Identification of Rubus yellow net virus as a distinct badnavirus and its detection by PCR in Rubus species and in aphids

Rubus yellow net virus (RYNV) infects Rubus species and cultivars worldwide and is an essential component of raspberry veinbanding mosaic (RVBMD), a virus disease complex that causes serious decline in plant vigour and productivity. The virus is transmitted, probably in a semi‐persistent manner, by the large raspberry aphid, Amphorophora idaei in Europe, and A. agathonica in North America. The particles of RYNV are bacilliform in shape and measure 80–150 × 25–30 nm, similar to those of badnaviruses. A1.7 kb fragment of the viral DNA was amplified by PCR and then directly sequenced. Analysis of this sequence suggests that RYNV is possibly a distinct species in the genus Badnavirus and is most closely related to Gooseberry vein banding associated virus (GVBAV) and Spiraea yellow leaf spot virus, two other badnaviruses described recently. Using the sequence derived from the PCR‐amplified viral DNA fragment, RYNV‐specific primers were designed and used in PCR to assay for RYNV in a range of Rubus germplasm infected with RYNV, with other unrelated viruses and virus‐like diseases found in Rubus, and in healthy plants. RYNV was detected in all glasshouse cultures of RYNV‐infected plants, whether alone or in complex infections with other viruses, but not from healthy Rubus plants, nor from plants infected with other viruses. It was also detected in field‐grown raspberry plants with and without symptoms of RVBMD and in raspberry plants infected with RYNV by viruliferous A. idaei. RYNV was also detected by PCR in A. idaei following access feeds on RYNV‐infected plants of 1 h or more. PCR failed to amplify DNA from gooseberry infected with GVBAV confirming the specificity of the RYNV analysis. PCR detection of RYNV in dormant raspberry buds allows assays to be made outside the natural growing season, providing a useful application for plant introduction and quarantine programmes.     

Association of different kinds of bacilliform particle with vein chlorosis and mosaic diseases of raspberry (Rubus idaeus)

Thin sections of diseased raspberry (Rubus idaeus) were examined by electron microscopy. Plants of the cv. Baumforth's B and of an aphid (Amphorophora rubi)-resistant breeding selection (6820/54), both infected with raspberry vein chlorosis virus (RVCV) but not with other detectable viruses, contained large bacilliform particles c. 430 × 65 nm. Particles occurred in the cytoplasm and perinuclear space of a small proportion of xylem parenchyma cells. They had an inner core c. 25–30 nm in diameter with cross-banding of periodicity 4·5 nm, and were bounded by an outer membrane. They are probably the particles of RVCV. Plants of cv. Mailing Jewel and of a selection (M14) both showing symptoms of raspberry mosaic (veinbanding) disease contained smaller bacilliform particles c. 125 × 30 nm, which occurred singly or in clusters in the cytoplasm of a small proportion of vascular parenchyma cells. It is not known which, if any, of the viruses associated with raspberry mosaic are represented by the particles.     

Rubus host range of rubus yellow net virus and its involvement with other aphid-borne latent viruses in inducing raspberry veinbanding mosaic disease

After graft inoculation with rubus yellow net virus (RYNV), 12 of 34 Rubus species and cultivars developed noticeable symptoms. R. macraei developed the most conspicuous symptoms and is recommended as an improved indicator plant. In attempts to determine the cause of raspberry veinbanding mosaic, a disease in which RYNV is involved, several European and North American red raspberry cvs were graft-inoculated with RYNV and three other aphid-borne viruses, black raspberry necrosis (BRNV), raspberry leaf mottle (RLMV) and raspberry leaf spot, singly and in all combinations. In periods of up to 4 yr, classical veinbanding mosaic symptoms developed in sensitive cvs only when they contained both RYNV and RLMV. These symptoms were intensified in plants co-infected with additional viruses. Veinbanding mosaic disease did not develop in any of 11 cvs infected with RYNV + BRNV, the combination of viruses previously assumed to be responsible for this disease in Britain and North America.     

