Search Results (22)

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

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

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. 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

Hop latent viroid (HLVd), a subviral pathogen from the family Pospiviroidae, is a major threat to the global cannabis industry and is the causative agent for “dudding disease”. Infected plants can often be asymptomatic for a period of growth and then develop symptoms such as malformed and yellowing leaves, as well as stunted growth. During flowering, HLVd-infected plants show reduced levels of valuable metabolites. This study was undertaken to expand our basic knowledge of HLVd infectivity, transmission, and host range. HLVd-specific primers were used for RT-PCR detection in plant samples and were able to detect HLVd in as little as 5 picograms of total RNA. A survey of hemp samples obtained from a diseased production system proved sole infection of HLVd (72%) with no coexistence of hop stunt viroid. HLVd was infectious through successive passage assays using a crude sap or total RNA extract derived from infected hemp. HLVd was also highly transmissible through hemp seeds at rates of 58 to 80%. Host range assays revealed new hosts for HLVd: tomato, cucumber, chrysanthemum, Nicotiana benthamiana, and Arabidopsis thaliana (Col-0). Sequence analysis of 77 isolates revealed only 3 parsimony-informative sites, while 10 sites were detected among all HLVd isolates available in the GenBank. The phylogenetic relationship among HLVd isolates allowed for inferring two major clades based on the genetic distance. Our findings facilitate further studies on host–viroid interaction and viroid management. Full article

Trialeurodes vaporariorum, commonly known as the greenhouse whitefly, severely infests important crops and serves as a vector for apple scar skin viroid (ASSVd). This vector-mediated transmission may cause the spread of infection to other herbaceous crops. For effective management of ASSVd, it is important to explore the whitefly’s proteins, which interact with ASSVd RNA and are thereby involved in its transmission. In this study, it was found that a small heat shock protein (sHsp) from T. vaporariorum, which is expressed under stress, binds to ASSVd RNA. The sHsp gene is 606 bp in length and encodes for 202 amino acids, with a molecular weight of 22.98 kDa and an isoelectric point of 8.95. Intermolecular interaction was confirmed through in silico analysis, using electrophoretic mobility shift assays (EMSAs) and northwestern assays. The sHsp22.98 protein was found to exist in both monomeric and dimeric forms, and both forms showed strong binding to ASSVd RNA. To investigate the role of sHsp22.98 during ASSVd infection, transient silencing of sHsp22.98 was conducted, using a tobacco rattle virus (TRV)-based virus-induced gene silencing system. The sHsp22.98-silenced whiteflies showed an approximate 50% decrease in ASSVd transmission. These results suggest that sHsp22.98 from T. vaporariorum is associated with viroid RNA and plays a significant role in transmission. Full article

Viroids are a unique type of infectious agent, exclusively composed of a relatively small (246–430 nt), highly base-paired, circular, non-coding RNA. Despite the small size and non-coding nature, the more-than-thirty currently known viroid species infectious of higher plants are able to autonomously replicate and move systemically through the host, thereby inducing disease in some plants. After recalling viroid discovery back in the late 60s and early 70s of last century and discussing current hypotheses about their evolutionary origin, this article reviews our current knowledge about these peculiar infectious agents. We describe the highly base-paired viroid molecules that fold in rod-like or branched structures and viroid taxonomic classification in two families, Pospiviroidae and Avsunviroidae, likely gathering nuclear and chloroplastic viroids, respectively. We review current knowledge about viroid replication through RNA-to-RNA rolling-circle mechanisms in which host factors, notably RNA transporters, RNA polymerases, RNases, and RNA ligases, are involved. Systemic movement through the infected plant, plant-to-plant transmission and host range are also discussed. Finally, we focus on the mechanisms of viroid pathogenesis, in which RNA silencing has acquired remarkable importance, and also for the initiation of potential biotechnological applications of viroid molecules. Full article

Viroids are single-stranded, circular RNA molecules (234-406 nt) that infect a wide range of crop species and cause economic losses in agriculture worldwide. They are characterized by the existence of a population of sequence variants, attributed to the low fidelity of RNA polymerases involved in their transcription, resulting in high mutation rates. Therefore, these biological entities exist as quasispecies. This feature allows them to replicate within a wide range of host plants, both monocots and dicots. Viroid hosts include economically important crops such as tomato, citrus, and fruit trees such as peach and avocado. Given the high risk of introducing viroids to viroid disease-free countries, these pathogens have been quarantined globally. As discussed herein, Mexico represents a geographical landscape of viroids linked to their origin and comprises considerable biodiversity. The biological features of viroid species endemic to Mexico are highlighted in this communication. In addition, we report the phylogenetic relationships among viroid and viroid strains, their economic impact, geographical distribution, and epidemiological features, including a broad host range and possible long-distance, seed, or insect-mediated transmission. In summary, this review could be helpful for a better understanding of the biology of viroid diseases and future programs on control of movement and spread to avoid economic losses in agricultural industries. Full article

