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.

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.

A viroid-derived small interfering RNA targets bHLH transcription factor MdPIF1 to regulate anthocyanin biosynthesis in Malus domestica.

Fruit colour is a critical determinant for the appearance quality and commercial value of apple fruits. Viroid-induced dapple symptom severely affects the fruit coloration, however, the underlying mechanism remains unknown. In this study, we identified an apple dimple fruit viroid (ADFVd)-derived small interfering RNA, named vsiR693, which targeted the mRNA coding for a bHLH transcription factor MdPIF1 (PHYTOCHROME-INTERACTING FACTOR 1) to regulate anthocyanin biosynthesis in apple. 5' RLM-RACE and artificial microRNA transient expression system proved that vsiR693 directly targeted the mRNA of MdPIF1 for cleavage. MdPIF1 positively regulated anthocyanin biosynthesis in both apple calli and fruits, and it directly bound to G-box element in the promoter of MdPAL and MdF3H, two anthocyanin biosynthetic genes, to promote their transcription. Expression of vsiR693 negatively regulated anthocyanin biosynthesis in both apple calli and fruits. Furthermore, co-expression of vsiR693 and MdPIF1 suppressed MdPIF1-promoted anthocyanin biosynthesis in apple fruits. Infiltration of ADFVd infectious clone suppressed coloration surrounding the injection sites in apple fruits, while a mutated version of ADFVd, in which the vsiR693 producing region was mutated, failed to repress fruit coloration around the injection sites. These data provide evidence that a viroid-derived small interfering RNA targets host transcription factor to regulate anthocyanin biosynthesis in apple.

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.

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.

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.

Association of apple scar skin viroid (ASSVd) infection with an emerging disease in 'Saiwaihong' apples.

A novel disease affecting small immature fruits has surfaced in 'Saiwaihong' apples (Malus pumila), a recently developed variety extensively cultivated across more than 20,000 hectares in China. In an effort to pinpoint the causal agent(s) responsible for this ailment, RNA sequencing analysis was conducted on four diseased and four healthy apple samples. The results revealed a diverse range of viruses and viroids, indicating a mixed viral infection in diseased samples. However, a more focused examination involving 152 diseased and 122 healthy fruit samples, utilizing RT-PCR and dot-blotting hybridization techniques, highlighted a close association between the disease and the presence of apple scar skin viroid (ASSVd). Among the obtained ASSVd variants from diseased 'Saiwaihong' apples, 20 were identified, and they were either identical or closely related to isolates from various apple varieties cultivated in different regions and countries. This suggests that ASSVd isolates in 'Saiwaihong' might have been introduced from other apple varieties. Furthermore, the analysis indicates the possibility of two separate introductions, as the ASSVd 'Saiwaihong' isolates demonstrated two distinct phylogenetic groups. These insights provide valuable guidance for disease control strategies and emphasize the significance of ongoing monitoring for ASSVd, both in its familiar forms and potential new variants.

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.

[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.

Expression of symptoms elicited by a hammerhead viroid through RNA silencing is related to population bottlenecks in the infected host.

Chlorosis is frequently incited by viroids, small nonprotein-coding, circular RNAs replicating in nuclei (family Pospiviroidae) or chloroplasts (family Avsunviroidae). Here, we investigated how chrysanthemum chlorotic mottle viroid (CChMVd, Avsunviroidae) colonizes, evolves and initiates disease. Progeny variants of natural and mutated CChMVd sequence variants inoculated in chrysanthemum plants were characterized, and plant responses were assessed by molecular assays. We showed that: chlorotic mottle induced by CChMVd reflects the spatial distribution and evolutionary behaviour in the infected host of pathogenic (containing a UUUC tetranucleotide) and nonpathogenic (lacking such a pathogenic determinant) variants; and RNA silencing is involved in the initiation of the chlorosis in symptomatic leaf sectors through a viroid-derived small RNA containing the pathogenic determinant that directs AGO1-mediated cleavage of the mRNA encoding the chloroplastic transketolase. This study provides the first evidence that colonization of leaf tissues by CChMVd is characterized by segregating variant populations differing in pathogenicity and with the ability to colonize leaf sectors (bottlenecks) and exclude other variants (superinfection exclusion). Importantly, no specific pathogenic viroid variants were found in the chlorotic spots caused by chrysanthemum stunt viroid (Pospiviroidae), thus establishing a clear distinction on how members of the two viroid families trigger chlorosis in the same host.

Integrative time-scale and multi-omics analysis of host responses to viroid infection.

