Line pattern in Limonium latifolium caused by tobacco rattle virus

Tobacco rattle virus (TRV) was isolated from plants ofLimonium latifolium showing bright yellow or red line patterns and ringspots on the leaves. It was proved that this virus, designated TRV-Lim, was the causal agent of the disease. In its reactions onNicotiana clevelandii it resembled a yellow strain of TRV from Oregon (USA), but the symptoms inN. glutinosa, N. megalosiphon, N. tabacum andPetunia hybrida were more comparable to those caused by socalled unstable variants of TRV. Dilution end-point was 10^–6–10^–7, thermal inactivation at 76–80°C, and ageing in vitro 55–60 days. The purified virus suspension contained particles of three normal lengths, 70, 102, and 194 nm. The virus sedimented as three components with average sedimentation coefficients of 129, 161 and 206 S, respectively. In purified suspensions TRV-Lim had two different buoyant densities. A serological relationship was found with TRV isolated from Europe and Brazil.Samenvatting Tabaksratelvirus (TRV) werd geïsoleerd uitLimonium latifolium planten die heldergeel of rood figuurbont op de bladeren vertoonden. Er werd aangetoond dat dit virus, aangeduid als TRV-Lim, de ziekteverwekker was. De reacties van dit virus opNicotiana clevelandii deden denken aan die van een gele stam van TRV afkomstig uit Oregon (VS), maar de symptomen opN. glutinosa, N. megalosiphon, N. tabacum enPetunia hybrida vertoonden meer gelijkenis met die welke veroorzaakt worden door de zogenaamde onstabiele varianten van TRV. De verdunningsgrens was 10^–6–10^–7, de inactiveringstemperatuur 75–80°C en de houdbaarheid in vitro 55–60 dagen. De gezuiverde virussuspensie bevatte deeltjes met drie normale lengtes, nl. 70, 102 en 194 nm. Het virus sedimenteerde, als drie componenten met gemiddelde sedimentatiecoëfficiënten van respectievelijk 129, 161 en 206 S. In gezuiverde suspensie vertoonde TRV-Lim twee verschillende zweefdichtheden. Het virus was serologisch verwant aan TRV-isolaten uit Europa en Brazilië.     

Tobacco rattle virus serotypes and associated nematode vector species of Trichodoridae in the bulb-growing areas in the Netherlands

Soil samples from the coastal bulb-growing areas in the provinces of North- and South-Holland and the North-East Polder in the Netherlands were examined for trichodorid nematodes and tobacco rattle virus (TRV) serotypes. At least one of a total of eight species of Trichodoridae, of whichParatrichodorus pachydermus was most prevalent, was found in 93% of the samples from the provinces of North- and South-Holland and TRV, including four serotypes, was obtained from 49% of these samples. In the North-East Polder one of three species of trichodorids, of whichP. teres occurred most frequently, was present in 72% of the samples, and TRV of one serotype was obtained from 28% of these samples. The TRV isolates recovered from these samples reacted serologically with one of four antisera to strains of TRV. Virus transmitted byP. pachydermus reacted to the PRN-, byTrichodorus viruliferus to the RQ-, byP. teres to the N5- and byT. similis, to the TS-antiserum, respectively.     

Selective recovery of the virus-vector trichodorid nematode <Emphasis Type="Italic">Paratrichodorus anemones</Emphasis> from soil samples by immunomagnetic capture

Lectins and polyclonal antiserum that bind specifically and reproducibly to the overall surface of Paratrichodorus anemones were identified and bound to monodisperse superparamagnetic particles (Dynabeads) to assess their efficiency as probes for capturing target nematodes from test suspensions. In recovery experiments, while both types of probe isolated nematodes, antibody-coated beads recovered them more efficiently than beads coated with lectins. When immunomagnetic capture was used to isolate P. anemones from mixtures of naturally occurring populations of nematodes extracted in bulk, from soil samples, 80% of the target nematodes were recovered.     

