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Transgenic Nicotiana tabacum plants expressing a single-chain variable region antibody fragment derived from a broad-spectrum monoclonal antibody 3-17 showed suppression of virus infection following challenge by two distinct potyviruses: potato virus Y strain D, and clover yellow vein virus strain 300. Monoclonal antibody 3-17, which was raised against the potyvirus Johnsongrass mosaic virus, was shown to react strongly with 14 potyvirus species. Two different single-chain antibody constructs were used to produce chimeric genes encoding recombinant proteins designed to be targeted either to the apoplasm or to the cytoplasm. Transgenic plant lines showed reduced numbers of local lesions and systemic symptoms when challenged with potato virus Y, strain D and reduced local lesions following challenge with clover yellow vein virus, strain 300. The level of suppression conferred by the transgene when plants were challenged under laboratory conditions with high concentrations of virus, together with the ability of the transgene to partially protect plants against distinct viruses suggest that one single-chain gene construct might be used to protect plants from distinct potyviruses.

Viral diseases, such as Alfalfa mosaic virus (AMV), cause significant reductions in the productivity and vegetative persistence of white clover plants in the field. Transgenic white clover plants ectopically expressing the viral coat protein gene encoded by the sub-genomic RNA4 of AMV were generated. Lines carrying a single copy of the transgene were analysed at the molecular, biochemical and phenotypic level under glasshouse and field conditions. Field resistance to AMV infection, as well as mitotic and meiotic stability of the transgene, were confirmed by phenotypic evaluation of the transgenic plants at two sites within Australia. The T(0) and T(1) generations of transgenic plants showed immunity to infection by AMV under glasshouse and field conditions, while the T(4) generation in an agronomically elite 'Grasslands Sustain' genetic background, showed a very high level of resistance to AMV in the field. An extensive biochemical study of the T(4) generation of transgenic plants, aiming to evaluate the level and composition of natural toxicants and key nutritional parameters, showed that the composition of the transgenic plants was within the range of variation seen in non-transgenic populations.

Subterranean clover stunt virus (SCSV) was previously found to be representative of a new type of single-stranded DNA virus. Purified SCSV particles did not infect subterranean clover when various attempts were made to transmit them to this host using aphids ( Aphis craccivora ) which had previously been fed on preparations of the virus. To demonstrate that purified preparations of SCSV are capable of replication, pea and subterranean clover protoplasts were inoculated with the virus using PEG treatment or electroporation, and maintained for up to 13 days. Up to 40% of the protoplasts survived after PEG treatment but less than 10% survived after electroporation. Both enzyme-linked immunosorbent assay (ELISA) and nucleic acid hybridisation detected de novo synthesis of SCSV in the inoculated protoplasts. The amount of SCSV coat protein detected by ELISA decreased from day 0 to day 3 post-inoculation but increased thereafter over several days to a maximum at about 10 days. Nucleic acid hybridisation studies using strand-specific probes showed that the kinetics of synthesis of the virion and its complementary strand nucleic acid corresponded with the kinetics of accumulation of SCSV antigens. These results suggest that purified SCSV particles are capable of replication in transient protoplast systems. The protoplast assay could be used to characterise new or altered segments or functional domains of the SCSV genome and for the development of SCSV genome as a vector for foreign gene expression in plants.

Subterranean clover stunt disease is an economically important aphid-borne virus disease affecting certain pasture and grain legumes in Australia. The virus associated with the disease, subterranean clover stunt virus (SCSV), was previously found to be representative of a new type of single-stranded DNA virus. Analysis of the virion DNA and restriction mapping of double-stranded cDNA synthesized from virion DNA suggested that SCSV has a segmented genome composed of 3 or 4 different species of circular ssDNA each of about 850-880 nucleotides. To further investigate the complexity of the SCSV genome, we have isolated the replicative form DNA from infected pea and from it prepared putative full-length clones representing the SCSV genome segments. Analysis of these clones by restriction mapping indicated that clones representing at least 4 distinct genomic segments were obtained. This method is thus suitable for generating an extensive genomic library of novel ssDNA viruses containing multiple genome segments such as SCSV and banana bunchy top virus. The N-terminal amino acid sequence and amino acid composition of the coat protein of SCSV were determined. Comparison of the amino acid sequence with partial DNA sequence data, and the distinctly different restriction maps obtained for the full-length clones suggested that only one of these clones contained the coat protein gene. The results confirmed that SCSV has a functionally divided genome composed of several distinct ssDNA circles each of about 1 kb.

In subcellular extracts of Kunjin virus-infected cells prepared by lysis and differential centrifugation, the viral RNA polymerase, RNA and proteins were associated mainly with cytoplasm. When the cytoplasmic extract (500g supernate) of infected cells labelled for 3 h from 24 h post-infection was further fractionated by rapid centrifugation through a sucrose density gradient, all viral products were located only in dense or “heavy membrane” fractions, which contained three types of virus-induced morphologically distinct membrane structures. These dense fractions were treated with 0.5% NP 40 and the soluble material was again centrifuged through a sucrose gradient for analyses as before. Viral RNA polymerase activity was retained and was associated with replicative intermediate RNA and some replicative form RNA in the peak enzyme fractions sedimenting at 20 S to 40 S. Enrichment of NS 3 and of the small nonstructural proteins NS 2 A and NS 2 B/NS 4 A was apparent in these fractions which were well separated from the slow sedimenting structural proteins. No detergent-resistant structures in the “heavy membrane” fractions other than ribosome-like particles were visible. The data show that the RNA polymerase complex cosedimented with virus-induced membrane structures and remained associated with specific nonstructural proteins and replicative intermediate RNA after detergent treatment.

