Detection of Sap-Transmissible Viruses by ELISA in Tissue-Propagated Plants of Red Raspberry during in vitro Culture

Apple mosaic virus and raspberry bushy dwarf virus were detected by ELISA in plantlets of red raspberry still growing in vitro. The plantlets were derived from explants which were excised from plants infected by either of the viruses mentioned. Detection by ELISA of prune dwarf virus in 4-month-old in vitro cultures of sour cherry was reported earlier. Thus, application of ELISA to tissue cultured plants in vitro seems to be an appropriate method for early detection of virus-infected plant cultures.     

Detection of Prune Dwarf Virus by ELISA in Meristem-Propagated Sour Cherry Plants During in vitro Culture

Tissue-cultured plantlets which were derived from meristems of a sour cherry plant infected by prune dwarf virus were examined by enzym-linked immunosorbent assay (ELISA). The virus could be detected with sufficient reliability in 17-week-old rooted plant-lets still growing in vitro.     

The incidence of virus diseases in English sweet-cherry orchards and their effect on yield

Two thousand sweet-cherry trees (Prunus avium) in English orchards were tested for virus infection by using Lambert and Mazzard F 12/1 as indicators. Most trees of varieties commonly grown before 1920 were infected with more than one virus, usually little cherry (69%) and necrotic ringspot/prune dwarf (56%). Other infection was less prevalent, 35% of these trees having European rusty mottle, 30% ring mottle and 3% necrotic rusty mottle. Most trees of varieties introduced since 1920 were virus-free (61%) but some had become infected with each of these viruses except necrotic rusty mottle. In a field trial of 12 years duration the yield of three varieties was diminished by infection with necrotic ringspot/prune dwarf, rugose mosaic, rusty mottle, ring mottle and necrotic line pattern. The effect of rusty mottle was due to growth suppression resulting in smaller trees, but that of other viruses was also due to impaired fertility. The yield of one variety (Merton Heart) was greatly enhanced by infection with rugose mosaic, rusty mottle and necrotic ringspot/prune dwarf viruses. The high incidence of virus infection and consequent yield depression has probably diminished the potential yield by at least 30% and contributed to the decline in acreage of sweet cherries in England.     

The isolation and identification of rhubarb viruses occurring in Britain

Virus-like symptoms were common in British crops of rhubarb. All plants tested of the three main varieties, ‘Timperley Early’, ‘Prince Albert’ and ‘Victoria’, were virus-infected. Turnip mosaic virus and a severe isolate of arabis mosaic virus (AMV) were obtained from ‘Timperley Early’; and ‘Prince Albert’ contained turnip mosaic virus, cherry leaf roll virus (CLRV), a mild isolate of AMV and, infrequently, cucumber mosaic virus (CMV). The main commercial variety ‘Victoria’ contained turnip mosaic virus, CLRV, a mild isolate of AMV and, infrequently, strawberry latent ringspot virus (SLRV). All the viruses were identified serologically. The rhubarb isolates did not differ markedly from other isolates of these viruses in herbaceous host reactions, properties in vitro or particle size and shape. A rhubarb isolate of CLRV was distinguished serologically from a cherry isolate of the virus. Turnip mosaic virus, CLRV and SLRV, were transmitted with difficulty, but AMV isolates were readily transmitted by mechanical inoculation. Turnip mosaic virus was also transmitted to rhubarb by Myzus persicae and Aphis fabae. CLRV was transmitted in 6–8% of the seed of infected ‘Prince Albert’ and ‘Victoria’ rhubarb and in 72% of the seed of infected Chenopodium amaranticolor. Mild isolates of AMV were also transmitted in 10–24% of the seed of infected ‘Prince Albert’ and ‘Victoria’ plants.     

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.     

