Download A suspected virus from soybean infecting cocoa seedlings

A suspected virus from soybean infecting cocoa
seedlings
G. K. OWUSU
Cocoa Research Institute, P.O. Box 8, Tafo, Ghana
SUMMARY
A disorder with virus-like symptoms was mechanically
transmitted from soybean plants to cocoa seedlings in
the greenhouse. The causative agent was present in the
testa tissue but not in the perisperm or embryo of seeds
produced by infected soybean plants. It is suggested that
the condition was due to an unidentified virus carried in
the testa tissue of soybean seed.
Introduction
During the course of studies on the mechanical
transmission of cocoa swollen shoot virus (CSSV)
virulent strain A, by inoculating leguminous seeds
with preparations
of the virus, two soybean
(Glycine max (L.) Merr. var. Bragg) seedlings
developing from inoculated seeds produced pale
leaves with prominent green veins. Back test to
Amelonado cocoa beans resulted in cocoa seedlings
with virus-like symptoms quite distinct from those
of CSSV. This note reports attempts to find out
the cause and source of the condition.
Materials and methods
The soybean seeds were obtained from Mr E. V.
Doku of the University of Ghana, Legon.
Soybean seeds and cocoa beans were mechanically
inoculated by brushing the exposed, intact
cotyledons with the inoculum containing Celite 545
as abrasive, in a manner similar to that of Brunt &
Kenten (1960). Dry soybean seeds were softened
before inoculation by placing them on moist
blotting paper overnight. Soybean seedlings were
RESUME
G. K. Owusu: Un virus de soja soupr;onnede s'attaquer
aux semis de cacaoyer. Une degenerescence des tissus,
offrant I'apparence des symptomes d'une maladie a
virus, a ete transmise mecaniquement a partir de plantes
de soja, a des semis de cacaoyer, en serre. L'agent de
cette maladie etait present dans I'enve!oppe des grains
infectes de soja, mais pas dans Ie perisperme ni dans
I'embryon. L'auteur suggere que ces resultats sont dus
a un virus non identifie, qui se trouvait dans les tissus
de I'enveloppe de graines de soja.
inoculated by rubbing both the cotyledons and
leaves with the forefinger wet in the inoculum
containing extra fine carborundum (about 300 grit
powder).
Crude soybean extracts were prepared either by
grinding leaves or seed parts in distilled water or
a medium (pH 8) containing 0·01 M disodium
hydrogen phosphate and 0 ·01 M thioglycollic acid
(P-T8).
In attempts partially to purify a virus from
infected cocoa, 15 g young first flush leaves from
Amelonado cocoa seedlings with well developed
symptoms were ground in 300 ml of a solution
(pH 6,5) containing 0·05 M disodium hydrogen
phosphate,
0 .05 M thioglycollic acid, 0 ·01
M sodium diethyldithiocarbamate and 4·5 g hide
powder (Hopkins & Williams Ltd.), as described
for CSSV by Brunt & Kenten (1963). The homogenate was immediately squeezed through cotton
cloth and clarified by centrifuging at 10 000 g for
10 min. The supernatant was centrifuged at
75 000 g for 90 min and the pellets suspended in
P-T8.
G. K. Owusu (1971) Ghana Jnl agric. Sci. 4, 201-204
TABLE
Transmission from soybean to cocoa
A virus was apparently transmitted to cocoa
seedlings when crude extracts from infected soybean leaves were inoculated to whole Amelonado
cocoa beans (Table 1).
In cocoa seedlings, symptoms of the disorder
were present only on first flush leaves, subsequent
flushes, including those produced when infected
seedlings were coppiced, being free of symptoms.
Young, first flush leaves showed transient red
vein-banding followed by diffuse clearing of the
very fine veins. Next appeared either large, bright
yellow chlorotic patches, or small, bright yellow,
chlorotic spots. In hardened leaves, the chlorotic
spots sometimes coalesced to form large, irregular
patches. Symptoms may be uniformly scattered
over the lamina or may be more numerous on one
side of the midrib than the other. There was severe
crinkling of leaves, and hardened ones were
leathery (Fig. 1).
1
Infection of Cocoa Beans by Sap Extracts from Leaves of
Infected Soybean Plants
Age of soybean source
Extractant
plantst
(days)
t
I Infection
rate
%
infection
38
P-T8
21/32*
66
71
"
38/175
22
71
Distilled
water
15/39
38
2 g of each material.