Grafting cultivars onto rootstocks is a widely used practice by the apple industry predominantly aimed at faster fruit bearing. Using high-throughput sequencing, we revealed the presence of recently described viral agents, namely apple hammerhead viroid (AHVd), apple luteovirus 1 (ALV-1), and citrus concave gum-associated virus (CCGaV), in germplasm collections and production orchards in the Czech Republic and Hungary. The HTS results were validated with RT-(q)PCR, and Northern blotting techniques. To obtain further insight about the presence of these agents, RT-PCR based surveys were carried out and showed their widespread presence alone or in mixed infections. The pathogens were present both in production areas and in feral samples. In addition, rootstock-to-scion transmission of ALV-1 and CCGaV was confirmed using commercial rootstock materials. Phylogenetic relationships based on partial sequences of distinct variants were also investigated. Furthermore, the rosy apple aphid was found to be ALV-1-positive, suggesting that it might be a potential vector of the virus. Full article

Viroid and viroid-like satellite RNAs are infectious, circular, non-protein coding RNAs reported in plants only so far. Some viroids (family Avsunviroidae) and viroid-like satellite RNAs share self-cleaving activity mediated by hammerhead ribozymes (HHRzs) endowed in both RNA polarity strands. Using a homology-independent method based on the search for conserved structural motifs of HHRzs in reads and contigs from high-throughput sequenced RNAseq libraries, we identified a novel small (550 nt) viroid-like RNA in a library from a Citrus reticulata tree. Such a viroid-like RNA contains a HHRz in both polarity strands. Northern blot hybridization assays showed that circular forms of both polarity strands of this RNA (tentatively named citrus transiently-associated hammerhead viroid-like RNA1 (CtaHVd-LR1)) exist, supporting its replication through a symmetric pathway of the rolling circle mechanism. CtaHVd-LR1 adopts a rod-like conformation and has the typical features of quasispecies. Its HHRzs were shown to be active during transcription and in the absence of any protein. CtaHVd-LR1 was not graft-transmissible, and after its first identification, it was not found again in the original citrus source when repeatedly searched in the following years, suggesting that it was actually not directly associated with the plant. Therefore, the possibility that this novel self-cleaving viroid-like RNA is actually associated with another organism (e.g., a fungus), in turn, transiently associated with citrus plants, is proposed. Full article

We detected tobacco mosaic virus (TMV), a member of the genus Tobamovirus and one of the most significant plant-infecting viruses, for the first time in a chrysanthemum in Thailand using reverse-transcription polymerase chain reaction (RT–PCR). The TMV-infected chrysanthemum leaves exhibited mosaic symptoms. We conducted a sequence analysis of the coat protein (CP) gene and found that the TMV detected in the chrysanthemum had 98% identity with other TMV isolates in GenBank. We carried out bioassays and showed that TMV induced mosaic and stunting symptoms in inoculated chrysanthemums. We observed the rigid rod structure of TMV under a transmission electron microscope (TEM). To enhance the speed and sensitivity of detection, we developed a colorimetric RT loop-mediated isothermal amplification (LAMP) technique. We achieved LAMP detection after 30 min incubation in isothermal conditions at 65 °C, and distinguished the positive results according to the color change from pink to yellow. The sensitivity of the LAMP technique was 1000-fold greater than that of RT–PCR, and we found no cross-reactivity with other viruses or viroids. This is the first reported case of a TMV-infected chrysanthemum in Thailand, and our colorimetric RT–LAMP TMV detection method is the first of its kind. Full article

Grapevine leafroll disease (GLD) is one of the most economically damaging virus diseases in grapevine, with grapevine leafroll-associated virus 1 (GLRaV-1) and grapevine leafroll-associated virus 3 (GLRaV-3) as the main contributors. This study complements a previously published transcriptomic analysis and compared the impact of two different forms of GLD to a symptomless control treatment: a mildly symptomatic form infected with GLRaV-1 and a severe form with exceptionally early leafroll symptoms (up to six weeks before veraison) infected with GLRaV-1 and GLRaV-3. Vine physiology and fruit composition in 17-year-old Pinot noir vines were measured and a gradient of vigor, yield, and berry quality (sugar content and berry weight) was observed between treatments. Virome composition, confirmed by individual RT-PCR, was compared with biological indexing. Three divergent viromes were recovered, containing between four to seven viruses and two viroids. They included the first detection of grapevine asteroid mosaic-associated virus in Switzerland. This virus did not cause obvious symptoms on the indicators used in biological indexing. Moreover, the presence of grapevine virus B (GVB) did not cause the expected corky bark symptoms on the indicators, thus underlining the important limitations of the biological indexing. Transmission of GLRaV-3 alone or in combination with GVB by Planococcus comstocki mealybug did not reproduce the strong symptoms observed on the donor plant infected with a severe form of GLD. This result raises questions about the contribution of each virus to the symptomatology of the plant. Full article