New mite-borne virus isolates from rakkyo,shallot and wild leek species

Flexuous viruses were transmitted from rakkyo (Allium chinense) and wild leek species (especiallyA. commutatum) to plants of crow garlic (A. vineale), by transfer of dry bulb mites. By electron microscope decoration tests using three antisera and by inoculations onto test plants, it was concluded that from each of the two natural host species at least two viruses were isolated. The viruses from wild leeks are both pathogenic onAllium spp. and may be of economic importance. Decoration tests on a virus mixture from shallot obtained earlier, revealed another new mite-borne virus in this species. The mite-borne viruses ofAllium spp. appear to be very common; they are largely diverse and their identification remains difficult.     

Molecular analysis and virus transmission tests place Olpidium virulentus, a vector of Mirafiori lettuce big-vein virus and tobacco stunt virus, as a distinct species rather than a strain of Olpidium brassicae

The rDNA-ITS sequences of ten single-sporangium isolates of Olpidium virulentus (a noncrucifer strain of Olpidium brassicae), which transmits Mirafiori lettuce big-vein virus (MLBVV) and tobacco stunt virus (TStV), were compared with those of six single-sporangium isolates of O. brassicae. The sequence similarity within isolates of O. virulentus or O. brassicae was almost identical (98.5%–100.0%), but was low between the two species (79.7%–81.8%). In a phylogenetic analysis of the rDNA-ITS region, O. virulentus and O. brassicae fell into two distinct clusters, indicating that O. virulentus, a vector of MLBVV and TStV, is a distinct species rather than a strain of O. brassicae.     

Infection and necrosis of cowpea mesophyll cells by tobacco necrosis virus and two strains of tobacco mosaic virus

When cowpea mesophyll tissue with or without any epidermal layer was inoculated with tobacco necrosis virus (TNV), local necrotic lesions were produced. In epidermal strips isolated after inoculation of intact leaves local lesions were never observed. Homogenates of epidermal strips removed within 30 min after inoculation of the leaf with the cowpea strain of tobacco mosaic virus (Cp-TMV) or with TNV and incubated on agar for 2 or 4 days were not infectious. However, when clusters of mesophyll cells or vein pieces were still attached to the epidermal strips after stripping, the homogenates showed virus activity. When cowpea leaves were inoculated with Cp-TMV or a common strain of TMV (TMV-U) infective virus material was present in the mesophyll tissue as measured in the homogenates, at the moment of stripping, i.e. within 10 min after inculation.It may be concluded that cowpea mesophyll cells can act as primary sites of viral ingress into the leaf and that the epidermis is not required for necrosis production after virus inoculation.Samenvatting De mogelijkheid werd onderzocht om cowpea-mesofylcellen zonder de aanwezigheid van epidermiscellen met TNV te infecteren. Kleine lokale necrotische lesies werden 40–72 uur na inoculatie zichtbaar waaruit blijkt, dat bij cowpea de epidermis niet noodzakelijk is voor de vorming van TNV-lesies. Geïsoleerd epidermisweefsel vertoonde nooit lokale lesies. Homogenaten van met TNV geïnoculeerde en daarna geïsoleerde cowpea-epidermisstukjes werden getoetst op virusactivitiet. Als de stukjes volledig vrij waren van mesofylcellen of nerfweefsel, dan vond daarin geen virusvermeerdering plaats tijdens een incubatie van 2 of 4 dagen op agar. Als na het strippen nog enkele mesofylcellen of nerfstukjes aanwezig waren, kon wel enige virusactiviteit in de homogenaten worden aangetoond.In cowpeabladeren die geïnoculeerd werden met de cowpea-stam van TMV of de normale stam van TMV had infectieus virusmateriaal al binnen 10 min na inoculatie het mesofyl bereikt. Blijkbaar is in cowpeabladeren de epidermis niet noodzakelijk voor de binnenkomst van virus of voor de necroseproduktie na virusinoculatie.     

Molecular diagnostics of some trichodorid nematodes and associated Tobacco rattle virus

Several Paratrichodorus and Trichodorus (trichodorid) nematode species are natural vectors of Tobacco rattle virus (TRV) and cause economically important diseases, especially in potato and ornamental bulbous crops. Identification of trichodorid species based on morphological characters is laborious, time-consuming, and requires the services of highly trained personnel. Molecular diagnostics for trichodorid nematodes, using the ribosomal DNA repeat unit, were successfully developed to distinguish two Paratrichodorus and two Trichodorus species. The complete sequences of the 18S genes and the ITS-1 regions for these species were obtained and species-specific primers successfully designed for them. An RT-PCR assay was developed utilizing isolate-specific primers that amplify serologically distinguishable strains of TRV in individual trichodorid nematodes. The primers were based on the highly conserved RNA-1 segment of the bipartite genome and also on different parts of the RNA-2 segment of the virus genome.     

