Chrysanthemum Stunt Viroid Research Articles

Multiplex reverse transcription loop-mediated isothermal amplification for the simultaneous detection of CVB and CSVd in chrysanthemum

A multiplex reverse transcription loop-mediated isothermal amplification (mRT-LAMP) assay was developed for the simultaneous detection of Chrysanthemum Virus B (CVB) and Chrysanthemum stunt viroid (CSVd), which are the major viral pathogens of chrysanthemum worldwide. Two sets of mRT-LAMP primers were designed for the coat protein gene of CVB and the complete nucleotide sequence of CSVd, and a restriction enzyme cleavage site was inserted into two pairs of species-specific primers. The mRT-LAMP assay was designed by combining these two sets for a total of eight primers. The mRT-LAMP method distinguished between CVB and CSVd due to the subsequent restriction enzyme analysis. The sensitivity of the mRT-LAMP method was 103 times higher than classical PCR regarding the detection limits for CVB and CSVd. No positive results were observed when RNA from other chrysanthemum pathogens were used as mRT-LAMP templates. The method was verified by testing chrysanthemum samples collected from Beijing and Henan Province and showed high reliability and sensitivity. The developed mRT-LAMP assay also offers an efficient, convenient, and rapid tool for screening chrysanthemum virus and viroid, especially CVB and CSVd, and can be diagnosed in a single reaction. These results suggest that the new mRT-LAMP method may be used routinely for virus and viroid surveys.

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.

CHRYSANTHEMUM STUNT VIROID IN BRAZIL: SURVEY, IDENTIFICATION, BIOLOGICAL AND MOLECULAR CHARACTERIZATION AND DETECTION METHODS

In Brazil, the ornamental flowers and plants market moves in the wholesale and retail more than two billion US dollars annually, and chrysanthemum stands out as one of the most valuable commercial species. The stunting disease induced by Chrysanthemum stunt viroid (CSVd) has become a serious problem in chrysanthemum production systems worldwide. CSVd incites also colour breaking and retards flowering, but in many situations it does not induce visible symptoms, facilitating its spread in the field and passing international borders unnoticed. In Brazil there are few studies on this pathogen, with a single report of its possible occurrence in chrysanthemum in the State of Sao Paulo. In this work we aimed at surveying, identifying and characterizyng viroids in chrysanthemum crops in the State of Sao Paulo; challenging chrysanthemum varieties with a Brazilian CSVd isolate; and establishing diagnostic strategies to strengthen quarantine and indexing programs. Our survey showed that CSVd is widely disseminated in chrysanthemum crops in the State of Sao Paulo. All evaluated chrysanthemum varieties were susceptible of infection, although infection was sometimes symptomless. The complete genome sequencing of seven CSVd isolates showed high nucleotide identity when compared with other isolates from databases. Specific oligonucleotides were designed for conventional RT-PCR and RT-qPCR CSVd analysis. Results show that CSVd can be identified with a sensitivity about 100,000 times higher than PAGE. Dot-blot proved also to be highly sensitive and, consequently, most useful for large scale diagnosis. This is the first biological and molecular identification and characterization of CSVd isolates in Brazil.

Construction of Infectious cDNA Clone of a Chrysanthemum stunt viroid Korean Isolate.

Chrysanthemum stunt viroid (CSVd), a noncoding infectious RNA molecule, causes seriously economic losses of chrysanthemum for 3 or 4 years after its first infection. Monomeric cDNA clones of CSVd isolate SK1 (CSVd-SK1) were constructed in the plasmids pGEM-T easy vector and pUC19 vector. Linear positive-sense transcripts synthesized in vitro from the full-length monomeric cDNA clones of CSVd-SK1 could infect systemically tomato seedlings and chrysanthemum plants, suggesting that the linear CSVd RNA transcribed from the cDNA clones could be replicated as efficiently as circular CSVd in host species. However, direct inoculation of plasmid cDNA clones containing full-length monomeric cDNA of CSVd-SK1 failed to infect tomato and chrysanthemum and linear negative-sense transcripts from the plasmid DNAs were not infectious in the two plant species. The cDNA sequences of progeny viroid in systemically infected tomato and chrysanthemum showed a few substitutions at a specific nucleotide position, but there were no deletions and insertions in the sequences of the CSVd progeny from tomato and chrysanthemum plants.

