Characterization of cowpea stunt isolates 59 PARTIAL MOLECULAR CHARACTERIZATION OF COWPEA STUNT ISOLATES OF CUCUMBER MOSAIC VIRUS AND BLACK EYE COWPEA MOSAIC VIRUS FROM ARKANSAS AND GEORGIA (USA) A. DIALLO1.H., R.C. GERGERICH2 and E.J. ANDERSON3. 1University of Abobo-Adjame, UFR-SN, B.P. 801 Abidjan, COTE D’IVOIRE. 2University of Arkansas, Department of Plant Pathology, 217 PTSC Bldg, Fayetteville, ARKANSAS (USA) 72701. 3Pioneer Hi-Bred International, Inc., 6900 NW 62nd Ave., Johnston, IA 50131-0256. ABSTRACT Partial molecular characterization of the coat protein of the cowpea stunt-causing isolates of Cucumber Mosaic Virus (CMV) from Arkansas and Georgia revealed that both isolates of CMV belong to CMV subgroup I and differ at eight nucleotides positions, resulting in two amino acids difference. There was only one amino acid difference for the Blackeye Cowpea Mosaic Virus (BlCMV) isolates from both locations. Differences in the coat protein genes of CMV and BlCMV isolates from Arkansas and Georgia could partially be responsible for the variation in the virus accumulation pattern. This is the first report on the taxonomic classification of the cowpea stunt disease-causing isolates of CMV and BlCMV in Arkansas and Georgia based on nucleotides and amino acid sequences analysis. Keywords : cowpea, Vigna unguiculata (L.) Walp. subsp. unguiculata, USA, Côte d’Ivoire. RESUME CARACTERISATION MOLECULAIRE PARTIELLE D’ISOLATS DE CMV ET BICMV ASSOCIES A LA MALADIE DU NANISME DU NIEBE DANS L’ARKANSAS ET LA GEORGIE (USA) La caractérisation partielle des gènes de la protéine de capside des isolats de CMV associés à la maladie du nanisme du niébé provenant de l’Arkansas et de la Georgie a révélé que les deux isolats de CMV appartiennent au sous-group I et qu’ils diffèrent au niveau de huit nucléotides, ce qui se traduit par une différence de deux acides aminés. Pour BlCMV, la différence entre les deux isolats porte seulement sur un acide aminé. Ces différences dans les gènes de la capside des isolats de CMV et de BlCMV des deux différentes localités pourraient être partiellement responsables de la différence entre le mode d’accumula- tion de ces virus. Ceci constitue le premier travail sur la classification taxonomique , basé sur l’analyse des séquences d’acides nucléiques et d’acides aminés des isolats de CMV et de BlCMV responsable du nanisme du niébé dans l’Arkansas et la Georgie. Mots clés : Niébé, Vigna unguiculata (L.) Walp. sesp. unguiculata, USA, Côte d’Ivoire. INTRODUCTION 1980), is caused by a synergistic interaction between blackeye cowpea mosaic virus Cowpea (Vigna unguiculata (L.) Walp. subsp. (BlCMV) and cucumber mosaic virus (CMV). Unguiculata) is an important crop worldwide with Cowpea plants doubly infected with both a global production covering approximately 12.5 viruses are severely stunted with small, million ha of which, 8 million ha are in west and blistered and malformed leaves.Stems and central Africa (Singh et al., 1997). petioles of these infected plants become Cowpea stunt, a severe disease of cowpea, first necrotic and show a significant reduction in the reported in Georgia, Alabama and South Carolina number of leaves and pods. Cowpea stunt (Pio-Ribeiro et al., 1978 ; Pio-Ribeiro and Kuhn, caused important yield loss of 86.4 % Agronomie Africaine 16 (1) : 59-69 (2004) 60 Diallo A. et al. whereas only 2.5 and 14.2 % reduction of cytological inclusions and host range, these occurred with single infections with CMV and viruses have been shown to be different BlCMV, respectively (Pio-Ribeiro et al., 1978). (Edwardson et al., 1972 ; Zettler and Evans, Cowpea stunt was more recently found in 1972). BlCMV and cowpea aphid-born mosaic Magnolia County, Arkansas (Anderson et al., virus (CaBMV), sometimes considered closely 1994). related or similar (Bock and Conti, 1974), have been differentiated based on their reactions on Cucumber mosaic virus is the type virus of the resistant cowpea cultivars (Taiwo et al., 1982). cucumovirus group. It has a wide host range For a better differenciation and classification of mostly composed of dicots. CMV can infect potyviruses, methods based on coat protein and/ more than 800 plant species (Palukaitis et al., or 3'-UTR region sequences seem to give good 1992). The virus is characterized by small, results (Frenkel et al., 1989, 1991; Khan et al., icosahedral particles of about 30 nm in diameter. 1990, 1993 ; Lana et al., 1988 ; Shukla and It genome is composed of three genomic, Ward, 1988 ; Shukla and Ward, 1989 ; Van der positive-sense RNA molecules (RNAs 1, 2 and Vlugt et al., 1993). 