Viral diseases such as alfalfa mosaic virus (AMV) cause significant reductions in dry matter yield and persistency of white clover (Trifolium repens L.). Transgenic white clover plants expressing the AMV coat protein (AMV-CP) gene and showing immunity to AMV infection were evaluated in multi-site small-scale field releases. Two transformation events showing field immunity to aphid-mediated AMV infection This article outlines the development of transgenic elite white clover germplasm with AMV immunity, involving the world’s first breeding nursery for transgenic white clover.were selected for elite transgenic germplasm development. Following top crosses with elite parental breeding lines, diallel crosses of heterozygous offspring plants and identification of AMV-CP homozygous T2 lines through quantitative PCR-based high-throughput screening, a breeding nursery with 1,300 transgenic white clover plants was established. Agronomically superior transgenic white clover plants were selected as parents for the production of world’s first AMV immune transgenic white clover cultivars.

Viruses such as alfalfa mosaic alfamovirus (AMV), white clover mosaic potex virus (WCMV) and clover yellow vein potyvirus (CYVV), which are members of the three largest groups of plant viruses, have been found to have significant adverse effects on forage legumes. Each of these viruses individually infects a large number of plant species, has a worldwide distribution, and causes significant losses, especially in pasture and grain legumes.

Over the past several years, increased emphasis has been placed on conducting comprehensive ecological-risk assessments of virus-resistant genetically modified organisms (GMOs) prior to their release into the environment. In this paper we report on the first stage in our assessment of the level of risk posed by virus-resistant transgenic Trifolium repens L. (white clover) to native plant communities in south-eastern Australia. We investigated the distribution, abundance and phytosociological characteristics of naturalised T. repens populations in two areas in the subalpine region of New South Wales (NSW) and the Australian Capital Territory (ACT), and determined the distribution and abundance of Alfalfa mosaic virus, Clover yellow vein virus and White clover mosaic virus in 31 populations of white clover in this region. We found that T. repens is a significant component of Poa grasslands and Eucalyptus–Poa woodlands in the subalpine region, but is absent or rare in Eucalyptus species forests and Carex–Poa species bogs. Clover yellow vein virus was by far the most common virus in the study area, being present in 18% of T. repens plants across a wide range of plant communities. Alfalfa mosaic virus and White clover mosaic virus were each recorded in only one white-clover population growing in a native plant community. We conclude that white clover is a significant constituent of subalpine grasslands and woodlands in the region studied, and that of the viruses investigated, Clover yellow vein virus is the most abundant and widespread.

A reverse transcription polymerase chain reaction (RT-PCR) assay has been developed that can simultaneously test a sample, in one tube, for the presence of five seedborne legume viruses that are of great concern to legume germ plasm banks: alfalfa mosaic alfamovirus, bean yellow mosaic potyvirus, clover yellow vein potyvirus, cucumber mosaic cucumovirus, and subterranean clover mottle sobemovirus. RT-PCR assays were also developed for the detection of subterranean clover redleaf luteovirus and subterranean clover stunt virus, thus providing a detection system for all well-characterized viruses known to infect subterranean clover. Primers were designed so that the size of the RT-PCR product was indicative of the virus amplified and, where sequences of more than one strain of virus were available, conserved regions were given preference as primer targets

A new mechanically transmissible viruslike disease, subterranean clover distortion, was found in subterranean clover (Trifolium subterraneum) in a pasture in New South Wales. The pathogen has a wide experimental host range. Symptoms varied from thickening and distortion of young leaves, reddening of older leaves, and production of axillary shoots on subterranean clover to chlorosis of leaves, flower malformation, flower abortion, and stem necrosis on other hosts. Symptomless shoots often developed on infected plants. No local lesion host was identified (.)

SUMMARY An enzyme-linked immunosorbent assay (ELISA) system for the rapid detection of the plant rhabdovirus, lettuce necrotic yellows virus (LNYV) in plant hosts and individual aphid vector is described. The method has been shown to be reliable and sensitive, and to have a number of advantages over the conventional methods of detecting LNYV in plants and insects by infectivity tests.

In this review, ways of detecting plant pathogens are divided into specific and non-specific methods. Specific methods are those which are used to detect a particular species or group of pathogens after preliminary diagnosis suggests the presence of that pathogen. Specific methods are also used when the assay involves a known pathogen, for instance as in epidemiological surveys. On the other hand, non-specific methods include those used to detect unknown pathogens or when detection of a number of pathogens is desired

SUMMARY Transgenic lines of subterranean clover were constructed that contained three different Bean yellow mosaic virus (BYMV) coat protein (CP) gene constructs; full-length CP, the core region of the CP, and full-length CP plus the 3′ untranslated region of the viral genome. Transgenic plants containing the full-length and core CP gene constructs showed high and moderate levels of BYMV resistance. Resistance was measured as a lack or amelioration of viral disease symptoms, which was correlated with a reduction in virus levels and yield loss. A range of different resistance phenotypes was observed. They included reduced infection rates, delay and reduction in local lesion development, and delay and reduction in severity of systemic symptom development. Resistance levels were not correlated with transgene mRNA levels and no transgene-encoded protein was detected in any of the transgenic lines. This is the first example of genetically engineered virus resistance in a clover.