Detection of strains of African cassava mosaic virus by nucleic acid hybridisation and some effects of temperature on their multiplication

DNA probes, made by cloning double-stranded forms of each of the genome parts (DNA-1 and DNA-2) of the Kenyan type isolate of African cassava mosaic virus (ACMV-T), reacted strongly with extracts from Nicotiana benthamiana plants infected with ACMV-T, or with Angolan or Nigerian isolates that are closely serologically related to the type isolate. However, only the DNA-1 probes reacted with extracts of TV. benthamiana infected with a Kenyan coast isolate (ACMV-C), which is serologically less closely related to ACMV-T. DNA-1 and DNA-2 probes also reacted with extracts of mosaic-affected Angolan cassava plants, including some which have not yielded ACMV particles detectable by immunosorbent electron microscopy and from which virus isolates have not been transmitted to TV. benthamiana. These anomalous plants, unlike other naturally infected cassava plants, showed mosaic symptoms on all their leaves which, however, contained only traces of virus particle antigen detectable by enzyme-linked immunosorbent assay. They contain isolates of ACMV that are probably defective for particle production. ACMV-T particles accumulated optimally in N. benthamiana at 20–25°C. At 30°C fewer particles, which apparently had a slightly greater specific infectivity, were produced. At 15°C, considerable quantities of virus particle antigen, virus DNA and virus particles were produced but the particles were poorly infective, and the few that could be purified contained an abnormally large proportion of polydisperse linear DNA molecules, and fewer circular molecules than usual. Angolan isolates, whether particle-producing or not, likewise replicated better in cassava plants at 23 °C than at 30 °C. In contrast, ACMV-C attained only very low concentrations in N. benthamiana, but these were greater at 30 °C than at 23°C.     

Purification and Characterization of Indian Cassava Mosaic Virus

The Indian cassava mosaic virus (ICMV) was transmitted by the whitefly Bemisia tabaci and sap inoculation. ICMV was purified from cassava and from systemically infected Nicotiana benthamiana leaves. Geminate particles of 16–18 × 30 nm in size were observed by electron microscopy. The particles contained a single major protein of an estimated molecular weight of 34,000. Specific antiserum trapped geminate particles from the extracts of infected cassava and N. benthamiana plants in ISEM test. The virus was detected in crude extracts of infected cassava, ceara rubber, TV. benthamiana and N. tabacum cv. Jayasri plants by ELISA. ICMV appeared serologically related to the gemini viruses of Acalypha yellow mosaic, bhendi yellow vein mosaic, Croton yellow vein mosaic, Dolichos yellow mosaic, horsegram yellow mosaic, Malvastrum yellow vein mosaic and tobacco leaf curl.     

African Cassava Mosaic Virus (ACMV): Stability of Purified Virus and Improved Conditions for its Detection in Cassava Leaves by ELISA

African Cassava Mosaic Virus (ACMV) was purified by a method which allowed the separation of monomer from dimcr virus particles. Optimal conditions for storing purified virus to be used for immunization were determined by ELISA and inoculation on Nicotiana benthamiana. Purified virus could be stored without loss of infectivity and serological activity for more than 145 days at 4 °C or frozen at –20 °C, but not longer than 40 days in the presence of 50 % redistilled glycerol. Rabbit and chicken immunoglobulins were used to detect ACMV in cassava leaves by direct and indirect ELISA. To obtain the same absorbance values, it was necessary to use longer incubation times with the indirect method, but the virus detection end-point m sap from infected plants was the same for the two methods (1/512). Conditions for improving virus detection tn cassava samples were determined. The virus was better detected when leaves from diseased plants were ground in 100 mM Tris-HCl containing 1 % polyvinylpyrrolidone at pH 8.5 than in phosphate buffer. Plant inhibitors were the restricting factor in the detection of virus by ELISA, but this difficulty was avoided when leaves to be tested were harvested from the top of the cassava plants.     

