Use of Clq enzyme-linked immunosorbent assay to detect plant viruses and their serologically different strains

Clq was prepared from bovine serum using a simple method involving repeated dialysis at low ionic strength in the presence of chelating agents (yield c. 3 mg/100 ml serum). It was viable when stored at -18°C for up to 2 months, and at 4°C for at least 10 wk in a storage buffer containing 10% sucrose. When used in Clq ELISA this test was as sensitive as the direct double antibody sandwich form of ELISA (direct ELISA) in detecting purified potato virus Y (PVY), with a limit of detection in both methods of c. 15 ng/ml, and slightly more sensitive in detecting purified cocksfoot mild mosaic virus (CMMV), with limits of detection of c. 15 ng/ml and c. 15–60 ng/ml respectively. Using an antiserum to one strain of each virus, Clq ELISA readily detected strains of PVY, CMMV, Andean potato latent virus (APLV) and barley yellow dwarf virus (BYDV). This included detection of APLV-Hu by APLV-Caj antibodies and CMMV(G) by PMV(S) antibodies, neither of which system gives detection in direct ELISA. Clq ELISA was therefore less specific than direct ELISA in detecting serologically different virus strains. Virus detection by Clq ELISA was inhibited when sap of tobacco, Nicotiana clevelandii and Setaria italica was used at low dilution. Inhibition by N. clevelandii sap was alleviated by using increased concentrations of virus specific antibody to detect APLV and plum pox virus. Also, extracting APLV infective N. clevelandii or CMMV infective S. italica saps in a minimum of buffer, centrifuging at low speed and diluting the supernatant before testing, partially overcame the inhibition. The inhibitory substance(s) in sap may act by preventing the binding of Clq to virus-antibody aggregates. Sap of wheat, oat and barley did not appear to have an inhibitory effect and BYDV was readily detected in naturally infected field grown plants of these species.     

Effect of Fungicides on the Viability of Phylophthora cactorum Propagules m the Soil

Vein-clearing followed by downward rolling and necrosis of leaves and severe stunting of groundnut (Arachis hypogaea) plants were caused by cowpea mild mottle virus (CMMV). The virus was readily transmitted by mechanical sap inoculations to groundnut and to 10 plant species belonging to Leguminosae, Chenopodiaceae and Solanaceae. Chenopodium quinoa and Beta vulgaris were good diagnostic hosts. Diseased sap remained infective at 10^–3 but not 10^–4, when stored 8 to 9 days at 25 °C; for 10min at 75 °C but not 80°C. In limited tests, virus was not seed-transmitted m groundnut or soybean. Virus was transmitted by Bemisia tabaci but not by Aphis craccivora or Myzus persicae. An antiserum for CMMV was produced and virus was serologically related to CMMV reported on cowpea and groundnut crinkle virus (GCV) from West Africa. Employing carbon diffraction grating replica as a standard the modal length of virus particles to be 610 nm. Infected cells contained large number of virus particles associated with endoplasmic reticulum.     

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.     

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.     

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/*.     

Purification and some properties of Tephrosia symptomless virus

Tephrosia symptomless virus (TSV), isolated from Tephrosia villosa, is widely distributed in coastal districts of Kenya. The virus was readily transmitted by inoculation of sap, but not by Aphis craccivora or Apion sp. (Curculionidae) or through soil. Host range was very restricted and it infected only 10 of 70 species tested in one of nine plant families; susceptible species were confined to five genera within the Papilionaceae. The virus was cultured, propagated and assayed in soybean. TSV remained infective after 10 min at 85°C, 3 wk at 20°C and 26 wk at -12°C; crude infective sap of Glycine max retained infectivity when diluted 10^-6 but not 10^-7. Virus was purified from systemically infected soybean by clarifying sap extracted in 0.06 m phosphate buffer containing 0.001 m EDTA and 0.1% thioglycollic acid (pH 7.5) with equal volumes of 1:1 n-butanol/chloroform followed by two cycles of differential and one of sucrose density gradient centrifugation. Purified preparations contained c. 33 nm isometric particles. TSV contained RNA and one protein of molecular weight 1.53. 10⁶ and c. 42 000, respectively. Analytical centrifugation indicated a single component with a sedimentation coefficient (s.20, w) of 127 S; in Cs₂SO₄ and CsCl isopycnic gradients a single virus band formed; buoyant density in CsCl was 1.361. TSV was not related serologically to any of 44 viruses in nine plant virus groups but it resembled the tombusviruses and other ungrouped viruses such as carnation mottle in some of its properties.     

