African Crop Science Journal African Crop Science Society ISSN: 1021-9730 EISSN: 2072-6589 Vol. 4, Num. 2, 1996, pp. 215-222

Occurrence of southern bean mosaic virus (SVMV) in Togo and its interaction with some cowpea cultivars

M. Y. D. GUMEDZOE, G. THOTTAPPILLY^1 and A. ASSELIN^2

Universite du Benin, Ecole Superieure d'Agronomie, BP 1515 Lome - Togo
^1 International Institute of Tropical Agriculture (IITA),
Biotechnology Unit, Oyo Road, PMB 5320 Ibadan Nigeria
^2 Universite Laval, Faculte des Sciences de l'Agriculture et de l'Alimentation (FSAA), Cite Universitaire Sainte-Foy (Quebec) G1K7P4, Canada

(Received 28 July, 1994; accepted 16 September, 1995)

Code Number: CS96059
Sizes of Files:
    Text:
    Graphics: Line drawing (gif) - 9.2K
              Photograph (jpg) - 79.4K
              Tables (gif) - 26.9K

Cowpea (Vigna unguiculata L. Walp) is one of the major grain legumes in tropical Africa. This leguminous plant is susceptible to many diseases including those caused by viruses. This article reports results from surveys conducted from the main cowpea producing areas in Togo for the occurrence of Southern Bean Mosaic Virus (SBMV) and search for cowpea cultivars that are resistant to this virus. Serological tests (double diffusion in agarose gel and DAS-ELISA) with polyclonal antiserum to intact SBMV were used for the identification of the SBMV isolates. Results indicated that SBMV is widespread in the main cowpea producing areas in Togo. Cassia hirsuta, naturally infected by SBMV, may constitute a carryover host for SBMV between two cowpea growing seasons. Two SBMV isolates (18-10 and 10-19) cloned by local lesions transfers on susceptible cowpea were used for the screening of 58 cowpea cultivars. Cowpea cultivars IT82D-703, IT82D-786, IT83S-818, TVx1193-9F and TVx1850-01E were resistant to both isolates. These resistant cowpea cultivars could be recommended for farmers where SBMV is a serious problem for cowpea production.

Key Words: Serological tests, viruses, Vigna unguiculata

RESUME

Le niebe (Vigna unguiculata L. Walp) est une legumineuse e graines cultivee dans les regions tropicales d'Afrique. Cette legumineuse est tres sensible e un grand nombre de maladies dont celles causees par des virus. Ce papier rapporte les resultats de prospections phytosanitaires dans les principales regions productrices de cette legumineuse au Togo et portant sur la presence du SBMV et aussi sur la recherche de cultivars de niebe resistants e ce virus. Des tests serologiques (immunodiffusion dans l'agarose et le test DAS-ELISA) utilisant l'antiserum polyclonal e l'egard du SBMV ont permis d'identifier les isolats de ce virus. Les resultats indiquent que le SBMV est tres repandu dans les principales regions productrices du niebe au Togo. Le Cassia hirsuta a ete trouve infecte naturellement par le SBMV, il peut servir de reservoir de virus entre deux saisons de culture du niebe. Deux isolats du SBMV, (18-10, 10-19) clones par transfert de lesions locales sur du niebe sensible ont ete utilises pour cribler par test 58 cultivars de niebe. Les cultivars de niebe suivants : IT82D-703, IT82D-786, IT83S-818, TVx1850-01E se sont reveles resistants e l'egard des deux isolats du SBMV, et ceux-ci peuvent tre recommandes aux producteurs surtout dans les regions ou le SBMV est une contrainte majeure pour la production du niebe.

Mots Cles: Tests serologique, Virus, vigna unguiculata

INTRODUCTION

Cowpea (Vigna unguiculata L. Walp) is cultivated in most countries in West Africa, especially in the Guinea and Sudan Savanna regions.

The average yield from farmers' fields is very low (between 200 and 300 kg ha^-1). Susceptibility to diseases and insect pests, and several other constraints, are responsible for such low yields (Singh and Allen, 1979). Many viruses have been reported as infecting cowpea in West Africa : Cowpea (yellow) mosaic virus (CPMV), Cowpea Aphid-borne Mosaic Virus (CAMV), Blackeye Cowpea Mosaic Virus (BlCMV), Cowpea Mottle Virus (CMeV), Cucumber Mosaic Virus (CMV), Cowpea Golden Mosaic Virus (CGMV), Cowpea Mild Mottle Virus (CMMV), Southern Bean Mosaic Virus Cowpea Strain (SBMV-CS) and Tobacco Mosaic Virus Cowpea Strain (TMV-CS) (Thottappilly and Rossel, 1985, 1992).

