Chenopodium necrosis: a distinctive strain of tobacco necrosis virus isolated from river water

A distinctive strain of tobacco necrosis virus (TNV) of unknown source was repeatedly isolated from water of the River Avon (Warwickshire) and two of its tributaries (R. Swift and R. Alne) using a technique developed for the concentration and isolation of water-borne bacteriophages. The same strain was isolated from the rivers Cam and Thames and from Lake Esthwaite (Cumbria) together with tomato bushy stunt virus. The TNV strain, designated Chenopodium necrosis (TNV-CN) was mechanically transmissible to C. amaranticolor and C. quinoa in both of which it caused local lesions and systemic infection. TNV-CN caused no infection when inoculated to tobacco (Nicotiana tabacum cv. White Burley) plants. The virus was not adsorbed to soil, could be isolated from leachate of soil in which systemically infected C. quinoa were grown and C. quinoa plants became infected when grown in soil watered with suspensions of the virus. The virus was not transmitted by Myzus persicae but was vectored by the zoospores of a lettuce isolate of Olpidium brassicae. TNV-CN was infective after 10 min at 85 °C., 3 wk at 20 °C and when diluted to 10^-8 but not 10^-9. Purified virus preparations contained c. 26 nm isometric virus particles. TNV-CN contained single-stranded RNA (mol. wt 1·5 × 10⁶) and one protein (mol. wt c. 26·4 × 10³) which co-electrophoresed in polyacrylamide gels with the protein of the D strain of TNV (TNV-D). Analytical centrifugation of TNV-CN indicated a single component virus with the same sedimentation coefficient (s₂₀, w= 115S) and buoyant density (1·385) in a CsCl gradient as those of TNV-D. TNV-CN and TNV-D were indistinguishable serologically.     

Studies on the occurrence of tomato bushy stunt virus in English rivers

Tomato bushy stunt virus (TBSV) of unknown source was isolated from water of the River Thames, near Oxford. The isolate designated TBSV-T was mechanically transmissible to several tomato (Lycopersicon esculentum) cvs and to other species including Petunia hybrida, pepper (Capsicum annuum). eggplant (Solanum melongena), Nicotiana clevelandii, Chenopodium amaranticolor and C. quinoa in which it caused systemic symptoms. It caused no infection of globe artichoke (Cynara scolymus) or Pelargonium domesticum. The virus was not adsorbed to soil and could be isolated from leachate of soil in which systemically-infected tomato or C. quinoa plants were grown. Tomato plants became infected when grown in soil watered with virus suspensions. TBSV-T was infective after 10 min at 80°C but not at 90°C and when diluted to 10^-5 but not to 10^-6. Purified virus preparations contained C. 30 nm isometric particles. In gel-diffusion serological tests, TBSV-T reacted with homologous anti-serum and with antiserum to petunia asteroid mosaic virus but not to pelargonium leaf curl virus. Seed-borne infection (50–65%) of TBSV was demonstrated in plants grown from seed of symptomlessly-infected tomato fruit. TBSV was isolated from symptomlessly-infected tomato fruit imported from Morocco during October-April 1981. One of the isolates (TBSV-M) was indistinguishable from TBSV-T in host range, symptomatology and serological reactions. TBSV was also found in tomato plants growing extraneously in primary settlement beds at sewage works; such plants having been derived from undigested seeds in sewage. Because of its ‘alimentary-resistance’ in man, it is possible that one ecological route whereby TBSV enters rivers is by man's consumption of TBSV-infected tomatoes and eventual sewage dispersal into rivers.     

Viral twig necrosis of sweet cherry. Modes of transmission and spread of petunia asteroid mosaic virus (PeAMV)