Some properties of a previously undescribed virus from cassava: Cassava American Latent Virus

A virus with spherical particles c. 28 nm in diameter was sap-transmitted from different cassava (Manihot esculenta) cultivars to a limited range of species in the families Chenopodiaceae and Solanaceae. Cassava seedlings infected by inoculation with sap or with purified virus preparations did not show any symptom, although the virus was readily detected by ELISA or by further inoculations. Leaf extracts from infected Nicotiana benthamiana were infective after dilution of 10^--³but not 10^--⁴, and after heating for 10 min at 70°C, but not at 72°C. The virus was purified from N. benthamiana, N. clevelandii or from cassava. On sucrose gradients, the virus particles sediment as three components all containing a protein of mol. wt c. 57000. The genome of the virus is composed of two RNAs of mol. wt c. 2.54 times 10⁶(RNA-1) and 1.44 times 10⁶(RNA-2). RNA-2 was detected in the middle and the bottom nucleoprotein components, and RNA-1 only in the bottom component. An antiserum prepared to purified virus particles was used to readily detect the virus in cassava and other host plants by ELISA and by ISEM. No serological relationship was shown between this virus and eight nepoviruses, including the recently described cassava green mottle nepovirus infecting cassava in the Solomon Islands (Lennon, Aiton & Harrison, 1987). The virus described here is the first nepovirus isolated from cassava in South America, and is named cassava American latent virus.     

Serological studies on cassava latent virus

Particles of cassava latent virus (CLV) were purified by a method that yielded up to 3 mg per 100 g of systemically infected Nicotiana benthamiana leaf. Specific antiserum was prepared and used for enzyme-linked immunosorbent assay (ELISA), which detected purified virus at 5 ng/ml. As estimated by ELISA, CLV antigen reached a greater concentration in leaves of N. benthamiana plants kept at 20–25 °C than in those at 15 °C or 30 °C. CLV was also detected in leaf extracts of naturally infected cassava plants kept at 25 C but its concentration was only 1–7% of that in comparable extracts from N. benthamiana. Staining sections of N. benthamiana leaves with fluorescent antibody indicated that CLV particle antigen accumulates in the nuclei of many phloem cells and of some cells in other tissues. In tests on mosaic-affected cassava plants of Angolan origin, three plants were found in which CLV could not be detected by either ELISA or immunosorbent electron microscopy, or by transmission to indicator plants. This suggests that the mosaic symptoms were caused by a pathogen other than CLV, but no such agent was detected by electron microscopy of leaf extracts. Three kinds of serological test indicated that CLV is related to bean golden mosaic virus. Evidence was also obtained of a distant relationship to beet curly top virus but none was detected to four other geminiviruses.     

Some properties of cocksfoot mottle virus

Cocksfoot mottle virus (CFMV) was transmitted by manual inoculation of sap to cocksfoot (Dactylis glomerata L.), wheat, oats and barley, but not to nineteen other monocotyledonous and thirteen dicotyledonous plant species. The virus was also transmitted by cereal leaf beetles (Lema melanopa L.). Adult beetles infected plants more frequently than larvae, and remained infective for up to 2 weeks after they had fed on infected plants. Seed from infected cocksfoot and oat plants produced virus-free seedlings. The infectivity of sap was lost during 10 min. at 65° C., and 2 weeks at 20° C., but survived many months at — 15° C. Purified virus preparations, made by various methods, contained numerous nearly spherical particles, about 30 mμ in diameter. In electron micrographs some of the particles were penetrated by negative stain though most appeared intact. However, all the particles migrated together in a centrifugal (sedimentation coefficient = 118 S) or electrophoretic field. The ultraviolet absorption spectrum, and the phosphorus and nitrogen contents of the virus preparations, were typical of a nucleoprotein containing about 25 % nucleic acid. Serological tests failed to show any relationship between CFMV and eleven other viruses with particles of similar shape and size.     