Serological and biological characterisation of isolates of barley yellow dwarf virus in Sweden in 1983

In 1983, cereal plants showing symptoms of barley yellow dwarf virus (BYDV), collected from 15 localities in Sweden, were tested for BYDV using enzyme-linked immunosorbent assay (ELISA). Antisera against two Swedish isolates of BYDV were used, a mild isolate (27/77) transmitted specifically by Sitobion avenae and a severe one (39/78) transmitted mainly by Rhopalosiphum padi. No virus was detected in 57 of 607 plants of oats and barley tested. Of the 550 plants in which virus was detected, 366 were infected with viruses similar to isolate 27/77, 116 with viruses similar to 39/78 and the remaining 68 reacted strongly with both antisera. When tested, the latter isolates were shown to be mixtures. Thirty-nine selected samples were also tested with antisera against the USA isolates RPV, RMV, MAV and PAV, and for transmission by S. avenae and R. padi. Twenty-six of these samples were transmitted specifically by S. avenae, one was transmitted only by R. padi and the remaining 12 samples were shown to be infected with a mixture of an S. avenae-specific isolate and one transmitted mainly by R. padi. Antisera against PAV and MAV each detected all isolates tested and the results were very similar to those with the antisera to the 39/78 and 27/77 isolates, respectively. None of the field isolates reacted with antisera against RMV or RPV. It was concluded that 1983 was an epidemic year for BYDV in Sweden and that isolates specifically transmitted by S. avenae predominated. Symptoms of infection by these isolates on oat plants ranged from mild to severe.     

Hedgerow hawthorn (Crataegus spp.) and blackthorn (Prunus spinosa) as hosts of fruit tree viruses in Britain

Apple chlorotic leafspot virus (CLSV) was detected in 27 of 109 hawthorn and three of 67 blackthorn plants sampled in various parts of Britain. The CLSV isolates possessed similar properties to those isolated from other rosaceous species but differed in the severity of symptoms they induced in woody indicators. No seed or aphid transmission of CLSV was detected. Prunus necrotic ringspot (PNRV) and prune dwarf (PDV) viruses were detected in four and three respectively of 67 blackthorn plants. The PNRV and PDV isolates were serologically closely related to isolates from cherry. Arabis mosaic virus was detected in one blackthorn plant, but plum pox virus was not found in any of the tested plants.     

Seed-transmission of nematode-borne viruses

Transmission through seed of crop and weed plants seems to be characteristic of nematode-borne viruses. It occurred with tomato black ring virus (TBRV) in nineteen species (thirteen botanical families), with arabis mosaic virus (AMV) in thirteen species (eleven families), with raspberry ringspot virus (RRV) in six species (five families), and also, in more limited tests, with tomato ringspot, cherry leaf roll and tobacco rattle viruses. A remarkable feature was that infected seedlings, except those containing tobacco rattle virus, often appeared healthy. The occurrence and extent of seed-transmission depended on both the virus and the host plant. In many progenies more than 10%, and in some 100%, of seedlings were infected. The viruses were transmitted through at least two or three generations of seed of those host species tested. After 6 years' storage, TBRV- and RRV-containing seed of Capsella bursa-pastoris and Stellaria media germinated to give infected seedlings. In controlled crossing experiments with strawberry and raspberry, virus was transmitted to seed from both male and female parents but, at least in raspberry, the presence of competing virus-free pollen much decreased the ability of pollen from infected plants to set seed. There was no evidence that healthy mother plants became infected when their flowers were pollinated with infected pollen.     

A yellow mosaic disease of horse chestnut (Aesculus spp.) caused by apple mosaic virus

A severe foliar yellow mosaic disease was observed in horse chestnut trees (Aesculus carnea and A. hippocastanum). Reactions in woody indicator plants grafted with diseased horse chestnut suggested the presence of an ilarvirus. Virus isolates obtained by mechanical inoculation of herbaceous test plants reacted with antisera to apple mosaic virus but not with antisera to its serotype prunus necrotic ringspot virus, or to prune dwarf virus. Yellow mosaic was induced in horse chestnut seedlings grafted with tissues from herbaceous hosts infected with horse chestnut isolates or with the European plum line pattern isolate of apple mosaic virus. Virus was detected by enzyme-linked immunosorbent assay (ELISA) in embryo and endosperm of immature seed from infected trees but not in mature seed, or progeny seedlings. Strawberry latent ringspot virus was detected in one of six A. hippocastanum trees with a leaf vein yellows disease.     