Most of these viruses often occurred in mixed infections and had detrimental effects on cowpea yields Rossel and Thottappilly, 1985; Thottappilly and Rossel, 1985, 1992; Gumedzoe et al., 1990). Southern Bean Mosaic Virus (SBMV) has been reported in West Africa (Lamptey and Hamilton, 1974; Shoyinka et al., 1979; Givord, 1981; Fauquet and Thouvenel, 1987; Gaikwad and Thottappilly, 1988; Gumedzoe et al., 1990). In preliminary studies, SBMV was identified in Togo (Gliem, 1984; Gumedzoe et al., 1990) but it was not well characterised. As an adequate knowledge of SBMV and its isolates or strains occurring in the main cowpea growing areas in Togo is a prerequisite for effective control measures to be developped against this virus, it was necessary to describe SBMV isolates identified in Togo and to search for cowpea cultivars resistant to SBMV.

This article reports results from surveys in the main cowpea production areas in Togo for the prevalence of SBMV and the characterisation of the isolates of the virus. Cowpea cultivars were screened for resistance to SBMV.

MATERIALS AND METHODS

Antisera and plant materials. Polyclonal antiserum to intact SBMV (1mg/ml) was obtained from the Biotechnology Unit at the International Institute of Tropical Agriculture (IITA, Ibadan, Nigeria). All cowpea lines were grown and maintained in insect-proof cages.

Surveys. Both farmers' fields and experimental plots in major cowpea production areas in Togo were surveyed for cowpea plants showing mosaic or mottling symptoms of the virus. One trifoliate leaf was collected from each of the virus-infected plants and put in separate plastic bags. The plastic bags were labelled, stored in a cool box for one or two days prior to their transfer to the laboratory where they were frozen.

Identification of SBMV isolates. Serological tests (double diffusion in agarose gels : 0.7 % agarose, 1 % NaCl, 0.1 % sodium azidin) 0.01 M potassium phosphate buffer at pH 7.5) were used for the identification of SBMV isolates as described elsewhere (Gumedzoe et al., 1990). The gels were incubated in a moist chamber at room temperature for 24hr. After the completion of the test, the gels were photographed. In order to better visualise the precipitin lines, a simple procedure of staining these agarose gels was used as described elsewhere ( Crowle and Cline, 1977; Asselin et al., 1980; Mink, 1988). In this case, the gels consisted of 1.5 % agarose, 1 % NaCl, 0.1 % sodium azide and 0.01 M potassium phosphate buffer of pH 7.5. A double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) was also used for the identification of SBMV isolates (Clark and Adams, 1977). Precoated plates from the Biotechnology Unit of the International Institute of Tropical Agriculture were used for this assay.

Reactions of some cowpea cultivars to SBMV isolates. Fifty eight cowpea cultivars were screened using local-lesion selected SBMV isolates 10-19 and/or 18-10. Inoculum was prepared by grinding leaves of infected cowpea cultivars in a cold sterilized mortar with a few drops of 0.01 M potassium phosphate buffer (pH7.5). Inoculations were carried out by rubbing primary leaves of one week-old cowpea seedlings dusted with 600 mesh silicon dioxide (carborandum) with the extract prepared in the phosphate buffer. Healthy seedlings inoculated with buffer served as controls. Inoculated leaves were immediately washed with tap water. At least five cowpea seedlings (including the control) were inoculated at each time. Inoculated plants were scored for infection between seven and thirty days after inoculation by counting the number of plants showing symptoms on inoculated primary leaves and younger trifoliate leaves (mottle, mosaic).

The scale used to rate the symptom of SBMV on cowpea cultivars inoculated with this virus was as follows.

Score 1: No symptom in plants (no visible mottle or mosaic).

Score 2: Slight mosaic symptom (1 to 25% of the cowpea cultivars inoculated were infected).

Score 3: Moderate mosaic symptom (25 to 50 % of plants infected).

Score 4: Severe symptoms with stunting (50 to 75 % of plants infected).

Score 5: Highly severe symptoms with stunting (75 to 100% of plants infected).

RESULTS

Surveys, identification and prevalence of SBMV in various cowpea producing areas in Togo. The aim of this study was to establish the distribution and incidence of SBMV in various cowpea producing areas in Togo. From the 705 field samples examined, 63.12 % reacted positively to at least one of the antiserum of the seven major cowpea viruses currently studied in our laboratory (BlCMV, CAMV, CMeV, CMMV, CMV, CPMV and SBMV). The incidence of SBMV in the samples assayed was 13.86 % on cowpeas and non-cultivated plants from the following prefectures : Bassar, Golfe, Haho, Kloto, Kozah, Ogou, Oti, Sotouboua, Tone, Vo, Yoto and Zio (Fig. 1). Virus mixtures were identified in single plants and ranged from three viruses to two of the cowpea viruses in various combinations (i.e. SBMV + CPMV + CAMV and SBMV + CPMV). These mixed infections were recorded using serological tests (DAS-ELISA and double diffusion in agarose gels) and they included two or more cowpea viruses. Various non-cultivated plants, including Cassia hirsuta, were also found infected with SBMV. The SBMV isolate 10-19 was recovered from cowpea cultivar IT86D-901 in experimental plots of the "Societe Togolaise du Coton" (SOTOCO) at the locality Adza Yao in Ogou prefecture.

