1.11.7
COMPARISON OF WHITEFLY TRANSMITTED GEMINIVIRUSES INFECTING COWPEA AND LIMA BEAN IN NIGERIA

G THOTTAPPILLY1, S WINTER2 and DP MAXWELL3

1Biotec Research Unit, IITA, PMB 5320, Ibadan, Nigeria; 2DSMZ Plant Virus Collection, Messeweg 11/12, 38104 Braunschweig, Germany; 3Department of Plant Pathology, University of Wisconsin, Madison, WI 53706-1598, USA

Background and objectives
Two whitefly-transmitted legume viruses, cowpea golden mosaic virus (CGMV) and lima bean golden mosaic (LGMV) are reported infecting Vigna spp. and Phaseolus lunatus, respectively, in Nigeria [1]. The aetiology of these diseases is not yet reported. A golden mosaic disease is also found in wild relatives from cowpea, Vigna unguiculata subsp. dekindtiana var. dekindtiana (Vud-GMV), a common perennial weed growing all over Nigeria and West Africa. In order to elucidate the aetiology of these diseases, to study the relationships between these viruses and to determine the relationship with other whitefly-transmitted geminiviruses, genomic components of the viruses were amplified by PCR and subjected to sequence analysis.

Materials and methods
Vigna unguiculata infected with CGMV was obtained from cowpea fields near Nsukka in the south-eastern part of Nigeria, where virus incidence is high. A sample from the wild perennial cowpea showing bright golden mosaic (Vud-GMV) symptoms was collected from abandoned land in the Ibadan area. Lima bean plants infected with LGMV were collected from P. lunatus in Ibadan. Putative geminiviral DNA was amplified from total plant DNA preparations by PCR using a set of primers described by Briddon and Markham [2], for amplification of near full-length DNA A of whitefly-transmitted geminiviruses. Sequence information was obtained from the uncloned, gel-purified PCR amplicons by dyedeoxy cycle sequencing with specific primers deduced from the sequence obtained. All residues in the sequence were confirmed by sequencing in both directions, by sequencing separate PCR fragments obtained by amplification of ambiguous genome positions and the missing region in the initial amplification. Sequence was assembled and analysed with DNAsis version 2.5 (Hitachi) and multiple sequence alignments were generated using CLUSTAL W.

Results and conclusions
The genomic structure of the nucleotide sequence deduced from DNA A of all viruses sequenced resembles that of DNA A of bipartite geminiviruses with DNA A encoding five putative genes. Comparison of amino-acid sequences of all ORFs clustered the viruses sequenced within subgroup III - old world geminiviruses, forming a separate branch. Comparison of the most conserved geminiviral genes, AC1 and AV1, confirmed an amino-acid sequence homology of 90 and 95% for AC1 and AV1 between CGMV and Vud-GMV, in contrast to an 80 and 81% homology for AC1 and AV1 from the amino-acid sequence comparison of CGMV and LGMV. Despite the difficulties in transmitting Vud-GMV to cowpea, our sequence data suggest that the geminivirus infecting Vigna unguiculata subsp. dekindtiana can be considered a strain of CGMV. Sequence data obtained for LGMV DNA A clearly define this virus as a distinct geminivirus infecting lima bean in Nigeria. However, its relationship to viruses causing a similar disease in lima beans and occurring in South America is still to be determined.

References
1. Anno-Nyako FO, Vetten HJ, Allen DJ, Thottappilly G, 1983. Annals of Applied Biology 102, 319-323.
2. Briddon RW, Markham PG, 1994. Molecular Biotechnology 1, 202-205.