Viroids are circular RNAs of minimal complexity compelled to subvert plant-regulatory networks to accomplish their infectious process. Studies focused on the response to viroid-infection have mostly addressed specific regulatory levels and considered specifics infection-times. Thus, much remains to be done to understand the temporal evolution and complex nature of viroid-host interactions. Here we present an integrative analysis of the temporal evolution of the genome-wide alterations in cucumber plants infected with hop stunt viroid (HSVd) by integrating differential host transcriptome, sRNAnome and methylome. Our results support that HSVd promotes the redesign of the cucumber regulatory-pathways predominantly affecting specific regulatory layers at different infection-phases. The initial response was characterised by a reconfiguration of the host-transcriptome by differential exon-usage, followed by a progressive transcriptional downregulation modulated by epigenetic changes. Regarding endogenous small RNAs, the alterations were limited and mainly occurred at the late stage. Significant host-alterations were predominantly related to the downregulation of transcripts involved in plant-defence mechanisms, the restriction of pathogen-movement and the systemic spreading of defence signals. We expect that these data constituting the first comprehensive temporal-map of the plant-regulatory alterations associated with HSVd infection could contribute to elucidate the molecular basis of the yet poorly known host-response to viroid-induced pathogenesis.

Down-regulation of tomato STEROL GLYCOSYLTRANSFERASE 1 perturbs plant development and facilitates viroid infection.

Potato spindle tuber viroid (PSTVd) is a plant pathogen naturally infecting economically important crops such as tomato (Solanum lycopersicum). Here, we aimed to engineer tomato plants highly resistant to PSTVd and developed several S. lycopersicum lines expressing an artificial microRNA (amiRNA) against PSTVd (amiR-PSTVd). Infectivity assays revealed that amiR-PSTVd-expressing lines were not resistant but instead hypersusceptible to the viroid. A combination of phenotypic, molecular, and metabolic analyses of amiRNA-expressing lines non-inoculated with the viroid revealed that amiR-PSTVd was accidentally silencing the tomato STEROL GLYCOSYLTRANSFERASE 1 (SlSGT1) gene, which caused late developmental and reproductive defects such as leaf epinasty, dwarfism, or reduced fruit size. Importantly, two independent transgenic tomato lines each expressing a different amiRNA specifically designed to target SlSGT1 were also hypersusceptible to PSTVd, thus demonstrating that down-regulation of SlSGT1 was responsible for the viroid-hypersusceptibility phenotype. Our results highlight the role of sterol glycosyltransferases in proper plant development and indicate that the imbalance of sterol glycosylation levels favors viroid infection, most likely by facilitating viroid movement.

Analysis of Coconut cadang-cadang viroid variants on field samples exhibiting variation in orange spotting symptom expression and severity.

BACKGROUND: Sequence variation has been attributed to symptom variations but has not been investigated in Orange Spotting-Coconut cadang-cadang viroid (OS-CCCVd) infected palms. Likewise, the relationship between Coconut cadang-cadang viroid (CCCVd) variants, Orange Spotting (OS) severity and the accumulation of the viroid in the palms have not been elucidated. This paper describes the characterization of CCCVd variants by cloning and sequencing, followed by correlation with symptom expression. METHODS AND RESULTS: Total nucleic acids were extracted from leaf samples harvested from frond 20 of seven Dura × Pisifera (D × P) African oil palm (Elaeis guineensis Jacq.) aged between 13 and 21 years old collected from local plantations. The nucleic acids were fractionated using 5% non-denaturing polyacrylamide gel electrophoresis (PAGE) before being subjected to detection by reverse transcribed polymerase chain reaction (RT-PCR). The PCR products were cloned into a plasmid vector and the sequence of the clones was analyzed. CCCVd variants were quantified using real-time qPCR assay with CCCVd specific primers. Sixteen randomly selected clones of (OP246) had an arbitrary 100% identity with CCCVdOP246 (GeneBank Accession No: HQ608513). Meanwhile, four clones had >93% similarity with several minor sequence variations forming variants of OP234, OP235, OP251 and OP279. CONCLUSION: The OS symptoms observed in the field were characterized into three categories based on the size and morphology of the orange spots on the affected fronds. In addition, there was no direct correlation between disease severity and the accumulation of CCCVd variants in oil palm. This finding is the first report describing the sequence variation of the CCCVd RNA and symptom variation in OS oil palm field samples.

Long Noncoding RNAs in Plant-Pathogen Interactions.

Long noncoding RNAs (lncRNAs) are commonly defined as transcripts that lack protein-coding capacity and are longer than 200 nucleotides. Since the emergence of next-generation sequencing technologies in this century, thousands of lncRNAs have been identified from nearly all living organisms. Notably, various pathogens also express their own lncRNAs in host cells during infection. In plants, many lncRNAs exhibit dynamic expression patterns in response to environmental stimuli, including pathogen attacks. In contrast to well-established methods in identifying such lncRNAs, the current understanding of lncRNAs' functional mechanisms is in its infancy. Some lncRNAs serve as precursors for generating small RNAs or serve as target mimics to sequester functional small RNAs, which have been extensively reviewed in the literature. This review focuses on the emerging evidence supporting that certain lncRNAs function as negative or positive regulators of plant immunity. A common theme is that those regulations rely on specific interactions between lncRNAs and key regulatory proteins. Viroids as single-stranded circular noncoding RNAs provide a handle to investigate how RNA local motifs render interaction specificity between lncRNAs and regulatory proteins. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Occurrence and Pathogenicity of Hop Stunt Viroid Infecting Mulberry (Morus alba) Plants in China.

