Biological Control of Basal Stem Rot of Chrysanthemum By Antagonists*

The basal stem rot of chrysanthemum caused by Rhizoctonia solani is an important chrysanthemum disease in Taiwan, Control of the disease using the antagonists of R.solani, including members of the genera Aspergillus, Gliocladium, Paecilomyces, Penicillhium, Tricboderma and Bacillus, was studied. The antagonists were grown in solid media or culture broths for preparations of rhe inoculums which were u.sed as soil amendments or coating materials for applications on the disease control. Sawdusts, rice husks and sawdust composts were used as supporting material to prepare the solid media. By planting of the chrysanthemums, the solid media growth with antagonists were put in the nursery sand beds, or the chrysanthemum cutting seedlings were coated with antagonists of culture broths; whilst the sand beds were inoculated with R. solani. The results indicated that all antagonists could protect the chrysanthemum from the infection of R. solani, and the effects of the solid media were higher than the culture broths. When the chrysanthemum seedlings were treated with the extracts of the antagonists, the disease could also be depressed. The different control effects on Rhizactonia disease of chrysanthemums resulted from the species of the antagonists and their delivery systems to be used.     

A Codon-Optimized Nucleic Acid Hydrolyzing Single-Chain Antibody Confers Resistance to Chrysanthemums Against Chrysanthemum Stunt Viroid Infection

Chrysanthemum stunt viroid (CSVd), the smallest plant pathogen known to infect chrysanthemums, is a single-stranded circular RNA viroid that induces stunting that results in an overall height reduction of 30–50 % in mature plants. A catalytic single-chain variable antibody, 3D8 scFv, which exhibits intrinsic DNase and RNase activities, was expressed in chrysanthemums to generate transgenic plant resistance to CSVd infection. Moreover, a codon-optimized version of the 3D8 scFv gene for chrysanthemums was also transformed into plants; these codon-optimized transgenic chrysanthemums expressed twice as much 3D8 scFv and displayed 60 % more resistance to CSVd infection, compared with transgenic chrysanthemums harboring the original 3D8 scFv gene. CSVd challenge experiments with codon-optimized and original 3D8 scFv-transgenic chrysanthemums showed that CSVd in newly produced leaves of both codon-optimized and original 3D8 scFv-transgenic plants was not detected by RT-PCR. This is the first report describing the development of a CSVd-resistant chrysanthemum harboring a catalytic single-chain antibody, 3D8 scFv, which has intrinsic RNase activity.     

The role of the helper virus, anthriscus yellows, in the transmission of parsnip yellow fleck virus by the aphid Cavariella aegopodii

Transmission of parsnip yellow fleck virus (PYFV) by the aphid Cavariella aegopodii occurs only when the aphids are also carrying the helper virus, anthriscus yellows (AYV). None of five other viruses tested was able to act as helper. In experiments in which aphids were allowed to feed through membranes on crude or treated extracts from infected plants, aphids already carrying AYV acquired PYFV, but virus-free aphids failed to acquire either AYV or PYFV. PYFV was not transmitted by insects injected with haemolymph from aphids carrying both viruses, or with purified preparations of PYFV. PYFV was transmitted when AYV-carrying aphids, except those whose stylets had been removed, were contaminated externally with PYFV preparations. Ultraviolet irradiation of infected leaves did not prevent aphids from acquiring AYV, presumably because it is confined to deeply-lying tissues. AYV-carrying aphids could acquire PYFV from u.v.-irradiated leaves after acquisition access times of 2 h but not after feeds of only 2 or 15 min (which are adequate on unirradiated leaves), suggesting that PYFV is present in all parts of the leaf. No ‘helper agent’ distinct from AYV itself was detected in these experiments or in experiments on minimum acquisition feeding time or maximum period of persistence in the aphid. U.v.-inactivated PYFV competed with infective PYFV for retention sites in AYV-carrying aphids, whereas AYV apparently did not. It is suggested that there is no helper agent for PYFV, other than AYV particles. The possibility that there is one for AYV is not excluded.     

