Chrysanthemum Chlorotic Mottle Viroid Research Articles

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 chrysanthemumstunt viroid (Pospiviroidae), thus establishing a clear distinction on how members of the two viroid families trigger chlorosis in the same host.

Occurrence of Viruses and Viroids in Chrysanthemum Plants (Dendranthema morifolium) Cultivated in Yesan-gun, Chungcheongnam-do in Korea

Chrysanthemum plants are one of the most economically important plants in South Korea. Both virus and viroid can cause diseases and economic damage to the plants. In this study, we investigated the detection of seven viruses and two viroids in 350 chrysanthemum plants cultivated in Yesan-gun, Chungcheongnam-do. Two viruses, chrysanthemum virus B (CVB) and tomato aspermy virus (TAV), and two viroids, chrysanthemum chlorotic mottle viroid (CChMVd) and chrysanthemum stunt viroid (CSVd), were detected in this study. The two viruses were detected in six samples and one sample, respectively. The two viroids were detected in 97 samples and 21 samples, respectively. The nucleotide sequences of the CVB-CN-Y, TAV-CN-Y, CChMVd-CN-Y, and CSVd-CN-Y obtained in this study showed 83.7–86.9%, 99.2–100.0%, 94.4–99.5%, and 95.7–99.7% identity, respectively, compared to their other strains/isolates. The CVB-CN-Y and TAV-CN-Y showed the greatest nucleotide sequence homology to CVB-GS1 and three TAV isolates (TAV-V, TAV-P, and TAV-ChJ), respectively. The CChMVd-CN-Y and CSVd-CN-Y showed the greatest nucleotide sequence homology to CChMVd-Horst and four CSVd isolates (Au1.1, K4pop, Sagae, and Tochigi), respectively. This study is the report on the infection rate of viruses and viroids in chrysanthemum plants cultivated in Yesan-gun in 2021.

On the early identification and characterization of pear blister canker viroid, apple dimple fruit viroid, peach latent mosaic viroid and chrysanthemum chlorotic mottle viroid

In the 90′s, pear blister canker viroid (PBCVd), apple dimple fruit viroid (ADFVd), peach latent mosaic viroid (PLMVd) and chrysanthemum chlorotic mottle viroid (CChMVd) were identified and characterized in the Ricardo Flores’ laboratory. In these studies, the autonomous replication of these infectious RNAs and their involvement in the elicitation of diseases in their natural hosts were also shown. Their discovery was achieved by classical approaches based on the physical purification of the viroid RNAs from polyacrylamide gels followed by the sequencing of their genomic RNAs and by bioassays to assess their autonomous replication and the fulfillment of Koch's postulates. The molecular characterization of these four viroids, including the study of their sequence variability, contributed to the establishment of the concept of quasispecies for viroids and to the development of reliable molecular diagnostic methods that have facilitated the control of the diseases they caused. Most importantly, some of these viroids became valuable experimental model systems that are still used nowadays to study structural-functional relationships in RNAs and to dissect evolutionary and pathogenic pathways underlying plant-viroid interaction. The differences between early viroid discovery strategies, relying on biological and pathogenic issues, and the current high-throughput sequencing-based approaches, that frequently allow the discovery of new viroids and viroid-like RNAs in symptomless hosts, is also discussed, clarifying why the traditional molecular and biological studies mentioned above are still required to conclusively define the nature of any novel viroid-like RNA.

Development of Colorimetric Loop-Mediated Isothermal Amplification (LAMP) Technique for Rapid, Sensitive and Convenient Detection of Chrysanthemum Chlorotic Mottle Viroid (CChMVd) in Chrysanthemum

Chrysanthemum chlorotic mottle viroid (CChMVd) is a serious pathogen infecting chrysanthemum worldwide. To improve and enhance the detection procedure, the colorimetric loop-mediated isothermal amplifi cation (LAMP) technique was developed. Six LAMP primers were newly designed and tested the optimal conditions using a recombinant plasmid of CChMVd as a DNA template. The optimal conditions for colorimetric LAMP were incubation at 65°C for 60 min. At these conditions, the ladder-liked pattern LAMP products were detected along with the change of color from pink to yellow in the positive reactions. The sensitivity of colorimetric LAMP was up to 2.18 × 103 copies of recombinant plasmid concentration which was 104-fold greater than that of polymerase chain reaction (PCR). The developed colorimetric LAMP was not cross reacted to other viruse s and viroid. Effi ciency of colorimetric reverse transcription (RT)-LAMP for detecting CChMVd in chrysanthemum plantlets obtained from meristem tip culture was evaluated to verify and ensure CChMVd-free plantlets production. The results showed that 18 chrysanthemum plantlets were free of CChMVd contamination. Therefore, the colorimetric RT-LAMP can be used to detect CChMVd routinely due to its accuracy, rapidness and sensitivity.