= number of infected cocoa seedlings;
Denominator = total number of cocoa beans inoculated.
* Numerator
Transmission from cocoa to cocoa
Crude and clarified extracts from cocoa leaves
were usually not infective. Of 120 cocoa beans
Fig. I. Two leaves from an infected cocoa seedling showing vein clearing. chlorotic patches and crinkling. Leaf at the
extreme left is from a healthy control seedling.
A suspected virus from soybean infecting cocoa seedlings
inoculated with partially purified extracts from
cocoa leaves, only one seedling developed characteristic symptoms.
Grafting of 4-week-old and 6-month-old Amelonado cocoa seedlings with bark and petiole
patches from affected cocoa seedlings did not induce
symptons, neither did the transfer by the CSSV
vector Planococcoides njalensis (Laing). In the latter
test, the mealybugs were starved for 48 h and then
given an acquisition feed for 72 h on the shoot
tips and young leaves of cocoa seedlings with welldeveloped symptoms. Nymphs were then transferred to 80 dissected Amelonado cocoa beans
(Posnette & Strickland, 1948) at the rate of ten
insects per bean.
Seed transmission studies
Mature but green seeds produced by the two
affected soybean plants were dissected into testa,
perisperm (remains of nucellar tissue) and embryo:
each seed tissue was bulked and the extract
inoculated to 40 cocoa beans. Those inoculated
with extract from the testa, but not that from
perisperm or embryo tissues, developed into
seedlings with symptoms.
Seedlings were grown from a second lot of
seeds produced by the two affected soybean plants,
and the first trifoliate leaves bulked. Extracts made
from them were tested for the presence of a virus
by inoculating cocoa beans. No virus-like symptoms appeared on 40 cocoa seedlings grown from
the inoculated cocoa beans.
Seeds of five varieties of soybean (vars. Bragg,
Bilon, Hardy, Hill and Lee) were tested in an
attempt to find out whether they carried a virus
that would infect cocoa. They were dissected as
above into testa, perisperm and embryo, and
different seed tissues from each soybean variety
tested separately, by inoculating to 40 cocoa beans.
No virus-like symptoms appeared on any of the
seedlings from the inoculated cocoa beans.
Serological studies
Tests were conducted to determine if a virus was
present that was serologically related to cocoa
yellow mosaic virus (CYMV) since the disease
symptoms in cocoa seedlings caused by the former
resemble those of the latter. Antiserum to CYMV
was provided by Mr R. H. Kenten of this
Institute. In tube precipitin tests, 0·5 ml of
203
antiserum or antigen (partially purified extract
from cocoa leaves) at various 2-fold dilutions in
0·85% saline were mixed with 0·5 ml of antigen
or antiserum, respectively, at a fixed dilution. The
reaction mixtures were incubated overnight at
37°C in a water bath. No positive reactions were
obtained.
Possible implication of CSSV
Since the two soybean plants from which the
agent was transmitted were among plants inoculated
as seeds with CSSV, the possibility existed that
CSSV might be implicated in the disease symptoms produced in cocoa seedlings. This was
investigated by mechanically inoculating 50 soybean (var. Bragg) seeds with a strong partiallypurified preparation of CSSV virulent strain A.
(At a dilution of 1 : 5 this inoculum infected all
20 Amelonado cocoa seedlings grown from
inoculated beans). Three weeks later, the resulting
seedlings were tested for infection, by inoculating
cocoa beans with their leaf extracts. In another
experiment, 33 soybean (var. Bragg) seedlings,
14 days old, were mechanically inoculated with
CSSV virulent strain A, and the plants tested for
infection 3 weeks later, as above. None of the
seedlings grown from the inoculated cocoa beans
showed characteristic symptoms.
Discussion
In nature, a few viruses are known to infect
cocoa; it was therefore interesting to determine the
source of this virus-like disorder. The evidence
presented above, apparently insufficient, suggests
a virus-causal agent for the disorder. The failure
to demonstrate graft transmission from cocoa to
cocoa might have been due to the fact that cocoa
seedlings beyond the first flush of growth did not
show symptoms of the disorder. That only a single
plant developed symptoms after inoculation with
preparations from diseased cocoa seedlings was
probably because the methods of extraction and
partial purification were not good enough. It is
also possible that the virus was present at a very
low concentration in cocoa seedlings. In many of
its characteristics, however, the suspected virus
differed from known cocoa viruses (Thresh &
Tinsley, 1959; Blencowe, Brunt, Kenten & Lovi,
1963; Brunt, Kenten, Gibbs & Nixon, 1965).