Host range and some properties of potato mop-top virus

Potato mop-top virus (PMTV) was transmitted by inoculation of sap to twenty-six species in the Solanaceae or Chenopodiaceae and to Tetragonia expansa; species in eleven other plant families were not infected. The virus was cultured in inoculated leaves of Nicotiana tabacum cv. Xanthi-nc or in N. debneyi. Diagnostic local lesions were produced in Chenopodium amaranticolor. In winter, ten solanaceous species were slowly invaded systemically but the first leaves infected were those immediately above inoculated leaves. When transmitted to Arran Pilot potato by the vector Spongospora subterranea, PMTV induced all the main types of shoot and tuber symptoms found in naturally infected plants. Isolates of PMTV from different sources differed considerably in virulence. PMTV-containing tobacco sap lost infectivity when heated for 10 min at 80 °C, diluted to 10^-4, or stored at 20 °C for 14 weeks. Infectivity was partially stabilized by 0·02% sodium azide. When sap was centrifuged for 10 min at 8000 g, infectivity was mainly in the sediment. Infective sap contained straight rod-shaped particles about 20 nm wide, with lengths up to 900 nm and crossbands at intervals of 2·5 nm. Many of the particles were aggregated side-to-side, and the ends of most seemed damaged. The slight infectivity of phenol-treated leaf extracts was abolished by pancreatic ribonuclease. The present cryptogram of PMTV is R/*:*/*:E/E:S/Fu.     

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.     

Cytopathology of Plgeonpea sterility mosaic virus in pigeonpea and Nicotiana benthamiana: similarities with those of eriophyid mite-borne agents of undefined aetiology

Pigeonpea sterility mosaic virus (PPSMV) is transmitted by the eriophyid mite, Aceria cajani, and is very closely associated with sterility mosaic disease (SMD) of pigeonpea (Cajanus cajah) in the Indian subcontinent. Antiserum produced to purified PPSMV preparations detected a virus‐specific 32 kDa protein in sap of SMD‐affected pigeonpea plants by ELISA and Western blotting. PPSMV was transmitted mechanically in sap of SMD‐affected pigeonpea leaves to Nicotiana benthamiana. Ultrastructural studies of symptom‐bearing leaves of two pigeonpea cultivars, (ICP8863 and ICP2376) and N. benthamiana infected with PPSMV, detected quasi‐spherical, membrane bound bodies (MBBs) of c. 100–150 nm and amorphous electron‐dense material (EDM). These structures were distributed singly or in groups, in the cytoplasm of all cells, except those in conductive tissues. Fibrous inclusions (FIs), composed of randomly dispersed fibrils with electron lucent areas, were present in the cytoplasm of palisade cells and rarely in mesophyll cells of the two pigeonpea cultivars but were not detected in infected TV. benthamiana plants. In the PPSMV‐infected pigeonpea cultivars and TV. benthamiana, immuno‐gold labelling, using antiserum to PPSMV, specifically labelled the MBBs and associated EDM, but not the FIs. The MBBs and associated inclusions are similar in appearance to those reported for plants infected with the eriophyid mite‐transmitted High Plains virus and the agents of unidentified aetiology associated with rose rosette, fig mosaic, thistle mosaic, wheat spot chlorosis and yellow ringspot of budwood. The nature of these different inclusions is discussed.     

Pepino mosaic virus, a new potexvirus from pepino (Solanum muricatum)

