Chrysanthemum Stunt Viroid Research Articles

Development of the colorimetric loop-mediated isothermal amplification technique for rapid and sensitive detection of chrysanthemum stunt viroid in chrysanthemum

Development of the colorimetric loop-mediated isothermal amplification technique for rapid and sensitive detection of chrysanthemum stunt viroid in chrysanthemum

Application of Low Temperature and Antiviral for Elimination of Chrysanthemum stunt viroid and Its Effect on the Growth of Shoot Tip Culture of Chrysanthemum

Chrysanthemum stunt viroid (CSVd) telah banyak dilaporkan menjadi salah satu faktor pembatas dalam usaha produksi bunga potong krisan. Penelitian bertujuan untuk mengetahui pengaruh perlakuan suhu rendah (5 ℃) dan antiviral ribavirin terhadap pertumbuhan dan pembebasan CSVd pada planlet krisan yang dikultur dari ujung tunas. Tanaman terinfeksi CSVd diperoleh dari kebun percobaan milik pemerintah di Cianjur, Jawa Barat. Penelitian dilakukan dengan beberapa tahap, yaitu 1) pengambilan sampel tanaman, 2) deteksi viroid dengan metode RT-PCR, 3) inisiasi eksplan dan perbanyakkan bahan tanaman secara in vitro, 4) perlakuan suhu rendah 5 ℃ dengan tiga taraf waktu inkubasi (1, 3, dan 5 bulan), 5) perlakuan antiviral ribavirin dengan tiga taraf konsentrasi (25, 100, dan 125 ppm), dan 6) konfirmasi bahan tanaman bebas viroid dengan RT-PCR. Hasil penelitian menunjukkan bahwa penyimpanan suhu rendah dan perlakuan ribavirin menurunkan laju pertambahan tinggi tunas dan jumlah daun. Sampai pada konsentrasi 125 ppm, walaupun menurunkan laju pertumbuhan tunas dan jumlah daun, ribavirin tidak menimbulkan toksisitas terhadap tanaman. Akan tetapi, kombinasi perlakuan suhu rendah (1, 3, dan 5 bulan) dengan ribavirin (25, 100, dan 125 ppm) belum mampu mengeliminasi CSVd dari jaringan pada semua taraf perlakuan.

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.

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.

Evaluation of the pathogenicity of chrysanthemum stunt viroid variants against dahlia under different conditions

AbstractChrysanthemum stunt viroid (CSVd) is an important pathogen that decreases dahlia yields due to stunting symptoms. Here, we evaluated the effect of nucleotide sequence of CSVd, cultivation conditions, inoculum type and infection timing on CSVd‐infected dahlia growth. Inoculation with CSVd after planting resulted in reduced plant height in the second cut flower, but not the first, indicating that the infection may affect the growth of dahlias after approximately 3–5 months. Additionally, there was no significant difference in the degree of symptom severity among the plants infected with the two types of inoculum, synthesized CSVd RNA and CSVd‐infected plant sap. All three CSVd variants affected the growth of dahlias. The reduction in plant height was almost similar among the three variants. However, the reduction in plant fresh weight and compound leaf size in variant JP1 was significantly higher than that in the other variants in the spring experiment. The rates of blown centre in JP1 were also higher than those in the other variants in the spring and winter experiments under the 14.5‐h photoperiod. These results suggest that JP1 is a more severe variant than JP2 and JP3. Dark period interruption improved the reduction in plant height and weight and flower weight, as well as the rate of blown centre in CSVd‐infected dahlias in the winter experiment compared with the 14.5‐h photoperiod. Our results indicate that the three CSVd variants incite stunting symptoms in dahlias and nucleotide sequence of CSVd, photoperiod and infection timing affect the degree of symptom severity.

Correction to: Genetic diversity and pathogenicity of potato spindle tuber viroid and chrysanthemum stunt viroid isolates in Russia

Correction to: Genetic diversity and pathogenicity of potato spindle tuber viroid and chrysanthemum stunt viroid isolates in Russia

Detection of Avocado Sunblotch and Other Viroids Using RNA Filter Paper Capture and RT-PCR.

