Groundnut rosette assistor virus
A. F. Murant
Scottish Crop Research Institute, Invergowrie, Dundee, DD2 5DA, Scotland, UK
First recognised and named by Hull & Adams (1968)
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
Infects groundnut (peanut; Arachis hypogaea)
causing symptoms, but acts as a helper virus (Hull & Adams,
) for aphid transmission of groundnut rosette virus (Storey
& Bottomley, 1928)
and its satellite RNA (Murant et al.,
. 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
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,
). 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 four species in other families
(Capsella bursa-pastoris, Gomphrena globosa, Montia perfoliata
and Spinacia oleracea)
; Hull & Adams,
; 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
- 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
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
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 &
). 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
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
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
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.
The virus is serologically related to several members of the
. 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
luteoviruses; however, these antisera coated the
particles only weakly. Reddy et al. (1985)
& Murant (1988)
confirmed these results and in addition found
weak trapping with antisera to beet mild yellowing
clover red leaf
and tobacco necrotic dwarf
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)
groundnut rosette assistor virus did not react in DAS-ELISA or
-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
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
Stability in Sap
The following procedure was developed by Rajeshwari & Murant
, using TAS-ELISA with a monoclonal antibody to potato leafroll
(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
, 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
. 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
Properties of Particles
(Rajeshwari & Murant, 1988
): Virus particle
contain a single sedimenting component with a sedimentation
) of 115 S (not corrected for infinite
Buoyant density in Cs2SO4: 1.34 g/cm3.
Amax: 259 nm; Amin: 237 nm;
In uranyl acetate negative stain the particles are isometric,
28 nm in diameter, with hexagonal outlines (Fig.2
(Rajeshwari & Murant, 1988
Particle CompositionNucleic 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 &
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
, are probably confined to phloem tissue.
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 &
Resistance to rosette disease was first found in groundnut germplasm
from the region between Cote dIvoire 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).
- Adams, Rhodesia, Zambia Malawi J. agric. Res. 5: 145, 1967.
- Casper, Meyer, Lesemann, Reddy, Rajeshwari, Misari & Subbarayadu, Phytopath. Z. 108: 12, 1983.
- De Berchoux, Oléagineux 13: 237, 1958.
- Falk, Duffus & Morris, Phytopathology 69: 612, 1979.
- Hull & Adams, Ann. appl. Biol. 62: 139, 1968.
- Murant, Rajeshwari, Robinson & Raschke, J. gen. Virol. 69: 1479, 1988.
- Okusanya & Watson, Ann. appl. Biol. 58: 377, 1966.
- Rajeshwari & Murant, Ann. appl. Biol. 112: 403, 1988.
- Rajeshwari, Murant & Massalski, Ann. appl. Biol. 111: 353, 1987.
- Reddy, Murant, Duncan, Ansa, Demski & Kuhn, Ann. appl. Biol. 107: 57, 1985.
- Sauger & Catharinet, Agron. trop. 11: 28, 1954.
- Storey & Bottomley, Ann. appl. Biol. 15: 26, 1928.
- Storey & Ryland, Ann. appl. Biol. 42: 423, 1955.
- Waterhouse & Helms, J. virol. Meth. 8: 321, 1984.
- Waterhouse & Murant, Ann. appl. Biol. 103: 455, 1983.
- Watson & Okusanya, Ann. appl. Biol. 60: 199, 1967.
- Zimmermann, Der Pflanzer 3: 129, 1907.
Supported in part by the Overseas Development Natural Resources
Institute (Research Project No. X0011).
Symptoms of chlorotic rosette in field-infected groundnut.
Particles of groundnut rosette assistor virus from a
purified preparation, stained in 1% uranyl acetate. Bar represents
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).