Invasion of shoot apical meristems by Chrysanthemum stunt viroid differs among Argyranthemum cultivars.

Chrysanthemum stunt viroid (CSVd) is a damaging pathogen attacking Argyranthemum plants. Our study attempted to reveal distribution patterns of CSVd in shoot apical meristems (SAM) and to explore reasons for differential ability of CSVd to invade SAM of selected Argyranthemum cultivars. Symptom development was also observed on greenhouse-grown Argyranthemum plants. Viroid localization using in situ hybridization revealed that the ability of CSVd to invade SAM differed among cultivars. In diseased ‘Yellow Empire’ and ‘Butterfly’, CSVd was found in all tissues including the uppermost cell layers in the apical dome (AD) and the youngest leaf primordia 1 and 2. In diseased ‘Border Dark Red’ and ‘Border Pink’, CSVd was detected in the lower part of the AD and elder leaf primordia, leaving the upper part of the AD, and leaf primordia 1 and 2 free of viroid. Histological observations and transmission electron microscopy showed similar developmental patterns of vascular tissues and plasmodesmata (PD) in the SAM of ‘Yellow Empire’ and ‘Border Dark Red’, while immunolocalization studies revealed a major difference in the number of callose (β-1, 3-glucan) particles deposited at PD in SAM. A lower number of callose particles were found deposited at PD of SAM of ‘Yellow Empire’ than ‘Border Dark Red’. This difference is most likely responsible for the differences in ability of CSVd to invade SAM among Argyranthemum cultivars.

Evaluation of chrysanthemum stunt viroid (CSVd) infection in newly-expanded leaves from CSVd-inoculated shoot apical meristems as a method of screening for CSVd-resistant chrysanthemum cultivars

SummaryA total of 199 chrysanthemum (Chrysanthemum × morifolium Ramat.) cultivars were used in screening for resistance to chrysanthemum stunt viroid (CSVd). Plants were inoculated with CSVd by attaching leaf primordia-free, shoot apical meristems to CSVd-infected root tips of a CSVd-sensitive cultivar. CSVd titres in the first and third youngest expanded leaves were analysed after 2 months in culture, and a systematic classification into four groups was conducted. Long-term cultivation records provided by a breeding nursery confirmed our findings of those screened cultivars classified in vitro as “non-detected-type” (i.e., those expected to have a high level of resistance). In addition, of the 15 candidate cultivars selected as “non-detected-type”, four cultivars were shown to have a high level of CSVd-resistance by ex vitro grafting experiments. From the results of our in vitro and ex vitro experiments, the advantages of our newly-developed in vitro-screening system are discussed.