Grafting symptomless scions, derived from petunia asteroid mosaic virus (PeAMV)-infected trees, to healthy rootstocks resulted in only 3.3% infection in the resulting trees. Up to 90% of seeds from infected sweet cherries contained high quantities of PeAMV, but the virus was not transmitted to the seedlings apparently because of low virus content in the embryo and loss of infectivity during seed maturation and storage. Replanting healthy cherry trees cv. Sam, grafted to different rootstocks, into contaminated soils resulted in new infections. Eight of 13 trees on rootstocks derived from Prunus avium (F 12/1 and cv. Sam on its own roots) were infected with PeAMV within a period of four years but only one of 16 trees on Weiroot-rootstocks (selections from Prunus cerasus) became infected. The detection of PeAMV in naturally contaminated soil samples by the bait plant procedure, using Nicotiana clevelandii, was superior to testing soil eluates by enzyme-linked immunosorbent assay (ELISA) and immuno electron microscopy (IEM). Wild plants may contribute to virus propagation and maintenance of virus contamination of the soil as 25 of 310 samples from 712 herbaceous plants growing in the vicinity of infected trees contained PeAMV; the contaminated samples represented 12 species. The perpetuation of PeAMV by infected scion wood is probably of minor significance, and infection via the soil probably represents the most important means of spread of viral twig necrosis in northern Bavaria.     

Dissemination and detection of peanut clump virus in groundnut seed

Experiments were done to assess the role of seed-transmission in the dissemination of peanut clump virus (PCV) in groundnut (Arachis hypogea L.), and the usefulness of enzyme-linked immunosorbent assay (ELISA) for detecting the virus in infected groundnut seed. The virus was present in 7.5% of seedling progeny from infected plants and could be detected in 16.5% of the seeds by ELISA. When groundnut seeds were grown in a field contaminated by the virus, it was shown that by roguing the infected plants, only 0.1% of the seeds from the remaining plants contained the virus. It was also established that the level of contamination of seeds by the virus was inversely proportional to the seed size.     

Development of potato blight Phytophthora infestans after planting infected seed tubers

About 1000 blight-infected seed potato tubers, usually of the cultivar King Edward, were planted for 9 yr and the subsequent plants examined until the disease had developed in the plots. Haulm infection originated each year from the seed tubers and occurred first on basal leaves. When tubers were inoculated with a complex race of P. infestans this race was recovered from the leaves and from the soil near the seed tuber. Transmission of infection from soil to leaves was demonstrated by splash of artificially contaminated soil to leaves suspended above the soil. In 4 yr, plants were grown on flat rows as well as on ridges. In 2 yr, when emergence was almost complete, infected stems were observed on otherwise normal plants. In the first year 0.6% grew on ridges and 3.0% on the flat and in the second all grew on the flat (5.3%). Only seven of the 43 plants had more than one infected stem. Flat plots had a significantly higher number of stemdplant than ridge plots, but this bore no relation to numbers of infected stems. When flat plots which had developed affected plants had soil replaced as ridge plots, no further infected stems were observed. Such stems continued to develop on flat plots. No prematurely dead stems were observed below soil level when all plants were dug. Underground portions of most infected stems showed little evidence of P. infestans which was found only at about soil level. Infection appeared to occur first in this area.     

Augusta disease in tulip - a reassessment

In an experiment in which the roots of field-grown tulip were commonly infected with tobacco necrosis virus (TNV), Augusta disease did not develop in the year of infection or when progeny bulbs were grown in the field or glass-house. When tulip bulbs of other stocks, including grades of 11 and 12 cm circumference, were forced, the disease developed sporadically, in some instances as the result of infection with TNV from the soil in which they were planted and in others as a result of infection by bulb-borne virus. The incidence of disease produced by current year infection was increased by warming the plunge bed. Different strains of TNV were obtained from field-grown plants with Augusta disease and different strains of the virus produced the disease when inoculated to tulip. Some, but not all, naturally diseased plants contained satellite virus, which therefore does not cause or prevent disease development. The disease was produced in some plants by TNV transmitted by Olpidium brassicae, but neither a vector nor a non-vector isolate of O. brassicae completed its life cycle in tulip. However, Olpidium-like zoospores were observed in some washings of tulip roots from TNV-infested soils. TNV was not obtained from all tulip plants with necrotic leaf symptoms resembling Augusta disease. Some were infected with tomato bushy stunt virus or cucumber mosaic virus, or with another agent that was transmitted by inoculation of sap to Nicotiana clevelandii and Chenopodium quinoa, and carried by bulbs of up to 11 cm circumference.     