Chrysanthemum virus B and chrysanthemum chlorotic mottle viroid infect chrysanthemum in South Africa

This is the first report of chrysanthemum virus B (CVB) and chrysanthemum chlorotic mottle viroid (CChMVd) on Chrysanthemum × morifolium cultivars, in South Africa. Samples were collected from plants showing vein clearing, mosaic and chlorosis, in the Western Cape province. RNAtag-seq was applied and indicated the presence of CVB in all four samples and the co-infection with CChMVd in one sample. The presence of CVB and CChMVd were confirmed using RT-PCR and Sanger sequencing.

Phylogenetic analysis and molecular characterization of chrysanthemum chlorotic mottle viroid and chrysanthemum stunt viroid from chrysanthemum in Thailand

AbstractChrysanthemum chlorotic mottle viroid (CChMVd) and chrysanthemum stunt viroid (CSVd) are limiting factors for chrysanthemum cultivation and cause economic losses throughout the world. Chrysanthemum leaves were collected from Chiang Mai and Chiang Rai provinces of Thailand during 2019–2021. Both viroids were detected by using reverse transcription polymerase chain reaction (RT‐PCR) and molecularly characterized. Six CChMVd and five CSVd amplicons were detected, cloned and sequenced. BLAST analysis revealed that CChMVd shared 93%–99% identical and CSVd shared 95%–99% identical with the representative species retrieved from GenBank, Moreover, the nucleotides at the position 82–85 of the tetraloop region were keys for CChMVd clusters resulting to the distinct clade of either symptomatic or non‐symptomatic strains. The phylogenetic analysis of individual viroid represented that six CChMVd variants were closely related to Korea and China variants and clustered in the symptomatic clade of the Asia cluster. For CSVd, five variants in the present study were grouped with CSVd from Dahlia spp. of Japan. Predicted secondary structure of both viroids showed the distribution of sequence variations. The predicted secondary structure showed sequence variants of CChMVd were distributed on most of the secondary structure, whereas sequence variants of CSVd were frequently found in pathogenic, variable and terminal right domains. This study provides phylogenetic relationship of CChMVd and CSVd Thai variants along with their sequence variability.

Molecular characterization of sequence variants of Chrysanthemum chlorotic mottle viroid from Himachal Pradesh, India

Molecular characterization of sequence variants of Chrysanthemum chlorotic mottle viroid from Himachal Pradesh, India

Distribution of chrysanthemum chlorotic mottle viroid in shoot meristem and flower buds of chrysanthemum

Detailed information on viroid invasion of the shoot apical meristem is important for the efficient production of viroid-free plants by meristem tip culture. Additionally, knowledge on viroid distribution in plant reproductive tissues, especially the embryo, is crucial to assess seed transmission of viroids. In this study, we analyzed histochemically the distribution of chrysanthemum chlorotic mottle viroid (CChMVd) in the shoot meristems and flower buds of CChMVd-infected chrysanthemum plants (cv. ‘Okayama-heiwa’). In situ hybridization revealed that CChMVd invaded the shoot apical meristems and leaf primordia in the shoot meristem at a high frequency (ca. 70%). In flower buds, CChMVd was found in the bract, receptacle vascular, petals, ovules, and immature stamens and pistils. Furthermore, we showed that seed transmission of CChMVd occurred at a high frequency when CChMVd-infected plants were used as female parents (♀). Overall, our findings suggest that CChMVd infection of ovules in flower buds is involved in seed transmission of the viroid.

