crops.

Sustainable production of pome fruit crops is dependent upon having virus-free planting materials. The production and distribution of plants derived from virus- and viroid-negative sources is necessary not only to control pome fruit viral diseases but also for sustainable breeding activities, as well as the safe movement of plant materials across borders. With variable success rates, different in vitro-based techniques, including shoot tip culture, micrografting, thermotherapy, chemotherapy, and shoot tip cryotherapy, have been employed to eliminate viruses from pome fruits. Higher pathogen eradication efficiencies have been achieved by combining two or more of these techniques. An accurate diagnosis that confirms complete viral elimination is crucial for developing effective management strategies. In recent years, considerable efforts have resulted in new reliable and efficient virus detection methods. This comprehensive review documents the development and recent advances in biotechnological methods that produce healthy pome fruit plants. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Comparative Performance Evaluation of Double-Stranded RNA High-Throughput Sequencing for the Detection of Viral Infection in Temperate Fruit Crops.

There is limited information on the compared performances of biological, serological. and molecular assays with high-throughput sequencing (HTS) for viral indexing in temperate fruit crops. Here, using a range of samples of predetermined virological status, we compared two performance criteria (inclusivity and analytical sensitivity) of enzyme-linked immunosorbent assay (ELISA), molecular hybridization, reverse transcription (RT)-PCR, and double-stranded RNA (dsRNA) HTS for the detection of a total of 14 viruses (10 genera) and four viroids (three genera). When undiluted samples from individual plants were used, ELISA had the lowest performance, with an overall detection rate of 68.7%, followed by RT-PCR (82.5%) and HTS (90.7%; 100% if considering only viruses). The lower performance of RT-PCR reflected the inability to amplify some isolates as a consequence of point mutations affecting primer-binding sites. In addition, HTS identified viruses that had not been identified by other assays in nearly two-thirds of the samples. Analysis of serial dilutions of fruit tree samples allowed comparison of analytical sensitivities for various viruses. ELISA showed the lowest analytical sensitivity, but RT-PCR showed higher analytical sensitivity than HTS for most of the samples. Overall, these results confirm the superiority of HTS over biological indexing in terms of speed and inclusivity and show that while the absolute analytical sensitivity of RT-PCR tends to be higher than that of HTS, PCR inclusivity is affected by viral genetic diversity. Taken together, these results make a strong case for the implementation of HTS-based approaches in fruit tree viral testing protocols supporting quarantine and certification programs.

Molecular detection of Citrus exocortis viroid (CEVd), Citrus viroid-III (CVd-III), and Citrus viroid-IV (CVd-IV) in Palestine.

Citrus hosts various phytopathogens that have impacted productivity, including viroids. Missing data on the status of viroids in citrus in Palestine were not reported. This study was aimed to detect any of Citrus exocortis viroid (CEVd), Citrus viroid-III (CVd-III), and Citrus viroid-IV (CVd-IV) in the Palestinian National Agricultural Research Center (NARC) germplasm collection Field inspections found symptoms such as leaf epinasty; vein discoloration, and bark cracking on various citrus varieties. RT-PCR revealed a significant prevalence of CVd-IV; CEVd and CVd-III (47%, 31%, and 22%; respectively). CVd-III variants with 91.3% nucleic acid sequence homology have been reported. The sequence of each viroid were deposited in GenBank as (OP925746 for CEVd, OP902248 and OP902249 for CVd-III-PS-1 and -PS-2 isolates, and OP902247 for CVd-IV). This was the first to report three of citrus viroids in Palestine, appealing to apply of phytosanitary measures to disseminate healthy propagating materials free from viroids.