PM 3/24 (2)Chrysanthemum stunt viroid:inspection

EPPO BulletinVolume 43, Issue 3 p. 396-396 Phytosanitary procedures Méthodes phytosanitairesFree Access PM 3/24 (2) Chrysanthemum stunt viroid: inspection First published: 19 November 2013 https://doi.org/10.1111/epp.12072Citations: 1AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Abstract Specific scope This standard describes the inspection for Chrysanthemum stunt viroid. Specific approval and amendment First approved in September 1988. Edited as EPPO Standard in 1998. Revised in 2013-09. Introduction Chrysanthemum stunt viroid is on the EPPO A2 List of pests recommended for regulation and details about its biology, distribution and economic importance can be found in EPPO Datasheet (EPPO/CABI, 1997). Phytosanitary measures at import may include provisions that countries exporting plants of chrysanthemum certify either that they are derived, at not more than the third generation, from mother plants which have been tested and found free from the viroid, or that they are directly derived from mother plants of which a representative sample has been inspected visually at flowering. Methods To obtain satisfactory results by visual inspection, it is necessary to examine at least 10% of the mother plants because of the high proportion of symptomless carriers (30%) in some cultivars. It may be difficult to diagnose accurately in cases when a mild strain affects a high percentage of a batch and there are no distinct differences between healthy and affected plants. However, even with these strains, expression is not uniform and inspection will show some variation in growth. It is desirable, if such a situation is suspected, to make a confirmatory laboratory or glasshouse test. Visual inspection A representative sample of 30 plants or 10% of the mother plants whichever is the greater, should be inspected at flowering. In infected plants the flowers are smaller and fewer and, in fact, infected plants bloom earlier, between a few days and several weeks, than healthy plants. The flower colour may be somewhat lighter (see photos in PQR EPPO, 2013). Laboratory testing For laboratory testing see PM 7/6 Diagnostic Protocol for Chrysanthemum stunt viroid. References EPPO (2013) PQR-EPPO database on quarantine pests (available online) http://www.eppo.int/DATABASES/pqr/pqr.htm. EPPO/CABI. (1997) Chrysanthemum stunt viroid. In: Quarantine Pests for Europe, 2nd edn, (Eds Smith IM, McNamara DG, Scott PR, Holderness M) pp. 1227– 1230. CAB International, Wallingford (GB). Citing Literature Volume43, Issue3December 2013Pages 396-396 ReferencesRelatedInformation

Nested PCR Detection of Chrysanthemum stunt viroid (CSVd) from Chrysanthemum Cultivar Seedlings

Chrysanthemum stunt viroid (CSVd) is a serious pathogen affecting chrysanthemum that has caused significant economic losses to Chrysanthemum flower production worldwide. Control of CSVd disease is difficult due to its contagious nature and long latent period in the field. As chrysanthemum is most often produced by implanting seedlings, it is necessary to diagnose CSVd infection before cultivation. In this study, we screened CSVd infection in seedlings from 30 varieties including 5 domestic, 6 Japanese, and 19 European varieties. Molecular diagnosis of the combination of RT-PCR and nested PCR showed that CSVd was not detected by the first RT-PCR but detected by the second nested PCR analysis in 10 varieties, including 1 domestic, 2 Japanese, and 7 European varieties. Further comparison of 10 identified CSVd nucleotide sequences showed that those are highly conserved (99-100%) and the most similar to an isolate (AB006737) identified in Hokkaido, Japan. Our study suggests that the combination of RT-PCR and nested PCR analysis is successful for the CSVd diagnosis of seedlings and the molecular diagnosis is necessary to prevent the introduction and propagation of viroid disease into the fields.

First Report of a 16SrIX Group ('Candidatus Phytoplasma phoenicium'-Related) Phytoplasma Associated with a Chrysanthemum Disease.

In November 2010, approximately 2% of the chrysanthemum (Chrysanthemum morifolium) cv. Paniz plants showed numerous small leaves in the top and stunting in a field collection of the National Research Center of Ornamental Plants in Mahallat, Iran. Next to these plants, some plants of the same collection showed leaves with a reddish and/or chlorotic discoloration around the veins. The observed symptoms were believed to represent infection by a phytoplasma and/or a viroid. Two plants with each type of the symptoms were individually analyzed. Using a total RNA extract from diseased leaves, RT-PCR with primer pairs targeting all known pospiviroids, including Chrysanthemum stunt viroid (CSVd) (3), were negative. Purified DNA was examined for the highly conserved phytoplasma 16S rRNA gene by nested-PCR using the universal primer sets P1/P7 and R16F2n/R16R2 (2). Fragments of 1.2 kb, obtained only from the plants with the small leaves and stunting, were sequenced and one of these sequences, which were identical, was deposited in GenBank (Accession No. KC176800). BLAST analysis of the chrysanthemum phytoplasma sequence exhibited 99% identity to Candidatus Phytoplasma phoenicium (Ca. P. phoenicium) species of the 16SrIX group. Subsequently, in silico RFLP analysis of the nested PCR product with the pDRAW32 program using AluI and TaqI restriction sites used for 16SrIX subgroups A, B, C, D, and E indicated that the 16SrIX chrysanthemum isolate belonged to subgroup D (1). Recently, based on GenBank sequences, several strains of Ca. P. phoenicium have been isolated and identified from diverse host species like Lactuca serriola, L. sativa, Solanum lycopersicon, Sonchus sp. [16SrIX-E], Carthamus tinctorius, and Prunus amygdalus [16SrIX-B] (4) in Iran. The vector species transmitting Ca. P. phoenicium to C. morifolium still needs to be identified. The leafhopper Neoaliturus fenestratus may be a potential vector as it is an often encountered efficient transmitter vector of 16SrIX group phytoplasmas in Iran (2). Next to the susceptibility of chrysanthemum to members of aster yellows, stolbur, and Ca. P. aurantifolia phytoplasma groups, this is, to our knowledge, the first report of a 16SrIX group member infecting chrysanthemum. The detection of this phytoplasma in chrysanthemum can form a new threat to this crop and other ornamentals in the Mahallat flower production region.