Tests for transmission of four potato viruses through potato true seed

The Andean potato calico strain of tobacco ringspot virus (TRSV-Ca) was detected in 2–9% of potato seedlings grown from true seed from plants of cv. Cara and clone G5998(6) infected with TRSV-Ca. Similarly, a potato isolate of the oca strain of arracacha virus B (AVB-O) was detected in 4–12% of progeny seedlings of cv. Cara and clone D42/8 infected with AVB-O. Potato virus T (PVT) passed through 33–59% of seed from PVT-infected cv. Cara, but only 0–2% infection was detected in seedlings from seed of PVT-infected clone D42/8. By contrast, no infection was detected in seedlings grown from seed from plants of G5998(6), D42/8 or cv. Cara infected with Andean potato latent virus strains Hu (APLV-Hu) or Caj (APLV-Caj), although both strains passed through seed of Nicotiana clevelandii. AVB-O, PVT and TRSV-Ca were detected in all tests of pollen from flowers of infected potato plants, but APLV-Hu and APLV-Caj were detected less frequently. AVB-O and PVT were transmitted through 2% and 8% respectively, of seed from healthy potato plants pollinated with pollen from infected plants. However, no transmission through seed was detected when pollen from TRSV-Ca infected plants was used. None of the four viruses were transmitted to healthy potato plants pollinated with pollen from infected plants. APLV-Hu caused exceptionally severe symptoms in the cv. Cara plants used for seed production, but the Bolivian strain of PVT induced only mild symptoms rather than the severe systemic necrosis previously reported for the type of strain of PVT in this cultivar. No symptoms developed in potato seedlings infected with TRSV-Ca, AVB-O or PVT through the seed.     

A strain of cassava latent virus occurring in coastal districts of Kenya

A strain of cassava latent geminivirus (CLV) was isolated from mosaic-affected cassava plants from coastal districts of Kenya. This virus (CLV-C) did not infect Nicotiana clevelandii, a diagnostic host of the type strain (CLV-T); experimental host range was very restricted and CLV-C only infected N. benthamiana and N. rustica out of several solanaceous hosts readily infected by CLV-T. CLV-C was also isolated from naturally infected Jatropha multifida (Euphorbiaceae) and Hewittia sublobata (Convolvulaceae). CLV-C was propagated in N. benthamiana with difficulty and only those isolates derived from cassava plants infected with severe mosaic symptoms were maintained more or less successfully; these sources usually contained a higher concentration of CLV than plants with mild symptoms. Symptom variants generally remained unchanged when grafted into a highly susceptible South American cassava variety. CLV-C and CLV-T seemed to occur respectively only in coastal and western districts but their ranges overlapped in central Kenya where they could have been introduced in infected material. CLV-C could be purified satisfactorily with the method used for CLV-T but only after modifying the procedure by substituting phosphate for borate in the extraction buffer, n-butanol for n-butanol/chloroform in clarification of extracts, and phosphate for borate buffer when resuspending concentrated virus. A virus serologically indistinguishable from CLV-T was isolated from mosaic- affected material obtained from Nigeria; East African and Nigerian isolates were essentially similar in host range and symptomatology. In gel-diffusion serology tests, pronounced precipitation spurs developed between CLV-T and CLV-C indicating that the isolates were related but not identical serologically. Symptoms typical of cassava mosaic disease appeared in only three of 105 plants in experiments on transmission of CLV-C and CLV-T by whiteflies, when attempted acquisition of either clarified CLV-infective sap or purified CLV was made through ‘Parafilm’ membranes. Because it is possible that the three infections resulted from contamination, they cannot constitute proof of transmission. The presence of CLV in relation to the etiology of cassava mosaic thus remains unresolved.     

Studies on the seed transmission of cucumber mosaic virus in chickweed (Stellaria media) in relation to the ecology of the virus

Cucumber mosaic virus (CMV) was transmitted in the seed of infected Stellaria media plants. The rate of seed transmission varied both in manually infected plants (3–21%) and in plants grown from infected seed (21–40%). In naturally infected plants the rates of transmission found were 4–29%. Seeds recovered from field soil carried 4–5% infection and in infected seed placed in the soil the virus persisted for at least 5 months. Seed transmission of CMV also occurred in infected Lamium purpureum (4%), Cerastium holosteoides (2%) and Spergula arvensis (2%) but it could not be demonstrated in six other more common weed species in five botanical families. Seed transmission in Stellaria media occurred with a British (W) and an American (Y) strain of CMV. The virus was shown to occur in S. media pollen. The importance of CMV-infected S. media seed in the soil in relation to the epidemiology of the virus is discussed.     

