Oat chlorotic stunt virus

Neil Boonham
Central Science Laboratory, Sand Hutton, York, YO41 1LZ.

K. Roger Wood
The School of Biosciences, The University of Birmingham, Edgbaston, Birmingham, B15 2TT.

Contents

Introduction Main Diseases Geographical Distribution Host Range and Symptomatology Strains Transmission by Vectors Transmission through Seed Transmission by Grafting Transmission by Dodder Serology Nucleic Acid Hybridization Relationships Stability in Sap

Purification Properties of Particles Particle Structure Particle Composition Properties of Infective Nucleic Acid Molecular Structure Genome Properties Satellites Relations with Cells and Tissues Ecology and Control Notes References Acknowledgements Figures

Introduction

The disease was first described by Catherall (1986) and the virus identified by Thomas (1987).

The virus possesses isometric particles c. 35nm in diameter and the genome is composed of single stranded +ve sense RNA. The virus accumulates to high concentrations in infected plants, but is poorly sap transmissible. It is known naturally to cause symptoms only in winter oats (Avena sativa) and has been identified at only a few sites in England and Wales. No vector has so far been identified.

Main Diseases

The symptoms in infected oat plants consist of severe stunting and bright yellow chlorotic streaking of the leaves (Fig 1). Yellow areas often become necrotic as the leaves age. Emerging leaves exhibit distinctive twisting and are broader and darker green than those of healthy oat plants.

Geographical Distribution

The virus has only been identified at three sites; two in Wales (Aberystwyth and Brecon) and one in England (Shropshire) (Boonham et al., 1997).

Host Range and Symptomatology

Under field conditions, systemic symptomatic infection has been identified only in winter oats (Avena sativa) (Fig.1), although most plants found to be infected in the field were symptomless. Under field conditions, symptomless infections were also identified in winter wheat (Triticum aestivum), winter barley (Hordeum vulgare) and annual meadow grass (Poa annua). However, the virus was present at low titre and localised only in the roots. Standard mechanical inoculation of virus to leaves or roots of susceptible hosts using infected sap or purified virus did not result in infection. Using purified virus and an embryo wounding technique, oats (Avena sativa), wheat (Triticum aestivum), barley (Hordeum vulgare) and maize (Zea mays) could be infected, although symptoms were observed only in oats (Avena sativa). No dicotyledonous hosts are known.

Propagation hosts

The virus could be propagated by inoculating healthy oat seeds using an embryo wounding technique (Boonham et al., 1997).

Transmission by Vectors

Initial observations were consistent with transmission by zoosporic fungi, such as Polymyxa or Olpidium (Catherall, 1986). However, the vector of the virus, if any, remains unknown. The predominance of root infection suggests that OCSV, in common with other members of the Tombusviridae, is transmitted via the soil, and experiments in controlled environments have shown that roots of oat plants could be infected (symptomlessly) by growing seedlings in soil from infected sites (Boonham et al., 1997).

Transmission through Seed

None found (Boonham et al., 1997).

Serology

The virus is strongly immunogenic, with both rabbit polyclonal (Welsh Plant Breeding Station, Aberystwyth) and rat monoclonal antibodies (Central Science Laboratory, York) being prepared. The polyclonal antiserum has been used to detect the virus in gel diffusion assays and, when conjugated to alkaline phosphatase, it has been used in DAS ELISA (Boonham et al., 1997).

Relationships

In gel diffusion assays, the virus was found to be serologically unrelated to Carnation mottle virus (Carmovirus), Tomato bushy stunt virus (Tombusvirus) or Maize chlorotic mottle virus (Machlomovirus), all members of the Tombusviridae.

Purification

Virus was purified by grinding plant material in 10 volumes of grinding buffer (0.013M Tris HCl, pH 8, 0.067M K₂HPO₄ containing 0.1% thioglycolic acid). The sap extract was passed through muslin, and clarified with either n-butanol or chloroform. The solvent layer was discarded and the virus purified by differential centrifugation at 110,000 g and 10,000 g. The virus was resuspended in 0.05M Tris HCl pH 7.4 (Boonham et al., 1995).

Particle Structure

The particles are isometric and about 35nm in diameter (Fig 2). Some particles are penetrated by uranyl acetate negative stain.

Particle Composition

RNA

The genome is monopartite, positive-sense single stranded RNA consisting of 4114 nucleotides, without a poly-A tail. A single 3' coterminal sub-genomic RNA, calculated to be 1772 nt in length, was observed in RNA extracted from infected plants and from purified virus (Boonham et al., 1998).

Protein

The major protein in virus preparations was digested with CNBr and the fragments sequenced directly. The resulting amino acid sequence had homology with the 48kDa predicted protein product of ORF3. This, along with significant sequence homology with motifs found within the coat proteins of other tombusviruses, identifies the 48kDa protein as the coat protein (Boonham et al., 1995).

Genome Properties

A complete nucleotide sequence has been determined for the genome of the Brecon isolate (accession number X83964) (Boonham et al., 1995). The RNA has three predicted major ORFs (Fig.3). ORF1 encodes a short protein (23 kDa) with unknown activity which concludes with a UAG (amber) termination codon. The amber codon could potentially be read through to generate a larger product (84 kDa) incorporating ORF2, which encodes RNA-dependent RNA polymerase (RdRp) motifs, indicating that the product is involved in virus replication. ORF3 is in a different reading frame to ORF1/2 and encodes the 48 kDa coat protein (Boonham et al., 1995). Only the 23kDa product was observed following in vitro translation of genomic RNA in rabbit reticulocyte lysates (Boonham et al., 1998).

Relations with Cells and Tissues

In thin sections, virus was observed to fill the cytoplasm around the organelles in mesophyll cells. No particles were found in the mitochondria, chloroplasts or nuclei which appeared unaltered when compared to uninfected tissue (Thomas 1987).

References

1. Boonham, Henry & Wood, Journal of General Virology 76: 2025, 1995.
2. Boonham, Harju, Wood & Henry, Plant Pathology 46: 795, 1997.
3. Boonham, Henry & Wood, Virus Genes 16: 143, 1998.
4. Catherall, Welsh Plant Breeding Station Annual Report, 132, 1986.
5. Thomas, Welsh Plant Breeding Station Annual Report, 15, 1987.

Figure 1

An infected oat plant (cv. Aintree) from the Brecon site, exhibiting typical symptoms of OCSV infection.

Figure 2

35nm isometric virus particles of OCSV, from a leaf squash preparation, negatively stained with uranyl acetate. Bar represents 100nm.

Figure 3

Genome organisation of OCSV.