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.     

Setting confidence limits for the detection of prune dwarf virus in Prunus avium with a monoclonal antibody-based triple antibody-sandwich ELISA†

A triple antibody sandwich enzyme-linked immunosorbent assay (TAS-ELISA) with a monoclonal antibody was developed and evaluated for the detection of prune dwarf virus (PDV) in sweet cherry trees (Prunus avium). An independent reverse transcribed polymerase chain reaction test was also developed to establish, in conjunction with a bioassay, the incidence of PDV in 40 sweet cherry trees and to confirm the absence of virus in 15 control trees. Trees with two-thirds of their leaves positive for PDV would be identified with 99% probability by testing four leaves per tree with TAS-ELISA. The monoclonal antibody did not cross-react with Prunus necrotic ringspot virus in the TAS-ELISA.     

Barley Yellow Striate Mosaic Virus as the Cause of a Major Disease of Wheat and Millet in Iran

Barley yellow striate mosaic virus (BYSMV) was identified in Iran by electron microscopy and serology. The virus was widespread in the Fars province causing mosaic, stunting and head sterility in wheat and mosaic symptoms in Setaria spp. In 1989, about 1/3 of wheat plants in the Bajgah Experiment Station, 15 km north of Shiraz, were infected. The yield in individual plants was drastically affected. Rhabdovirus particles were consistently observed in leaf-dip preparations and thin sections from the infected plants. The virus was transmitted by Laodelphax stritellus to wheat. It reacted with BYSMV antisera from Italy and Morocco but not with antisera to several other rhabdoviruses of gramineous plants.     

Increased sensitivity of detection of plant viruses obtained by using a fluorogenic substrate in enzyme-linked immunosorbent assay

The fluorogenic substrate 4-methylumbelliferyl phosphate (MUP) of alkaline phosphatase was compared with the chromogenic substrate p-nitrophenyl phosphate (NPP) in tests for plant viruses by enzyme-linked immunosorbent assay (ELISA). In tests on leaf extracts of squash infected with prune dwarf virus, Chenopodium quinoa and apple infected with apple mosaic virus (ApMV), and potato infected with potato leafroll virus (PLRV), MUP increased sensitivity 2–16 times, the smallest and greatest increases being obtained with ApMV (in apple) and PLRV respectively. In similar tests on 21 dormant PLRV-infected potato tubers, sensitivity was increased 2–4 times with 13 tubers, but the two substrates gave the same detection end-points with eight tubers. When individual seeds of potato plants infected with the Andean potato calico strain of tobacco ringspot virus were tested, the virus was detected in virtually all seeds by MUP-ELISA, but detection by NPP-ELISA was inefficient unless absorbance values were measured after overnight incubation at 4 °C, instead of after 2 h at room temperature. In tests on Myzus persicae carrying PLRV and Sitobion avenae carrying barley yellow dwarf virus (BYDV), both viruses were consistently detected in a greater proportion of individual aphids by MUP-ELISA than NPP-ELISA irrespective of whether incubation was for 2 h at room temperature or overnight at 4 °C. The effeciency of detection of virus in single viruliferous aphids by MUP-ELISA was not decreased by grouping with one or four non-viruliferous aphids but was decreased (PLRV) or greatly decreased (BYDV) by grouping with nine. MUP-ELISA and transmission tests to Physalis floridana seedlings (2–3 day inoculation access periods) both detected PLRV in most individual M. persicae, but the results obtained with the two methods did not correlate completely. In similar tests for BYDV in individual S. avenae, virtually all aphids transmitted BYDV to oat seedlings during a 3-day inoculation access period but it was subsequently detected by MUP-ELISA in less than half of them. By contrast, MUP-ELISA detected PLRV in most viruliferous M. persicae even after they had fed for 3 days on Chinese cabbage, a non-host for this virus.     