SBMV isolate 18-10 was recovered on cowpea at Vogan in Vo prefecture. These two viral isolates were cloned by local lesion transfers on the cowpea cultivar IT81D-985 before being used to screen the cowpea cultivars.

In the immune double diffusion assay, precipitin lines normally appeared about 16 to 24 hr after leaf extracts and the SBMV antiserum were added to wells (Fig. 2). Precipitin lines stained with Crocein scarlet in combination with Coomassie blue were quite distinct and remained visible after long storage. The immunoprecipitin lines stained with the combination of Crocein scarlet 7B and Coomassie brillant blue were more distinct than those stained with Coomassie blue alone. There were some black smears around the reactant wells with SBMV isolates.

Figure 1

Figure 2

Symptoms observed on inoculated leaves of most cowpea cultivars included the development of necrotic or chlorotic local lesions. Systemic reactions included varying degrees of mosaic leaf distortion and plant stunting. Severity of symptoms varied with SBMV isolates infecting the same cowpea cultivar. For example, the cowpea cultivar Locale Blanche is susceptible to isolate 10-19 but was found resistant to isolate 18-10. Likewise, isolate 10-19 readily infected the cowpea cultivar IT81D-985, whereas isolate 18-10 did not easily infect this cultivar. SBMV isolate 10-19 failed to infect the following plant species : Cajanus cajan, Phaseolus vulgaris cv. Saint-Fiacre and Saxa, Chenopodium amaranticolor, Crotalaria sp. and Glycine max cv Jupiter (Data not shown). One species of Mucuna reacted with local lesions when inoculated with SBMV isolate 10-19.

DISCUSSION

Among isometric viruses infecting cowpea, SBMV is one of the more important. It has been identified in Togo and in other areas of Africa (Lamptey and Hamilton, 1977; Shoyinka et al., 1979; Givord, 1981; Thottappilly and Rossel, 1985; Fauquet and Thouvenel, 1987). Results from the present study confirmed this wide distribution of SBMV throughout the cowpea producing areas in Togo. Similar observations were made earlier in this country but no research was conducted to search for cowpea cultivars that are resistant to this virus (Gliem, 1984; Gumedzoe et al., 1990). Most of the cowpea cultivars that are popular in major cowpea producing areas in Togo (58-146, IT81D-985 or VITOCO and VITA5) were highly to moderately susceptible to the SBMV isolates studied. A number of cowpea cultivars (IT82E-16, IT82D-889, IT83S-818, TVu19) were resistant to these viral isolates as previously reported (Ladipo and Allen, 1979; Singh and Ntare, 1985; Singh et al., 1992). The use of these resistant cowpea cultivars could help reduce considerably the losses caused by this virus in cowpea growing areas where SBMV is a common problem (O' Hair et al., 1981; Collins et al., 1985). Various non-cultivated plants, including Cassia hirsuta were found infected with SBMV. This finding may have some epidemiological implications if the two leguminous plants (cowpea and Cassia hirsuta) occur in the same field. The non-cultivated Cassia hirsuta may constitute a carryover host for SBMV between two cowpea growing seasons in some areas.

Table 1 Reactions of 36 cultivars to SBMV isolate 18- 10

Table 2 Reactions of cowpea cultivars to SBMV isolate 10-19

This serological test, however, has the following disadvantages: it uses much antiserum and is not sensitive, but modifications have been devised to overcome these difficulties. The immune double diffusion assay also cannot be used with sap from natural hosts because of low virus concentration, or presence of factors (such as mucilages, tannins etc.) in sap which interferes with the reaction. Moreover, precipitation lines disappear quickly in few days and it is necessary to photograph the gel before the precipitation lines disappear.

To overcome these problems, especially the preservation of precipitation lines, some improvements like staining the precipitation lines were developed by different workers (Crowle and Cline,1977; Mink,1988). We applied successfully the staining technique of Mink (1988) to the SBMV isolate 18-10.

ACKNOWLEDGEMENT

The authors acknowledge the financial support of the International Development Research Centre (IDRC, Ottawa, Canada) and the International Institute of Tropical Agriculture (IITA, Ibadan, Nigeria) throughout the duration of this study (Grant 3-P-88-1029-01).

