From 1999 to 2002, field surveys were conducted in the legume-growing areas of Spain including Ávila, Badajoz, Cádiz, Córdoba, León, Málaga, Murcia, Salamanca, and Zamora provinces. Leaf tissue from 35 asymptomatic and 224 virus symptomatic plants was sampled and analyzed by indirect-ELISA with a specific monoclonal antibody against the potyvirus group (Adgia, Elkhart, IN). All symptomatic plants of bean (Phaseolus vulgaris L.), broad bean (Vicia faba L.), lentils (Lens culinaris L.), and chickpea (Cicer arietinum L.) were positive for potyvirus infection. Identification as Bean yellow mosaic virus (BYMV) was obtained by double-antibody sandwich (DAS)-ELISA with a polyclonal antiserum (Loewe Biochemica Gmbh, Sauerlach, Germany). To analyze the genetic variability of BYMV Spanish isolates, 33 Spanish isolates were selected at random from our BYMV collection, and extracts from these plants were used with primers 1985 (5′-gagagaatgatacacatactgaa-3′) and 1984 (5′-caaggtgagtggacaatgatgg-3′) to amplify by immunocapture (IC)-reverse transcription (RT)-PCR a 524-nt fragment of the BYMV genome that includes the C-terminal 417 nt of the coat protein and 107 nt from the 3′ untranslated region. The IC-RT-PCR products were cloned into pGEM-T easy vector (Promega, Madison, WI) and a minimum of three clones from each PCR amplification were sequenced. BLAST analysis showed that the sequences of 30 samples were 96 to 98% identical to BYMV, but three samples (GenBank Accession Nos. EU860364--66) from bean, broad bean, and lentils had a high (98%) identity with Clover yellow vein virus (ClYVV). Sequence alignments of the ClYVV Spanish isolates and 14 ClYVV isolates from the GenBank (Accession Nos. AB03308, AB004545, AB011819, AF185959, AF203536, D86044, S77521, D95538--94) were obtained using the Clustal X software. Genetic distances were estimated using the Kimura two-parameter method. Within-population and between-population nucleotide diversities were estimated from the genetic distances (2). ClYVV sequences were phylogenetically separated into two clades: one with the three isolates from Japan (Accession Nos. D89542, D89543, and D89544) and the other with the remaining isolates. Molecular clustering coincides with biology and serological variations of strains 1 and 2 (3). Phylogenetic distances were independent of geographic origin, host, or time of sampling. The nucleotide diversity value among populations (0.18) was higher than within the subpopulations (0.017 and 0.029). dNS/dS in the ClYVV population was 0.031 (<1) and we can conclude that negative selection is occurring in the gene in study and that the population of ClYVV present in Spain is homogenous. In Spain, ClYVV was reported infecting borage (Borago officinalis L.) (1). To our knowledge, this is the first report of natural infection of bean, broad bean, and lentils with ClYVV in Spain. ClYVV might cause important economic losses in grain legumes since it causes an important viral disease of legumes worldwide.
References: (1) M. Luis-Arteaga et al. Plant Pathol. 45:38, 1996. (2) M. Nei and T. Gojobori. Mol. Biol. Evol. 3:418, 1986. (3) T. Sasaya et al. Virology 87:1014, 1987.