Viroid Infection Research Articles

TOR Inhibition Enhances Autophagic Flux and Immune Response in Tomato Plants Against PSTVd Infection.

Viroids are small, non-coding RNA pathogens known for their ability to cause severe plant diseases. Despite their simple structure, viroids like Potato Spindle Tuber Viroid (PSTVd) can interfere with plant cellular processes, including transcriptional and post-transcriptional mechanisms, impacting plant growth and yield. In this study, we have investigated the role of the Target Of Rapamycin (TOR) signaling pathway in modulating viroid pathogenesis in tomato plants infected with PSTVd. Our findings reveal that PSTVd infection induces the accumulation of the selective autophagy receptor NBR1, potentially inhibiting autophagic flux. Pharmacological inhibition of TOR with AZD8055 mitigated PSTVd symptomatology by reducing viroid accumulation. Furthermore, TOR inhibition promoted the recovery of autophagic flux through NBR1. It primed the plant defense response, as evidenced by enhanced expression of the defense-related gene PR1b and S5H, a gene involved in the salicylic acid catabolism. These results suggest a novel role for TOR in regulating viroid-induced pathogenesis and highlight the potential of TOR inhibitors as tools for enhancing plant resistance against viroid infections.

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 ha in China. In an effort to pinpoint the causal agent(s) responsible for this ailment, RNA sequencing analysis was conducted on four symptomatic and four asymptomatic apple samples. The results revealed a diverse range of viruses and viroids, indicating mixed viral infection in diseased samples. However, a more focused examination involving 152 symptomatic and 122 asymptomatic fruit samples, using 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 ASSVd variants obtained 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 exhibited 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.

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.

Features of the immunopathogenesis of infections caused by viroids and satellite viruses

One of the main molecular mechanisms of viroids that cause disease in plants is the blocking of ribonucleic acid functions of host cells by viroid ribonucleic acid. The variety of splicing options is due to viroid replication, which requires ribonucleic acid polymerase, ribonuclease, ligase, and deoxyribonucleic acid working on ribonucleic acid matrices. Viroids with faster replication are preferred. Identifying host proteins that interact with viroid ribonucleic acid is critical in the pathogenesis of viroid infections, which leads to gene expression changes of host plants. The study of the pathogenesis of plant infections caused by satellite viruses has shown that they have a common ancestor and that the satellite viruses suppress the reproduction of the helper virus. Satellite viruses require accessory viral proteins to encode capsid proteins for genome encapsulation and reproduction. Replication occurs in the cytoplasm and is induced by ribonucleic acid polymerase of the helper virus. Hepatitis D virus ribonucleic acid replication requires cell ribonucleic acid polymerase II. When the viral and endosomal membranes fuse, the ribonucleic acid of the hepatitis D virus moves into the nucleus, and antigenomic ribonucleic acid is produced, which is the template for the synthesis of matrix ribonucleic acids encoding delta protein. The minor delta antigen regulates ribonucleic acid editing, and the large delta antigen inhibits viral replication and induces a signal for the transport of ribonucleic acid from the cell nucleus to the cytoplasm, which ensures the assembly of new viral particles. Large and small delta antigens have been detected in the brain, liver, lungs, kidneys, and spleen of snakes by reverse transcriptase polymerase chain reaction, Western blotting, and immunohistochemical assays, indicating that all delta viruses possibly share a common ancestor that arose before the divergence of reptiles and mammals.

A Multiplex RT-PCR for the Detection of Three Viruses and One Viroid Infecting Hemp

