Hibiscus latent ringspot virus, a newly recognised virus from Hibiscus rosa-sinensis (Malvaceae) in western Nigeria

Hibiscus latent ringspot virus (HLRV) was prevalent in Hibiscus rosa-sinensis in Ibadan, Nigeria. It was readily transmitted mechanically to 22 of 73 species from seven of 20 families, but was best propagated in Nicotiana clevelandii or Hibiscus cannabinus and assayed in Chenopodium murale. HLRV was readily purified from systemically infected hosts by differential centrifugation of leaf extracts clarified with 8.0% n-butanol, followed by molecular permeation chromatography on controlled-pore glass beads (700 Å, 120–200 mesh). The virus has isometric particles c. 28 nm in diameter which sedimented as three components (T, M and B), with sedimentation coefficients (s°_{20, w}) of 51; 114 and 132 S and buoyant densities in caesium chloride of 1.32, 1.49 and 1.52 g/cm³, respectively. All three components contained a single polypeptide of rnol. wt 53.6 × 10³. T component particles contained only protein but M and B components also contained single-stranded RNA of rnol. wt 2.2 × 10⁶ and 2.5 × 10⁶, respectively. The properties of HLRV suggest affinities with nepoviruses but no serological relationship was detected between HLRV and 15 recognised or possible members of the nepovirus group.     

Crimson clover latent virus - a newly recognised seed-borne virus infecting crimson clover (Trigolium incarnatum)

Crimson clover latent virus (CCLV) was detected in five seed lots of crimson clover (Trifolium incarnatum) from Europe and in one from the United States of America. Ninety-seven per cent of all crimson clover plants examined were found to be infected but were without symptoms. Keeping crimson clover plants at 32–38°C for 34 days failed to free them from CCLV. The virus was not transmitted by Myzus persicae, but was transmitted by inoculation of sap to Chenopodium album, C. amaranticolor and C. quinoa. Twenty-four other plant species from seven families were not infected. CCLV was best propagated in C. quinoa in which it caused stunting and systemic chlorosis. Sap from infected C. quinoa was infective after dilution to 10^-2 but not 10^-3, after 10 min at 60°C but not 65°C, and after 20 days at 20°C. In neutral phosphotungstate, CCLV had isometric particles c. 26 nm in diameter with a hexagonal profile. About 20 to 80 A_1cm,260 units of purified virus were obtained from 1 kg of infected C. quinoa or C. amaranticolor leaves by extraction in 0.5 M phosphate buffer, pH 7.5, containing 0.01 M ethylene diamine tetra-acetate and 0.4% 2–mercaptoethanol and clarification with chloroform-butanol followed by two precipitations with polyethylene glycol (mol. wt 6000) and several cycles of differential centrifugation. Purified virus sedimented as three components with sedimentation coefficients (s°_{20, w}) of 52S, 101S and 122S. The 101S and 122S components had buoyant densities in CsCl of 1.438 and 1.495 g/cm³ respectively. From these values the nucleic acid content of the 101S and 122S components was estimated to be 32–35% and 40–41% respectively. The virus contained a single protein with an estimated mol. wt of 52 000 and two single-stranded RNA species of estimated mol. wt 1.6 × 10⁶ and 2.2 × 10⁶. CCLV was serologically unrelated to 31 other morphologically similar viruses. Although its vector is unknown, CCLV seems to have affinities with nepoviruses. The cryptogram of CCLV is R/1:2.2/40–41 + 1.6132–35:S/S:S/*.     

Ullucus virus C, a newly recognised comovirus infecting Ullucus tuberosus (Basellaceae)

Ullucus virus C (UVC) is a comovirus prevalent in Ullucus tuberosus grown at high altitudes in the Bolivian and Peruvian Andes. It was transmitted mechanically to U. tuberosus (Basellaceae) and to five of 26 species from three of eight other families, infecting U. tuberosus symptomlessly but inducing conspicuous systemic infection in Chenopodium amaranticolor and C. quinoa. Sap from infected C. quinoa was usually infective after 10 min at 70 but not 75 °C, after dilution to 10^-7 but not 10^-8, and after 8 but not 16 wk at 20 °C. UVC was not transmitted by either of two aphid species (Aphis gossypii and Myzus persicae) or through seed of C. quinoa, but it was transmitted by leaf contact between infected and healthy plants. UVC has isometric particles which, in neutral phosphotungstate, are c. 28 nm in diameter. The particles sediment as three components (T, M and B) with sedimentation coefficients (s^?_{20, w}) of 51 S (T), 95 S (M) and 116 S (B). M component particles have a buoyant density (g cm^-3) in caesium chloride of 1.404, and B component particles separated into minor and major sub-components with densities of 1.409 and 1.463, respectively. T, M and B particles were serologically indistinguishable, and each contained similar relative amounts of two polypeptides of mol. wts 20 700 and 45 100. T particles contained only protein, but M particles also contained c. 30% ss-RNA of mol. wt 1–45 ×10⁶ and B particles c. 38% ss-RNA of mol. wt 2·2 × 10⁶. The virus is serologically distantly related to cowpea mosaic virus but, as it showed no relationship to any of 11 other similar viruses, it is probably a distinct member of the comovirus group.     