This method originated due to the need to quickly and sensitively detect Avocado sunblotch viroid (ASBVd) in nursery and field trees in California. Optimum sampling protocols were developed for leaf collection from different sized trees based on size and branching as well as for fruit. An ethanol containing buffered extract from 1g of ground leaf tissue was used as the source of RNA. The extract was absorbed onto small pieces (disks) of Whatman No. 1 filter paper which were then washed and dried. RNA was eluted from the filter paper using sterile water and used as a template in a standard single-tube RT-PCR reaction. The RNA adsorbed on the filter paper disks was quite stable, and the disks could be stored for over 1year and shipped worldwide at ambient temperature with no noticeable decline in the quality or quantity of the resulting RT-PCR products. The filter paper capture method was expanded to the detection of other viroids including Potato spindle tuber viroid, Peach latent mosaic viroid, and Chrysanthemum stunt viroid and was tested with some viruses as well with minor modifications of the standard protocol.

The effects on adventitious root formation caused by chrysanthemum stunt viroid in Chrysanthemum morifolium and C. seticuspe

AbstractChrysanthemum stunt viroid (CSVd) is a disease agent caused by a viroid from the genus Pospiviroid. The CSVd pathogen infects chrysanthemum plants and causes severe symptoms of stunting and significant yield loss in cultivation. Although many studies have focussed on this issue, the mechanism of CSVd‐infection remains to be clarified. In particular, the effects of CSVd on the underground parts of infected plants are not yet fully understood in Chrysanthemum. Here, we investigated whether the effects of CSVd on the root formation of infected plants varies among cultivars and wild Chrysanthemum species, C. morifolium and C. seticuspe, using transcribed RNA and in vitro assays. Furthermore, to compare infected plants with uninoculated plants, we assessed the symptoms and measured the CSVd RNA accumulation levels in several tissues using RT‐qPCR. Our findings indicated that CSVd‐infected plants exhibited severe root formation symptoms in both C. morifolium and C. seticuspe. RT‐qPCR revealed CSVd accumulation in the root tips and similar patterns of CSVd accumulation in each organ of C. morifolium and C. seticuspe. We propose that in vitro assays are useful for determining root formation in CSVd‐infected chrysanthemum plants. Such investigations are necessary to better understand CSVd infection in Chrysanthemum species and plant defences against such diseases.

Genetic diversity and pathogenicity of potato spindle tuber viroid and chrysanthemum stunt viroid isolates in Russia

Genetic diversity and pathogenicity of potato spindle tuber viroid and chrysanthemum stunt viroid isolates in Russia

Molecular and biological characterisation of an Indian variant of Chrysanthemum stunt viroid

Chrysanthemum stunt viroid (CSVd), is known to cause severe stunting symptoms in chrysanthemum. Here, we have identified a CSVd strain that infects chrysanthemum in India and have biologically characterised it by generating in vitro RNA transcripts from the cloned viroid genome. Transcripts inoculated on chrysanthemum caused severe stunting (characteristic of the viroid) and mottling in chrysanthemum and stunting symptoms in cucumber var. Summer green is also reported. Sequence changes in the Terminal Conserved region (TCR) corroborated with change in RNA secondary structure might have implications associated with the viroid pathogenicity. This is the first report of CSVd causing symptoms on cucumber plants.

The distribution and nucleotide sequences of chrysanthemum stunt viroid in dahlia fields in Japan and the effect of its infection on the growth of dahlias

Chrysanthemum stunt viroid (CSVd) is known to infect a wide range of Dahlia spp; however, to the best of our knowledge, no detailed study has been conducted on the distribution of CSVd variants, the infection efficacy of sap inoculations, and the symptoms caused by infection of CSVd in dahlias. We have detected CSVd in 94 of 214 dahlia samples, from across eight different Japanese Prefectures, using RT-PCR. Six sequence variants of CSVd were detected from 32 CSVd-infected dahlia samples including 3 variants which have not been previously reported and three previously isolated variants from dahlias or chrysanthemums. Five CSVd variants infected and replicated in both dahlia and chrysanthemum plants by sap and RNA inoculation three months post inoculation. The sequence mutations among those variants did not significantly affect the infection efficacy. Infection of CSVd significantly reduced fresh plant weight, plant height, size of compound leaves, flower diameter, and fresh flower weight of dahlias in two cultivars, though it did not affect the number of nodes. In addition, it altered the color of ray florets and increased the rate at which flowers exposed their disks and that of disk florets. The altered color of ray florets in the ‘Magokoro’ cultivar were darker pink and those of the ‘Portlight pair beauty’ cultivar were darker orange than the uninfected samples. Our results show that CSVd infects a wide range of dahlias in the field and is an important pathogen to manage for their cultivation as it negatively affects their productivity.