Some coat protein constituents from strawberry latent ringspot virus expressed in transgenic tobacco protect plants against systematic invasion following root inoculation by nematode vectors

The coding sequences in RNA2 for the coat proteins (CP) of strawberry latent ringspot virus (SLRSV) were modified and amplified using polymerase chain amplification reactions (PCR) to facilitate their expression inAgrobacterium tumefaciens-transformedNicotiana tabacum Xanthi-nc. The coding sequences for the smaller capsid protein (S, 29kDa) and that for the theoretical precursor of L and S (P, 73kDa) had ATG initiation codon sequences added at the 5-proximal Ser/Gly (S/G) cleavage site in the unmodified sequence. The sequence coding for the larger of the two proteins of mature SLRSV capsids (L, 44kDa) had an ATG codon added at its 5 S/G site and a TAG stop codon sequence added at the 3-proximal S/G site. The P, L and S proteins were expressedin planta to a maximum concentration of 0.01 % of total extractable proteins but did not assemble into virus-like particles. When challenged by mechanical inoculation with virus particles or viral RNA, and compared with control plants, tobacco plants (primary transgenic clones or S1 and S2, kanamycin-resistant seedlings) expressing the virus capsid subunits separately, or their precursor, decreased the accumulation of SLRSV particles in inoculated leaves and fewer plants became invaded systemically. In experiments in which the roots of seedlings were exposed to SLRSV-carrying vector nematodes (Xiphinema diversicaudatum), SLRSV was detected in the roots of non-transformed control tobacco plants (6/20) and in transgenic tobacco expressing the L protein (7/40), but not in any of 25 tobacco plants expressing the S protein or in 35 expressing the P protein. This is the second example of CP-mediated resistance to virus inoculation by nematode vectors.     

Differentiation of Bean pod mottle virus isolates based upon host symptoms

Accessions from Glycine, Phaseolus, and Vigna genera were screened for their reactions to different subgroups of isolates of Bean pod mottle virus (BPMV) in order to establish a differential host system. Screening results indicated that the BPMV isolates differed in pathogenic aggressiveness but not in virulence. No major resistance genes were found in soybean (Glycine max) or G. soja since all screened accessions showed mosaic or necrotic symptoms to BPMV inoculation. However, these accessions expressed differences in severity of symptoms when challenged by various BPMV isolates. The inoculation of G. tomentella accessions did not result in mosaic symptoms, and some accessions did not support systemic infection of some of the isolates. Resistance, presented as a hypersensitive reaction, was observed in some of Phaseolus and Vigna genotypes, and resistant response or susceptibility was stable to all the isolates used in the screening. In conclusion, the selected G. soja genotypes PI 407019, PI 464889A, and PI 464928, and ‘Amsoy 71’ soybean may help to separate severe (reassortant) from mild isolates of BPMV based upon their phenotypic reactions.     

Interaction between the helicase domain of the tobacco mosaic virus replicase and a tobacco arginine decarboxylase

In a yeast two-hybrid screening test for tobacco proteins that interact with TMV replicase using the helicase (H) domain as bait, a cDNA clone was selected that encodes a polyamine biosynthetic enzyme, arginine decarboxylase (ADC). In yeast cells, the C-terminal internal region of ADC interacted with the H domain. This observation was confirmed in vitro by far-Western blotting. Inhibition of the binding between the H domain and the IRnHEL (I region and N-terminus of helicase domain) region by ADC using a yeast three-hybrid assay suggested possible interference of the heterodimerization of 126K and 183K by ADC.The nucleotide sequence data of pADCF reported in this study is available in the DDBJ/EMBL/GenBank databases under accession number AB110952     

The transmission by nematodes of tobraviruses is not determined exclusively by the virus coat protein

The coat protein gene of the nematode non-transmissible, SP5 isolate of pea early-browning tobravius was replaced with that of the highly nematode transmissible, PPK20 isolate of tobacco rattle tobravirus. Plants were infected with the recombinant virus when mechanically inoculated and the virus invaded the plants systemically. However, although the PPK20 isolate of TRV was transmitted by nematodes from these plants, the recombinant virus was not transmitted. Therefore, the virus coat protein is not the exclusive determinant of nematode transmission.     