Properties and partial purification of infective material from plants containing groundnut rosette virus*

Groundnut plants with chlorotic rosette disease contain a manually transmissible virus, groundnut rosette (GRV), which is also transmitted in the persistent (circulative) manner by aphids (Aphis craccivora), but only from plants that are co-infected with a manually non-transmissible luteovirus, groundnut rosette assistor virus (GRAV). Strains of GRV from plants with chlorotic or green forms of rosette are called GRV(C) and GRV(G) respectively. An isolate of GRV(C) from Nigeria remained infective in Nicotiana clevelandii leaf extracts for 1 day at room temperature and for 15 days at 4d?C, but lost infectivity after 1 day at -20d?C or after dilution to 10^--⁴. Its infectivity and longevity in vitro were not altered by addition of 1 mg/litre bentonite to the extraction buffer. Infectivity in leaf extracts was abolished by treatment with 50% (v/v) ether, 10% (v/v) chloroform or 8% (v/v) n-butanol, but not by treatment for 30 min with RNase A at up to 100 ng/ml. In attempts to purify GRV(C), nearly all the infectivity from N. clevelandii extracts was found in the pellets from centrifugation at 65 000 g for 1. 5 h; infectivity also occurred in a cell membrane fraction that collected at the top of a 30% sucrose ‘cushion’ containing 4% polyethylene glycol and 0.2 M NaCI. However, no virus-like particles were found in either type of preparation by electron microscopy. Nucleic acid preparations made directly from GRV(C)-infected N. clevelandii leaves were very infective; this infectivity was totally inactivated by treatment for 30 min with RNase A at 10 ng/ml in buffers of both low and high ionic strength and was therefore attributed to ssRNA. When nucleic acid preparations were electrophoresed in gels no virus-specific bands were visible but the position of the infectivity indicated that the infective ssRNA has an apparent mol. wt of c. 1.55 × 10⁶. A similar mol. wt was indicated by the rate of sedimentation of the infective ssRNA in sucrose gradients. Preparations of dsRNA made from GRV(C)-infected N. clevelandii leaves contained a species of mol. wt c. 3.0 × 10⁶; in addition some dsRNA preparations contained an abundant component of mol. wt c. 0.6 × 106 together with several other components of intermediate mol. wt. Similar patterns of bands were observed in dsRNA preparations made from Nigerian-grown groundnut material infected with GRV(C) alone, or with GRV(C) + GRAV, or with GRV(G) + GRAV. The properties of GRV closely resemble those of two other viruses that depend on luteoviruses for transmission by aphids, carrot mottle virus and lettuce speckles mottle virus.     

Attempts to eliminate chrysanthemum stunt from chrysanthemum by meristem-tip culture after heat-treatment

‘Mistletoe’ chrysanthemums infected with stunt were grown at 35°C for 14–37 weeks, and meristem-tips cultured from them at intervals. Of 337 meristems, seventy-two survived to plants but their development took 50% longer than did that of stunt-free meristem-tips. All plants were symptomless for at least 5 weeks after planting in 75 mm pots of soil-mix, and only three showed symptoms after 9 weeks. No more plants developed symptoms during the next 5 (winter) months, but between mid-March and late May sixty-seven did so. Only two plants were freed from stunt. ‘Mini-cuttings’ rooted from shoot tips of infected chrysanthemums grown at 35°C for 37 weeks all developed stunt symptoms within 27 weeks.     

Ornamental Chrysanthemums: Improvement by Biotechnology

The in vitro tissue culture and micropropagation of chrysanthemums, important floricultural (cut-flower) and ornamental (pot and garden) plants, have been well studied. An increase in genetic transformation studies aimed at improving aesthetic and growth characteristics of the plants has been hampered by low transformation efficiencies and genotype dependence of protocols. As a result chrysanthemum regeneration studies have once again emerged as an essential complement of transformation studies. This review highlights the impact that biotechnology has had on the improvement of chrysanthemum in vitro cell, tissue and organ culture, micropropagation and transformation.     