A current overview of two viroids that infect chrysanthemums: Chrysanthemum stunt viroid and Chrysanthemum chlorotic mottle viroid.

The chrysanthemum (Dendranthema X grandiflorum) belongs to the family Asteraceae and it is one of the most popular flowers in the world. Viroids are the smallest known plant pathogens. They consist of a circular, single-stranded RNA, which does not encode a protein. Chrysanthemums are a common host for two different viroids, the Chrysanthemum stunt viroid (CSVd) and the Chrysanthemum chlorotic mottle viroid (CChMVd). These viroids are quite different from each other in structure and function. Here, we reviewed research associated with CSVd and CChMVd that covered disease symptoms, identification, host range, nucleotide sequences, phylogenetic relationships, structures, replication mechanisms, symptom determinants, detection methods, viroid elimination, and development of viroid resistant chrysanthemums, among other studies. We propose that the chrysanthemum and these two viroids represent convenient genetic resources for host–viroid interaction studies.

Elimination of Chrysanthemum stunt viroid (CSVd) from meristem tip culture combined with prolonged cold treatment

Chrysanthemum production in Korea has recently been greatly affected by the spread of Chrysanthemum stunt viroid (CSVd) infection, necessitating the use of CSVd-free stocks to ensure successful chrysanthemum cultivation. We investigated the effects of low temperature (4°C), antiviral chemicals (ribavirin and amantadine) and a combination of these treatments on CSVd elimination by meristem tip cultures using plantlets that originated from CSVd-infected chrysanthemum ‘Ency’. Neither antiviral agents led to CSVd elimination, despite the suppression of meristem tip growth in a concentration dependent manner. However, the CSVd elimination rate increased up to 42.8% when meristem tips were excised after storage at 4°C for two months. The most effective results were obtained from a combination of low temperature for three months at 4°C followed by meristem tip culture on media containing 50 and 100 mg·L−1 ribavirin. These results suggest that antiviral agents can also be useful for CSVd elimination if their treatment is combined with prolonged periods of low temperature. This is the first report of eradication of viroids from spray type chrysanthemum bred in Korea.

Dahlia latent viroid: a recombinant new species of the family Pospiviroidae posing intriguing questions about its origin and classification

A viroid-like RNA has been detected in two asymptomatic dahlia accessions by return and double PAGE. It appeared smaller than Chrysanthemum stunt viroid and Potato spindle tuber viroid, the two members of the genus Pospiviroid, family Pospiviroidae, reported in this ornamental previously. RT-PCR with primers designed for amplifying all pospiviroids produced no amplicons, but RT-PCR with random primers revealed a 342 nt RNA. The sequence of this RNA was confirmed with specific primers, which additionally revealed its presence in many dahlia cultivars. The RNA was named Dahlia latent viroid (DLVd) because it replicates autonomously, but symptomlessly, in dahlia and shares maximum sequence identity with other viroids of less than 56 %. Furthermore, DLVd displays characteristic features of the family Pospiviroidae: a predicted rod-like secondary structure of minimum free energy with a central conserved region (CCR), and the ability to form the metastable structures hairpins I and II. Its CCR is identical to that of Hop stunt viroid (HSVd, genus Hostuviroid). However, DLVd: (i) has the terminal conserved region present in members of the genus Pospiviroid, but absent in HSVd, and (ii) lacks the terminal conserved hairpin present in HSVd. Phylogenetic reconstructions indicate that HSVd and Pepper chat fruit viroid (genus Pospiviroid) are the closest relatives of DLVd, but DLVd differs from these viroids in its host range, restricted to dahlia so far. Therefore, while DLVd fulfils the criteria to be a novel species of the family Pospiviroidae, its recombinant origin makes assignment to the genera Pospiviroid or Hostuviroid problematic.