Hemp ( Cannabis sativa L.) is an emerging industrial crop in the United States. In recent surveys of hemp growers, disease identification and management were determined to be significant priority areas for future research. From previous studies on disease identification in Western U.S. hemp production systems, hop latent viroid (HLVd), beet curly top virus (BCTV), Cannabis sativa mitovirus 1 (CasaMV1), and citrus yellow vein associated virus (CYVaV) were detected at high incidences. In the present study, a multiplex reverse transcription PCR protocol was developed to detect this viroid and viruses from hemp leaf samples simultaneously. Using the developed protocol, the multiplex reverse transcription PCR could detect each virus and viroid specifically in 10−1-diluted cDNA. The protocol was validated on 6 hemp metavirome leaf samples from field-grown hemp grown in 2019 and 23 uncharacterized leaf samples in 2021 from Colorado. Of the samples tested, 95% tested positive for CasaMV1 in 2021. This was greater than infections of BCTV (4%), HLVd (0%), and CYVaV (0%). Coinfections accounted for 4% of samples in 2021. This method offers sensitive, specific, and fast simultaneous detection of three viruses and one viroid from hemp, contributing to large-scale pathogen-free hemp certification schemes. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY 4.0 International license .

Composting and fermentation: mitigating hop latent viroid infection risk in hop residues

Hop cultivation, integral to the brewing industry, faces challenges from viroids, especially the citrus bark cracking viroid (CBCVd) but also the hop latent viroid (HLVd) influences hop cone quality. We focused on the degradation kinetics of HLVd thereby covering compost, silage, and digestate made from hop residues. In this study, HLVd serves as a model for understanding CBCVd, which causes significant stunting and yield losses in European hop crops. Composting experiments revealed that although composting significantly lowers HLVd levels, complete degradation within 7 weeks is not guaranteed, with loose compost showing a more rapid reduction than compacted variants. Infectivity experiments conducted using inocula obtained from HLVd-infected hop plant residues exposed to composting, ensiling, and biogas digestate did not result in the transmission of HLVd to viroid-free plants. Also extracting and analyzing the soil-root mixture of plants inoculated with HLVd-infected hop residues did not show evidence for viroid persistence. Degradation experiments further differentiated between the physiochemical and biological influences on viroid and viroid-like random RNA stability, showing that higher temperatures of 50 °C enhance degradation over 40 °C, and pH levels of 5 or 7 are slowing degradation. In contrast deionized water or a pH of 4 or 9 enhances viroid degradation. Adding extracts from digestate accelerated the process indicating a role of biological activity. Interestingly, a viroid-like random RNA with similar physiochemical properties, showed to degrade faster compared to HLVd, suggesting high robustness of the actual viroid secondary structure. These findings offer valuable insights into managing HLVd in hops and potentially other crops, highlighting effective strategies to mitigate viroid spread, and contributing to broader understanding of RNA degradation in agriculture.

Assessment of the health status of citrus propagation material against hop stunt viroid through biological and molecular diagnosis

Hop stunt viroid (HSVd) is the causal agent of citrus cachexia, which can lead to significant crop losses. As part of a preventive management strategy, it is essential to use healthy plants from a certification program. This study aimed to, healthiness of 17 citrus cultivars was evaluated against HSVd using both biological and molecular diagnostics. For biological diagnosis, Etrog citron (Citrus medica L.) clone Arizona 861 S1 was used as an indicator plant, incorporating negative and positive controls for HSVd infection. For molecular diagnosis, reverse transcription polymerase chain reaction (RT-PCR) technique was employed with specific primers for viroid detection. Results indicated that six months after inoculation, the positive control exhibited symptoms associated with viroid infection, such as necrosis in the petiole and stem, leaf epinasty, and necrosis in the midvein. In contrast, both the negative control and evaluated citrus cultivars showed no symptoms. These findings were supported by molecular analysis, which allowed the amplification of a viroid genome region only in the positive control. Both biological and molecular diagnosis confirmed the absence of HSVd in the citrus varieties assessed. This study will allow the evaluation of the citrus plant collections used in the foundation category as a source of buds for plant multiplication of registered and certified seeds for citrus propagation nurseries in Colombia.

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.