3) and a subgenomic RNA (RNA 4) coding for the 24.5 Kilodalton (Kd) coat protein (Matthews, Since cowpea stunt was first discovered in 1991). The CMV coat protein (CP) is involved Georgia and later in Arkansas, it was important not only in symptom development, but also in to compare the disease-causing isolates of CMV the encapsidation of the RNAs as well as in and BlCMV from both geographical locations. aphid transmission (Mossop and Francki, 1977). In previous studies where the stunt-causing Several strains of CMV have been isolated isolates of CMV and BlCMV from Arkansas and (Kapper and Waterworth, 1981; Rizos et al., Georgia were compared biologically, it was found 1992). In order to differentiate and classify these that although all four types of mixed infections strains, several methods have been used: produced similar symptoms on cowpea plants, serology (Devergne and Cardin, 1983), nucleic the viruses behaved differently based on their acid hybridation (Gould and Symons,1978; accumulation patterns in the leaves and stems Piazzolla et al., 1979 ; Owen and Palukaitis, (Diallo, 1998). The objective of this study was to 1988) and peptide mapping of the coat protein molecularly characterize the Arkansas and gene (Edwards and Gonsalves, 1983). However, Georgia cowpea stunt isolates of CMV and only nucleotide sequence analysis of the CP BlCMV. gene and the 3' untranslated region was able to provide an accurate alternative differentiation MATERIALS AND METHODS between CMV strains and separate them into two subgroups, I and II (Quemada et al., 1989; Rizos et al., 1992). VIRUSES AND PLANTS Blackeye cowpea mosaic virus belongs to the The Arkansas cowpea isolates of BlCMV most economically important group of plant (BlCMVAR) and CMV (CMVAR) were originally viruses, the potyvirus group. These viruses obtained from field samples taken in Columbia particles are long and flexuous rod-shape, County in 1994. The Georgia isolates of cowpea measuring approximately 12 x 900 nm. stunt viruses (BlCMVGA and CMVGA) were Potyviruses have their genome composed of a provided by Dr. A. G. Gillaspie, Jr., USDA-ARS linear, single-stranded, positive-sense RNA, Genetic Resources Unit, University of Georgia, approximately 9.500 nucleotides (nt) in length. Griffin, GA. All viruses were maintained as dried A genome-linked protein (Vpg) is located at the infected tissues at 4 °C or in the cowpea culti- 5' terminus and at the 3'-end, there is a 200 nt var ‘Coronet’ (Brantley, 1976) under greenhouse untranslated region (3'-UTR), followed by a poly- conditions with temperatures ranging from 20- A tail (Matthews, 1991). The virus genome 30 °C. ‘Coronet’ cowpea plants were grown consists of a large open reading frame. The coat individually in 3-inch pots containing Redi-Earth protein gene is located at the 3'-end. BlCMV 3CP potting mixture (Grace Sierra, Milpitas, CA). has been reported as the cowpea strain of bean yellow mosaic virus (BYMV) based on CHARACTERIZATION OF CMV AND microagglutination test (Gay and Winstead, 1970 BICMV COAT PROTEIN GENES ; Harrison and Gudauskas, 1968a ; Harrison and Gudauskas, 1968b ; Kuhn, 1964 ; Kuhn et al., Total nucleic acids from plants infected with 1965). However, based on comparative studies CMVAR, CMVGA, BlCMVAR, BlCMVGA and healthy Agronomie Africaine 16 (1) : 59-69 (2004) Characterization of cowpea stunt isolates 61 cowpea plants were extracted according to the sequence of BlCMV strain W (Khan et al., 1993) procedure described by Pappu et al. (1993). corresponding to positions (1-17) on the viral Each plant sample was frozen in liquid nitrogen polymerase gene portion of the published and ground in 300µl of extraction buffer (2 % sequence and an oligo-dT primer. The predicted SDS, 0.1 M Tris, 0.002M EDTA, pH 8.0). The length of the RT-PCR fragment was appro- viral RNAs were extracted in phenol/ chloroform- ximately 1200 bp. Total nucleic acids were isoamyl alcohol (1:1). Crude RNA extracts were denatured at 70 °C for 3 min, and 55.3 µl added purified on a sephadex G 50 column equilibrated to tubes containing ρ36.9 µl of PCR mix (same with TE buffer (10 mM Tris, pH 7.5, 1 mM EDTA, as before) and 100 mol of each primer. The pH 8.0). The nucleic acid eluants were collected reactions were incubated at 42 °C for 1 h for the and used for reverse transcriptase polymerase synthesis of the first strand cDNA. Thirty cycles chain reaction (RT-PCR) amplification and were run in a thermal cycler with periods of 45 s cloning of the coat protein (CP) genes of all four at 93.5 °C for denaturation, 45 s at 36 °C for isolates of CMV and BlCMV according to the annealing and 1 min at 72 °C for extension. The procedure described by Pappu et al. (1993). For RT-PCR fragments were analysed by a 1 % CMV, two oligonucleotide primers were designed agarose gel electrophoresis. based on the published sequence of CMV strain Q and FNY (Quemada et al., 1989). The entire CP gene including the 3' flanking sequence was CLONING AND SEQENCING OF CMV amplified using the following upstream primer AND BLCMV COAT PROTEIN GENES EA 39 (5'-TTC TCC GCG AGA TTG C-3') corresponding to positions 1167-1182 of The RT-PCR fragments of CMV and BlCMV the published sequence of FNY-CMV isolates from Arkansas and Georgia were (C M V FNY).