A physical map covering the nsv locus that confers resistance to Melon necrotic spot virus in melon (Cucumis melo L.)

Melon necrotic spot virus (MNSV) is a member of the genus Carmovirus, which produces severe yield losses in melon and cucumber crops. The nsv gene is the only known natural source of resistance against MNSV in melon, and confers protection against all widespread strains of this virus. nsv has been previously mapped in melon linkage group 11, in a region spanning 5.9 cM, saturated with RAPD and AFLP markers. To identify the nsv gene by positional cloning, we started construction of a high-resolution map for this locus. On the basis of the two mapping populations, F₂ and BC1, which share the same resistant parent PI 161375 (nsv/nsv), and using more than 3,000 offspring, a high-resolution genetic map has been constructed in the region around the nsv locus, spanning 3.2 cM between CAPS markers M29 and M132. The availability of two melon BAC libraries allowed for screening and the identification of new markers closer to the resistance gene, by means of BAC-end sequencing and mapping. We constructed a BAC contig in this region and identified the marker 52K20sp6, which co-segregates with nsv in 408 F₂ and 2.727 BC1 individuals in both mapping populations. We also identified a single 100 kb BAC that physically contains the resistance gene and covers a genetic distance of 0.73 cM between both BAC ends. These are the basis for the isolation of the nsv recessive-resistance gene.     

Studies on melon necrotic spot virus disease of cucumber and on the control of the fungus vector (Olpidium radicale)

Melon necrotic leaf spot virus (MNSV) caused a major outbreak of a leaf necrosis disease of hydroponically-grown cucumber plants at Humberside in 1983. The virus had c. 33 nm diam. particles which reacted serologically with MNSV antiserum of Dutch or American origin. Virus particles, which contained a single polypeptide (mol. wt 45 × 10³) and a presumed RNA species (mol. wt 1.5 × 10⁶), had a sedimentation coefficient (s_20.w) of 134 S and a buoyant density in caesium chloride of 1.35 g/cm³. The virus was mechanically transmissible, confined to species of Cucurbitaceae, transmitted by zoospores of Olpidium radicale and retained in the resting spores of the fungus. MNSV is thus both water-borne and soil-borne. O. radicale zoospores were killed in <5 min in suspensions containing 20 μg/ml of the surfactant Agral (alkyl phenol ethylene oxide). The disease did not reappear in 1984 when the cucumber crops were fed with nutrients containing 20μg/ml Agral.     

Zucchini Yellow Fleck Virus is an Emergent Virus on Melon in Sicily (Italy)

Since 2006, winter melon plants (Cucumis melo L. var inodorus) showing symptoms of pin‐point yellow spots were noticed in Sicily (Italy). Leaf samples were tested by enzyme‐linked immunosorbent assay to the most important viruses‐infecting cucurbits. Zucchini yellow fleck virus (ZYFV, genus Potyvirus) was the only virus detected. Surveys in 2007 and 2008 revealed an increasing number of sites in Sicily with ZYFV‐infected winter melon plants. To confirm the identity of the virus as ZYFV, two isolates from different locations were sequenced and shown to be approximately 85% identical to the published sequences of isolates previously identified in Italy and France. This is the first report of ZYFV occurring on melon in Italy.     

Epidemiological study of Cucumber green mottle mosaic virus in greenhouses enables reduction of disease damage in cucurbit production