ANTIOXIDANT STATUS OF CALLUS CULTURE AND NATIVE PLANTS OF POTATO UNDER VIRAL INFECTION CONDITIONS

Potato (Solanum tuberosum L.) is a vital tuberous crop consumed around the globe following rice, wheat, and maize. The biotic factors, such as fungi, viruses, bacteria, and viroid infections, significantly impact the potato plant's metabolism, altering its physiological, biochemical, and intermolecular reactions to these stress factors. To investigate the activity levels of antioxidant enzymes (AOEs) in the oxidative stress of potato plants and assess their resistance levels to viral infections, callus culture is often employed as a primary model. This study examines the activity profiles of AOEs, specifically peroxidase (POD), catalase (CAT), and superoxide dismutase (SOD), in two entities – potato callus tissue and native plants – under potato virus X (PVX) virus infection conditions. The research results demonstrate distinct AOE profiles in callus tissues and leaves of infected potato plants. The POD activity in callus tissue is over 50% higher on the 3 dpi compared to plant leaves. The dynamics of increased AOE activity follow a pattern, peaking on the 3 dpi and declining by the 7 dpi. CAT activity remains consistent until the 7 dpi across all five studied varieties. SOD activity as the primary enzyme in hydrogen peroxide deactivation, increases by 16-42% post infection. The AOE activity exhibits a consistent pattern during PVX virus infection in callus and leaf tissues, with the preinfection activity level being over 100 units/gram dry weight lower in callus tissue than in leaf tissue. Moreover, the nature of increased activity differs between the two entities.

New Insights into Hop Latent Viroid Detection, Infectivity, Host Range, and Transmission.

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.

Cellular roadmaps of viroid infection.

Viroids are single-stranded circular noncoding RNAs that infect plants. According to the International Committee on Taxonomy of Viruses, there are 44 viroids known to date. Notably, more than 20 000 distinct viroid-like RNA sequences have recently been identified in existing sequencing datasets, suggesting an unprecedented complexity in biological roles of viroids and viroid-like RNAs. Interestingly, a human pathogen, hepatitis delta virus (HDV), also replicates via a rolling circle mechanism like viroids. Therefore, knowledge of viroid infection is informative for research on HDV and other viroid-like RNAs reported from various organisms. Here, we summarize recent advancements in understanding viroid shuttling among subcellular compartments for completing replication cycles, emphasizing regulatory roles of RNA motifs and structural dynamics in diverse biological processes. We also compare the knowledge of viroid intracellular trafficking with known pathways governing cellular RNA movement in cells. Future investigations on regulatory RNA structures and cognate factors in regulating viroid subcellular trafficking and replication will likely provide new insights into RNA structure-function relationships and facilitate the development of strategies controlling RNA localization and function in cells.

PSTVd infection in Nicotiana benthamiana plants has a minor yet detectable effect on CG methylation.

Viroids are small circular RNAs infecting a wide range of plants. They do not code for any protein or peptide and therefore rely on their structure for their biological cycle. Observed phenotypes of viroid infected plants are thought to occur through changes at the transcriptional/translational level of the host. A mechanism involved in such changes is RNA-directed DNA methylation (RdDM). Till today, there are contradictory works about viroids interference of RdDM. In this study, we investigated the epigenetic effect of viroid infection in Nicotiana benthamiana plants. Using potato spindle tuber viroid (PSTVd) as the triggering pathogen and via bioinformatic analyses, we identified endogenous gene promoters and transposable elements targeted by 24 nt host siRNAs that differentially accumulated in PSTVd-infected and healthy plants. The methylation status of these targets was evaluated following digestion with methylation-sensitive restriction enzymes coupled with PCR amplification, and bisulfite sequencing. In addition, we used Methylation Sensitive Amplification Polymorphism (MSAP) followed by sequencing (MSAP-seq) to study genomic DNA methylation of 5-methylcytosine (5mC) in CG sites upon viroid infection. In this study we identified a limited number of target loci differentially methylated upon PSTVd infection. These results enhance our understanding of the epigenetic host changes as a result of pospiviroid infection.

Chloroplast clustering around the nucleus induced by OMP24 overexpression unexpectedly promoted PSTVd infection in Nicotiana benthamiana.