Induction of male sterility in transgenic chrysanthemums (Chrysanthemum morifolium Ramat.) by expression of a mutated ethylene receptor gene, Cm-ETR1/H69A, and the stability of this sterility at varying growth temperatures

Chrysanthemum (Chrysanthemum morifolium Ramat.) is one of the most popular ornamental flowers in the world, and many agronomic traits have recently been introduced to chrysanthemum cultivars by gene transformation. Concerns have been raised, however, regarding transgene flow from transgenic plants to wild plants. In early studies, ethylene receptor genes have been used for genetic modification in plants, such as flower longevity and fruit ripening. Recently, overexpression of ethylene receptor genes from melon (CmETR1/H69A) caused delayed tapetum degradation of the anther sac and a reduction in pollen grains. We therefore introduced the ethylene receptor gene into chrysanthemums to induce male sterility and prevent transgene flow via pollen. The chrysanthemum cultivar Yamate shiro was transformed using a disarmed strain of Agrobacterium tumefaciens, EHA105, carrying the binary vector pBIK102H69A, which contains the CmETR1/H69A gene. A total of 335 shoots were regenerated from 1,282 leaf discs on regeneration medium (26.1%). The presence of the Cm-ETR1/H69A gene was confirmed in all of the regenerated plantlets by Southern blot analysis. These genetically modified (GM) plants and their non-GM counterparts were grown in a closed greenhouse and flowered at temperatures between 10 and 35°C. In 15 of the 335 GM chrysanthemum lines, the number of mature pollen grains was significantly reduced, particularly in three of the lines (Nos. 91, 191 and 324). In these three lines, pollen grains were not observed at temperatures between 20 and 35°C but were observed at 10 and 15°C, and mature pollen grains were formed only at 15°C. In northern blot analyses, expression of the CmETR1/H69A gene was suppressed at low temperatures. This phenomenon was observed as a result of both the suppression of CmETR1/H69A expression at low temperatures and the optimal growth temperature of chrysanthemums (15–20°C). Furthermore, the female fertility of these three GM lines was significantly lower than that of the non-GM plants. Thus, the mutated ethylene receptor is able to reduce both male and female fertility significantly in transgenic chrysanthemums, although the stability of male and/or female sterility at varying growth temperatures is a matter of concern for its practical use.     

Nuclear DNA content as an indicator of inflorescence colour stability of in vitro propagated solid and chimera mutants of chrysanthemum

In chrysanthemum, breeders seek for desirable characteristics of the inflorescence, which can first be established once the plant is mature. The present study aims to determine whether measurement of DNA content can be useful in the detection of somaclonal variants and/or separation of chimera components in chrysanthemum at the early in vitro multiplication stage. Eleven Chrysanthemum?×?morifolium (Ramat.) Hemsl. cultivars of the Lady group (a mother cultivar and ten of its radiomutants obtained by X-ray- or γ-irradiation; solid and periclinal chimeras) were propagated in vitro. Single-node explants were cultured in Murashige and Skoog (MS) medium, either without plant growth regulators (PGRs) or supplemented with 6-benzyladenine (BA) and indole-3-acetic acid (IAA). The nuclear DNA content was measured by flow cytometry (FCM) in the shoots produced in vitro. After acclimatization and growth of the plants in a glasshouse, inflorescence colour was recorded. The addition of PGRs to the medium almost doubled the mean number of shoots produced in vitro per explant, but caused a change in inflorescence colour of all (‘Lady Apricot’; periclinal chimera) or part of the plants (‘Lady Amber’; solid mutant and ‘Lady Salmon’; periclinal chimera). All radiomutants contained less DNA than the mother cultivar ‘Richmond’. There were significant differences in DNA content between plants of the same cultivar grown in media with or without PGRs for ‘Lady Apricot’ and ‘Lady Salmon’, but no phenotype alternation occurred in chrysanthemums produced in PGR-free medium compared to the original cultivars. Conversely, in medium with PGRs, chimeras produced flowers different from the original colour. In all except one cultivar (‘Lady Amber’; solid mutant) a lack of differences in genome size between plants grown in either medium coincided with a stable inflorescence colour. The occurrence of some plants of ‘Lady Amber’ with different inflorescence colour may be due to small DNA changes, undetectable by FCM. It can be concluded that FCM analysis of DNA content in young plantlets can be indicative of the stability of inflorescence colour in chrysanthemum, especially chimeric cultivars, and for mutant detection.

