345
December 1989
Family: Luteoviridae
Genus: Unassigned Luteoviridae
Species: Groundnut rosette assistor virus
Acronym: GRAV


Groundnut rosette assistor virus

A. F. Murant
Scottish Crop Research Institute, Invergowrie, Dundee, DD2 5DA, Scotland, UK

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

First recognised and named by Hull & Adams (1968) as a component of groundnut rosette disease. Identified as a luteovirus by Casper et al. (1983) and Reddy et al. (1985). Virus purified and characterized by Rajeshwari & Murant (1988).

A virus with isometric particles about 28 nm in diameter containing a single nucleic acid species (presumed to be RNA) and forming a single sedimenting and buoyant density component. Infects several species of Leguminosae (symptomlessly) and a few in other families. Transmitted in a persistent circulative manner by aphids (Aphis craccivora) but not by inoculation of sap or through seed. Important as the helper for aphid transmission of groundnut rosette virus. Known only from sub-Saharan Africa and Madagascar.

Main Diseases

Infects groundnut (peanut; Arachis hypogaea) without causing symptoms, but acts as a helper virus (Hull & Adams, 1968) for aphid transmission of groundnut rosette virus (Storey & Bottomley, 1928) and its satellite RNA (Murant et al., 1988). The entire complex is responsible for groundnut rosette disease (Zimmermann, 1907), the most important virus disease of groundnut in Africa. Groundnut plants infected with the virus complex exhibit various degrees of stunting or rosetting and of reduction in leaf size, and the leaves may be yellow or show a mosaic (Fig.1).

Geographical Distribution

The isolates of groundnut rosette assistor virus reported by Hull & Adams (1968) were found in rosetted groundnut plants from Malawi and Nigeria, and these are still the only countries from which the virus has been identified unequivocally by serological testing (Rajeshwari et al., 1987; Rajeshwari & Murant, 1988). However, the virus is assumed to occur wherever groundnut rosette disease has been reported, i.e. from east, west, central and southern Africa, and Madagascar. The disease has not been reliably reported outside the African continent; scattered early reports from India, Java and Australia have not been confirmed (D. V. R. Reddy, personal communication).

Host Range and Symptomatology

Groundnut is the only natural host known, but experimentally the virus has been shown to infect seven other species of Leguminosae (Pisum sativum, Stylosanthes gracilis, S. hamata, S. mucronata, S. sundaica, Trifolium incarnatum and T. pratense) and four species in other families (Capsella bursa-pastoris, Gomphrena globosa, Montia perfoliata and Spinacia oleracea) (Adams, 1967; Hull & Adams, 1968; Okusanya & Watson, 1966; Rajeshwari & Murant, 1988). All these hosts are infected symptomlessly except C. bursa-pastoris, which may show a generalised chlorosis at high light intensities.


In aphid transmission experiments with the rosette virus complex, usually some plants become infected with either of the viruses alone. Groundnut rosette assistor virus can also be obtained alone by aphid transmission to Pisum sativum, or to ‘rosette-resistant’ groundnut lines such as RG1 or RRI/6 (see Notes).

Diagnostic species
Arachis hypogaea (groundnut, peanut), Pisum sativum (pea). Symptomless systemic infection detectable only by serological tests.

Glycine max (soybean), Phaseolus aureus (mung bean), P. mungo (urd bean, black gram), P. vulgaris (French bean) and Vicia faba (broad bean) are immune.

Propagation species
A. hypogaea.

Assay species
None.

Strains

No strains reported. Isolates from plants with green or chlorotic rosette in Nigeria, or with chlorotic rosette in Malawi, appear identical in biological and serological properties (Rajeshwari et al., 1987; Rajeshwari & Murant, 1988).

Transmission by Vectors

The groundnut rosette virus complex, and therefore presumably groundnut rosette assistor virus itself, is transmitted by Aphis craccivora in a persistent manner, the aphids retaining ability to transmit for up to at least 10 days (Storey & Ryland, 1955; Watson & Okusanya, 1967). Four cultures of what must now be regarded as the virus complex were transmitted by a Nigerian population of A. craccivora but only the two East African strains were transmitted by a Kenyan population (Watson & Okusanya, 1967). However, in recent studies (A. F. Murant, unpublished data), A. craccivora from Malawi transmitted cultures of groundnut rosette assistor virus from both countries. Watson & Okusanya (1967) found that most individuals of A. craccivora needed longer than 24 h to acquire the virus complex from infected groundnut and many needed to feed for 2 - 3 days on healthy plants to cause infection. However, the frequencies of transmission in their experiments were much lower than in those of Storey & Ryland (1955), which suggests that the strain of vector or environmental conditions might not have been optimal.