Rhabdovirus-like and closterovirus-like particles in ultrathin sections of Ribes species with symptoms of blackcurrant reversion and gooseberry veinbanding diseases

In attempts to determine the causal agents of blackcurrant reversion (BCRD) and gooseberry veinbanding (GVBD) diseases of Ribes species, details of the ultrastructure of different kinds of tissue from plants affected with these different diseases were studied. In three of 12 blackcurrant plants affected with BCRD, leaves and flowers of plants showing symptoms typical of the severe (R) form of the disease, contained rhabdovirus-like particles c. 65–80 nm × 215–485 nm. They were seen most often in the nucleus of cells as single particles but were also found in clusters or rafts. In leaves, these virus-like particles (VLPs) were present only in cells associated with the xylem parenchyma where they occurred as membrane-bound clusters within the nucleus. In flowers, they were also found in phloem parenchyma cells in the peripheral cytoplasm and very occasionally in the cytoplasm of epidermal cells. All non-nuclear VLPs were membrane-bound, either singly or in groups and the membrane seemed to be part of the endoplasmic reticulum. The proportion of vascular cells containing these VLPs was very low (< 1%). In a few cells, smaller bacilliform particles, c. 40–50 nm × 200–250 nm, were found in the nucleus together with the larger particles. Double-membrane bodies, detected in fig leaves affected with fig mosaic (the agent of which is also mite-transmitted), were not detected in any BCRD-affected plants. In leaf tissue of one of three gooseberry and one of two blackcurrant plants affected with GVBD, two kinds of VLPs were found. Rhabdovirus-like particles, similar to those in BCRD-affected material, were present in the nuclei, perinuclear space and cytoplasm of xylem parenchyma cells. They were c. 60–72 nm × 155–230 nm but there was no evidence of the smaller rhabdovirus-like particles detected in a few cells of BCRD-affected tissues. The second kind of VLP was found in noncrystalline masses, with a mean centre-centre spacing of c. 10 nm, in the cytoplasm of phloem cells. These particles, together with other ultrastructural changes, were typical of those reported for aphid-transmitted closteroviruses. No badnavirus-like particles, reported previously from GVBD-affected plants, were observed in any of the plants studied. The significance of these findings in relation to these two important diseases of commercial Ribes species is discussed.     

Unusual filamentous virus-like particles in symptomless blackberry associated with severe leaf curling in the virus indicator Rubus macraei, and anomalous data using a degenerate badnavirus primer in PCR of Rubus species

Electron microscopy of ultrathin sections of leaves of symptomless Himalaya Giant blackberry and of the virus indicator species, Rubus macraei, showing severe leaf curl symptoms following graft inoculation with scions from this blackberry, detected highly flexuous virus‐like particles with an unusual ‘beaded’ structure. Such particles were restricted to a few vascular cells and were distinct from P‐protein common in some such cells. This virus, provisionally named Hawaiian rubus leaf curl virus (HRLCV), symptomlessly infected a wide range of Rubus species and cultivars. Badnavirus‐like bacilliform particles were observed in some cells of a single R. macraei plant showing leaf curl symptoms following graft inoculation with the causal agent of this disease symptom from Himalaya Giant blackberry after passage through red raspberry, but not in any other material. PCR with primer sets for the badnaviruses Rubus yellow net virus and Gooseberry veinbanding associated virus, showed that no Rubus sources studied contained these viruses. However, using a sequence‐specific primer set designed from the sequence of the product generated with a badnavirus degenerate primer set, a specific product was amplified from healthy plants of all of 16 raspberry cultivars and two Rubus species, but not from 16 blackberry cultivars (including cv. Himalaya Giant). All of these sources were free from viruses known to occur in Rubus. Sequence analysis of this product showed no homology with any known badnavirus, or with any other published sequences. It seems most likely therefore that a region of the raspberry genome has been amplified using the degenerate badnavirus primer set and that it is absent from the blackberry genome.     

Rubus host range, symptomatology and ultrastructural effects of a British isolate of black raspberry necrosis virus

Scions from red raspberry (Rubus idaeus) plants naturally infected with an aphid- and sap-transmissible virus, code-named 52V, always induced apical necrosis in R. occidentals signifying the presence of black raspberry necrosis virus (BRNV), whereas plants free from 52V did not. These and other findings provide strong circumstantial evidence that 52V is an isolate of BRNV, the heat-labile member of the pair of viruses that together cause raspberry veinbanding mosaic disease. On grafting with R. idaeus scions containing a 52V culture of BRNV free from other detectable viruses, all of twenty-two red raspberry cultivars and four other Rubus species were infected symptomlessly but apical necrosis developed in R. henryi and R. molaccanus. Electron microscopy of thin sections of 52V-infected Chenopodium quinoa, R. henryii and R. occidentalis showed areas of dead cells in the vascular tissue and leaf blade. Some of the cells adjacent to these areas had cell wall outgrowths and many of the plasmodesmata contained virus-like particles c. 25 nm in diameter arranged in a single file.     