Pepino mosaic virus (PepMV), a previously undescribed virus, was found in fields of pepino (Solanum muricatum) in the Canete valley in coastal Peru. PepMV was transmitted by inoculation of sap to 32 species from three families out of 47 species from nine families tested. It caused a yellow mosaic in young leaves of pepino and either a mild mosaic or symptomless infection in 12 wild potato species, five potato cultivars and potato clone USDA 41956 but S. stoloniferum and potato cultivars Merpata and Revolucion reacted with severe systemic necrotic symptoms. The virus was transmitted by plant contact but not by Myzus persicae. It was best propagated and assayed in Nicotiana glutinosa. Sap from infected N. glutinosa was infective after dilution to 10^-1 but not 10^-6, after 10 min at 65°C but not 70°C and after 3 months at 20°C. PepMV had filamentous particles with a normal length of 508 nm; the ends of some seemed damaged. Ultra-thin sections of infected leaves of N. glutinosa revealed many inclusions containing arrays of virus-like particles some of which were banded or whorled; small aggregates of virus-like particles were also common. The virus was purified by extracting sap from infected leaves in a solution containing 0·065 M disodium tetraborate, 0·435 M boric acid, 0·2% ascorbic acid and 0·2% sodium sulphite at pH 7·8, adding silver nitrate solution to the extract, and precipitating the virus with polyethylene glycol followed by two cycles of differential centrifugation. Particles of PepMV normally yielded two proteins with molecular weights of 26 600 and 23 200, but virus obtained from infective sap aged overnight yielded only the smaller protein suggesting that it was a product of degradation of the larger one. The virus is serologically related to two potexviruses, narcissus mosaic and cactus X and its properties are typical of the potexvirus group.     

Biological and biochemical properties of an isolate of cherry rasp leaf virus from red raspberry

A mechanically transmissible virus obtained from symptomless plants of a red raspberry selection imported into Scotland from Quebec, Canada was indistinguishable serologically from a cherry isolate of cherry rasp leaf virus (CRLV). The raspberry isolate, CRLV-R, was graft transmitted to several virus indicator species and cultivars of Rubus without inducing noticeable symptoms. In Chenopodium quinoa sap, CRLV-R lost infectivity after dilution to 10^-5 or heating for 10 min at 60°C but was infective after 16 days (the longest period tested) at 18°, 4° or - 15°C. The virus particles are isometric, c. 28 nm in diameter, and were purified with difficulty from infected C. murale and C. quinoa plants. The particles comprise two nucleoprotein components with sedimentation coefficients of 89 and 115 S and are prone to aggregate during purification. When centrifuged to equilibrium in CS₂SO₄ solution, purified virus preparations formed two major components with p= 1·28 and 1·36 g/cm³. Virus particles contained two RNA species which, when denatured in glyoxal and electrophoresed in agarose gels, had estimated mol. wt of 2·56 × 10⁶ (RNA-1) and 1·26 × 10⁶ (RNA–2). Infectivity of CRLV-R RNA was abolished by treatment with proteinase K, suggesting that the RNA is linked to protein necessary for infectivity; RNA molecules contained polyadenylate. In reticulocyte lysates, CRLV-R RNA stimulated the incorporation of ³H-leucine, mainly into two polypeptides of estimated mol. wt 200 000 and 102 000. When electrophoresed in polyacrylamide gels, protein obtained from CRLV-R particles purified by centrifugation to equilibrium in Cs₂SO₄ separated into three bands with estimated mol. wt 26 000 , 23 000 and 21 000.     

Mechanical Transmission, Purification and Properties of an Isolate of Maize Stripe Virus from Venezuela

A Venezuelan isolate of maize stripe virus (MStpV) was successfully transmitted mechanically and by the leafhopper Peregrinus maidis from field infected plants to sweet cv. Iochief. After purification of maize infected with MStpV, fine spiral filamentous particles about 4 nm in diameter and with variable lengths were consistently associated with a nucleoprotein band present in CsCl or Cs₂SO₄ isopycnic gradients. Purified preparations exhibited a typical nucleoprotein absorption spectrum with a maximum at 260–263 nm and a minimum at 240–243 nm and a 260–280 ratio of 1.38. The density of the nucleoprotein in CsCl gradients was estimated at 1.29 g/ml. The sedimentation coefficient was calculated at 62 S. The nucleoprotein consisted of 5 % single stranded RNA and a capsid protein of molecular weight 33.500 daltons. Large quantities of non-capsid proteins were isolated from infected tissue with a molecular weight of 17.500 and 16.500 daltons. Peregrinus maidis, injected with purified MStpV preparation failed to transmit the disease to healthy plants. However, they were infectious when injected with clarified infected plant sap. Antisera against capsid and non-capsid proteins from MStpV-Florida strain reacted positively with the Venezuelan antigens.     