     

A new chrysanthemum potyvirus: molecular evidence

Abstract

A number of viruses are known to infect chrysanthemum plants, however in the present study a previously unknown potyvirus was detected using techniques such as ELISA, RT-PCR and hybridization. The ELISA-positive samples were amplified using a potyvirus group-specific primer which gave an amplification of ～850 bp. The amplified product was cloned and sequenced, and shows 72 – 73% homology with known potyviruses that infect chrysanthemums such as Potato virus Y potyvirus, Soyabean mosaic virus and Turnip mosaic potyvirus when compared to the sequence available in the database. However, present potyvirus isolates show 93% homology with Chilli veinal mottle virus and Pepper vein banding virus. The results were further confirmed by Northern hybridization. This is the first report of a potyvirus similar to Chilli veinal mottle virus, and Pepper vein banding virus infecting chrysanthemums.     

Comparison of Chemical Constituents and Pharmacological Effects of Different Varieties of Chrysanthemum Flos in China

Chrysanthemum Flos is the prestigious traditional Chinese medicinal material and the popular health drink. This article comprehensively evaluated the chemical constituents, antioxidant activity, and hepatoprotective effects of 25 common chrysanthemum varieties in China. Firstly, we analyzed the chemical compositions of water extracts of chrysanthemum using UPLC/Q-TOF-MS, and identified 29 chemical components. The results displayed that chrysanthemum was rich in chemical constituents, but there were significant differences in the contents of four phenolic acids and five flavonoids among different varieties, and the coefficient of variation (CVs) ranged from 35.96 % to 114.62 %. Then, the antioxidant activities of different chrysanthemums were investigated, respectively via 1,1-diphenyl-2-picrylhydrazyl (DPPH), 2,2’-azinobis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), and Ferric Reducing Antioxidant Power (FRAP) assays. The spectrum-effect relationships between nine main components and antioxidant activities were investigated to identify the antioxidant constitutes in chrysanthemums. Meanwhile, H₂O₂-induced hepatocyte injury testing showed wide variation in cultivar antioxidant capacity, with Tongchengju (TCJ) producing the best effect (90.32 %), followed by Chuju (CJ; 85.78 %). In addition, the hepatoprotective effects of 8 mainstream varieties were determined by the model of acute alcoholic liver injury. They protected liver from injury by affecting relevant liver function and antioxidant indexes. Huangshangongju (HSG) could decrease aspartate aminotransferase (AST) activity by 39.27 % in liver tissue; Hangju-Fubaiju (HJ-FBJ), Jinsihuangju (JSH), and Chuju (CJ) significantly decreased the malondialdehyde (MDA) content of liver tissue, which reduced by more than 40 %; Jinsihuangju (JSH) of used for tea could double the content of glutathione (GSH) and had the similar effect on superoxide dismutase (SOD) as the positive group, showing significant antioxidant capacity. Therefore, this study confirmed that chrysanthemums are potential resources as antioxidants, functional foods, and medicinal materials. Importantly, it may provide a scientific support for further development and utilization of chrysanthemum, and screen excellent varieties for different demands.     