Pepper veinal mottle virus-a new member of the potato virus Y group from peppers (Capsicum annuum L. and C. frutescens L.) in Ghana

Pepper veinal mottle virus (PVMV), a previously undescribed virus widespread in Capsicum annuum and C. frutescens in the Eastern Region of Ghana, is acquired and inoculated in 2 min feeding periods by aphids (Myzus persicae and Aphis gossypii); it is transmissible by inoculation of sap to eleven of fifteen Solanaceae and to five of forty-six other species within three of seventeen other families. The virus was propagated in Nicotiana clevelandii and Petunia hybrida, and assayed in Chenopodium quinoa, C. amaranticolor and C. murale. Sap from Capsicum annuum was infective after dilution to 10^-3 but not 10^-4, after 10 min at 55 but not 60^oC, and after 7 but not 8 days at 25^oC. Lyophilized sap from P. hybrida was infective after 6 years in vacuo. Yields of 10–25 mg of virus per kg of leaf tissue were consistently obtained from P. hybrida or N. clevelandii by extracting systemically infected leaves in 0.5 M borate (pH 7.8) containing 0.2% mercaptoethanol and chloroform, followed by repeated precipitation with 50 g polyethylene glycol (M.W. 6000) per l, several cycles of differential centrifugation and centrifugation in sucrose density-gradient columns. Virus preparations had ultraviolet absorption spectra typical of a nucleoprotein containing c. 6% nuclei acid (A 260/280 = 1.25; A 260/246 = 1.27) and contained numerous unaggregated and unbroken filamentous particles c. 770 times 12 nm which sedimented as a single component with a sedimentation coefficient (s^o_20,w) of 155 S. PVMV contained RNA (moles %: G = 24, A = 23, C = 27, U = 26), and a single protein species with a molecular weight of 32000–33000 daltons. PVMV was not serologically related to potato virus Y (three strains), or to twelve other morphologically similar viruses, and seems to be a distinct member of the potato virus Y group. The cryptogram of PVMV is R/(I):*/(6):E/E:S/Ap.     

Tulip chlorotic blotch virus, a second potyvirus causing tulip flower break

Tulip chlorotic blotch virus (TCBV), an apparently undescribed potyvirus found in field grown tulips in Australia, causes symptoms in tulip leaves and flowers identical to those induced by tulip breaking virus (TBV). TCBV was transmitted mechanically to 14 of 34 species in four of 13 families. Nicotiana clevelandii is a suitable propagation host and Chenopodium amaranticolor a local-lesion assay host. TCBV was transmitted from tulip to tulip and TV. clevelandii by the aphid Myzus persicae. Unlike TBV it was not transmitted to Lilium formosanum either by M. persicae or by manual inoculation. Leaf extracts from TCBV-containing TV. clevelandii were infective after dilution to l0^-3 but not 10-⁴ and after heating for 10 min at 50°C but not 60°C; infectivity and particle recovery were adversely affected by freezing at -20°C. TCBV particles were purified (c. 1 mg/100g g N. clevelandii leaf) from tissue extracts in 0·3 M citrate buffer containing 10 mM EDTA and 0·2% (v/v) 2-mercaptoethanol at pH 7·4 by clarification with 8·5% (v/v) n-butanol followed by differential centrifugation and sucrose density gradient centrifugation. Purified particles measured c. 720 × 12 nm. Virus particle antigen was readily detected in leaf and tepal extracts of tulip by enzyme-linked immunosorbent assay. A distant serological relationship was found between particles of TCBV and those of bean yellow mosaic virus but no serological relationship was found to TBV or four other potyviruses.     

Purification and properties of cassava green mottle, a previously undescribed virus from the Solomon Islands

A sap-transmissible virus obtained from cassava with a green mottle disease occurring at Choiseul, Solomon Islands, was transmitted to 30 species in 12 plant families and was readily seed-borne in Nicotiana clevelandii. In cassava plants infected by inoculation with sap, the first leaves to be infected systemically developed a mottle with some necrosis whereas leaves produced subsequently were symptomless but contained the virus. Most other species developed chlorotic or necrotic local lesions and systemic mottle or necrosis. This was followed, in several species, by production of small symptomless virus-containing leaves. The virus was cultured in N. clevelandii; Chenopodium quinoa was used for local-lesion assays. Leaf extracts from infected N. clevelandii were infective after dilution to 10–⁵ but usually not at 10–⁶, after heating for 10 min at 60°C but not at 65°C, and after storage at 20°C for at least 12 days. The virus has isometric particles of 26 nm diameter which sediment as three components, all containing a protein of mol. wt c. 53000. The two fastest sedimenting components respectively contain single-stranded RNA of mol. wt, estimated after glyoxylation, c. 2.9 × 10⁶ and 2.3 × 10⁶. Both RNA species are needed for infection of plants. In tests with antiserum prepared to purified virus particles, the virus was detected in cassava and N. clevelandii by gel-diffusion precipitin tests, by immunosorbent electron microscopy and by ELISA. Despite its similarity to nepoviruses, the virus did not react with antisera to 18 members of the group. It was named cassava green mottle virus and is considered to be a previously undescribed nepovirus.     