Studies on broccoli necrotic yellows virus

Electron microscopy of infected D. stramonium cells showed that the virus particles occurred in the cytoplasm. Particles were mostly bacilliform and measured 297 ± 18 times 64 ± 4 nm. In negatively stained leaf homogenates, particles were mostly disrupted; intact particles measured 267 ± 20 times 69 ± 6 nm. In brussels-sprout cells containing BNYV and CIMV, BNYV particles were rarely found compared with those of CIMV, and they occurred within and near CIMV inclusion bodies. BNYV particles were also found in extracts of virus-carrying Brevicoryne brassicae. Broccoli necrotic yellows (BNYV) and cauliflower mosaic (CIMV) viruses occurred together in naturally infected Brussels sprout plants, which showed conspicuous symptoms, and in cauliflower. BNYV was transmitted to and maintained in Datura stramonium and Hyoscyamus niger. It was partially purified from D. stramonium. Using these preparations, from which inhibitor had been removed, BNYV was manually transmitted to cauliflower, causing mild symptoms, and to Brussels sprout, causing a symptomless infection. BNYV also infected Sinapis alba but not cabbage, lettuce, Sonchus oleraceus or Plantago major. BNYV was transmitted by Brevicoryne brassicae but not by Myzus persicae, Hyperomyzus lactucae or Aleyrodes proletella.     

Particle purification and properties of cassava Ivorian bacilliform virus

A virus found in cassava from the north-west of the Ivory Coast was transmitted by inoculation with sap extracts to herbaceous species in six plant families. Chenopodium quinoa was used as a propagation host and C. murale was used for local lesion assays. The virus particles are bacilliform, c. 18 nm in diameter, with predominant lengths of 42,49 and 76 nm and a structure apparently similar to that found in alfalfa mosaic virus. Purified preparations of virus particles had A₂₆₀/A₂₈₀ of 1.7 ±0.05, contained one protein of M_rc. 22 000, and yielded three species of RNA with M_r (× 10^-6) of c. 0.7, 0.8 and 1.2. Although the virus particles were poorly immunogenic, an antiserum was produced and the virus was detected by enzyme-linked immunosorbent assay (DAS-ELISA) in leaf extracts at concentrations down to c. 6 ng/ml. Four other field isolates were also detected, including a strain which caused only mild systemic symptoms in C. quinoa instead of necrosis. The naturally infected cassava source plants were also infected with African cassava mosaic virus (ACMV) but when the new virus was cultured in Nicotiana benthamiana, either separately or together with ACMV, its concentration was the same. The new virus did not react with antisera to several plant viruses with small bacilliform or quasi-bacilliform particles, and alfalfa mosaic virus reacted only weakly and inconsistently with antiserum to the cassava virus. The new virus, for which the name cassava Ivorian bacilliform virus is proposed, is tentatively classified as the second member of the alfalfa mosaic virus group.     

A probable rhabdovirus infection of lemon-scented thyme

Bacilliform particles were found in the perinuclear space of phloem parenchyma cells of Thymus xcitriodorus (lemon-scented thyme) showing a marked leaf chlorosis. They were of typical rhabdovirus morphology, of mean dimensions 219 X72nm and composed of a bullet-shaped internal component surrounded by an envelope. A few unenveloped internal components were seen in the nucleus and the perinuclear space. The bacilliform particles were concentrated and partially purified from leaf material but were not easily detected by negative staining of leaf squashes. They were not transmitted by inoculation of infected sap and the evidence indicates that they were not the causal agent of the leaf chlorosis.     

Morphology and development of rhabdovirus-like particles inCuscuta odorata (Convolvulaceae) simultaneous infected with virus and mycoplasma-like organisms

Summary Electron microscopic examination ofCuscuta odorata, used for transmission trials, revealed mycoplasma-like organisms (MLO) as well as rhabdovirus-like particles, unknown toCuscuta. The virus infection is confined to certain phloem-parenchyma cells and a 1–2 cell thick layer of parenchyma cells with thickened walls surrounding the central cylinder. Virus particles, mostly bacilliform, could be detected mainly in the nucleus but also in the cytoplasm. They reach a length of 350–400 nm and a diameter of approximately 75 nm. Virus assembly takes place exclusively in the nucleus. Virus maturation occurs in membrane bound areas within the nucleus, which have no connection with the perinuclear space. Formation of nucleocapsids is always associated with a nuclear viroplasm. Envelopment of virus particles occurs in these membrane bound areas. Budding into the perinuclear space does not occur. Virus infection leads to degeneration and finally to death of the protoplast.Abbreviations cy cytoplasm - m membrane stacks - mt mitochondria - my mycoplasma-like organisms - nc nucleocapsid - ncp nucleocapsid particles - nf nuclear filaments - np nucleoplasm - nu nucleus - nvp nuclear viroplasm - oc obliterated cells - p plastid - pc passage cells - ph phloem - ps perinuclear space - spc strand of parenchymatous cells - v virus particle - x xylem     