Since 2007, the tobamovirus Cucumber green mottle mosaic virus (CGMMV) has become widespread in Israel, causing severe damage to trellised cucumber and melon in greenhouses and watermelon grown in open fields. To reduce disease damage below the economic threshold, this study focused on four objectives: (a) monitoring the patterns of virus distribution within commercial cucumber greenhouses; (b) studying the potential transmission of CGMMV by agrotechnical activities; (c) virus localization in plant tissues; and (d) searching for techniques that might be adapted for mitigating the disease in trellised cucurbit growth. The results of our surveys demonstrated the role of contaminated seeds and soil as primary inoculum sources, and secondary spread caused by agrotechnical activities. The patterns of secondary disease spread were demonstrated in a series of inoculation experiments involving contaminated knives, shears or hands on wet and dry plants, conducted under research‐greenhouse conditions. In parallel experiments using CGMMV‐specific antibody and secondary antibody conjugated to Alexa fluor 488, the viral coat protein was visualized in several plant tissues: phloem, xylem, trichomes and grasping tendrils. In addition, commercial‐greenhouse experiments were aimed at reducing the number of inoculum sources by identifying and removing infected plants from the plots (early monitoring) prior to agrotechnical activities and/or by adding intermediate medium (IM), such as virus‐free compost, to the planting pits at the planting stage. It is suggested that the use of IM combined with early monitoring, awareness of worker mobility (from contaminated structures to young planting areas) and proper sanitation (e.g. disinfection of agrotechnical tools) may reduce the yield losses caused by CGMMV below the economic threshold.     

Transmission of the hop strain of arabis mosaic virus by Xiphinema diversicaudatum*

Hop plants became infected with the hop strain of arabis mosaic virus (AMV(H)) when grown in hopfield and woodland soil in which infected plants had been growing. Infection occurred in soil infested with the dagger nematode Xiphinema diversicaudatum, but neither in uninfested soil nor in soil previously heated to kill nematodes. X. diversicaudatum transferred direct from hop soils transmitted AMV(H) to young herbaceous plants and to hop seedlings; some of the hop seedlings developed nettlehead disease. A larger proportion of plants was infected using X. diversicaudatum obtained from a woodland soil and then given access to the roots of hop or herbaceous plants infected with AMV(H). AMV(H) was transmitted by adults and by larvae, in which the virus persisted for at least 36 and 29 wk, respectively. Difficulties were encountered in detecting AMV(H) in infected hop plants, due partly to the delay in virus movement from roots to shoots. Infection of hop shoots was seldom detected until the year after the roots were infested and sometimes nettlehead symptoms did not appear until the third year. Isolates of arabis mosiac virus from strawberry did not infect hop. The results are discussed in relation to the etiology and control of nettlehead and related diseases of hop.     

Complete Genome Sequence of a Chinese Isolate of Melon Necrotic Spot Virus and its Phylogenetic Relationship

The full‐length nucleotide sequence and genomic organization of a melon necrotic spot virus isolate from Haimen, China (MNSV‐HM), were determined. The MNSV‐HM genome consists of a positive sense single‐stranded RNA, 4267 nt in length, with at least five open reading frames (ORFs) encoding p29, p89, p42, and two small 7‐kDa proteins (p7A and p7B). p89 shares a common start codon with p29 and continues through the amber stop codon of p29 to produce an 89‐kDa protein. The p7A ORF terminates in an amber codon whose read‐through could generate a 14‐kDa protein. Phylogenetic analyses based on the p42 amino acid sequence and complete genomic sequence placed MNSV‐HM and Spanish isolates of the virus in a major cluster, indicating a close genetic relationship. In conclusion, we report the full‐length sequence of MNSV‐HM and its translation strategy. The obtained genomic organization and phylogenetic trees indicate that MNSV‐HM belongs to the MNSV genus Carmovirus. To the best of our knowledge, this is the first demonstration of the complete nucleotide sequence of an MNSV isolate from China.     

Transmission Efficiency of Cucumber green mottle mosaic virus via Seeds,Soil, Pruning and Irrigation Water

Cucumber green mottle mosaic virus (CGMMV; genus Tobamovirus) infects frequently the grafted watermelon and is widely distributed in China. Investigating the transmission modes and their efficiency is urgently needed to understand the factors contributing to the epidemiology of this viral disease. In the present study, we found that the occurrence of CGMMV in a bottle gourd seed production base reached 100%, while the contamination rate and transmission rate were 100 and 0.92%, respectively. The bottle gourd plants showed obvious mottle symptom on leaves starting 36 days after seed sowing. The long latent period of CGMMV in seedlings implies a potential risk to use contaminated seeds in the production of grafted watermelon. This virus could overwinter in soil with debris of infected plants, and the infection rate of CGMMV from contaminated soils was 10.30%. CGMMV could be transmitted from infected watermelon plants to healthy ones by pruning at least to the ninth plant during the whole growing season. The transmission distance was 1.87 m by drip irrigation and 2.31 m by flow irrigation. This study suggested that contaminated seeds, contaminated soil, pruning and irrigation could transmit CGMMV at different efficiency, and all contribute to the epidemiology of CGMMV.     