Cryopreservation of viroid-infected chrysanthemum shoot tips

Chrysanthemum stunt viroid (CSVd) and chrysanthemum chlorotic mottle viroid (CChMVd) are the two viroids most frequently infecting chrysanthemum. This study attempted to cryopreserve in vitro shoot tips of Chrysanthemum morifolium ‘Piato’ infected with high or low titers of CSVd, and co-infected with CSVd and CChMVd. By optimizing several key factors including the time and light regimes during cold-hardening of stock shoots, and the size of shoot tips, an encapsulation-vitrification procedure was established for cryopreservation of viroid-infected shoot tips. Viroid-infected stock shoots were cold-hardened in vitro at 4 °C in a 16-h photoperiod for 6 weeks. Shoot tips (1.5 mm in size) containing 3-4 LPs were excised from the cold-hardened stock shoots and subjected to encapsulation-vitrification cryopreservation. With this protocol, about 65%, 45% and 42% of shoot regrowth levels were obtained in cryopreserved shoot tips derived from in vitro stock shoots infected with low or high titers of CSVd, and co-infected with CSVd and CChMVd, respectively. All regenerants from cryopreservation maintained their viroid-infected status, identical to those of their in vitro stock shoots. Histological observations and in situ hybridization elucidated why cryopreservation could maintain viroids in cryopreserved shoot tips. Cryopreservation of viroid-infected plant materials has potential applications to all types of viroid-related basic and applied studies.

Different rates of spontaneous mutation of chloroplastic and nuclear viroids as determined by high-fidelity ultra-deep sequencing.

Mutation rates vary by orders of magnitude across biological systems, being higher for simpler genomes. The simplest known genomes correspond to viroids, subviral plant replicons constituted by circular non-coding RNAs of few hundred bases. Previous work has revealed an extremely high mutation rate for chrysanthemum chlorotic mottle viroid, a chloroplast-replicating viroid. However, whether this is a general feature of viroids remains unclear. Here, we have used high-fidelity ultra-deep sequencing to determine the mutation rate in a common host (eggplant) of two viroids, each representative of one family: the chloroplastic eggplant latent viroid (ELVd, Avsunviroidae) and the nuclear potato spindle tuber viroid (PSTVd, Pospiviroidae). This revealed higher mutation frequencies in ELVd than in PSTVd, as well as marked differences in the types of mutations produced. Rates of spontaneous mutation, quantified in vivo using the lethal mutation method, ranged from 1/1000 to 1/800 for ELVd and from 1/7000 to 1/3800 for PSTVd depending on sequencing run. These results suggest that extremely high mutability is a common feature of chloroplastic viroids, whereas the mutation rates of PSTVd and potentially other nuclear viroids appear significantly lower and closer to those of some RNA viruses.

Molecular diversity among viroids infecting chrysanthemum in India.

Association of Chrysanthemum stunt viroid (CSVd) and Chrysanthemum chlorotic mottle viroid (CChMVd) with the Chrysanthemum plants exhibiting severe stunting, distinct yellow leaf mottling, and chlorosis was detected in the main chrysanthemum-growing regions of India. Sequence analysis of 90 cDNA clones obtained for CSVd and CChMVd, representing the chrysanthemum-growing regions of India, revealed the high degree of sequence variation throughout the genome under natural conditions. Additionally, all the analyzed CChMVd clones revealed the presence of UUUC in the tetraloop, a signature of symptomatic variants in susceptible cultivars. Phylogenetic analysis revealed that Indian CSVd is closely related to European isolates from ornamentals, whereas CChMVd clustered along with the isolates reported from the East Asian countries.

Molecular Identification of Chrysanthemum chlorotic mottle viroid Infecting Chrysanthemum in Karnataka, India