Cytopathology of Plants Infected with Viruses. Ultrastructure of the Leaf Cells of Cereals Affected by Cereal Rhabdoviruses

Ultrathin sections of oat, wheat, and ryegrass leaves from healthy plants and plants infected with rhabdoviruses by leafhoppers Laodelphax striatellusFallen were studied under the electron microscope. The bacilliform virions often surrounded by endoplasmic reticulum (ER) membranes, viroplasm, and tubular structures conforming, in diameter and structure, to the rhabdovirion nucleocapsid were observed in the cytoplasm of leaf cells of the diseased plants. The cereal pseudorosette virus [(165–200) × (63–70) nm, CPV] is the causative agent of the disease of cereals in Siberia. The mycoplasma-like organisms were found in the phloem cells of plants infected with CPV. The cereal mosaic virus [(360–420) × (56–64) nm, CMV] is the causative agent of the disease of cereals in the Russian Far East. CMV appears to be a strain of the northern cereal mosaic virus.     

Effects of mist irrigation on the physiology of sugar beet

Bacilliform particles characteristic of plant rhabdoviruses were found in negatively-stained leaf sap and in thin sections of Laburnum anagyroides in England showing vein yellowing. The particles were detected principally in the perinuclear space of parenchyma cells. They were not transmitted by sap inoculation to twelve herbaceous species. The affected trees also showed mosaic symptoms but there was no evidence of an association between these and the bacilliform particles.     

The Abutilon Mosaic Virus (AbMV) in Different Leaf Tissues of Several Host Species of Malvaceae

Cytological changes occurring after infection by Abutilon Mosaic Virus (AbMV) have been investigated in the host plants Abutilon sellovianum Rgl., Malva parviflora L., Malva silvestris L., and Sida micrantha Schr. Two types of virus-like inclusions were found in different tissues: particles of 16–18 nm in diameter and elongated filaments with 6–8 nm in diameter. The particles are detectable in the nuclei (Abutilon sellovianum, Malva spec.) and cytoplasm (Malva spec.) of cells in the bundle area, and the filaments accumulate in the nuclei (Sida micrantha) and the plastids (Malva spec.) of leaf mesophyll cells.     

Genetic control of the reactions of raspberry to black raspberry necrosis, raspberry leaf mottle and raspberry leaf spot viruses

Black raspberry necrosis virus (BRNV) induces a severe apical necrosis in black raspberry (Rubus occidentalis) but fails to induce diagnostic symptoms in red raspberry. However, BRNV infection of F₁, F₂ and F₃ hybrids from the cross black raspberry × red raspberry induced mosaic symptoms of varying intensity but no typical apical necrosis. In a survey of 28 red raspberry cultivars, a few developed severe angular chlorotic leaf spots when infected with raspberry leaf mottle virus and a few others did so when infected with raspberry leaf spot virus. These reactions were determined by single dominant genes designated Lm and Ls respectively. The value of the different host reactions for controlling the effects and spread of these viruses is discussed.     

Leafhopper transmission of a virus causing maize wallaby ear disease

A virus causing maize wallaby ear disease was transmitted experimentally by Cicadulina bimaculata to fourteen species of monocotyledonous plants. It was also transmitted by Nesoclutha pallida, and by grafting. The symptoms obtained resemble closely those reported for maize leaf gall disease in the Philippines and maize rough dwarf virus in Italy and Israel. About 85% of C. bimaculata caught in the field carried maize wallaby ear virus (MWEV), and many of their progeny were viruliferous even when not allowed access to infected plants. The proportion of infective individuals in clones bred for nine generations from selected non-transmitting adults decreased from 85% in the first nymphs to less than 1%; such individuals were difficult to rear, as their fecundity and longevity decreased greatly. N. pallida transmitted MWEV after injection with partially purified extracts of infected plants. Spherical particles c. 85 nm in diameter were found in the salivary glands of viruliferous C. bimaculata, but not in those of non-transmitting individuals. The particles occurred in tubules in the cytoplasm and each had a densely stained core c. 50 nm in diameter. Particles similar in size to the core were found in extracts of infected but not uninfected maize, and in extracts of viruliferous but not in non-viruliferous C. bimaculata and N. pallida.     