A systematic and extensive literature search on crop production of host plants of seven organisms harmful to horticulture

EFSA Supporting PublicationsVolume 10, Issue 3 401E External scientific reportOpen Access A systematic and extensive literature search on crop production of host plants of seven organisms harmful to horticulture M.P.M. Derkx, M.P.M. Derkx Stichting Dienst Landbouwkundig Onderzoek (DLO), Droevendaalsesteeg, 1 6708 PB Wageningen, The NetherlandsSearch for more papers by this authorJ.H.D. Brouwer, J.H.D. Brouwer Stichting Dienst Landbouwkundig Onderzoek (DLO), Droevendaalsesteeg, 1 6708 PB Wageningen, The NetherlandsSearch for more papers by this authorP.J.M. van Breda, P.J.M. van Breda Stichting Dienst Landbouwkundig Onderzoek (DLO), Droevendaalsesteeg, 1 6708 PB Wageningen, The NetherlandsSearch for more papers by this authorF.P.M. van der Helm, F.P.M. van der Helm Stichting Dienst Landbouwkundig Onderzoek (DLO), Droevendaalsesteeg, 1 6708 PB Wageningen, The NetherlandsSearch for more papers by this authorM.E.C.M. Hop, M.E.C.M. Hop Stichting Dienst Landbouwkundig Onderzoek (DLO), Droevendaalsesteeg, 1 6708 PB Wageningen, The NetherlandsSearch for more papers by this authorK.M. Landzaat, K.M. Landzaat Stichting Dienst Landbouwkundig Onderzoek (DLO), Droevendaalsesteeg, 1 6708 PB Wageningen, The NetherlandsSearch for more papers by this authorC.F.M. Wubben, C.F.M. Wubben Stichting Dienst Landbouwkundig Onderzoek (DLO), Droevendaalsesteeg, 1 6708 PB Wageningen, The NetherlandsSearch for more papers by this author M.P.M. Derkx, M.P.M. Derkx Stichting Dienst Landbouwkundig Onderzoek (DLO), Droevendaalsesteeg, 1 6708 PB Wageningen, The NetherlandsSearch for more papers by this authorJ.H.D. Brouwer, J.H.D. Brouwer Stichting Dienst Landbouwkundig Onderzoek (DLO), Droevendaalsesteeg, 1 6708 PB Wageningen, The NetherlandsSearch for more papers by this authorP.J.M. van Breda, P.J.M. van Breda Stichting Dienst Landbouwkundig Onderzoek (DLO), Droevendaalsesteeg, 1 6708 PB Wageningen, The NetherlandsSearch for more papers by this authorF.P.M. van der Helm, F.P.M. van der Helm Stichting Dienst Landbouwkundig Onderzoek (DLO), Droevendaalsesteeg, 1 6708 PB Wageningen, The NetherlandsSearch for more papers by this authorM.E.C.M. Hop, M.E.C.M. Hop Stichting Dienst Landbouwkundig Onderzoek (DLO), Droevendaalsesteeg, 1 6708 PB Wageningen, The NetherlandsSearch for more papers by this authorK.M. Landzaat, K.M. Landzaat Stichting Dienst Landbouwkundig Onderzoek (DLO), Droevendaalsesteeg, 1 6708 PB Wageningen, The NetherlandsSearch for more papers by this authorC.F.M. Wubben, C.F.M. Wubben Stichting Dienst Landbouwkundig Onderzoek (DLO), Droevendaalsesteeg, 1 6708 PB Wageningen, The NetherlandsSearch for more papers by this author First published: 11 March 2013 https://doi.org/10.2903/sp.efsa.2013.EN-401Citations: 1 The present document has been produced and adopted by the bodies identified above as author(s). This task has been carried out exclusively by the author(s) in the context of a contract between the European Food Safety Authority and the author(s), awarded following a tender procedure. The present document is published complying with the transparency principle to which the Authority is subject. It may not be considered as an output adopted by the Authority. The European food Safety Authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. Published date: 11 March 2013 Question number: EFSA-Q-2012-00498 AboutPDF ToolsExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat References http://www.cabi.org/ http://www.info.sciverse.com/scopus/ http://www.icpa.ro/AgroWeb/AIC/RACC/Agris.htm Citing Literature Volume10, Issue3March 2013401E ReferencesRelatedInformation