Altered expression of CmNRRa changes flowering time of Chrysanthemum morifolium

Flowering time is an important ornamental trait for chrysanthemum (Chrysanthemum morifolium, Dendranthema x grandiflorum) floricultural production. In this study, CmNRRa, an orthologous gene of OsNRRa that regulates root growth in response to nutrient stress in rice, was identified from Chrysanthemum and its role in flowering time was studied. The entire CmNRRa cDNA sequence was determined using a combinatorial PCR approach along with 5′ and 3′ RACE methods. CmNRRa expression levels in various tissues were monitored by real‐time RT‐PCR. CmNRRa was strongly expressed in flower buds and peduncles, suggesting that CmNRRa plays a regulatory role in floral development. To investigate the biological function of CmNRRa in chrysanthemums, overexpression and knockdown of CmNRRa were carried out using transgenic Chrysanthemum plants generated through Agrobacterium‐mediated transformation. CmNRRa expression levels in the transgenic plants were assayed by real‐time RT‐PCR and Northern blot analysis. The transgenic plants showed altered flowering times compared with nontransgenic plants. CmNRRa‐RNAi transgenic plants flowered 40–64 days earlier, while CmNRRa‐overexpressing plants exhibited a delayed flowering phenotype. These results revealed a negative effect of CmNRRa on flowering time modulation. Alteration of CmNRRa expression levels might be an effective means of controlling flowering time in Chrysanthemum. These results possess potential application in molecular breeding of chrysanthemums that production year‐round, and may improve commercial chrysanthemum production in the flower industry.     

Application of Genomic SSR Locus Polymorphisms on the Identification and Classification of Chrysanthemum Cultivars in China

The Chinese traditional chrysanthemum is a notable group of chrysanthemums (Chrysanthemum×morifolium Ramat.) in which the phenotypic characteristics richly vary. At present, there is a serious controversy regarding homonyms and synonyms within this group. Moreover, the current international chrysanthemum classification systems are not comprehensive enough to be used on Chinese traditional chrysanthemums. Thus, we first identified a broad collection of 480 Chinese traditional chrysanthemum cultivars using the unique DNA fingerprints and molecular identities that were established by 20 simple sequence repeat markers. Five loci, which distinguished all of the selected cultivars, were identified as the core loci to establish unique fingerprints and molecular identities with 19 denary digits for each cultivar. A cluster analysis based on Nei''s genetic distance indicated that the selected cultivars were clustered according to their horticultural classification. Population structure analysis was subsequently performed with K values ranging from 2 to 14, and the most likely estimate for the population structure was ten subpopulations, which was nearly consistent with the clustering result. Principal component analysis was further performed to verify the classification results. On the basis of the Q-matrices of K = 10, a total of 19 traits were found to be associated with 42 markers. Taken together, these results can serve as starting points for the identification and classification of chrysanthemums based on the polymorphism of microsatellite markers, which is beneficial to promote the marker-assisted breeding and international communication of this marvelous crop.     

Some hosts and properties of dahlia mosaic virus

Dahlia mosaic virus (DMV) infected twenty-five of the eighty-five plant species from four of eighteen families inoculated, but only dahlias were found naturally infected. DMV infected fourteen members of the Solanaceae, Amaranthaceae and Chenopodiaceae, and eleven of twenty-nine Compositae. Verbesina encelioides was the best plant for diagnosis, assay and source of virus. Systemically infected hosts contained ovoid intracellular inclusions 2–5–10 μm in diameter which were shown by electron microscopy to consist of a finely granular, vacuolated matrix containing numerous virus particles. V. encelioides sap was sometimes infective after dilution to 1/2000 but not 1/3000, after heating for 10 min to 75 °C but not 80 °C, and after 4 days at 18 °C or 32 days at 2 °C. Sap from infected dahlia, Zinnia elegans or Ageratum houstonianum rapidly became non-infective, but extracts made with 0·05 M sodium thioglycollate or 0·03 M sodium diethyldithiocarbamate remained infective for 24–48 h at 18 °C. Some purified preparations remained infective for up to 3 years at 2 °C. DMV was best purified from V. encelioides by one or more cycles of differential centrifugation, followed by density-gradient centrifugation and further concentration. Composition, molarity, and pH of the extracting buffer had little effect on yield of virus. Best yields were obtained from extracts stored with 8-5% (v/v) n-butanol at 2 °C for 10–14 days. Purified preparations were infective at dilutions up to 1/5000, had ultraviolet absorption spectra typical of a nucleoprotein (Å 260/280 = 1·47), probably contained DNA, and had a single sedimenting component having isometric particles c. 50 nm in diameter with a sedimentation coefficient of 254 S. The cryptogram of DMV is (D)/*:*/(16):S/S:S/Ap. DMV is serologically closely related to cauliflower mosaic virus, but the viruses are distinct pathogens. The two viruses have similar properties, size, shape and other characteristics, and together with at least three others form a small but apparently homogeneous group of aphid-borne viruses.     