The effect of different virus isolates on the expression of tolerance to barley yellow dwarf virus in barley

A barley variety of Ethiopian origin, with a single Mendelian gene con-fering tolerance to barley yellow dwarf virus (BYDV), was equally tolerant to a number of isolates of the virus, whereas a susceptible European barley was more susceptible to isolates transmitted by Rhopalosiphum padi L. than to those transmitted by Macrosiphum (Sitobion) avenae (Fab). However, hybrids between these two varieties homozygous for the Ethiopian tolerance gene were more tolerant to ‘mild’ than to ‘severe’ isolates, irrespective of the vector specificity. The European variety was damaged more severely by all isolates when infected early than when infected late in its development, but the hybrids were damaged more severely by M. awraae-transmitted isolates when infected late. It is suggested that in susceptible plants the concentration, rather than the virulence, of the virus determines disease severity, whereas the reverse is true in plants possessing a gene which reduces virus multiplication. Virus concentration appears to determine the severity of R. padi-transmitted isolates, while virulence determines the severity of M. avenae-transmitted isolates. The latter would also seem to be adapted towards late infection.     

Subterranean clover mottle sobemovirus: its host range, resistance in subterranean clover and transmission through seed and by grazing animals

Subterranean clover mottle sobemovirus (SCMV) was transmitted by manual inoculation of sap to 27 cultivars belonging to three sub-species of subterranean clover. The virus readily infected systemically all inoculated plants of five susceptible cultivars of ssp. subterraneum. Ten others showed partial resistance as not all infected plants developed systemic infection; cold winter conditions further delayed or prevented systemic movement in four of them. Two cultivars of spp. brachycalycinum and four of spp. yanninicum failed to develop systemic infection following inoculation and were considered highly resistant. Resistance to SCMV in three of the spp. yanninicum was further confirmed by the failure to establish detectable primary infections in most of the inoculated leaves. Moreover, when the four ssp. yanninicum cultivars were graft-inoculated with SCMV, systemic infection eventually developed in them but the virus concentration was low. SCMV was also transmitted by manual inoculation of sap to a further 23 species of Trifolium, Medicago or Pisum. Three species were non-hosts, five were infected only in inoculated leaves and 18 others developed systemic infection in some or all plants. SCMV reached very high concentrations and was stable in subterranean clover sap. It was transmitted experimentally between subterranean clover plants by brushing infected leaves against healthy ones and in swards was readily transmitted by the trampling and grazing of sheep, but only poorly by mowing. Seed transmission of SCMV to seedlings of five cultivars of subterranean clover was low (0–0.12%). SCMV was not transmitted by Myzus persicae.     

The Natural Occurrence of Turnip Mosaic Potyvirus in Allium ampeloprasum

An isolate of turnip mosaic potyvirus (TuMV) was obtained from Allium ampeloprasum grown in commercial greenhouses in Israel. Symptoms on infected plants include systemic chlorosis and yellow stripes, accompanied by growth reduction. Leaves were distorted, often showing necrotic flecking. The virus was readily transmitted mechanically, and in a non-persistent manner by aphids, among Allium, Chenopodium. Gomphrena and some Nicotiana spp. Purified preparations contained numerous filamentous particles similar to those observed in crude extracts of infected leaves. Particles from crude plant extracts had a normal length of 806 nm. Cells of infected plants contained cylindrical cytoplasmic inclusions with pinwheel, scrolls and laminated aggregates which indicated the presence of a potyvirus of Edwardson's subgroup III. and which resemble those of turnip mosaic virus (TuMV), The virus reacted strongly with antiserum to typical isolates of TuMV in immunoelectron microscopy and western blotting but not with antisera to several other potyviruses. Based on serological reactivity, electron microscopy, aphid transmission and cytopathology, the virus was identified as an isolate of TuMV.     