In vitro Evaluation of Small RNA Accumulation in Chrysanthemum Mediated by Chrysanthemum Stunt Viroid

In vitro Evaluation of Small RNA Accumulation in Chrysanthemum Mediated by Chrysanthemum Stunt Viroid

Elimination of Chrysanthemum stunt viroid (CSVd) from an Viroidinfected Chrysanthemum through Shoot Tip Culture

As the increase of chrysanthemum demand on chrysanthemum increases in Korea, the production of high quality chrysanthemum is needed. Chrysanthemum stunt viroid (CSVd) is one of the important viroid, which infects chrysanthemum and induces diseases that affects the decrease of quality and yield. To solve this problem, we used different size of meristem of chrysanthemum ‘Ency’ for shoot tip culture and also that of combined with heat treatment at 37οC. The efficiency of CSVd elimination was influenced by the size of shoot tip. The small-sized of meristems with 1 or 2 leaf primodia were regenerated into the highest number of CSVd-free plantlets. By RT-PCR, the 214-bp band corresponding to CSVd was not detected in 22.2% of the total number of tested regenerants from shoot tips with 2 leaf primordia. While, shoot tip culture combined with heat treatment of one-month-old in vitro shoots was not effective for CSVd-elimination. The CSVd-free plants grew more vigorously than CSVd-infected plants in the greenhouse.

Scientific Opinion on the risk to plant health posed by Chrysanthemum stunt viroid for the EU territory, with identification and evaluation of risk reduction options

The Panel on Plant Health conducted a pest risk assessment for Chrysanthemum stunt viroid (CSVd) and identified and evaluated risk reduction options, particularly those listed in Council Directive 2000/29/EC. Three entry pathways were identified, with plants for planting being the most important and rated as moderately likely. CSVd is recorded in some EU Member States with a limited distribution and is regulated on chrysanthemum; host plants are widely cultivated in greenhouses and outdoors. Therefore, establishment is very likely. Short-distance spread within a crop is likely, whereas short-distance spread between different species is unlikely to moderately likely. For vegetatively propagated species, in the absence of an efficient certification system, long-distance spread is very likely and a major impact would be expected on chrysanthemum, with associated yield and quality losses. However, under the existing certification scheme for chrysanthemum plant propagation material, the probability of spread through infected cuttings is largely reduced and the direct consequences of viroid outbreaks are expected to be minor. Minimal impact is predicted for other ornamental hosts and a minor impact for solanaceous vegetable crops. The indirect effects of CSVd are considered to be limited, with the exception of eradication. Risk reduction options addressing the sanitary status of the propagation material are likely to be the most effective and feasible. These include the current EU measures, as well as a statutory certification system with associated statutory import requirements or, failing that, but potentially less effective, the use of voluntary industry standards. It is difficult to disentangle the benefits of the current legislation from those of the industry-developed chrysanthemum certification system. The high efficacy of current measures is supported by the absence or limited presence of CSVd in the EU and by the limited number of interceptions and findings. Possible improvements to current measures are described and uncertainties discussed.