The ability of groundnut rosette assistor virus to assist aphid transmission of groundnut rosette virus probably results from the packaging of the dependent virus RNA in the coat protein of the helper, as has been shown for two other helper luteoviruses, beet western yellows virus (which assists lettuce speckles mottle virus; Falk et al., 1979) and carrot red leaf virus (which assists carrot mottle virus; Waterhouse & Murant, 1983). Experimentally, groundnut rosette assistor virus has been shown also to assist transmission by Aphis craccivora of tobacco yellow vein virus, which normally depends on tobacco yellow vein assistor virus for transmission by Myzus persicae (Hull & Adams, 1968).

Transmission through Seed

Not found (Storey & Bottomley, 1928).

Serology

The virus is a good immunogen; antiserum with a titre of 1/256 in gel diffusion tests was raised in a rabbit given two intramuscular injections, each of 60 µg purified virus, at an interval of 15 days (Rajeshwari & Murant, 1988). The antiserum reacted well in immunosorbent electron microscopy (ISEM) tests and in enzyme- linked immunosorbent assay (ELISA), and has been used to detect the virus in leaf extracts from naturally and experimentally infected groundnut.

Relationships

The virus is serologically related to several members of the luteovirus group. Casper et al. (1983) showed that, in ISEM tests, virus particles presumed to be those of groundnut rosette assistor virus were trapped from crude extracts of rosette-diseased groundnut leaves on grids coated with antisera to bean leaf roll (= pea leaf roll), beet western yellows, potato leafroll or barley yellow dwarf luteoviruses; however, these antisera coated the particles only weakly. Reddy et al. (1985) and Rajeshwari & Murant (1988) confirmed these results and in addition found weak trapping with antisera to beet mild yellowing, subterranean clover red leaf and tobacco necrotic dwarf luteoviruses, though none with antiserum to carrot red leaf luteovirus. In the studies of Casper et al. (1983), only the antiserum to beet western yellows virus gave a positive reaction in double antibody sandwich ELISA (DAS-ELISA). Rajeshwari et al. (1987) found that groundnut rosette assistor virus did not react in DAS-ELISA or F(ab')2-ELISA when both the plate-coating and detecting antibodies were from polyclonal antisera to beet western yellows or potato leafroll viruses; however, the virus reacted with three out of ten monoclonal antibodies raised to potato leafroll virus when they were used as the second (detecting) antibody and polyclonal antiserum to beet western yellows or potato leafroll viruses was used as the first (plate-coating) antibody in triple antibody sandwich ELISA (TAS-ELISA). In F(ab')2-ELISA in which F(ab')2 fragments from antiserum to groundnut rosette assistor virus were used as the plate-coating antibody and various luteovirus antisera were used as the detecting antibody, Rajeshwari & Murant (1988) found a moderately close relationship to bean leaf roll and potato leafroll viruses, a distant relationship to tobacco necrotic dwarf virus, and no relationship to carrot red leaf virus.

Stability in Sap

No information.

Purification

The following procedure was developed by Rajeshwari & Murant (1988), using TAS-ELISA with a monoclonal antibody to potato leafroll virus (Rajeshwari et al., 1987) to track the virus through the steps of the purification procedure. Inoculate groundnut plants by grafting and after 15 - 20 days identify infected plants by ELISA. Harvest entire shoots of infected plants and grind each 100 g tissue with liquid nitrogen in a mortar. Homogenize the powder in a blender with 400 ml 60 mM phosphate buffer, pH 8.0, containing 10 mM disodium ethylenediamine-tetraacetate (EDTA) and 0.5% (v/v) of a 90% thioglycerol solution. Stir the extract, now at pH 6.8, for 2 h at 20°C in the presence of 5% (v/v) Celluclast, an industrial cellulase preparation (Nova Enzyme Products, Ltd., Windsor; Waterhouse & Helms, 1984). Pass the digest through two layers of cheesecloth and shake the filtrate thoroughly with half its volume of a 1:1 mixture of n-butanol and chloroform. Break the emulsion by centrifugation for 10 min at 13,000 g. Mix the supernatant fluid with polyethylene glycol (PEG; M. Wt 6000) at 80 g/l and NaCl at 0.2 M, and keep for 1.5 h at 4°C. Centrifuge the preparation for 20 min at 13,000 g, and shake the pellets overnight at 4°C in 40 ml 6 mM phosphate buffer, pH 7.2 (PB). Centrifuge the resuspended material for 10 min at 13,000 g, and add Triton X-100 to the supernatant fluid to 1% (v/v). Layer each 18 ml of the preparation over a ‘cushion’ of 7 ml 200 g/l sucrose in PB containing 80 g/l PEG and 0.2 M NaCl in Beckman 50.2 Ti rotor tubes, and centrifuge for 2 h at 50,000 rev./min. Resuspend the pellets in 16 ml PB and after 1 h at 4°C remove the insoluble material by centrifugation for 10 min at 13,000 g. Subject the virus to three cycles of centrifugation in sucrose density gradients made up in PB. If, in the first one or two cycles, the virus-containing zone is obscured by host material, identify the fractions containing virus particles by ELISA. Pool the fractions containing the virus particles and centrifuge for 1 h at 250,000 g, resuspending the pellets in 0.5 ml PB. Yields of virus particles are c. 0.5-1.0 mg/kg leaf material.