Dwarfed citrus trees for high-density plantings or mechanized production systems will be key for future sustainable citrus production. Citrus trees consist of two different species of scion and rootstock. Therefore, any observed phenotype results from gene expression in both species. Dwarfed sweet orange trees on trifoliate rootstock have been produced using citrus dwarfing viroid (CDVd). We performed RNA-seq transcriptome analysis of CDVd-infected stems and roots and compared them to non-infected controls. The identified differentially expressed genes validated with RT-qPCR corresponded to various physiological and developmental processes that could be associated with the dwarfing phenotype. For example, the transcription factors MYB13 and MADS-box, which regulate meristem functions and activate stress responses, were upregulated in the stems. Conversely, a calcium-dependent lipid-binding protein that regulates membrane transporters was downregulated in the roots. Most transcriptome reprogramming occurred in the scion rather than in the rootstock; this agrees with previous observations of CDVd affecting the growth of sweet orange stems while not affecting the trifoliate rootstock. Furthermore, the lack of alterations in the pathogen defense transcriptome supports the term “Transmissible small nuclear ribonucleic acid,” which describes CDVd as a modifying agent of tree performance with desirable agronomic traits rather than a disease-causing pathogen. Full article

Studies on the ways in which viroids are transmitted are important for understanding their epidemiology and for developing effective control measures for viroid diseases. Viroids may be spread via vegetative propagules, mechanical damage, seed, pollen, or biological vectors. Vegetative propagation is the most prevalent mode of spread at the global, national and local level while further dissemination can readily occur by mechanical transmission through crop handling with viroid-contaminated hands or pruning and harvesting tools. The current knowledge of seed and pollen transmission of viroids in different crops is described. Biological vectors shown to transmit viroids include certain insects, parasitic plants, and goats. Under laboratory conditions, viroids were also shown to replicate in and be transmitted by phytopathogenic ascomycete fungi; therefore, fungi possibly serve as biological vectors of viroids in nature. The term “mycoviroids or fungal viroids” has been introduced in order to denote these viroids. Experimentally, known sequence variants of viroids can be transmitted as recombinant infectious cDNA clones or transcripts. In this review, we endeavor to provide a comprehensive overview of the modes of viroid transmission under both natural and experimental situations. A special focus is the key findings which can be applied to the control of viroid diseases. Full article

Viroids are small, non-coding, parasitic RNAs that promote developmental distortions in sensitive plants. We analyzed pollen of Nicotiana benthamiana after infection and/or ectopic transformation with cDNAs of citrus bark cracking viroid (CBCVd), apple fruit crinkle viroid (AFCVd) and potato spindle tuber viroid (PSTVd) variant AS1. These viroids were seed non-transmissible in N. benthamiana. All viroids propagated to high levels in immature anthers similar to leaves, while their levels were drastically reduced by approximately 3.6 × 10³, 800 and 59 times in mature pollen of CBCVd, AFCVd and PSTVd infected N. benthamiana, respectively, in comparison to leaves. These results suggest similar elimination processes during male gametophyte development as in the Nicotiana tabacum we presented in our previous study. Mature pollen of N. benthamiana showed no apparent defects in infected plants although all three viroids induced strong pathological symptoms on leaves. While Nicotiana species have naturally bicellular mature pollen, we noted a rare occurrence of mature pollen with three nuclei in CBCVd-infected N. benthamiana. Changes in the expression of ribosomal marker proteins in AFCVd-infected pollen were detected, suggesting some changes in pollen metabolism. N. benthamiana transformed with 35S-driven viroid cDNAs showed strong symptoms including defects in pollen development. A large number of aborted pollen (34% and 62%) and a slight increase of young pollen grains (8% and 15%) were found in mature pollen of AFCVd and CBCVd transformants, respectively, in comparison to control plants (3.9% aborted pollen and 0.3% young pollen). Moreover, pollen grains with malformed nuclei or trinuclear pollen were found in CBCVd-transformed plants. Our results suggest that “forcing” overexpression of seed non-transmissible viroid led to strong pollen pathogenesis. Viroid adaptation to pollen metabolism can be assumed as an important factor for viroid transmissibility through pollen and seeds. Full article