A multiplex RT-PCR for rapid and simultaneous detection of viruses and viroids in chrysanthemum

Chrysanthemum plants are subject to serious virus diseases, so detection and identification of virus pathogens is important to prevent the virus spread. A reliable one-step multiplex RT-PCR was developed to simultaneously detect two viruses and two viriods: chrysanthemum virus B, tomato Aspermy virus, chrysanthemum stunt viroid and chrysanthemum chlorotic mottle viroid. In addition, we investigated the detection limit and the efficiency of single and multiplex RT-PCR assays. The results showed that the multiplex RT-PCR assay proved to be as sensitive as the single one. In conclusion, this technique is potentially useful in routine diagnosis of chrysanthemum viruses and viroids. The multiplex RT-PCR assay described in this study is the first report of simultaneous detection of virus and viroid in chrysanthemum, which provides a fast, convenient, cost-saving way to detect the virus and viroid mixed infections in plants.

ARE THERE STRAINS OF CHRYSANTHEMUM STUNT VIROID

Chrysanthemum stunt viroid (CSVd) has been detected or isolated from chrysanthemums or other host species in 26 countries. While strains (biological variants) have been described for many viroids, strains have not been described as such specifically for CSVd, despite the determination of 117 sequence variants of CSVd in 16 countries. Tomato has been described as an experimental host, either symptomless, or showing mild, or even severe symptoms, depending on the report; however, most of these studies were done with single isolates of CSVd and one cultivar of tomato plants. Thus, here we re-examined these issues by directly comparing the biological activity of three isolates of CSVd, one each from the USA, China and Australia, varying in their nucleotide sequence. We found that these isolates showed no differences in symptoms induced on either chrysanthemum or tomato plants, suggesting that much of the biological variation reported previously may have been due to cultivar differences and/or environmental effects.

Sequence comparisons of global chrysanthemum stunt viroid variants: multiple polymorphic positions scattered through the viroid genome

The nucleotide sequences of cDNA clones of three chrysanthemum stunt viroid (CSVd) isolates (one each from the USA, China, and Australia) were determined and analyzed. The sequences of CSVd cDNA clones of the US and Australian isolates were both quasi-species, while the cDNA clones of the Chinese isolate contained only a single variant. A comparison of the nucleotide sequences of 117 isolates and cDNA clones obtained from 16 countries showed that in some cases identical CSVd isolates were found in several countries and from multiple locations within the same country. CSVd isolates differed as much in sequence between countries as within countries. Sequence variation was observed at 103 sites scattered through the CSVd genome, and was not associated predominantly with a single variable region, as was the case with several other viroids. While some sequence changes were associated with CSVd found in other host species, it is unknown if these changes are required for infection of those species.

A single polyprobe for detecting simultaneously eight pospiviroids infecting ornamentals and vegetables

The spread of viroids belonging to the genus Pospiviroid (family Pospiviroidae), recorded recently in ornamentals and vegetables in several European countries, calls for fast, efficient and sensitive detection methods. Based on bioinformatics analyses of sequence identity among all pospiviroids, a digoxigenin-labeled polyprobe (POSPIprobe) was developed that, when tested by dot-blot and Northern-blot hybridization, detected Potato spindle tuber viroid, Citrus exocortis viroid, Columnea latent viroid, Mexican papita viroid, Tomato planta macho viroid, Tomato apical stunt viroid, Pepper chat fruit viroid and Chrysanthemum stunt viroid. The end-point detection limits of the POSPIprobe ranged from 5(-2) to 5(-4), and from 5(-1) to 5(-3) for nucleic acid preparations obtained by phenol extraction and silica-capture, respectively, similar to those of single probes. Based on sequence identity, the POSPIprobe is expected to detect also the two pospiviroid species not tested in this study (Tomato chlorotic dwarf viroid and Iresine viroid-1). Dot-blot assays with the POSPIprobe were validated by testing 68 samples from tomato, chrysanthemum and argyranthemum infected by different pospiviroids as revealed by RT-PCR, thus confirming the potential of this polyprobe for quarantine, certification and survey programs.