Chrysanthemum chlorotic mottle viroid (CChMVd, genus Pelamoviroid, family Avsunviroidae) has been reported to infect chrysanthemum (Dendranthema x grandiflorum). CChMVd was first reported in 1967 in the cultivar Yellow Delaware in New York State, USA (Dimock et al. 1971). Presence of CChMVd has previously been recorded, based on the symptoms and bioassay, in India (Singh et al. 1978). Karnataka State of India is the prominent chrysanthemum-growing region, with 19,161 ha cultivated under this crop and accounting for more than 40% of country’s flower production. During late summer 2010 (May and June), chrysanthemum leaves with yellow-green mottling and chlorotic spots were noted, indicating the possible association with CChMVd in Karnataka. Total RNA was extracted using a 2% CTAB buffer from leaf samples of symptomatic plants (Adkar-Purushothama et al. 2013). For the detection of CChMVd, reverse transcription PCR (RT-PCR) was performed using PIII/PIV as described previously (De la Pena et al. 1999). PCR products of the expected sizes (∼398 to 401 bp) were cloned and sequenced. BLAST analysis of the obtained sequences confirmed the presence of CChMVd in Karnataka. In order to estimate the incidence of CChMVd, the same RT-PCR assay was performed on 80 randomly selected chrysanthemum leaf samples of four different cultivars (Redgold, Bangalore, Sarval, and CO-1) collected from four chrysanthemum-growing districts of Karnataka. A PCR product of the size expected for CChMVd was detected in 8 of 80 samples analyzed, accounting for the infection incidence of 10%. To identify the prominent CChMVd variants, 10 clones obtained from the four representative samples were sequenced. To confirm the sequence of the primer regions, an additional set of primers CCh1/CCh2 were used for RT-PCR (Hosokawa et al. 2005) with high-fidelity LA-Taq polymerase (Takara-Bio, Japan). Alignment of all 40 sequences revealed the presence of at least two major sequence variants of CChMVd: CChMVd-Ind-1 (GenBank Accession No. KP262531) and CChMVd-Ind-3 (KP262533). Both the sequence variants had UUUC sequences (nucleotide position 82 to 85) in the tetraloop of the CChMVd, which is often associated with induction of symptoms in susceptible chrysanthemum cultivars (De la Pena et al. 1999). CChMVd-Ind-1was found to differ from CChMVd-Ind-3 by 17 nucleotide substitutions. BLAST analysis of CChMVd-Ind-1 showed 98% sequence identity with the CChMVd strain reported from Spain (GenBank Accession No. FJ647515) and from Himachal Pradesh state of India (FN646404), whereas CChMVd-Ind-3 showed 96% sequence identity with CChMVd strain W2-4, isolated from China (HQ891014). To our knowledge, this is the first molecular evidence for the presence of CChMVd infecting chrysanthemum in Karnataka State, India. The high degree of sequence diversity reported in the Karnataka isolates of CChMVd will help to clarify the evolution of this viroid and its possible threat to important crops.

Elimination of chrysanthemum stunt viroid and chrysanthemum chlorotic mottle viroid from infected chrysanthemum by cryopreservation

Chrysanthemum morifolium 'Borami' and 'Secret Pink' showing symptoms of stunt disease caused by chrysanthemum stunt viroid (CSVd) and 'Yellow Cap' showing chlorotic mottle disease caused by chrysanthemum chlorotic mottle viroid (CChMVd) were confirmed to be infected by the respective viroids by using reverse transcription polymerase chain reaction (RT-PCR). Real-time PCR results showed that the viroid concentrations in the infected cultivars varied between the different regions of origin (Chilgok, Gumi, and Gyeongsan). We applied a cryopreservation protocol for elimination of CSVd from naturally infected 'Borami' collected from Gumi, showing the lowest concentration of CSVd, by varying several factors such as plant vitrification solutions (PVS2 and PVS3), duration of exposure to liquid nitrogen, shoot-tip size, and low-temperature treatment. The solution (PVS2) and low-temperature treatment were found to be critical factors determining the efficacy of viroid elimination. We optimized the protocol by combining of all resulted optimal factors and tested the applicability of the protocol in 'Borami' collected from Chilgok and Gyeongsan and in 'Secret Pink' from Chilgok, Gumi, and Gyeongsan, which displayed different viroid concentrations. We found that the elimination rates varied depending on the cultivar and region of origin. Similar results were observed when the protocol was applied to eliminate CChMVd from the 'Yellow Cap' collected from the same regions. Finally, we found that nested PCR is more reliable for viroid detection than RT-PCR. Overall, cryopreservation can be used to eliminate viroids from infected chrysanthemums; however, the efficacy depends on genotype and initial viroid concentration.

A multiplex RT-PCR for simultaneous detection and identification of five viruses and two viroids infecting chrysanthemum

Pathogens causing significant economic losses in chrysanthemum include tomato aspermy virus (TAV), chrysanthemum virus B (CVB), cucumber mosaic virus (CMV), tobacco mosaic virus (TMV), potato virus Y (PVY), chrysanthemum stunt viroid (CSVd) and chrysanthemum chlorotic mottle viroid (CChMVd). A multiplex reverse transcription polymerase chain reaction (RT-PCR) method, using specific primer sets for each virus or viroid, was developed for simultaneous detection and differentiation of TAV, CVB, CMV, TMV, PVY, CChMVd, and CSVd. The RT-PCR method was validated by testing chrysanthemum samples collected from different regions of China. In this study, CVB, TAV, TMV, PVY, CSVd, CMV, and CChMVd were detected, respectively, in 24.7 %, 17.5 %, 4.4 %, 4.4 %, 2.9 %, 2.5 %, and 1.5 % of the samples tested. These results indicate that CVB and TAV (24.7 % and 17.5 %) are common, whereas CMV, TMV, CChMVd, CSVd, and PVY (all below 5 %) are less frequently encountered. This new multiplex RT-PCR method has potential to be used routinely in large-scale virus and viroid surveys.