Correction to: Screening of Chrysanthemum seticuspe accessions reveals different degrees of resistance to chrysanthemum stunt viroid

Correction to: Screening of Chrysanthemum seticuspe accessions reveals different degrees of resistance to chrysanthemum stunt viroid

Global transcriptome analyses reveal that infection with chrysanthemum stunt viroid (CSVd) affects gene expression profile of chrysanthemum plants, but the genes involved in plant hormone metabolism and signaling may not be silencing target of CSVd-siRNAs

Abstracts Chrysanthemum stunt viroid (CSVd) is a pathogen that induces stunted plant growth as a typical disease symptom in infected chrysanthemum plants. Because of its simple RNA genomic structure, the mechanism of how CSVd provokes pathogenicity in host plants remains elusive. Since small RNAs generated from CSVd genomic RNA accumulated in infected plants ( Tsuro et al., 2013 ), it has been suggested that forced gene silencing against intrinsic genes by CSVd-small interfering (si)RNAs is involved in its pathogenicity. In this study, we revealed that CSVd infection induced global fluctuation of gene expression in plants. Hypothetical CSVd-siRNAs (22 nucleotides long) from (+) and (−) strands of the CSVd RNA genome were surveyed to examine if they could target the down-regulated gene sequences. Six genes were identified as target candidates, but the causal link between their functions and disease symptoms was remained unclear. In contrast, the expression levels of genes involved in the biosynthesis of gibberellic acid (GA) and cytokinin (CK), indole-3-acetic acid (IAA) transport and cell wall growth in infected plants were down-regulated, but mRNAs of those genes were not found as possible target sequences of CSVd-siRNAs. On the other hand, endogenous levels of GA, CK, IAA, and abscisic acid and the rate of IAA movement in the stem were not different between healthy and infected plants. Growth promotion of infected plants by GA treatment was poorer than that of healthy plants, suggesting that CSVd infection suppressed the GA-signaling pathway. This was further supported by the findings of a reduced response of GA-regulated genes to GA treatment and up-regulation of the DELLA-protein gene, a negative regulator of GA-signaling, in infected plants. Thus, the down-regulation of GA responsiveness and inhibition of cell wall expansion are major causes of the stunted growth of CSVd-infected chrysanthemum plants.

Surveys in the Chrysanthemum Production Areas of Brazil and Colombia Reveal That Weeds Are Potential Reservoirs of Chrysanthemum Stunt Viroid.

The stunting disease, incited by chrysanthemum stunt viroid (CSVd), has become a serious problem in chrysanthemum production areas worldwide. Here we identified 46 weed species from chrysanthemum fields in two producing regions of the State of São Paulo, Brazil. The mechanical inoculation of these weeds with a Brazilian CSVd isolate revealed that this viroid was able to infect 17 of these species, in addition to chrysanthemum, tomato and potato. Plants of Oxalis latifolia and chrysanthemum naturally infected with CSVd were found in chrysanthemum fields in Colombia, which is the first CSVd report in that country. Therefore, weeds have the potential to act as reservoirs of CSVd in the field. These results are the first reports of experimental CSVd infection in the following species: Amaranthus viridis, Cardamine bonariensis, Chamaesyce hirta, Conyza bonariensis, Digitaria sanguinalis, Gomphrena globosa, Helianthus annuus, Lupinus polyphyllus, Mirabilis jalapa, Oxalis latifolia, Portulaca oleracea and Catharanthus roseus. The phylogenetic analyses of the CSVd variants identified herein showed three groups with Brazilian CSVd variants distributed in them all, which suggests that Brazilian CSVd isolates may have different origins through successive introductions of infected germplasm of chrysanthemum in Brazil.