Influence of rice black streaked dwarf virus on the ecological fitness of non-vector planthopper Nilaparvata lugens （Hemiptera： Delphacidae）

Rice black streak dwarf virus （RBSDV） is transmitted by the small brown planthopper （SBPH）, Laodelphax striatellus （Fallen）. Non-vector rice brown planthopper （BPH）, Nilaparvata lugens （Stal）, shares the same host rice plants with SBPH in paddy fields. The changes in nutritional composition of rice plants infected by RBSDV and the ecological fitness of BPH feeding on the infected plants were studied under both artificial climate chamber and field conditions. Contents of 16 detected amino acids and soluble sugar in RBSDV infected rice plants were higher than those in the healthy ones. On the diseased plants BPH had significantly higher nymphal survival rates, nymphal duration of the males, weight of the female adults, as well as egg hatchability compared to BPH being fed on healthy plants. However, there was no obvious difference in female nymph duration, longevity and fecundity. Defense enzymes （superoxidase dismutase, SOD and catalase, CAT） and detoxifying enzymes （carboxylesterase, CAE and glutathione S-transferase, GST） in BPH adults fed on diseased plants had markedly higher activities. The results indicate rice plants infected by RBSDV improved the ecological fitness of the brown planthopper, a serious pest but not a transmitter of the RBSDV virus.     

Transmission of broad bean stain virus and Echtes Ackerbohnenmosaik-Virus to field beans (Viciajaba) by weevils

Sitona lineatus and Apion vorax were the two most common species of weevil on field beans (Vicia faba minor) at Rothamsted between 1970 and 1974. In glasshouse tests, A. vorax was a much more efficient vector than 5. lineatus of broad bean stain virus (BBSV) and Echtes Ackerbohnenmosaik-Virus (EAMV), and both species transmitted EAMV more often than BBSV. Five other species of Apion transmitted the viruses infrequently or not at all. S. lineatus adults transmitted no more often after 8–16 days on infected plants than after 1–2 days. Some A. vorax adults transmitted EAMV, but not BBSV, after feeding on infected leaves for a few minutes. After 4 days on infected plants, A. vorax sometimes remained infective for the following 8 days. No A. vorax collected from woodland plants in spring was infective with BBSV or EAMV, but 4% from bean crops containing seed-borne infection carried BBSV and 17% carried EAMV. BBSV and EAMV were recovered from triturated weevils, but not from weevil haemolymph. Possibly the viruses are transmitted as contaminants of the mouthparts or by regurgitation during feeding, but A. vorax was observed to regurgitate only when anaesthetized. BBSV and EAMV were not transmitted by aphids (Aphis fabae and Acyrthosiphon pisum), nor by pollen beetles {Meligethes spp.). Field observations suggest that infected seed is the main source of BBSV and EAMV in spring-sown crops, and that crops grown from virus-free seed, and isolated from infected crops by 250–500 m, remain free of infection for most of the season.     

Decline and other effects of five virus infections on three varieties of plum (Prunus domestica L.)

The effects of five different virus inocula on three varieties of plum (Prunus domestica L.) were studied in a field trial of 10 years duration. The viruses causing line pattern, ringspot, prune dwarf and bark split had no effect on the growth of Marjorie's Seedling and Cambridge Gage, and only that causing prune dwarf stunted Oullins Gage trees. However, these viruses diminished the fruit yield of one or more varieties, and prune dwarf virus seriously decreased the yield of all three. Decline disease, probably caused by a strain of Prunus necrotic ringspot virus, developed in Marjorie's Seedling but not in the other varieties. The symptoms first appeared after 5 years and then the trees declined progressively with necrotic ‘incompatibility’ between rootstock and scion.