Properties of broad bean yellow band virus, a possible new tobravirus

A virus was transmitted from broad bean plants in Apulia (Southern Italy) with leaves showing yellow rings, line patterns or yellow vein banding and malformations and necrosis of pods. Symptoms in some, but not all, test plants were similar to those induced by tobraviruses. Purified virus preparations contained two classes of rod-shaped particles containing c. 5% nucleic acid with sedimentation coefficients of 186S and 276S. After centrifugation to equilibrium in CsCl gradients, two components were resolved, with buoyant densities of 1·298 and 1·316 g/cm³. Unfractionated virus preparations contained two species of single-stranded RNA with mol. wts of c. 1·06 × 10⁶ and 2·48 × 10⁶ and one species of coat protein with mol. wt of c. 21 300. The modal lengths of the two classes of particles, both in plant sap and in purified preparations, were 77 nm (S particles) and 202 nm (L particles). L particles accumulated in infected cells in paracrystalline aggregates, whereas S particles were randomly distributed in the cytoplasm of cells. The virus was serologically unrelated to two isolates of tobacco rattle virus and two isolates of pea early-browning virus. The virus, named broad bean yellow band, is considered a distinct tobravirus.     

Further properties of black raspberry latent virus, and evidence for its relationship to tobacco streak virus

Purified preparations of an isolate of black raspberry latent virus (BRLV) contained quasispherical particles with a mean diameter of 28·5 nm; these particles were resolved into three sedimenting components (s_{20, w}= 82S, 95S and 104S), but when centrifuged to equilibrium in caesium chloride solution they formed a single infective band (σ= 1·35 g/cm³). During electrophoresis in polyacrylamide gels, virus particles separated into three classes, and virus RNA was resolved into three major (mol. wt 1·35, 1·10 and 0·85 × 10⁶) and one minor (mol. wt 0·4 × 10⁶) component. The protein from virus particles had an estimated mol. wt of 28000. Isolates of BRLV were found to be serologically related but not identical to some strains of tobacco streak virus. No symptoms developed in black raspberry seedlings infected with BRLV by mechanical inoculation, nor in eight red raspberry cultivars infected by graft inoculation. However, graft inoculation of BRLV to Rubus henryi, R. phoenicolasius and Himalaya blackberry induced symptoms typical of necrotic shock disease.     

Virus-like particles in phloem tissue of chervil (Anthriscus cerefolium) infected with carrot red leaf virus

Isometric virus-like particles c. 22–25 nm in diameter were found in ultrathin sections of chervil leaves infected with carrot red leaf virus (CRLV). The particles were confined to the phloem and occurred in less than 5% of the cells in the vascular bundles. They were commonest in companion cells, occurred frequently in sieve elements and were also found in phloem parenchyma cells. The observations support other evidence that CRLV should be classified in the luteovirus group.     

Fine Structure of Healthy and TMV-Infected Tobacco Cells Grown in Tissue Culture

Three types of tobacco (Nicotiana tabacum cv. Havana 38) callus: 1) healthy stem callus, 2) TMV-infected stem callus, 3) TMV-infected leaf callus; and leaves differentiated from healthy stem callus, and from TMV-infected leaf callus were compared for fine structure. In addition, the fine structure was observed of plastids in cells of leaves differentiated from callus isolated from stem sections of TMV-infected hybrid tobacco plants (N. tabacum cv. Havana 38 ×N. glutinosa) grown under high temperature. The cytoplasmic organelles in tissue cultured cells were similar to those in cells of greenhouse-grown tobacco plants. Except for plastids, TMV infection did not noticeably affect morphologically other cellular organelles in tissue culture cells. In TMV-infected leaf callus, numerous small bodies were seen in plastid-like bodies, while vesicle-like structures were observed in the stroma of plastids in leaves differentiated from callus of hybrid tobacco inoculated with TMV. Morphological variations of mitochondria, such as swelling and vacuolization of the inner matrix, occurred frequently in TMV-infected leaf callus. Needle-like crystalline inclusions or looped inclusions composed of many fine, long filaments were considered TMV particles orientated parallel to each other. The TMV particles were detected in the cytoplasm of tissue culture cells.     

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.