Narcissus mosaic virus

Narcissus mosaic virus (NMV) is widespread in British crops of trumpet, large-cupped and double daffodils, but was not found in Narcissus jonquilla or N. tazzeta. Many commercial daffodil cultivars seem totally infected, and roguing or selection is therefore impracticable. Strict precautions by breeders and raisers to prevent infection of new cultivars is recommended. Healthy daffodil seedlings were readily infected with NMV by mechanical inoculation, but the virus was not detected in them until 17 months after inoculation, when a mild mosaic appeared. NMV infected twenty-eight of fifty-three inoculated plant species; only five (Nicotiana clevelandii, Gomphrena globosa, Medicago sativa, Trifolium campestre and T. incarnatum) were infected systemically, and NMV was cultured in these and assayed in Chenopodium amaranticolor and Tetragonia expansa. The virus was not transmitted to and from G. globosa or N. clevelandii by three aphid species, or through the seeds of Narcissus, G. globosa and N. clevelandii but was transmitted by handling. G. globosa sap was infective at a dilution of 10 ^-5 but not at 10^-6, when heated for 10 min. at 70° C. but not at 75° C, and after 12 weeks at 18° C, or 36 weeks at 0–4° C. NMV withstood freezing in infected leaves and sap, and purified preparations and freeze-dried sap remained infective for over 2 years. NMV was precipitated without inactivation by ammonium sulphate (313 g./l.) but was better purified by differential centrifugation of phosphate-buffer extracts treated with n-butanol. Such virus preparations from G. globosa, N. clevelandii, C. amaranticolor and T. expansa were highly infective, serologically active, produced a specific light-scattering zone when centrifuged in density-gradients and contained numerous unaggregated particles with a commonest length of 548–568 mμ. Antisera prepared in rabbits had precipitin tube titres of 1/4096. NMV was detected in three experimental hosts but not in narcissus sap. Unlike some viruses with elongated particles, NMV precipitates with antiserum in agar-gel. Purified preparations reacted with antiserum to a Dutch isolate of NMV but not with antisera to seven other viruses having similar particles and in vitro properties, or to narcissus yellow stripe virus.     

Efficacy of entomopathogenic nematode Steinernema feltiae (Rhabditida: Steinernematidae) as influenced by Frankliniella occidentalis (Thysanoptera: Thripidae) developmental stage and host plant stage

Entomopathogenic nematodes were investigated as an alternative biological control strategy for western flower thrips, Frankliniella occidentalis (Pergande) (Thysanoptera: Thripidae), in ornamental greenhouse crops, by using potted chrysanthemum as a model crop. The susceptibility of various life stages of F. occidentalis to different concentrations of the nematode Steinernema feltiae (Filipjev) (Rhabditida: Steinernematidae) was investigated in petri dish bioassays. This was followed with trials using potted chrysanthemums comparing the efficacy of nematode application to plants in vegetative (exposed habitat) versus flowering (cryptic habitat) stages. In both trials, the effect of the wetting agent Agral 90 (nonylphenoxy polyethoxyethanol), which is used in combination with the nematode spray, on F. occidentalis mortality was assessed. In petri dish trials, the prepupae and pupae were the most susceptible developmental stages of F. occidentalis to infection by S. feltiae. First and second instars were killed by very high rates of nematodes (> or =20,000 infective juveniles per milliliter), but corrected mortality was only approximately 28-37%. No significant mortality was observed for adult thrips. Results from the petri dish trials were confirmed on chrysanthemum plants. Foliar application of S. feltiae did not result in significant mortality in larvae or adults. No significant differences in efficacy were detected by application of nematodes on vegetative versus flowering chrysanthemum. Agral 90 had a significant impact on mortality on the first stage larvae and prepupae in the petri dish trials but not in the plant trials. Thrips control by S. feltiae in greenhouses may be partly or completely due to prepupal and pupal mortality.     