Pathology and properties of tulip virus X, a new potexvirus

Tulip virus X (TVX), a previously undescribed mechanically transmissible virus, causes chlorotic and necrotic lesions in leaves and streaks of intensified pigmentation in tepals of tulip plants. The virus infected 22 of 42 other plant species in 10 of 14 families, but most host species were infected only erratically. TVX is best propagated in Chenopodium quinoa and assayed in C. amaranticolor. Spindleshaped inclusions were observed in epidermal cells of C. amaranticolor leaves. Leaf extracts from C. quinoa contained flexuous filamentous particles measuring c. 495 ×13 nm. The extracts were infective after dilution to 10^-9, after heating for 10 min at 60 °C but not at 65 °C, and after storage at c. 20 °C for 30 days or at -20 °C for 6 months. TVX particles were purified (500 μg/g C. quinoa leaf) from tissue extracts in 0.067 M phosphate buffer containing 10 mM EDTA at pH 7, by twice precipitating the virus with 8% polyethylene glycol in 0.2 M NaCl followed by differential centrifugation. The virus particles have a sedimentation coefficient (s_{20, w}) of 102 S. They contain a protein of mol. wt c. 22 500 and a nucleic acid that, when glyoxalated, migrates in agarose gel like single-stranded RNA of mol. wt 2.05 × 10⁶. TVX particles tend to aggregate, and evidence was obtained that a 118 S component which was consistently observed in purified preparations and in infective sap is an end-to-end dimer. A distant serological relationship was found between particles of TVX and those of viola mottle and hydrangea ringspot viruses, but no serological relationship was detected to nine other potexviruses. TVX is considered to be a distinct and definitive member of the potexvirus group.     

Purification and properties of blackberry chlorotic ringspot, a new virus species in subgroup 1 of the genus Ilarvirus found naturally infecting blackberry in the UK

A mechanically transmissible virus was isolated from Bedford Giant blackberry plants showing chlorotic mottling and ringspot symptoms growing in Scotland. It infected several herbaceous test plants, many of them symptomlessly. This virus was also transmitted to several Rubus species and cultivars by graft inoculation with scions from the field‐infected Bedford Giant plant. Most grafted plants were infected symptomlessly, but Himalaya Giant blackberry and the hybrid berry Tayberry developed symptoms similar to those in the infected Bedford Giant plant. In the sap of infected Chenopodium quinoa, the virus lost infectivity when diluted 10^?4 but not 10^?3, after 6 h and 48 h when kept at 20°C and 4°C, respectively, but was infective for more than 8 days when kept at ?15°C. Preparations of purified virus from infected C. quinoa or spinach sedimented as three major nucleoprotein components and consisted of quasi‐isometric particles that varied in size from 24 to 32 nm in diameter and that were not penetrated by negative stain. Such virus particle preparations contained a major polypeptide of ca 28 kDa and three single‐stranded RNA species of estimated size 3.2, 2.8 and 2.1 kb. The complete sequence of the largest RNA (RNA 1, 3478 nt) and the partial sequence of the other RNAs (1863 and 2102 nt long, respectively) were determined and compared with sequences in databases. These findings, together with the biological and biochemical properties of this virus, indicate that it should be regarded as a distinct species in subgroup 1 of the genus Ilarvirus even though it was serologically unrelated to existing members of this subgroup. The virus showed a very distant serological relationship with prune dwarf virus (PDV) but differed significantly from it in the amino acid sequence of its coat protein, experimental host range and symptomatology and was unrelated to PDV at the molecular level. The virus, tentatively named blackberry chlorotic ringspot virus, is therefore a newly described virus and the first ilarvirus found naturally infecting Rubus in the UK.     