Purification and properties of Australian lucerne latent virus, a seed-borne virus having affinities with nepoviruses

An isolate of Australian lucerne latent virus (ALLV) from lucerne in New Zealand was mechanically transmitted to a few herbaceous hosts. It induced diagnostic symptoms in several species of the Chenopodiaceae, but was symptomless in most other hosts including lucerne and Trifolium subterraneum. It was seed transmitted in lucerne. When assayed to Chenopodium quinoa, infective C. quinoa sap lost infectivity after diluting to 10^-4, heating for 10 min at 55°C and storage for 4 days at 4°C. ALLV was purified from infected C. quinoa or pea plants by extracting sap in 0.1 m borate buffer (pH 7) containing 0.2% 2-mercaptoethanol and clarifying with 15% bentonite suspension, high and low speed centrifugation and sucrose density gradient centrifugation. Purified virus preparations contained isometric particles about 25 nm in diameter and sedimented as three virus components with sedimentation coefficients (s_20-w⁰) of 56 S, 128 S and 133 S. The 56 S component appeared to consist of nucleic acid-free protein shells. Polyacrylamide gel electrophoresis of virus preparations showed that ALLV contained a single protein species of mol. wt 55 000 and two RNA species of mol. wt 2.1 × 10⁶ and 2.4 × 10⁶. An antiserum to ALLV had an homologous titre of 1/256 to purified virus but failed to detect ALLV in infective sap of C. quinoa, pea or lucerne. Purified ALLV failed to react to antisera to 28 distinct isometric plant viruses including those to 10 nepoviruses.     

Some properties of pelargonium flower-break virus

A virus obtained from pelargonium cvs Irene and Paul Crampel appears to differ from any previously reported; although symptomless in most pelargonium cvs tested, it caused colour break in the flowers of two seedling clones. It seems uncommon in pelargoniums. The virus was readily transmitted by inoculation of sap, but not by Myzus persicae with short feeds, by dodder or through seed. It infected only fifteen of 100 species tested in six of thirty-five plant families. Pelargoniums were freed from the virus by heat-treatment. The virus remained infective after 10 min at 85 ^oC, 3 wk at 20 ^oC or 27 wk at 2 ^oC; it was infective at 1/500000 dilution of Nicotiana clevelandii or Chenopodium quinoa sap. Purified preparations were readily made by several methods, and contained isometric particles c. 30 nm diameter. Although a good antigen, the virus was serologically unrelated to any of forty-two isometric viruses. In immunoelectrophoresis, the virus moved as a single antigenic component towards the cathode. It gave a single, specific zone in density-gradient centrifugation, and one moving component (s⁰_{20 w}= 125 S) in analytical centrifugation. The virus contained one protein of mol. wt. c. 41000. The present cryptogram of the virus is (R)/*: */*:S/S:S/*, and the name pelargonium flower-break virus is proposed.     

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.     

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.     

Host plant reactions, physical properties and serology of three isolates of Andean potato latent virus from Peru

Three isolates of Andean potato latent virus (APLV) (Caj, Hu, Ay) each infected twenty-seven species of plants in the families Amaranthaceae, Chenopodiaceae, Cucurbitaceae and Solanaceae but differed somewhat in the symptoms they induced. Nicotiana bigelovii and N. clevelandii proved the most useful diagnostic hosts. Symptoms were sometimes produced by all three isolates in cultivated and wild potatoes. In sap from systemically infected N. bigelovii and N. clevelandii leaves, all three isolates remained infective when diluted to 10^-6 and when stored at room temperature for at least 3 wk. The thermal inactivation points were 65–70 °C for Hu and Ay, but 75–80 °C for Caj. All three isolates differed serologically from Col, the original isolate of APLV, forming spurs in gel diffusion tests. No serological difference was found between Hu and Ay, but both formed spurs in reciprocal reactions with Caj. The data from light absorption, particle morphology and protein molecular weight for Caj, Hu and Ay are similar to those reported for other tymoviruses. APLV was found widespread in Andean countries.     

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.     

Purification and properties of sweet potato mild mottle, a white-fly borne virus from sweet potato(Ipomoea batatas) in East Africa