Chloroplast clustering around the nucleus is a well-known mechanism that occurs in response to various biotic and abiotic stresses and is believed to be a mechanism of defence against pathogens in plants. This phenomenon is accompanied by increased production of reactive oxygen species (ROS), which can help to destroy invading pathogens. However, the function of chloroplast clustering during viroid infection is unclear. Here, we report that, although the infection by potato spindle tuber viroid (PSTVd) failed to induce chloroplast clustering, chloroplast clustering caused by the overexpression of the Nicotiana benthamiana chloroplast outer membrane protein 24 (NbOMP24) promoted the infection by PSTVd, a viroid pathogen, in N. benthamiana. Interestingly, H2 O2 treatment, which caused increased ROS accumulation, showed no significant effects on PSTVd infection. Moreover, NbOMP24 protein showed no direct interaction with PSTVd. We propose that perinuclear chloroplast clustering induced by NbOMP24 provides a favourable environment for PSTVd infection. These findings highlight the complexity of chloroplast clustering-mediated plant-pathogen interactions and the need for further research to fully understand these mechanisms.

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.

Influence of allelic polymorphism in the 3’ untranslated region of the <i>StTCP23</i> gene on the tolerance of potato cultivars to the potato spindle tuber viroid

Background. It is known that the pathological phenotype of potato plants can be mediated by complementary interactions between the genomic RNA of PSTVd and mRNA of some regulatory genes, which consequently lead to RNA interference, the synthesis of small interfering RNAs (vd-sRNA PSTVd), and impaired morphogenesis. At the same time, symptoms caused by the viroid may vary in different potato cultivars. Here we predict the interactions between the 3’ UTRs of various alleles of the StTCP23 transcription factor gene and the complementary regions in PSTVd genomic RNA.Materials and methods. We selected eight commercial potato cultivars with different symptoms of viroid infection and disease. For each cultivar, six clones of each cDNA amplicon of StTCP23 with a 3’ UTR were identified, and the allelic compositions of the target regions within their 3’ UTRs were characterized.Results. In total, 11 types of alleles of the 3’ UTR StTCP23 segment complementary to the vd-sRNA PSTVd were identified. Cultivars with the A allele (‘Gala’, ‘Colomba’, ‘Favorit’, and ‘Fioletovy’) identical to the reference genome or a high dose of the C allele with a deletion of four nucleotides (cv. ‘Impala’) were characterized by high susceptibility already at the primary (firstyear) infection with the PSTVd. Cvs. ‘Krepysh’, ‘Labadia’ and ‘Riviera’, classified as tolerant during primary inoculation, on the contrary, were characterized by the absence of the A allele and the presence of cultivar-specific mutant alleles.Conclusion. A high degree of polymorphism in the target site (3’ UTR region) of StTCP23 indicates a possible selection pressure on this locus. It can be assumed that cultivars with shorter alleles, which have fewer bases complementary to vd-sRNA in hypothetical duplexes and therefore less likely to induce target gene silencing, are more tolerant to the PSTVd upon primary viroid infection.

The multifaceted roles of R2R3 transcription factor HlMYB7 in the regulation of flavonoid and bitter acids biosynthesis, development and biotic stress tolerance in hop (Humulus lupulus L.)

Hop (Humulus lupulus) biosynthesizes the highly economically valuable secondary metabolites, which include flavonoids, bitter acids, polyphenols and essential oils. These compounds have important pharmacological properties and are widely implicated in the brewing industry owing to bittering flavor, floral aroma and preservative activity. Our previous studies documented that ternary MYB-bHLH-WD40 (MBW) and binary WRKY1-WD40 (WW) protein complexes transcriptionally regulate the accumulation of bitter acid (BA) and prenylflavonoids (PF). In the present study, we investigated the regulatory functions of the R2R3-MYB repressor HlMYB7 transcription factor, which contains a conserved N-terminal domain along with the repressive motif EAR, in regulating the PF- and BA-biosynthetic pathway and their accumulation in hop. Constitutive expression of HlMYB7 resulted in transcriptional repression of structural genes involved in the terminal steps of biosynthesis of PF and BA, as well as stunted growth, delayed flowering, and reduced tolerance to viroid infection in hop. Furthermore, yeast two-hybrid and transient reporter assays revealed that HlMYB7 targets both PF and BA pathway genes and suppresses MBW and WW protein complexes. Heterologous expression of HlMYB7 leads to down-regulation of structural genes of flavonoid pathway in Arabidopsis thaliana, including a decrease in anthocyanin content in Nicotiana tabacum. The combined results from functional and transcriptomic analyses highlight the important role of HlMYB7 in fine-tuning and balancing the accumulation of secondary metabolites at the transcriptional level, thus offer a plausible target for metabolic engineering in hop.