Attempts at Multiplication,Purification, Electron Microscopy,and Characterisation of Three Isolates of the Strawberry Mottle Agent

Three isolates of strawberry mottle agent (SMA) from strawberry plants were regularly maintained and multiplied by mechanical inoculation onChenopodium quinoa plants showing mosaic and mottle symptoms. The use of 5 mM borate buffer pH 8.6 or tap water pH 6.6-7.9 with 4 % (m/v) charcoal for homogenization resulted usually in 100 % infection. The total of 2090 plants were infected from 2264 inoculated ones under the same conditions. The infectivity of SMA isolates in crude sap ofC. quinoa was retained from 48 h to 72 h at 20 °C. The dilution end points of SMA isolates were 10^-3 while the inactivation temperatures were between 50 and 55 °C. The infectivity of SMA isolates in frozen leaves ofC. quinoa was detected still after six months. Purification procedure of SMA is based on using low molar 25 mM borate buffer pH 8.3 with cysteine hydrochloride, DIECA and Tween 20 for homogenization of infectedC. quinoa leaves, polyethyleneglycol precipitation, clarification with octanol, low and high speed centrifugation and sucrose density-gradient centrifugation. Partially purified preparations are highly infectious, causing mosaic, mottling and tip necrosis ofC. quinoa plants. The agent could not be completely separated from host proteins and it could not be concentrated to a high extent. Isometric virus-like particles 14-16 nm were observed in partially purified preparations.     

Carnation Italian ringspot virus

Carnation Italian ringspot virus (CIRV) was obtained only twice in tests on several thousand carnations in Britain during 15 yr. The two isolates, from cultivars ‘Dusty Sim’ imported from Italy and ‘Orchid Beauty’ from the U.S.A., were indistinguishable serologically and in host reactions. CIRV was cultured in Nicotiana clevelandii and assayed in Chenopodium amaranti-color; it was readily transmitted by leaf-rubbing inoculation to 62 of 104 plant species tested. Virus-free carnations were infected only by injecting purified preparations into the stem, and developed chlorotic spots and oval rings in the younger leaves. CIRV was eliminated from Nicotiana clevelandii plants grown for 8 weeks at 36°C. CIRV presents no threat to carnation growing in Britain. In N. clevelandii sap, CIRV was infective at a dilution of 1/50000 to 1/100000, after heating 10 min at 85 °C (but not 90 °C), and after 16 weeks at 16 °C or 23 weeks at 2 °C. After freeze-drying, the virus survived at least 7 yr storage under vacuum at room temperature. CIRV was still infective and antigenic after treatment for 30 min at 18 °C with ultraviolet radiation (750 μW/cm²), ultrasound, 2% formaldehyde or 0.2% tri-sodium ortho-phosphate (TSP). Infectivity was not wholly abolished in 30 min by 2% TSP. The virus was readily purified by overnight maceration of N. clevelandii leaves extracted in phosphate buffer + butanol, followed by differential centri-fugation. Purified preparations contained abundant isometric particles c. 29 nm diameter, and like other serotypes of the tomato bushy stunt-pelargonium leaf curl group, gave three or four specific bands in density-gradient centri-fugation. The bands corresponded to four Schlieren peaks in analytical centrifugation. Virus from the lower bands was usually less invasive in N. clevelandii than from the upper bands, although the material in the different bands contained similar amounts of nucleic acid. Only one antigenic component was found by Immunoelectrophoresis; different serotypes of the TBSV-PLCV group differed widely in immunoelectrophoretic behaviour. The present cryptogram of CIRV is */*:*/*:S/S:S/*.