Screening of Chrysanthemum seticuspe accessions reveals different degrees of resistance to chrysanthemum stunt viroid

The chrysanthemum stunt viroid (CSVd) causes severe stunting and significant reduction in flower yield and quality of commercial chrysanthemum (Chrysanthemum morifolium). The pattern of inheritance and the detailed mechanism underlying CSVd resistance remains unclear. Chrysanthemum morifolium shows unstable and variable chromosome numbers that form a hexaploid complex with aneuploidy. To overcome the issue of complex hybridity and polyploidy, a diploid wild chrysanthemum, C. seticuspe f. boreale was used here as an alternative model to C. morifolium. We screened 20 C. seticuspe accessions for resistance to CSVd and identified one resistant accession “AET-19” showing resistance. Tissue print hybridization analysis showed that CSVd concentration decreased in the uppermost leaves of the AET-19 after the scions were cut from the infected rootstocks, suggesting the resistance seems to be ‘CSVd-disappearance type’. In situ hybridization experiments revealed that CSVd does not invade the shoot tip of AET-19 plants after graft-inoculation onto infected rootstocks. Taken together, the results highlight the usefulness of C. setiscupe as a model for analyzing the mechanism of CSVd resistance and its pattern of inheritance.

First report of chrysanthemum stunt viroid isolated from potato (Solanum tuberosum) plants in Russia

A pospiviroid, detected from asymptomatic potato plants (Solanum tuberosum) from Russia, was identified as chrysanthemum stunt viroid (CSVd). The genomic sequence of this potato isolate showed 100% identity with that of the chrysanthemum isolates from Japan. The CSVd RNA transcribed from the cDNA, having the sequence of the isolate from Russia, was able to infect potato, tomato, and chrysanthemum plants. The infected chrysanthemum plants exhibited stunting symptom. This is the first report of the occurrence of CSVd in Russia, its natural infection of potato plants and the potential spread of CSVd through propagation of infected potato.

Chrysanthemum Stunt Viroid Resistance in Chrysanthemum.

Chrysanthemum stunt viroid (CSVd) is one of the most severe threats in Chrysanthemum morifolium production. Over the last decade, several studies have reported the natural occurrence of CSVd resistance in chrysanthemum germplasms. Such CSVd-resistant germplasms are desirable for the stable production of chrysanthemum plants. Current surveys include finding new resistant chrysanthemum cultivars, breeding, and revealing resistant mechanisms. We review the progress, from discovery to current status, of CSVd-resistance studies, while introducing information on the improvement of associated inoculation and diagnostic techniques.