Incidence and Epidemiology of Citrus Viroids in Greece: Role of Host and Cultivar in Epidemiological Characteristics.

Viroids represent a threat to the citrus industry and also display an intricate matter for citrus tristeza virus (CTV) control as most of the commercial citrus rootstocks that are resistant/tolerant to CTV appear to be highly susceptible to viroid infection. Therefore, a detailed knowledge of the viroid's incidence and distribution, along with the assessment of unexplored epidemiological factors leading to their occurrence, are necessary to further improve control measures. Herein, a large-scale epidemiological study of citrus viroids in five districts, 38 locations and 145 fields in Greece is presented, based on the analysis of 3005 samples collected from 29 cultivars of six citrus species. We monitored the occurrence of citrus exocortis (CEVd), hop stunt (HSVd), citrus dwarfing (CDVd), citrus bark cracking (CBCVd), and citrus bent leaf (CBLVd) viroids, and addressed their epidemiological patterns and factors shaping their population structure. Our results show a high frequency and wide distribution of four viroids in all areas and in almost all hosts, whereas CBLVd occurrence was restricted to Crete. Mixed infections were found in all districts in which a wide spread of viroids was observed. We identified a potential pathogens' different preferences that could be partially explained by the host and cultivar, including the type of infection (single or mixed) and the number of viroids in the mixed infections. Overall, this work provides the first detailed epidemiological study on citrus viroids, enriching our knowledge for the implementation, production, and distribution of certified citrus propagative material, and the development of sustainable control strategies.

First report of mume virus A infecting Prunus salicina worldwide and Prunus mume in Korea.