To investigate the presence of hop stunt viroid (HSVd) in mulberry (Morus alba) plants in China, HSVd was detected by reverse transcription (RT)-PCR using dsRNAs extracted from symptomatic or asymptomatic mulberry leaf samples collected from a mulberry field located in Zhenjiang, China, as a template and the primer pairs for HSVd detection. The primer pairs were designed based on the conserved sequence of 25 HSVd variants deposited in the GenBank database. Four out of a total of 53 samples were HSVd-positive, confirming that HSVd is present in mulberry plants in China. The consensus full-length nucleotide (nt) sequence of two HSVd variants determined by sequencing the HSVd variants in these four HSVd-positive samples consisted of 296 nt and shared the highest nt identity of 96.8% with that from plum in Turkey but relatively low identity with those from mulberry in Iran (87.3 to 90.8%). Phylogenetic analysis showed that these HSVd variants clustered together with those of the HSVd-hop group. Analysis of the infectivity and pathogenicity to hosts by the constructed Agrobacterium-mediated dimeric head-to-tail HSVd cDNA infectious clones demonstrated that one of the HSVd variants identified in this study infects the natural host, mulberry plants, and also infects experimental plants, cucumber, and tomato. It probably induces stunting symptoms in HSVd-infected tomatoes but does not induce symptoms on mulberry leaves or in cucumbers. Although HSVd infecting mulberry has been found in Iran, Italy, and Lebanon, this is the first study to report this viroid in naturally infected mulberry plants in China.

Challenges to Cannabis sativa Production from Pathogens and Microbes-The Role of Molecular Diagnostics and Bioinformatics.

The increased cultivation of Cannabis sativa L. in North America, represented by high Δ9-tetrahydrocannabinol-containing (high-THC) cannabis genotypes and low-THC-containing hemp genotypes, has been impacted by an increasing number of plant pathogens. These include fungi which destroy roots, stems, and leaves, in some cases causing a build-up of populations and mycotoxins in the inflorescences that can negatively impact quality. Viroids and viruses have also increased in prevalence and severity and can reduce plant growth and product quality. Rapid diagnosis of the occurrence and spread of these pathogens is critical. Techniques in the area of molecular diagnostics have been applied to study these pathogens in both cannabis and hemp. These include polymerase chain reaction (PCR)-based technologies, including RT-PCR, multiplex RT-PCR, RT-qPCR, and ddPCR, as well as whole-genome sequencing (NGS) and bioinformatics. In this study, examples of how these technologies have enhanced the rapidity and sensitivity of pathogen diagnosis on cannabis and hemp will be illustrated. These molecular tools have also enabled studies on the diversity and origins of specific pathogens, specifically viruses and viroids, and these will be illustrated. Comparative studies on the genomics and metabolomics of healthy and diseased plants are urgently needed to provide insight into their impact on the quality and composition of cannabis and hemp-derived products. Management of these pathogens will require monitoring of their spread and survival using the appropriate technologies to allow accurate detection, followed by appropriate implementation of disease control measures.

"Pathomorphogenic" Changes Caused by Citrus Bark Cracking Viroid and Transcription Factor TFIIIA-7ZF Variants Support Viroid Propagation in Tobacco.

Viroids are small, non-coding, pathogenic RNAs with the ability to disturb plant developmental processes. This dysregulation redirects the morphogenesis of plant organs, significantly impairing their functionality. Citrus bark cracking viroid (CBCVd) causes detrimental developmental distortions in infected hops (Humulus lupulus) and causes significant economic losses. CBCVd can infect cells and tissues of the model plant tobacco (Nicotiana tabacum), provided it is delivered via transgenesis. The levels of CBCVd in tobacco were enhanced in plant hybrids expressing CBCVd cDNAs and either the tobacco or hop variant of TFIIIA-7ZF, a viroid-mediated splicing derivative of transcription factor IIIA, which is important for viroid replication by DNA-dependent RNA polymerase II. The TFIIIA-7ZF variants can change the tobacco morphogenesis if expressed in leaves and shoots. In addition to the splitting of shoots, the "pathomorphogenic" network in hybrid plants expressing CBCVd and HlTFIIIA-7ZF induced leaf fusions and malformations. Moreover, CBCVd can dramatically change another morphogenesis into teratomic and petal-like tissues if propagated above some limit in young transgenic tobacco microspores and anthers. By comparative RNA profiling of transgenic tobacco shoots bearing TFIIIA-7ZFs and CBCVd-transformed/infected anthers, we found a differential expression of many genes at p < 0.05. As the main common factor showing the differential up-regulation in shoot and anther tissues, a LITTLE ZIPPER 2-like transcription factor was found. We propose that this factor, which can interact as a competitive inhibitor of the also dysregulated homeobox-leucin zipper family protein (HD-ZIPIII) in apical meristem, is essential for a network responsible for some morphological changes and modifications of plant degradome within shoot meristem regulation and secondary xylem differentiation.