Standards to be withdrawn

EPPO BulletinVolume 48, Issue 3 p. 538-539 CorrigendumFree Access Standards to be withdrawn This article corrects the following: PM 3/22 (2) Phytophthora fragariae: inspection Volume 43Issue 3EPPO Bulletin pages: 395-395 First Published online: November 19, 2013 First published: 07 December 2018 https://doi.org/10.1111/epp.12540AboutSectionsPDF 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 onFacebookTwitterLinked InRedditWechat The following Standards from the Series PM 3 are withdrawn: PM 3/22(2) Phytophthora fragariae: Inspection PM 3/23(1) Phoma exigua var. foveata. Inspection and test methods PM 3/24(2) Chrysanthemum stunt viroid. Inspection methods PM 3/31(1) Rubus viruses. Inspection and test methods PM 3/36(1) Phaeoisariopsis griseola. Inspection and test methods for bean seeds PM 3/37(2) Tilletia indica. Inspection methods for wheat seeds PM 3/46 (1) Methyl bromide fumigation of citrus and other hosts plants to control Aleurocanthus woglumi PM 3/47 and PM 3/50 have been revised to withdraw the schedule with methyl bromide. These are moved to series PM 10 and published in this issue of the EPPO Bulletin). The following Standard from the Series PP1 is withdrawn: PP 1/277 Insecticide co-formulated mixtures withdrawn as replaced by PP 1/306 (1) General principles for the development of co-formulated mixtures of plant protection products All the PP3 Standards Environmental risk assessment scheme for plant protection products are withdrawn: Decision-making scheme for the environmental risk assessment of plant protection products Chapter 1: Introduction Chapter 2: Guidance on identifying aspects of environmental concern Chapter 3: Soil Chapter 4: Ground water Chapter 5: Surface water Chapter 6: Aquatic organisms Chapter 7: Soil microflora Chapter 8: Earthworms Chapter 9: Arthropod natural enemies Chapter 10: Honeybees Chapter 11: Terrestrial vertebrates References PM3 Standards EPPO (2013) PM 3/22 (2) Phytophthora fragariae: inspection. EPPO Bulletin 43(3), 395– 395. Wiley Online LibraryGoogle Scholar EPPO (1989) PM 2/23 (1) Phoma exigua var. foveata. EPPO Bulletin 19(1), 157– 160. Wiley Online LibraryGoogle Scholar EPPO (2013) PM 3/24 (2) Chrysanthemum stunt viroid: inspection. EPPO Bulletin 43(3), 396– 396. Wiley Online LibraryGoogle Scholar EPPO (1991) PM 3/31 (1) Rubus viruses. Inspection and test methods. EPPO Bulletin 21(2), 241– 244. Google Scholar EPPO (1991) PM 3/36 (1) Phaeoisariopsis griseola. Inspection and test methods for bean seeds. EPPO Bulletin 21(2), 263– 264. Google Scholar EPPO (2013) PM 3/37 (2) Tilletia indica. Inspection methods for wheat seeds. EPPO Bulletin 43(3), 398– 398. Wiley Online LibraryGoogle Scholar EPPO (1994) PM 3/46 (1) Methyl bromide fumigation of citrus and other host plants to control Aleurocanthus woglumi. EPPO Bulletin 24(2), 315– 315. Wiley Online LibraryGoogle Scholar PP1 Standards EPPO (2012) PP 1/277 (1) Insecticide co-formulated mixtures. EPPO Bulletin 42(3), 353– 357. Wiley Online LibraryGoogle Scholar PP3 Standards EPPO (1993) Decision-making scheme for the environmental risk assessment of plant protection products - Chapter 1: Introduction. EPPO Bulletin 23(1), 3– 17. Wiley Online LibraryGoogle Scholar EPPO (1993) Decision-making scheme for the environmental risk assessment of plant protection products – Chapter 2: Guidance on identifying aspects of environmental concern. EPPO Bulletin 23(1), 19– 26. Wiley Online LibraryGoogle Scholar EPPO (1993) Decision-making scheme for the environmental risk assessment of plant protection products – Chapter 3: Soil. EPPO Bulletin 23(1), 27– 49. Wiley Online LibraryGoogle Scholar EPPO (1993) Decision-making scheme for the environmental risk assessment of plant protection products – Chapter 4: Ground water. EPPO Bulletin 23(1), 51– 71. Wiley Online LibraryGoogle Scholar EPPO (1993) Decision-making scheme for the environmental risk assessment of plant protection products – Chapter 5: Surface water. EPPO Bulletin 23(1), 73– 98. Wiley Online LibraryGoogle Scholar EPPO (1993) Decision-making scheme for the environmental risk assessment of plant protection products – Chapter 6: Aquatic organisms. EPPO Bulletin 23(1), 99– 130. Wiley Online LibraryGoogle Scholar EPPO (1994) Decision-making scheme for the environmental risk assessment of plant protection products – Chapter 7: Soil microflora. EPPO Bulletin 24(1), 1– 16. Wiley Online LibraryGoogle Scholar EPPO (1993) Decision-making scheme for the environmental risk assessment of plant protection products – Chapter 8: Earthworms. EPPO Bulletin 23(1), 131– 149. Wiley Online LibraryGoogle Scholar EPPO (1994) Decision-making scheme for the environmental risk assessment of plant protection products – Chapter 9: Arthropod natural enemies. EPPO Bulletin 24(1), 17– 36. Wiley Online LibraryGoogle Scholar EPPO (1993) Decision-making scheme for the environmental risk assessment of plant protection products – Chapter 10: Honeybees. EPPO Bulletin 23(1), 151– 165. Wiley Online LibraryGoogle Scholar EPPO (1994) Decision-making scheme for the environmental risk assessment of plant protection products – Chapter 11: Terrestrial vertebrates. EPPO Bulletin 24(1), 37– 88. Wiley Online LibraryGoogle Scholar Volume48, Issue3December 2018Pages 538-539 ReferencesRelatedInformation

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.