Wykrywanie i charakterystyka izolatów wirusa B chryzantemy (Chrysanthemum virus B, CVB) w Polsce

Summary In 2013 and 2014 in the Plant Disease Clinic of Institute of Plant Protection – National Research Institute in Poznan, chrysanthemum plants showing symptoms of stunted growth, mosaic and malformation of leaves and abnormal pigmentation of flowers were tested. All plants were analyzed using biological, electron microscopy and molecular biology methods. The plants were examined for the presence of the following pathogens: Chrysanthemum virus B (CVB), Tomato aspermy virus (TAV), Chrysanthemum stunt viroid (CSVd) and Chrysanthemum chlorotic mottle viroid (CChMVd). The presence of CVB was confirmed in the samples collected from 15 plants. The isolates were genetically identical and shared high level of similarity with isolates from India and Japan.

The discovery and eradication of potato spindle tuber viroid in Canada.

In 1960s, potato spindle tuber was thought to be a viral disease. In 1971, the agent of the disease was characterised as a low-molecular weight infectious ribonucleic acid (RNA), which was named as 'viroid', specifically Potato spindle tuber viroid (PSTVd)). Since then, more than 30 plant diseases in horticultural and ornamental plants have been shown to be caused by different viroids globally. Viroids are single-stranded RNA, covalently closed circular molecule, without any protein coat. They are the smallest known plant pathogen containing RNA genome ranging from 246 nucleotides (Coconut cadang-cadang viroid) to 399 nucleotides (Chrysanthemum chlorotic mottle viroid). Some viroids are located in the plant cell nucleus (pospiviroids) and others in the chloroplast (avsunviroids). With the recognition of pathogenic nature of viroid, specific detection methodologies were developed, which enabled detection of PSTVd in seed-potato tubers prior to their planting in the field, and thus PSTVd was prevented from spreading the disease. As a result, PSTVd was eradicated from Canada in late 1980s. Viroids similar to PSTVd (Pospiviroid) have been discovered and they are detected in symptomless ornamental plants. Although, PSTVd has been eradicated from Canada, there is a strong possibility of viroid introduction from other plants besides potato and tomato and causing PSTVd like diseases.

First Report of Potato virus Y Strain N-Wilga Infecting Chrysanthemum in China

Chrysanthemum (Chrysanthemum morifolium Ramat.) is a commercially important ornamental grown worldwide, and is also extensively used as an edible and medicinal plant. In the present work, viruses and viroids infecting chrysanthemum were investigated in China in 2012 and 2013. Typical viral symptoms were observed in field-grown chrysanthemum with leaf yellowing and mottled leaves in Wenjiang District, Sichuan Province, China. The incidence of these symptoms in the field was 12.3%. Chrysanthemum virus B (CVB), Tomato aspermy virus (TAV), Cucumber mosaic virus (CMV), Tobacoo mosaic virus (TMV), Chrysanthemum stunt viroid (CSVd), and Chrysanthemum chlorotic mottle viroid (CChMVd), which had previously been reported to infect chrysanthemum in China (2,3), were not detected by RT-PCR assay. Since these symptomatic chrysanthemum plants grew next to a tobacco field, viruses affecting tobacco were suspected as possible cause. Sixteen symptomatic leaves and 12 non-symptomatic leaves were collected and tested for Potato virus Y (PVY) presence using commercial PVY-specific DAS-ELISA kits (Catalog no. PSA20001, Agdia) Six samples were found positive for PVY. RT-PCR tests using specific primers for CP gene (CP-F 5'-ACTGTGATGAATGGGCTTATG-3'; CP-R 5'-GGCATATATGGTTCCTTTTTG-3') (4) amplified a single, expected 218-bp DNA fragment from chrysanthemum extracts from all six samples positive for PVY in ELISA. These six PCR fragments were sequenced and found 100% identical to each other. The sequence (GenBank Accession No. KJ174515) shared 99% identity with corresponding sequences of several PVY isolates (NC_001616, EF026076, HM590407, and JQ924288). The same six positive samples were subjected to a multiplex RT-PCR assay (1) to identify the PVY strain type, and all six PVY samples from Sichuan were found to belong to the PVYN-Wi strain. To our knowledge, this is the first report of the PVYN-Wi strain infecting chrysanthemum in Sichuan, China.