Properties of Particles

(Rajeshwari & Murant, 1988): Virus particle preparations contain a single sedimenting component with a sedimentation coefficient (s20,w) of 115 S (not corrected for infinite dilution).

Buoyant density in Cs2SO4: 1.34 g/cm3.

A260/A280: 1.86.

Amax: 259 nm; Amin: 237 nm; Amax/Amin: 1.96.

Particle Structure

In uranyl acetate negative stain the particles are isometric, c. 28 nm in diameter, with hexagonal outlines (Fig.2) (Rajeshwari & Murant, 1988).

Particle Composition

Nucleic acid: A single species, presumed to be RNA, of M. Wt 2.09 x 106. The nucleic acid did not bind to oligo(dT)-cellulose in high salt, indicating that there is no appreciable polyadenylate sequence (Rajeshwari & Murant, 1988).

Protein: A single species, M. Wt 24,500 (Rajeshwari & Murant, 1988).

Relations with Cells and Tissues

No information. However, the particles, like those of other luteoviruses, are probably confined to phloem tissue.

Notes

Groundnut rosette disease does not occur in South America, where the groundnut originated, or in any other regions of the world to which groundnut has been distributed except Africa, so the causal virus(es) must occur naturally in some native African plant. However, no such host has been discovered. The viruses probably survive between crops in ‘volunteer’ groundnut plants (Storey & Bottomley, 1928).


Resistance to rosette disease was first found in groundnut germplasm from the region between Cote d’Ivoire and Burkina Faso (Sauger & Catharinet, 1954; De Berchoux, 1958). This material was the source of the resistance in all rosette-resistant cultivars (Fig.3) developed since. The resistance is now known to be directed only against groundnut rosette virus: all rosette-resistant material is susceptible to groundnut rosette assistor virus (K. R. Bock, A. F. Murant & R. Rajeshwari, unpublished data).

References

  1. Adams, Rhodesia, Zambia Malawi J. agric. Res. 5: 145, 1967.
  2. Casper, Meyer, Lesemann, Reddy, Rajeshwari, Misari & Subbarayadu, Phytopath. Z. 108: 12, 1983.
  3. De Berchoux, Oléagineux 13: 237, 1958.
  4. Falk, Duffus & Morris, Phytopathology 69: 612, 1979.
  5. Hull & Adams, Ann. appl. Biol. 62: 139, 1968.
  6. Murant, Rajeshwari, Robinson & Raschke, J. gen. Virol. 69: 1479, 1988.
  7. Okusanya & Watson, Ann. appl. Biol. 58: 377, 1966.
  8. Rajeshwari & Murant, Ann. appl. Biol. 112: 403, 1988.
  9. Rajeshwari, Murant & Massalski, Ann. appl. Biol. 111: 353, 1987.
  10. Reddy, Murant, Duncan, Ansa, Demski & Kuhn, Ann. appl. Biol. 107: 57, 1985.
  11. Sauger & Catharinet, Agron. trop. 11: 28, 1954.
  12. Storey & Bottomley, Ann. appl. Biol. 15: 26, 1928.
  13. Storey & Ryland, Ann. appl. Biol. 42: 423, 1955.
  14. Waterhouse & Helms, J. virol. Meth. 8: 321, 1984.
  15. Waterhouse & Murant, Ann. appl. Biol. 103: 455, 1983.
  16. Watson & Okusanya, Ann. appl. Biol. 60: 199, 1967.
  17. Zimmermann, Der Pflanzer 3: 129, 1907.

Acknowledgements

Supported in part by the Overseas Development Natural Resources Institute (Research Project No. X0011).


Figure 1

Symptoms of chlorotic rosette in field-infected groundnut.

Figure 2

Particles of groundnut rosette assistor virus from a purified preparation, stained in 1% uranyl acetate. Bar represents 100 nm.

Figure 3

Part of a field trial of rosette-resistant groundnut lines at Chiredze, Malawi. Centre row, rosette-susceptible control cv. Spancross infected with both groundnut rosette and groundnut rosette assistor viruses. Outer rows, rosette-resistant breeding lines showing no symptoms; these plants were free from groundnut rosette virus but many were nevertheless infected with groundnut rosette assistor virus (photograph courtesy Dr K. R. Bock).