A virus obtained from sweet potatoes in Kenya, Uganda and Tanzania was transmitted by inoculation of sap and by whiteflies (Bemisia tabaci). It infected forty-five of 119 plant species in fourteen of thirty-six plant families. It was propagated in Nicotiana glutinosa and N. tabacum, in which diagnostic symptoms of vein clearing, leaf curling and distortion developed. Cheno-podium quinoa was a good local lesion host. Different seedling lines of sweet potato differed greatly in their susceptibility to infection and in symptoms produced; some developed leaf mottling and were stunted, some were symptomless, and some appeared immune. The virus was transmitted by dodder (Cuscuta campestris) but not by aphids, or through seed of Ipomoea nil or N. clevelandii. Sweet potato sap contained strong inhibitors of infection, and a low concentration of virus. Virus-free cuttings of sweet potato were obtained by thermotherapy (4–5 wk at 35 °C), or by meristem-tip culture. The virus remained infective in sap of N. tabacum after dilution to 10-³, or after 10 min at 55 °C (but not 60 °C), 3 but not 7 days at 18 °C, or 42 but not 49 days at 2 °C. Infectivity was abolished by sonication or u.v. irradiation, by 2% formaldehyde or 2% tri-sodium orthophosphate, and was greatly decreased by 20 % CHC1₃ or 20 % ether. Purified virus preparations were obtained from N. tabacum by clarifying phosphate buffer extracts with n-butanol, virus precipitation with polyethylene glycol, and differential centrifugation. The virus sedimented as one band in density gradients, and produced a single sedimenting boundary in analytical centrifugation (s°20, w = 1555)- It contained one polypeptide species of mol wt 37700, and preliminary digestion experiments suggested a single-stranded RNA. Antisera prepared against the virus reacted specifically in precipitin tube tests with titres of 1/16384, but no serological relationships could be found between the virus and fourteen viruses of the potato virus Y group. Electron micrographs showed straight, filamentous particles c. 950 nm long when mounted in MgCl_a, but 800–900 nra long in EDTA. The present cryptogram is: (R/i):*/*:E/E:S/Al. This virus is probably the same as Sheffield's virus B.     

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.     

Some hosts and properties of narcissus latent virus, a carlavirus commonly infecting narcissus and bulbous iris

Narcissus latent virus (NLV) is common in many cultivars of narcissus and bulbous iris, but was detected in only one of nineteen cultivars of nerine. It induced symptoms in some narcissus cultivars, but inconspicuous infection in bulbous iris and nerine. NLV was not seed-borne in narcissus or Nicotiana clevelandii but was transmitted readily by aphids (Acyrthosiphon pisum, Aphis gossypii and Myzus persicae) in the non-persistent manner and by sap-inoculation to twelve of fifty-three species from three of sixteen families. Sap from N. clevelandii was infective after dilution to io^-3 but not io*, after 10 min at 65 but not 70 ^oC or after 3–4 days at 20 ^oC or 16–32 days at 2 ^oC. Purified virus preparations were obtained from infected N. clevelandii by clarification of buffered leaf extracts with diethyl ether and carbon tetrachloride, followed by one or two cycles of differential centrifugation and molecular permeation chromatography. NLV has filamentous particles c. i3times65onm which sediment as a single component (i^o_{20, w}= 156S). They contain c. 5% nucleic acid and a single polypeptide of mol. wt 32·6 × 10³. The biological and physical properties of NLV place it in the carlavirus group; it is serologically related to lily symptomless virus, but not to fourteen other authentic carlaviruses. NLV has the cryptogram */*:*/(5):E/E:S/Ve/Ap.     

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.     

Vector relationships and other characteristics of barley yellow striate mosaic virus (BYSMV)

Barley yellow striate mosaic virus (BYSMV) was inoculated by its planthopper vector Laodelphax striatellus (Homoptera, Delphacidae) to 44 species of Gramineae, 26 of which in eight tribes were infected. The virus was not transmitted through wheat seed nor did it infect five dicotyledonous hosts of other rhabdoviruses. The most susceptible species were in the tribes Festuceae and Hordeae. Barley, Bromus spp., oats, Phalaris canariensis, Setaria italica, Sorghum spp., and sweet corn cv. Golden were diagnostic hosts. Electron microscopy of crude sap was also a sensitive diagnostic method. Properties of BYSMV were determined by injecting L. striatellus with crude sap from infected barley. Sap was infectious after 10 min at 50–55 °C but not after 10 min at 60 °C, when diluted with buffer to 10^--² but not to 10^--³, when stored for 2 but not 4 days at 5 °C or when kept for 1 but not 2 days at 22 °C. The planthopper Javesella pellucida was an experimental vector of BYSMV but the virus was not transmitted by the leafhoppers Macrosteles sexnotatus or Psammotettix striatus (Homoptera, Cicadellidae). The latent period of BYSMV in L. striatellus was most commonly 15 or 16 days (minimum, 9 days; maximum, 29 days). The minimum acquisition access period for transmission was between 1 h and 5 h, and the minimum inoculation feeding time was 15 min. After 24 h and 8 day acquisition feeds, 30.4% and 42.8% respectively of L. striatellus transmitted BYSMV. When transferred daily, infective hoppers transmitted virus intermittently. The maximum retention of infectivity by L. striatellus was 36 days. Two of five infective females transmitted BYSMV transovarially. Larvae became infective in the second wk after hatching and transmitted for up to 3 wk.