Stem apex detoxification culture markedly improved several physiological characters of chrysanthemum ‘YUTAI’

Chrysanthemums host many viruses, viroid species, and sequence variants thereof. In the present study, a stem apex detoxification culture system for chrysanthemum ‘YUTAI’, a cultivar used for chrysanthemum tea, was established by apical meristem culture in vitro on basal Murashige and Skoog medium supplemented with 0.5 mg L−1 benzyladenine and 0.1 mg L−1 naphthalene acetic acid (NAA) to stimulate shooting, and on medium containing 0.5 mg L−1 NAA to afford the highest frequency of rooting. Seven viruses [Chrysanthemum stunt viroid (CSVd), Chrysanthemum chlorotic mottle viroid (CChMVd), Chrysanthemum virus B, Cucumber mosaic virus, Tobacco mosaic virus, Tomato spotted wilt virus, and Tomato aspermy virus] present in seedlings were synchronously detected using a sensitive multiplex reverse transcription-polymerase chain reaction assay. Two of these viruses (CChMVd and CSVd) compromised plant growth and development (affecting both the photosynthetic rate and height attained), and their levels were investigated in annual plants. Compared with seedlings infected by either or both CChMVd and CSVd, the chlorophyll content, the net photosynthetic rate, and the stomatal conductance and transpiration rates were higher, but the concentration of intercellular CO2 slightly lower, in virus-free seedlings. Virus-infected seedlings were stunted, exhibiting a reduction of 27 % of the mean height of virus-free plants. We suggest that the lower net photosynthetic rate and stunted growth observed in virus-infected seedlings were caused by the virus-mediated degradation of leaf chlorophyll, which in turn compromised plant nutrition.

Inhibition by polyamines of the hammerhead ribozyme from a Chrysanthemum chlorotic mottle viroid

BackgroundViroids are the smallest pathogens known to date. They infect plants and cause considerable economic losses. The members of the Avsunviroidae family are known for their capability to form hammerhead ribozymes (HHR) that catalyze self-cleavage during their rolling circle replication. MethodsIn vitro inhibition assays, based on the self-cleavage kinetics of the hammerhead ribozyme from a Chrysanthemum chlorotic mottle viroid (CChMVd-HHR) were performed in the presence of various putative inhibitors. ResultsAminated compounds appear to be inhibitors of the self-cleavage activity of the CChMVd HHR. Surprisingly the spermine, a known activator of the autocatalytic activity of another hammerhead ribozyme in the presence or absence of divalent cations, is a potent inhibitor of the CChMVd-HHR with Ki of 17±5μM. Ruthenium hexamine and TMPyP4 are also efficient inhibitors with Ki of 32±5μM and IC50 of 177±5nM, respectively. ConclusionsThis study shows that polyamines are inhibitors of the CChMVd-HHR self-cleavage activity, with an efficiency that increases with the number of their amino groups. General significanceThis fundamental investigation is of interest in understanding the catalytic activity of HHR as it is now known that HHR are present in the three domains of life including in the human genome. In addition these results emphasize again the remarkable plasticity and adaptability of ribozymes, a property which might have played a role in the early developments of life and must be also of significance nowadays for the multiple functions played by non-coding RNAs.

Loop-mediated isothermal amplification for the rapid detection of Chrysanthemum chlorotic mottle viroid (CChMVd)

Loop-mediated isothermal amplification (LAMP) is an established nucleic acid amplification method offering rapid, sensitive, and convenient diagnosis of infectious diseases. Chrysanthemum chlorotic mottle viroid (CChMVd) causes one of the most serious viral diseases in chrysanthemum in Korea. A sensitive LAMP assay was developed for rapidly detecting CChMVd infection. The assay was based on a set of four primers matching the specific region of the CChMVd genome. The CChMVd LAMP primer sets were designed using the sequences from nonsymptomatic and symptomatic CChMVd isolates in Korea. The efficiency and specificity of this method were optimized using Bst DNA polymerase, which allowed for increased viroid detection sensitivity. The reaction was carried out at 65°C for 90min, and was improved by adding SYBR Green I dye to the inside of the reaction tube lid prior to amplification. The results indicate that this LAMP method will be useful for chrysanthemum viroid disease monitoring and detecting CChMVd infectious disease.