That the suspected virus was present in the
204
G. K. Owusu (1971) Ghana Jnl agric. Sci. 4. 201-204
testa tissue o~ seeds fr?~ infected. soy~ean. plants
sugg~sts t~at It was ongInally. earned ~ this way.
The mfectlon of soybean seedlIngs by a VIrUSwhich
is only present in the testa suggests that the virus
is highly infectious, although no infectivity tests
from soybean to soybean could be done before the
virus was lost in the greenhouse. Infection probably
resulted from contamination
of germinating
embryos and seedlings from the infected testa, as
reported for tobacco mosaic virus in tomato
(Taylor, Grogan & Kimble, 1961) and pungent
pepper (Crowley, 1957). The failure to detect the
suspected virus in seed dissection of five varieties
of soybean, induding the variety Bragg, indicates
that the incidence of testa tissue infection is very
low.
In the search for herbaceous hosts of CSSV
many plants have been tested by mechanically
inoculating them with partially purified preparations of the virus, but the results have largely been
negative (Legg & Lovi, 1968; Owusu, 1970).
Okusanya (1968), however, reported that three
varieties of cowpea were susceptible to two
Nigerian isolates of CSSV, but her results are yet
to be authenticated.
Quite a number of viruses are now known to be
seed-borne (Bennett, 1969), many in their leguminous and other herbaceous hosts. Evidence
presented in this report indicates that a seed-borne
virus from soybean can infect cocoa seedlings; this
may also apply to some of the other seed-borne
viruses of legumes and other plants. This phenomenon, hitherto assumed to be rare, should be
considered in the screening of plants for herbaceous hosts of cocoa viruses.
T~s paper is published by permission of the
DIrector, Cocoa Research Institute of Ghana, Tafo.
REFERENCES
Bennett, C. W. (1969) Seed transmission of plant
viruses. Advan. Virus Res. 14, 221-261.
Blencowe, J. W., Brunt, A. A., Kenten, R. H. & Lovi,
N. K. (1963) A new virus disease of cocoa in Sierra
Leone. Trop. Agric. Trin. 40, 233-236.
Brunt, A. A. & Kenten, R. H. (1960) Mechanical
transmission of cocoa swollen shoot virus. Virology
12, 328-330.
Brunt, A. A. & Kenten, R. H. (1963) The use of
proteins in the extraction of cocoa swollen shoot virus
from cocoa leaves. Virology 19, 388-392.
Brunt, A. A., Kenten, R. H., Gibbs, A. J. & Nixon,
H. L. (1965) Further studies on cocoa yellow mosaic
virus. J. gen. Microbiol. 38, 81-90.
Crowley, N. C. (1957) Studies on the seed transmission
of plant virus diseases. Aust. J. Biol. Sei. 10, 449-464.
Legg, J. T. & Lovi, N. K. (1968) Virus research. Search
for local lesion host of swollen shoot virus. In A. Rep.
Cocoa Res. Inst. Ghana, 1966-67, 22-24. Tafo: the
Institute.
Okusanya, B. A. M. (1968) Studies of cacao swollen
shoot disease. In A. Rep. Cocoa Res. Inst. Nigeria,
1966-67, 67-68. Ibadan: the Institute.
Owusu, G. K. (1970) Virus research. Inoculation of
leguminous bean seeds with cocoa swollen shoot virus.
In A. Rep. Cocoa Res. Inst. Ghana, 1968-69, 24-25.
Tafo: the Institute.
Posnette, A. F. & Strickland, A. H. (1948) Virus
diseases of cacao in West Mrica, III. Technique of
insect transmission. Ann. appl. Biol. 35, 53-63.
Acknowledgement
Taylor, R. H., Grogan, R. G. & Kimble, K. A. (1961)
Transmission of tobacco mosaic virus in tomato seed.
Phytopathology 51, 837-842.
The author is grateful to Dr J. T. Legg for
reading the manuscript, and to Messrs J. K.
Bonney and M. A. Donkor for technical assistance.
Thresh, J. M. & Tinsley, T. W. (1959) The viruses of
cacao. Tech. Bull. W. Afr. Cocoa Res. Inst. No.7.
Tafo: the Institute.