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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.