Mume virus A (MuVA) of the genus Capillovirus in the family Betaflexiviridae was first isolated from a Japanese apricot tree (Prunus mume) exhibiting symptoms of diffuse chlorotic spots (Marais et al. 2018). MuVA infection has been reported in Japanese apricot trees in Japan as well as in peach (P. persica) and Japanese apricot trees in China (Marais et al. 2018; Zhang et al. 2021; Zheng et al. 2020). In the present study, the diversity of viruses and viroids infecting Chinese plum trees (P. salicina) was investigated using high-throughput sequencing (HTS). Ten flowers each from 50 trees without obvious symptoms related to virus and/or viroid infection were randomly collected from five orchards in Gimcheon, Korea, in April 2020. The samples from each Chinese plum tree were pooled, and the same amounts of 50 individual samples prepared in advance were pooled for the extraction of total RNA using the RNeasy Plant Mini Kit (QIAGEN, Hilden, Germany). Removal of ribosomal RNA and construction of cDNA library from the extracted total RNA were conducted using the TruSeq Stranded Total RNA with Ribo-Zero Plant kit (Illumina, San Diego, CA, USA). Paired-end RNA sequencing using Illumina NovaSeq 6000 System (paired-end reads of 101 bp and a total of 162,845,322 reads) and data analysis were performed at Macrogen (Daejeon, Korea). Adaptor and low-quality sequences of reads were removed using Trimmomatic program. Trimmed reads were assembled into contigs using Trinity program, and several databases including NCBI Nucleotide and Kyoto Encyclopedia of Genes and Genomes were used for functional annotation. HTS identified plum bark necrosis stem pitting-associated virus (PBNSPaV; four contigs ranging from 2081 to 3202 nucleotides) and hop stunt viroid (HSVd; one contig of 618 nucleotides). PBNSPaV and HSVd were also detected by RT-PCR (PBNSPaV det-F and PBNSPaV det-R for PBNSPaV [Al Rwahnih et al. 2007]; VP-19 and VP-20 for HSVd [Astruc et al. 1996]) and confirmed by Sanger sequencing of the amplified products. Interestingly, one contig derived from MuVA, which was not previously reported in Korea, was also detected. The contig was 7,618-nucleotide long (15,205 reads), and NCBI BLASTN search revealed 98.74% homology (100% query coverage) with the MuVA isolate pm14 (GenBank accession number MG783575). To design diagnostic primers for reverse transcription (RT)-polymerase chain reaction (PCR), the contig sequence and MuVA sequences available in NCBI GenBank (GenBank accession numbers MG783575 and MN412555) were aligned using CLC Main Workbench 6.9.1 (QIAGEN, Redwood, CA, USA). The following primer set (expected size of 1,143 bp) was prepared: MuVA-2F (5'-CAGCTTTGTGACTCYAACCC-3') and MuVA-2R (5'-AATGGCTTGAGGRCCTGCAG-3'). The primers target a partial region (nt position 1185 to 2327 on the basis of the reference genome sequence of MuVA, GenBank accession no. NC_040568) of the polyprotein gene (ORF1). Each of the 50 samples was tested for the presence of MuVA using the above-mentioned RT-PCR primers with SuPrimeScript RT-PCR Premix (GeNet Bio, Daejeon, Korea). MuVA was detected in three samples collected from the same orchard. The three amplicons were inserted into a T&A cloning vector (RBC Bioscience, Taipei, Taiwan) and sequenced at Macrogen. Three consensus sequences obtained by Sanger sequencing were registered in NCBI GenBank under accession numbers MW589492, MW589493, and MW589494. NCBI BLASTN search revealed that the Korean isolates of MuVA shared high homology with isolate pm14 [98.16%, 98.08%, and 98.16% (100% query coverage), respectively]. To confirm additional MuVA infections, leaf samples of Chinese plum trees were collected from orchards in Uiseong (70 trees) and Seongju (50 trees) as well as a Japanese apricot tree in Chuncheon, from April to July 2021. RT-PCR confirmed additional MuVA infections from Uiseong (one tree) and Seongju (one tree) as well as from the Japanese apricot tree in Chuncheon. NCBI BLASTN search of the three additional amplicons (GenBank accession numbers OM210030, OM210031, and OM210032) revealed high homology with isolate pm14 [98.25%, 98.08%, and 97.90% (100% query coverage)]. To the best of our knowledge, this is the first report of MuVA infecting P. mume in Korea and P. salicina worldwide. Further research is needed to investigate MuVA infections on various Prunus spp. including P. persica in Korea.

Molecular characterization of divergent isolates of Citrus bent leaf viroid (CBLVd) from citrus cultivars of Punjab, Pakistan.

Citrus viroid infection is emerging as a serious threat because of its efficient systemic movement within the host plant and its quick spread due to contaminated pruning tools. A survey was conducted to investigate the primary distribution and molecular characterization of Citrus bent leaf viroid (CBLVd) and its variants in different citrus cultivars. A total of 154 symptomatic citrus samples were collected and detected by RT‒PCR with newly designed specific primers with the incidence of 36.33%. During biological indexing study on Etrog citron, expressions of reduced leaf size, yellowing with a light green pattern, and bending were observed. Amplified products were sequenced and analyzed using a nucleotide BLAST search, which showed 98% homology with other CBLVd isolates. The results of the phylogenetic tree analysis showed the presence of two main groups (A and B), with the predominant variants of CBLVd, i.e., CVd-I-LSS (Citrus viroid Low Sequence Similarity) sequences, clustering in subgroup A1 along with newly detected CVd-I-LSS from Palestinian sweet lime (Citrus limettioides), which has been identified as a new host of CVd-I-LSS in Pakistan. Further analysis of the sequences in subgroup A1 showed that the variant of CVd-I-LSS infecting citrus cultivars had a close relationship with isolates reported from China, Japan, and Iran, which may have resulted from the exchange of planting material. This study also unveiled the variability in nucleotide sequences of CBLVd, which made it unable to be detected by old primers. The results of this study indicate that the widespread presence of divergent variants of CBLVd is a major concern for the citrus industry in Pakistan and other countries where virulent isolates of CBLVd are prevalent. These findings suggest the need for future research on effective management and quarantine measures to stop the spread of CBLVd.