Purification and some properties of strawberry mottle virus

Strawberry mottle virus (SMoV) (three isolates: HJ, 3E and N) were transmitted to Chenopodium quinoa plants by sap inoculation. All three isolates induced very similar symptoms consisting of chlorotic spots and ringspots in inoculated leaves, and vein chlorosis, mottling, and dwarfing of the upper leaves. SMoV isolate HJ was purified from infected C. quinoa by homogenisation with 10 mM phosphate buffer, pH 7.2 containing 5% Triton X-100, followed by differential, sucrose density-gradient and CsCl equilibrium density-gradient centrifugations. A fraction with a buoyant density of 1.42g^- cm^-3 after CsCl density-gradient centrifugation was highly infectious to C. quinoa and contained many isometric virus-like particles c. 37 nm in diameter. These virus-like particles were never found in fractions from uninfected preparations. Electrophoretic analysis of a fraction containing virus-like particles revealed that these particles might have a single coat protein subunit with the apparent molecular mass of 26 K daltons and one nucleic acid of 6.6 kilobases. Double-stranded RNA analysis of isolate HJ-infected or uninfected C. quinoa and Fragaria vesca var. semperflorens seedling line ‘Alpine’ plants showed that both infected plants had two infection-specific dsRNA bands of mol. wts 4.5 and 3.9 × 10⁶.     

Leaf mottling of Spartina species caused by a newly recognised virus, spartina mottle virus

The cause of a previously undocumented leaf mottling of Spartina species was investigated. Negatively stained preparations of sap from mottled leaves revealed flexuous particles 725 × 12 nm. Pinwheels with associated laminar inclusion bodies were observed in thin sections of affected mesophyll cells. The virus was purified from infected Spartina anglica plants and had a sedimentation coefficient in 0·015 m borate of 150S. The virus was transmitted by inoculation of sap to healthy Spartina anglica, but not to a range of other graminaceous or dicotyledonous species tested. It was distantly serologically related to agropyron mosaic virus, but not to other viruses with similar morphology; the name spartina mottle virus is proposed.     

Partial purification and some properties of wineberry latent, a virus obtained from Rubus phoenicolasius

Wineberry latent virus (WLV) was obtained from a single symptomless plant of American wineberry (Rubus phoenicolasius) originally imported from the United States of America. On graft inoculation, WLV infected but induced no distinctive symptoms in several Rubus species including those used as indicators for known Rubus viruses. It was not seed-borne in wineberry. WLV was mechanically transmitted to several herbaceous species but induced local lesions in only a few; it was weakly systemic in some Chenopodium species. Infective C. quinoa sap lost infectivity after diluting to 10^-4, heating for 10 min at 70°C, and storage either for 8 days at 18°C or for 32 days at 4°C. Sap from infected plants contained flexuous filamentous particles c. 510°12 nm. WLV was partially purified by extracting infected C. quinoa leaves in 0·05 M tris-HCl buffer (pH ₇) containing 0·2% thio-glycerol and 10% (v/v) chloroform and concentrating virus by precipitation with 7% (w/v) polyethylene glycol (PEG, mol. wt 6000) and 0·1 NaCl. The virus was then pelleted through a 30% (w/v) sucrose pad containing 7% PEG+0·1 M NaCl and finally sedimented through a sucrose density-gradient. These preparations had A_260/280 ratios of 1·26, contained end to end aggregates of WLV particles and formed a partly polydispersed peak in the analytical ultracentrifuge. WLV did not react with antisera to four potex-viruses, or to apple chlorotic leaf spot or apple stem grooving viruses.     