A survey of tobacco viruses in tobacco crops and native flora in Greece

The most important tobacco producing areas in Greece were surveyed for virus presence, from 1997 to 2000. Tobacco seedlings or plants showing virus-like symptoms were randomly collected from seedbeds or fields, respectively, and tested by ELISA, and/or mechanical inoculation onto indicator plants. Potato virus Y (PVY), Cucumber mosaic virus (CMV) and Tobacco mosaic virus (TMV) were detected in all sampling areas, with TMV mainly found in oriental varieties. Tomato spotted wilt virus (TSWV) consisted a serious endemic virus in Northern Greece (Thrace, Central and Eastern Macedonia), whereas Alfalfa mosaic virus (AMV) was mainly found in regions, where alfalfa was cultivated in the vicinity of tobacco crops. Eggplant mottled dwarf virus (EMDV) was detected in several areas but always in very low incidence (<0.01%). Surveys were also conducted to assess the potential reservoir hosts of PVY, CMV and AMV among weeds collected from highly infected tobacco fields from 1998 to 2000. Among 3450 samples tested for PVY, plants from 17 species in 10 families were found infected. For CMV, 2891 weed samples were tested and 19 species in 12 families were positive. Assays for AMV infection were made on 961 samples and 12 species in 9 families were identified as hosts of this virus.     

Bionomics and importance of two species ofChaetocnema in rice yellow mottle virus transmission in lowland rice in Tanzania

Regular samplings were done of two important vectors in farmers’ fields during the 1999/2000 and 2000/01 rice seasons at crop stages susceptible to rice yellow mottle virus (RYMV) on a traditional rice variety (‘Supa’) under rainfed lowland conditions to provide information on the bionomics and importance of these vectors in the disease transmission. The population ofChaetocnema sp. (nr.varicornis Jacoby) (Coleoptera: Chrysomelidae) was significantly higher in hotspot than non-hotspot areas. However, there was no significant difference in theC. pulla Chapuis population between these two areas. In general, theChaetocnema sp. population was higher than that ofC. pulla, and both vectors reached the peak of their population at 63 days after planting. Early planting in the hotspot areas is suggested as a disease management strategy. Both vectors are naturally infective andChaetocnema sp. proved more efficient thanC. pulla in the transmission of RYMV.     

The effect of DD and dazomet treatments on nematode populations at various depths in the soil and on the transmission of soil-borne tobacco rattle virus to gladiolus

The relation between log dosage of DD injected at 15 cm depth or of dazomet applied to the soil surface (all in November 1971) and probit mortality ofRotylenchus and trichodorids in the top 20 cm of a field on sandy soil was found to be linear. Dosage increase efficiencies of both chemicals against both nematode species were medium to high. Superficial application of dazomet was very effective against the nematodes that would have survived if only a low dosage of DD had been injected at 15 cm depth. Injection of 40 ml or 80 ml DD per m² at 15 cm depth killed all nematodes between 20 cm and 60 cm deep. Gladiolus planted in the spring of 1972 grew better, flowered earlier and produced more weight of corms on treated than on untreated plots. The poor growth on the untreated plots cannot be ascribed to direct damage by nematodes or to the effect of TRV transmitted to the plants by the viruliferous trichodorids occurring in these plots in high densities. Symptoms of TRV infection in plants grown in 1973 from the corms harvested in the 1972 experimental field showed that only DD treatments had reduced the rate of TRV transmission considerably. However, even the highest dosages of DD had only reduced it from 26% (on untreated plots) to about 8%. Most probably, this residual TRV infection was due to transmission by trichodorids that had survived in soil layers below 60 cm depth. Therefore, soil treatment with nematicides, cannot prevent TRV transmission to gladiolus sufficiently where viruliferous trichodorids occur at great depths, as is the case in many sandy soils having a low water table.     