Transmission Studies with Cucumber Green Mottle Mosaic Virus

Cucumber green mottle mosaic virus may spread in bottlegourd under field conditions through soil contaminated with infected plant debris followed by contact. No seed transmission was noticed in bottlegourd (Lagenaria siceraria) or vegetable marrow (Cucurbita pepo) although pollen grains and cotyledons from infected bottlegourd flowers or seeds, respectively, contained negliginle amounts of virus. Cucumber leaf beerles (Raphidopalpa fevicollis) are probable vectors since their regurgitated fluid and excretes contained infective virus particles. No vector fungi were found in the soil around infected bottlegourd plants.     

Natural Infection of Tomato and Pelargonium in Germany by a Tombusvirus Originally Described from Pepper in Morocco

Two serologically apparently identical tombusviruses were isolated from glasshousegrown tomato plants showing mottle and malformation of the upper leaves and stunting of the shoot tips and, at a low rate, from a pelargonium plant, respectively. Both plant species were received from growers in southern Germany. In agar gel double diffusion tests and immuno-electrophoresis, the two virus isolates could not be distinguished from a tombusvirus (MPV) isolated from pepper in Morocco by F ischer and L ockhart (1977), although there were some minor differences in the reported host responses. In the tomato planting, strong symptoms were produced only temporarily. The majority of the 104 tomato plants of 15 different cultivars which had become infected by MPV following mechanical inoculation of the leaves showed only mild symptoms in contrast to a similar group of plants which developed severe stunting and foliar deformation when infected with the type strain of tomato bushy stunt virus. MPV was translocated only occasionally to the upper parts of tomato seedlings which had been grown in virus-containing soil, although root infections were more frequent. MPV is apparently not a major threat to tomato cultivation in Germany.     

Further studies on a British form of pea early-browning virus

An isolate of pea early-browning virus from Britain (PEBV (B)) has tubular particles most of which are either about 103 or 212 mμ long with sedimentation coefficients of 210 and 286 S respectively. Both types show cross-banding at intervals of 2.5 mμ. Virus preparations containing only the shorter particles were not infective. PEBV (B) was transmitted to pea seedlings by both adult and juvenile Trichodorus primitivus (de Man) (Nematoda) and persisted for 32 days in T. primitivus kept without plants. In two experiments T. primitivus failed to transmit a Dutch isolate (PEBV (D)), which is distantly related serologically to PEBV (B). PEBV (B) was transmitted by nematodes to cucumber roots more readily in soil at 20d? than at 24d? C., and more readily at 24d? than at 29d? C. When transmitted by inoculation of sap, PEBV (B) and PEBV (D) caused similar symptoms in some pea varieties but differed in virulence towards others. Thirty-one varieties resistant to natural infection with PEBV in The Netherlands were susceptible to PEBV (B) when manually inoculated with sap or when grown in naturally infested soil from one site; twenty-six of these varieties did not become infected in soil from a second site, in which several other varieties that are susceptible in The Netherlands were infected. Varieties should therefore be tested for resistance by growing them on many infested fields. All but one of the pea varieties resistant to PEBV in The Netherlands became infected with the English form of tomato black ring virus when grown in soil containing infective Longidorus attenuatus Hooper.     

Identification and Characterization of Pothos latent virus Causing Necrotic Ringspots and Line Patterns on Lisianthus (Eustoma grandiflorum) in Taiwan

Lisianthus (Eustoma grandiflorum) grown in screenhouses in Taiwan showed ringspots and concentric line patterns on leaves. A virus having isometric particles approximately 30–32 nm in diameter was isolated from affected lisianthus. Combined results of biological, cytological, serological, molecular and phylogenetic analyses show that the virus can be identified as Pothos latent virus (PoLV), genus Aureusvirus, family Tombusviridae. Inoculating the virus on non‐infected lisianthus plants reproduced the symptoms previously observed in the field. So, this is the first report of PoLV causing disease in lisianthus and the first report of the virus in Taiwan.