Olive latent virus-1, an isometric virus with a single RNA species isolated from olive in Apulia, Southern Italy

A mechanically transmissible virus isolated from some olive trees growing in Apulia, Southern Italy is described under the name of olive latent virus-1 (OLV–1). Virus particles (c. 30 nm in diameter) sedimented as a single component (11 IS) and contained a major single-stranded RNA species which when denatured had a size of about 4100 nucleotides and up to seven smaller RNAs. When centrifuged to equilibrium either in CsCl or CS2SO4 gradients, virus preparations were homogeneous. In polyacrylamide gel electrophoresis, about 50% of virus coat protein preparations formed a band of mol. wt 32 000 (32K protein) but two other bands of mol. wt 36 000 (36K protein) and 65 000 (65K protein) and some diffuse bands were also observed. The 32K protein, its probable dimer (65K protein) and diffuse bands are regarded as components of virus coat protein, whereas the 36K protein is probably a contaminant from the host plant. Virus particles were stabilised by divalent cations, pH-dependent bonds and salt links between protein and RNA. EDTA-treated virions were sensitive to ribonuclease and SDS. OLV-1 was serologically unrelated to four tombusviruses, seven sobemoviruses or putative sobemoviruses, and seven other single component isometric plant viruses.     

Arracacha virus A, a newly recognised virus infecting arracacha (Arracacia xanthorrhiza; Umbelliferae) in the Peruvian Andes

Arracacha virus A (AVA), a previously undescribed virus, is common in arracacha (Arracacia xanthorrhiza; Umbelliferae) in the Huanuco region of the Peruvian Andes. AVA was not transmitted by Myzus persicae, but was transmitted by inoculation of sap to 38 species from 10 families out of 63 species from 12 families tested. AVA was best propagated and assayed in Chenopodium quinoa and Nicotiana clevelandii in which it caused severe diseases. Sap from infected C. quinoa was occasionally infective after dilution to 10^-4 but not 10^-5, after 10 min at 65 °C but not 70 °C, and after 15 days at 20 °C. In neutral phosphotungstate, AVA has isometric particles c. 26 nm in diameter with a hexagonal profile, some of which were either fully or partially penetrated by the negative stain. Up to 50–200 E₂₆₀^1cm units of purified virus was obtained from 1 kg of infected N. clevelandii leaf by extraction in 0.05 M phosphate buffer at pH 7.5 containing 0.05 M ethylene diaminetetra-acetate, and clarification with chloroform, followed by differential precipitation with ammonium sulphate and three cycles of differential centrifugation. Purified virus sedimented as three components with sedimentation coefficients (S_20w^°) of 50 S, 92 S and 125 S and E₂₆₀/E₂₈₀ ratios of 0.65, 1.50 and 1.85 respectively. At equilibrium in CsCl gradients, buoyant densities of the 50, 92 and 125 S components were 1.32, 1.45 and 1.52 g/cm³ respectively. From the sedimentation coefficients and buoyant densities, the nucleic acid contents of the 92 S and 125 S components were estimated at 30–35% and 43–44% respectively. Only the 125 S component seemed to be infective but its infectivity was greater when mixed with the 92 S component. All three components contained a single protein with a molecular weight of 53 000. AVA was not serologically related to any of 33 other morphologically similar viruses. Although the vector is unknown, its properties suggest that it is a member of the nepovirus group. The cryptogram of AVA is */*: */43–44 +*/30–35: S/S:S/*.     

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.     