Characterization of two distinct Polish isolates of Pepino mosaic virus

In Poland in 2002 and 2005 two different isolates of Pepino mosaic virus signed PepMV-SW and PepMV-PK were obtained. Both isolates were compared on the basis of their symptomatology on a series of plant species. In addition, the isolates were characterized by the nucleotide sequence analysis of the triple gene block, coat protein and a part of the polymerase genes. The studies showed that the Polish isolates differ from each other and belong to two strains. PepMV-SW was highly similar to European isolates, showing extensive sequence identity, ca. 99%. Pairwise comparisons of PepMV-PK with other PepMV isolates from the GenBank database showed that the highest nucleotide sequence identity was with two isolates: Ch2 from Chile and US2 from the USA.     

Nigerian tobacco latent virus: a new Tobamovirus from tobacco in Nigeria

Field-grown tobacco plants in Nigeria showing chlorotic mottle and marginal veinbanding on the leaves apparently contained several viruses. One of them proved to be a new Tobamovirus for which we suggest the name Nigerian tobacco latent virus (NTLV), because it did not produce systemic symptoms on various cultivars of Nicotiana tabacum. Sequence analyses of the coat and movement protein genes and their translation products, as well as serological studies, revealed that NTLV is only distantly related to known Tobamoviruses from which it also differs in host range and symptomatology. Its closest relationship was found to Tobacco mild green mosaic virus (TMGMV). The percentages of amino acid sequence identity amounted to 73% for the coat proteins and to 64% for the movement proteins of the two viruses. The total sequence of 1415 nucleotides analysed share 63% identity with the corresponding region of TMGMV. In the immunoelectron microscopical decoration test using antisera at a dilution of 1 : 50, reactions of NTLV were observed only with its own antiserum and one out of two antisera to TMGMV. An antiserum to NTLV diluted 1 : 2 failed to react with TMGMV. NTLV induces the formation of characteristic inclusions in infected cells.     

Virus transmission by host-specific strains ofOlpidium bornovanus andOlpidium brassicae

Zoospores of 12 isolatesO. bornovanus from geographically diverse sites and representing the three host specific cucurbit strains were tested as vectors for seven viruses using watermelon bait plants and the in vitro acquisition method. All isolates of the cucumber, melon, and squash strains transmitted melon necrotic spot carmovirus (MNSV) and cucumber necrosis tombusvirus (CNV) but none transmitted petunia asteroid mosaic tombusvirus (PAMV) or tobacco necrosis necrovirus (TNV). The isolates varied as vectors of three other carmoviruses: cucumber leaf spot virus (CLSV); cucumber soil borne virus (CSBV); and squash necrosis virus (SqNV). All cucumber isolates transmitted CLSV and SqNV but not CSBV. Some of the melon isolates transmitted CLSV and SqNV but none transmitted CSBV. Two squash isolates transmitted CSBV and SqNV but not CLSV. Two isolates ofO. brassicae transmitted only TNV and a third did not transmit any of the viruses. The species of bait plant sometimes affected transmission. The most efficient vector strains ofO. bornovanus, as determined by reducing zoospores and virus in the inoculum, were the cucumber strain for CLSV; the cucumber strain for CNV if cucumber was the bait plant or melon strain if watermelon was the bait plant; and the squash strain for SqNV. The plurivorous strain ofO. brassicae was the most efficient vector of TNV.Olpidium bornovanus is the first vector reported for CSBV and is confirmed as a vector of SqNV. It is proposed that all carmoviruses may have fungal vectors.Ligniera sp. did not transmit any of the viruses in one attempt.Abbreviations CLSV cucumber leaf spot virus - CNV cucumber necrosis virus - CSBV cucumber soil borne virus - MNSV melon necrotic spot virus - PAMV petunia asteroid mosaic virus - SqNV squash necrosis virus - TNV tobacco necrosis virus - TBSV tomato bushy stunt virus     

A simple test for evaluation of transmission efficiency of barley yellow dwarf virus by aphids

Barley yellow dwarf virus (BYDV) transmission test systems involve the use of clip-cages or of whole plants in cages, which are both labor-intensive methods and require large controlled environment units. Employing detached leaves for assessment of the inoculation efficiency of aphids proved reliable for assessing transmission of a BYDV PAV-like isolate byRhopalosiphum padi. One use of the system could be for the rapid determination of the infectivity of field-collected aphids, an essential part of any epidemiological study of BYDV. http://www.phytoparasitica.org posting Aug. 14, 2002.