Development and application of reverse transcription droplet digital PCR assay for sensitive detection of apple scar skin viroid during in vitro propagation of apple plantlets.

Apple scar skin viroid (ASSVd), of the genus Apscaviroid, causes serious pome fruit diseases, such as apple scar skin, dapple apple, pear rusty skin, pear fruit crinkle, and pear dimple fruit. This study aimed at establishing a sensitive and accurate method for quantification of ASSVd in apple leaves and plantlets using a reverse transcription droplet digital polymerase chain reaction (RT-ddPCR) assay. The specificity was analyzed using other apple viruses, and the negative amplification of the cross-reaction assay demonstrated the high specificity of RT-ddPCR. The detection limit of ASSVd by RT-ddPCR was 1.75 × 102 copies/µL (0.14 concentration), and the sensitivity was ten-fold higher than that of RT-qPCR. Similarly, positive detection in apple plantlet samples by RT-ddPCR was higher than that by RT-qPCR. The RT-ddPCR assay represents a promising alternative for accurate quantitative detection and diagnosis of ASSVd infection in ASSVd-free certification programs.

Symptomatic plant viroid infections in phytopathogenic fungi.

Viroids are pathogenic agents that have a small, circular noncoding RNA genome. They have been found only in plant species; therefore, their infectivity and pathogenicity in other organisms remain largely unexplored. In this study, we investigate whether plant viroids can replicate and induce symptoms in filamentous fungi. Seven plant viroids representing viroid groups that replicate in either the nucleus or chloroplast of plant cells were inoculated to three plant pathogenic fungi, Cryphonectria parasitica, Valsa mali, and Fusarium graminearum By transfection of fungal spheroplasts with viroid RNA transcripts, each of the three, hop stunt viroid (HSVd), iresine 1 viroid, and avocado sunblotch viroid, can stably replicate in at least one of those fungi. The viroids are horizontally transmitted through hyphal anastomosis and vertically through conidia. HSVd infection severely debilitates the growth of V. mali but not that of the other two fungi, while in F. graminearum and C. parasitica, with deletion of dicer-like genes, the primary components of the RNA-silencing pathway, HSVd accumulation increases. We further demonstrate that HSVd can be bidirectionally transferred between F. graminearum and plants during infection. The viroids also efficiently infect fungi and induce disease symptoms when the viroid RNAs are exogenously applied to the fungal mycelia. These findings enhance our understanding of viroid replication, host range, and pathogenicity, and of their potential spread to other organisms in nature.

An Overview of Pathogens Associated with Biotic Stresses in Hemp Crops in Oregon, 2019 to 2020.

Hemp (Cannabis sativa) acreage in Oregon has increased by approximately 240 times in the last 5 years, and a greater number of hemp diseases have been observed. This special report documents pathogens, particularly those causing virus and virus-like diseases, that have been detected from field and greenhouse-grown hemp crops in Oregon, based on plant samples submitted to the Hermiston Agricultural Research and Extension Center Plant Clinic of Oregon State University in 2019 and 2020. Symptoms and signs were used to evaluate disease types and determine diagnostic assays used on each submission. Plants with signs or symptoms of fungal or oomycete infection were cultured to isolate pathogenic organisms and plants with symptoms suspected to be caused by virus infection were assayed for the presence of Beet curly top virus (BCTV), viroids, and phytoplasmas using PCR, or reverse transcription (RT)-PCR. Diseases with fungal or oomycete, and virus causes accounted for 26.5 and 42.9% of submissions, respectively; coinfection of viral and fungal or oomycete pathogens were detected from 6.1% of submissions between 2019 and 2020. BCTV, a curtovirus, and hop latent viroid (HLVd) were the predominant pathogens detected from field and indoor grown hemp. Worland-like strains of BCTV represented 93% of all curtovirus detections. Eighty percent of HLVd detections occurred from plants that originated from indoor growing facilities. Based on BCTV vector, beet leafhopper, prevalence, field-grown hemp in western production regions may be affected by curly top and increasing hemp acreage in the landscape may have potential implications on other crops affected by curtoviruses. Virus and virus-like diseases could be a limiting factor for hemp production in some regions of the United States.