Properties of a resistance-breaking strain of potato virus X

During indexing of a potato germplasm collection from Bolivia, a strain of potato virus X (PVX), X_HB, which failed to cause local lesions in inoculated leaves of Gomphrena globosa was found in 7% of the clones. X_HB was transmitted by inoculation of sap to 56 species from 11 families out of 64 species from 12 families tested. It was best propagated in Nicotiana glutinosa or N. debneyi; Montia perfolia and Petunia hybrida were useful as local lesion hosts. Inoculated leaves of G. globosa plants kept at 10°, 14°, 18°, 22°, or 26 °C after inoculation were always infected symptomlessly. X_HB caused a mild mosaic, systemic chlorotic blotching or symptomless infection in 16 wild potato species and eight Andean potato cultivars, systemic necrotic symptoms in clone A6 and cultivar Mi Peru, and bright yellow leaf markings in cultivar Renacimiento. It caused necrotic local lesions in inoculated leaves of British potato cultivars with the PVX hypersensitivity gene Nb but then invaded the plants systemically without causing further necrosis; with gene Nx systemic invasion occurred but no necrotic symptoms developed. These reactions resemble those of PVX strain group four. X_HB differed from other known strains of PVX in readily infecting PVX-immune clones 44/1016/10, G. 4298.69 and USDA 41956, cultivars Saphir and Saco, and Solanum acaule PI 230554. X_HB had slightly flexuous filamentous particles with a normal length of 516 nm. It was transmitted readily by plant contact and it partially protected G. globosa leaves from infection with X_CP, a group two strain of PVX. Sap from infected N. glutinosa was infective after dilution to 10^--⁶ but not 10^--⁷ after 10 min at 75° but not 80 °C and after 1 yr at 20 °C. X_HB was readily purified from infected N. debneyi leaves by precipitation with polyethylene glycol followed by differential centrifugation. Microprecipitin tests showed that X_HB and X_CP are closely related serologically.     

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.     

Pathology, soil transmission and characterization of cymbidium ringspot, a virus from cymbidium orchids and white clover (Trifolium repens)

Two strains of a virus, designated cymbidium ringspot virus (CyRSV), were isolated from cymbidium orchids and from Trifolium repens respectively in Britain. Experimentally infected cymbidiums developed slight chlorotic ring-mottle; T. repens developed flecks and mottling in the leaves, and slight stunting. Of 101 plant species tested, the cymbidium strain infected sixty-one (thirteen systemically) in twenty-three of thirty-five families; the clover strain infected sixty-four species (eighteen systemically) in twenty-two families. Both strains were propagated in Nicotiana clevelandii and assayed in Chenopodium quinoa. CyRSV was readily transmitted by inoculation of sap, and by foliage contact between plants, but not by the aphids Myzus persicae or Acyrtho-siphon pisum, nor through seed of T. incarnatum, Phaseolus vulgaris or N. clevelandii. Highly infective virus was released into soil from roots of infected N. clevelandii, and acquired by bait seedlings planted in such soil. Similar transmission occurred when purified virus was applied to the surface of sterilized soil containing bait plants; there was no evidence for any living soil vector. The virus was eliminated from 96 % of small cuttings taken from infected N. clevelandii plants grown at 35–37 °C for 9 wk. CyRSV was still infective in sap of N. clevelandii after dilution to 10?⁵-io–⁶ (only 2 × 10^_1 in cymbidium sap), or after 10min at 85–90 °C. It survived at least 10 months at c. 20 °C and more than 12 yr at 2 °C. Lyophilized sap was highly infective after over 13 yr at laboratory temperatures under high vacuum. Purified preparations made by clarification with n-butanol, followed by differential centrifugation and exclusion chromatography on controlled-pore glass beads, contained isometric particles c. 30 nm diam., with s°_20W= 137 S, and had a buoyant density in caesium chloride of 1–36 g/ml. The A 260/A 280 ratio was 1–55, and A max(26o)/A min(242) was 1–17. The virus contained c. 15 % of single-stranded RNA of mol. wt 1–7 × 10⁶; the nucleotide base ratios were: G27'8; A24/9; C2I-3; U26-I. There was one capsid polypeptide of mol. wt 43600. The virus was a good immunogen and a strongly reacting antigen in vitro; in Immunoelectrophoresis, each strain migrated as a single antigenic component towards the cathode. The cymbidium and clover strains were serologically closely related, although spurs were produced in immunodiffusion. No serological relationship was found to forty-three other isometric viruses, including eighteen tombusvirus isolates; CyRSV nevertheless shares many properties with tombusviruses, and we assign it provisionally to this group. The cryptogram is: R/r:1:7/15:S/S:S/O.