The occurrence, hosts and properties of lilac chlorotic leafspot virus, a newly recognised virus from Syringa vulgaris

Lilac chlorotic leafspot virus (LCLV), a hitherto undescribed virus, was isolated from three of 65 lilacs (Syringa vulgaris) with chlorotic leafspotting symptoms growing in S.E. England. The virus was transmitted readily by sap-inoculation to 21 of 52 species from eight of 20 families, but it was not seed-borne in four hosts or transmitted in the semi-persistent manner by any of four aphid species. The virus was moderately stable in vitro; sap from Chenopodium quinoa was infective after 10 min at 60 but not 65 ^oC, after 8–16 days at 20 ^oC or 25–30 wk at 2 ^oC, and after dilution to 10^-3 but not 10^-4. Up to 180 mg of purified virus per kg leaf tissue were obtained from C. quinoa by clarification of buffered leaf extracts with 8% (v/v) n-butanol, followed by one cycle of differential centrifugation and molecular permeation chromatography on controlled pore glass beads (700 Å, 120–200 mesh). LCLV has fragile flexuous filamentous particles which, when intact, mostly measured c. 12-5 times 1500–1600 nm; the helical substructure (pitch c. 3–7 nm) was clearly visible on some particles mounted in uranyl acetate. The particles sedimented as a single component (sedimentation coefficient 96 S; buoyant density 1–302 g cm^-3) and contained c. 5% nucleic acid and a single polypeptide of mol. wt 27 times 10³. Although these properties place LCLV in the closterovirus group, the virus showed no serological relationship to any of six closteroviruses (beet yellows, beet yellow stunt, carnation necrotic fleck, apple chlorotic leafspot, apple stem grooving and potato virus T) and differed from other recognised or possible members of this group in host range and/or symptoms induced in indicator species. The infrequent occurrence of LCLV in lilac in S.E. England indicates that the virus could probably be eradicated by selecting only virus-free plant material for propagation.     

Cowpea mild mottle, a newly recognized virus infecting cowpeas (Vigna unguiculata) in Ghana

Cowpea mild mottle virus (CMMV), a previously undescribed virus widespread in cowpeas (Vigna unguiculata) in the Eastern Region of Ghana, was seed-borne in V. unguiculata, Phaseolus vulgaris and Glycine max, but was not transmitted by twelve aphid species including Aphis craccivora, A. fabae, Acyrthosiphon pisum and Myzus persicae. CMMV was transmitted by inoculation of sap to eleven of seventeen members of the Papilionaceae causing very severe diseases in G. max and Arachis hypogaea, and to ten of fifty-one species within five of nineteen other families; it was best propagated in G. max and Nicotiana clevelandii, and assayed in Chenopodium quinoa. Sap from systemically infected G. max was infective after dilution to 10^-3 but not 10^-4, after 10 min at 65 °C but not at 70 °C, or after 4 days at 18 °C or 16 days at 2 °C. Lyophilized sap was infective after 3 years in vacuo. CMMV has straight to slightly flexuous, fragile filamentous particles, c. 13 × 650 nm which, in sap, are occasionally surrounded by a loose external spiral. About 5 mg of purified virus was obtained from 1 kg of leaf tissue of G. max or N. clevelandii by clarifying leaf extracts in 0.02 m borate buffer (pH 9.5) with chloroform, followed by two or three cycles of differential centrifugation, and density gradient centrifugation. Virus preparations had ultraviolet absorption spectra typical of a nucleoprotein containing c. 5 % nucleic acid, contained numerous particles without external spirals, which sedimented as a single component with a sedimentation coefficient (s°_{20, w}) of 165 × 4S, and contained a single polypeptide species with a molecular weight of 32000–33000. CMMV showed a distant serological relationship to carnation latent virus, but not to ten other morphologically similar viruses; it thus seems to be a distinct member of the carlavirus group, and has the cryptogram: */*:*/(5):E/E:S/*.     

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.     

The transmission of strawberry latent ringspot virus by Xiphinema diversicaudatutn (Nematoda)

Strawberry latent ringspot virus (SLRV) was found in diseased rose bushes growing in a glasshouse where the soil contained Xiphinema diversicaudatum (Micol). Adult female, adult male and juvenile X. diversicaudatum all transmitted the virus to cucumber seedlings, and nematodes kept without plants for 32 days after acquiring SLRV later transmitted it. When transferred to fresh plants every 2–4 days for 3 weeks, single nematodes transmitted up to three times; one nematode did not transmit until 19 days after the transfers began. One adult X. diversicaudatum, out of 141 tested, transmitted both SLRV and arabis mosaic virus. In all respects X. diversicaudatum behaved as a vector of SLRV as it does as a vector of arabis mosaic virus.     

Properties, relationships and transmission of a strain of raspberry ringspot virus infecting raspberry cultivars immune to the common Scottish strain*

Plants of Lloyd George and Seedling M raspberry (Rubus idaeus L.) were found in eastern Scotland infected with raspberry ringspot (RRV), a virus to which these varieties were previously considered immune. Most RRV isolates from affected plants caused milder symptoms in herbaceous test plants than did the type isolates of the common Scottish and English strains. In graft-transmission tests the Lloyd George strain of RRV infected all the raspberry cultivars tested, including those immune to the common Scottish strain. No consistent differences were found between isolates of the two strains in in vitro properties or serological behaviour. Both strains were transmitted in seed of Stellaria media and in soil containing Longidorus elongatus. Possible reasons why the new strain is uncommon in Scotland are discussed.     