REFERENCES

Asselin, A., Brisson, J. D. and Desjardins, S. 1980.Etude des diverses souches de TMV par microscopie electronique et par diverses techniques de separation par electrophorse. Phytoprotection 61:119-120.

Clark, M. F. and Adams, A. N. 1977. Characteristics of the microplate method on enzyme-linked immunosorbent assay for the detection of plant viruses. Journal of General Virology 34:475- 483.

Collins, M. H., Witcher, W. and Barnett, O. W. 1985.Reactions of 16 cowpea cultivars to six viruses. Plant Disease 69:18-20.

Crowle, A. J. and Cline, L. J. 1977. An improved staining for immunodiffusion tests. Journal of Immunological Methods 17:379-381.

Fauquet, C. and Thouvenel, J. C. 1987. Maladies virales des plantes en C™te d'Ivoire. Edition de l'ORSTOM. Collection: Initiations, Documentation Technique No. 46 (2e ed.) Paris, France. 243pp.

Gaikwad, D. G. and Thottappilly, G. 1988. Occurrence of southern bean mosaic virus on cowpea in Senegal. Phytopathology 121:366-369.

Givord, L. 1981. Southern bean mosaic virus isolated from cowpea (Vigna unguiculata) in the Ivory Coast. Plant Disease 65:755-756.

Gliem, G. 1984. Virus des plantes cultivees les plus importantes au Togo: expansion, reconnaissance, importance et possibilite de lutte. Pages 131-135. In: Maladies des Plantes Cultivees au Togo: Recherches et Observations. German Agency for Technical Cooperation. Eschborn. Federal Republic Germany.

Gumedzoe, M. Y., Sunu, D. Y., Thottappilly, G. and Asselin A. 1990. Importance du virus de la marbrure du niebe et du virus de la mosa•que jaune du niebe au Togo. Phytoprotection 71: 85-91.

Hamilton, R. I. 1992. The use of monoclonal antibodies and cDNA for detection of plant viruses. In: Biotechnology: Enhancing Research on Tropical Crops in Africa.

Thottappilly, G., Monti, L. M., Mohan Raj, D. R. and Moore, A. W. (Eds.), pp. 297- 303. CTA/IITA copublication. IITA, Ibadan, Nigeria.

Ladipo, J. L. and Allen, D. J. 1979. Identification of resistance to southern bean mosaic virus in cowpea. Tropical Agriculture (Trinidad) 56: 33-40.

Lamptey, P. N. L. and Hamilton, R. I. 1974. A new cowpea strain of southern bean mosaic virus from Ghana. Phytopathology 6:1100-1104.

Mink, G. I. 1988. A method for preparing dried double immunodiffusion gels for photographic slides. Phytopathology 76:114 - 117.

O'Hair, S. K., Miller, J. C. and Toler, R. W. 1981. Reaction of cowpea introductions to infections with the cowpea strain of southern bean mosaic virus. Plant Disease 65:251-252.

Rossel, H. W. and Thottappilly, G. 1985. Virus Disease of Important Food Crops in Tropical Africa. International Institute of Tropical Agriculture (IITA), Ibadan, Nigeria. 61pp.

Singh, B. B. and Ntare, B. R. 1985. Development of improved cowpea varieties in Africa. In: Cowpea, Research, Production and Utilization. Singh, S. R. and Rachie, K. O. (Eds.), pp. 105-115. John Wiley & Sons Ltd., New York.

Singh, S. R., Jackai, L. E. M., Thottappilly, G. Cardwell,K. F. and Myers, G. O. 1992. Status of research and constraints to cowpea production. In: Biotechnology: Enhancing, Research in Tropical Crops in Africa. Thottappilly, G., Monti, L. M.,Mohan, D. R. Raj and Moore, A. W. (Eds.), pp. 21-26. CTA/IITA, Ibadan, Nigeria.

Singh, S. R. and Allen, D. J. 1979. Cowpea Pests and Diseases, Manual series. IITA, Ibadan, Nigeria. pp. 21-29.

Shoyinka, S. A., Bozarth, R. F., Reese, J. and Okusanya, B.A. O. 1979. Field occurrence and identification of southern bean mosaic virus (cowpea strain) in Nigeria. Turrialba 29:11-16

Thottappilly, G. and Rossel, H. W. 1985. Worldwide occurrence and distribution of virus diseases. In: Cowpea Research, Production and Utilization. Singh, S. R. and Rachie, K. O. (Eds.), pp. 155 - 171. John Wiley & Sons Ltd, New York.

Thottappily, G. and Rossel, H. W. 1992. Virus diseases of cowpea in tropical Africa. Tropical Pest Management 38:337-348.

Copyright 1996 The African Crop Science Society

The following images related to this document are available:

Photo images

Line drawing images