Elderberry latent virus: its relationship to Pelargonium ringspot virus and its identification as a distinct member of the genus Carmovirus, family Tombusviridae

The properties of Elderberry latent virus (ELV) and Pelargonium ringspot virus (PelRSV) were compared. The viruses were largely indistinguishable in herbaceous host range and symptomatology, particle morphology, sedimentation coefficient and RNA profiles and size. They were also very closely related serologically with SDI differences in agarose gel double‐diffusion tests of 1 to 3. Purified virus particle preparations of each virus contained isometric particles c. 30 nm in diameter that sedimented as a major component with an s^O_20W of 112–115S. Purified virus particle preparations contained a major and a minor ssRNA species that in polyacrylamide gel electrophoresis (PAGE) had estimated sizes of c. 3.8 kb and c. 1.6 kb respectively. Plants of Chenopodium quinoa infected with ELV or PelRSV each contained three dsRNA species of c. 3.8, 2.6 and 1.8 kbp, although the smallest of these species was not evident in all preparations. Protein from purified virus particle preparations contained a major polypeptide that, in SDS‐PAGE, had an estimated M^r of 40 000 (40K). However, after storage of purified virus particles for 7–10 days, protein preparations from PelRSV particles also contained an additional major polypeptide of estimated M^r of 37 000 that is probably derived by degradation of the 40K protein; this additional component was not observed in freshly prepared preparations of ELV. Neither virus was found to be related serologically to 16 other viruses with isometric particles and similar properties. These data, together with the recent finding by other researchers that the smallest RNA species is a sub‐genomic RNA, suggests that both viruses are members of the genus Carmovirus, and that PelRSV is a minor variant of ELV. However, the taxonomic status of these two viruses is discussed in relation to recent brief reports comparing the nucleotide and amino acid sequences of these two viruses.     

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.     

Olive verticillium wilt or dieback of olive in Iran

During 2000--03, different areas in Zanjan, Golestan and Khorasan provinces were surveyed for the presence of olive dieback. Olive branches, leaves and roots showing typical symptoms and soil around the roots were collected for further study. Samples were surface-sterilized with sodium hypochlorite or ethanol and then cultured on PDA and Czapek media. Soil samples were diluted in ethanol-agar for fungal isolation and purification. Morphological characteristics of the fungal mycelium particularly phialide and spores identified the causal agent to be the soil-borne pathogen, Verticillium dahliae. The disease was present in all olive growing regions but it was severe in temperate and relatively humid regions such as Gorgan. Infection index of the disease varied between 5 to 30% with an average of 11.89+/-1.12 among various orchards in this area. The newly established orchards showed more infection than the older ones. A significant difference in disease incidence and severity were observed among olive cultivars of Michen, Roughani, Zard and Koronakei. The latter cultivar had the least amount of infection. Strains of V. dahliae isolated from olive trees had different morphological and pathogenicity characteristics. These strains had different growth rates in response to the optimum temperature of 20 or 25 degrees C. The number of fungal propagules per gram of air-dried soil ranged from 2 to 32 with an average number of 13.42+/-0.50. Regarding the number of propagules of V. dahliae in the soil and susceptibility of cultivars in the newly established orchards, it seems necessary to take serious control measures to prevent disease spread.     

Nucleotide sequence and newly formed phosphodiester bond of spontaneously ligated satellite tobacco ringspot virus RNA.

The satellite RNA of tobacco ringspot virus (STobRV RNA) replicates and becomes encapsidated in association with tobacco ringspot virus. Previous results show that the infected tissue produces multimeric STobRV RNAs of both polarities. RNA that is complementary to encapsidated STobRV RNA, designated as having the (-) polarity, cleaves autolytically at a specific ApG bond. Purified autolysis products spontaneously join in a non-enzymic reaction. We report characteristics of this RNA ligation reaction: the terminal groups that react, the type of bond in the newly formed junction and the nucleotide sequence of the joined RNA. The nucleotide sequence of the ligated RNA shows that joining of the reacting RNAs restored an ApG bond. The junction ApG has a 3'-to-5' phosphodiester bond. Thus the net ligation reaction of STobRV (-)RNA is the precise reversal of autolysis. We discuss this new type of RNA ligation reaction and its implications for the formation of multimeric STobRV RNAs during replication.