Begomoviruses (family Geminiviridae) are emergent pathogens of crops whose dissemination has been favoured by the increasing populations of their whitefly vectors [1]. In the Americas, a series of whitefly-transmitted begomoviruses have been reported to affect bean crops, and most have been associated to leaf mosaic symptoms and important yield reductions [1, 2]. In Colombia, distinct symptoms of leaf crumpling have been reported in fields of dry and snap bean varieties since 2002 [3]. Since then, there has been a drastic decrease (60-70%) in the cultivated area in localities where the symptoms have been reported [3, 4]. The most recent outbreak was recorded during December 2015 in Rozo, Department of ‘Valle del Cauca’ (Colombia), with a prevalence of 40% in fields of ‘Blue Lake’ beans, a popular but extremely susceptible commercial variety (Fig. 1A). The putative begomovirus causing the disease was originally named “virus del arrugamiento foliar” (Spanish for ‘leaf crumple virus’) [3], but the complete sequence was not available until now.

Fig. 1
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A) Uninfected (1) and BLCrV-infected (2) bean leaves of variety Blue Lake (sample 13, isolate HA) showing characteristic leaf crumple symptoms observed in the municipality of Rozo (see Table 1). B) A segment of the common region containing the iteron sequences (grey boxes), TATA boxes (underlined) and the invariant nonanucleotide sequence at the origin of replication (white box). An additional iteron sequence (TGGTGTC) is found at position 21, but only in DNA-A. The black arrow indicates the position of nucleotide 1, and the numbers flanking the sequences indicate their position in the genome. nt, nucleotide. C) Phylogenetic relationship of BLCrV (black circle) to other New World bipartite begomoviruses reported in the Americas. The tree was constructed using full genome sequences and the neighbor-joining algorithm in MEGA 5 [8]. A sequence of velvet bean severe mosaic virus was used to root the tree. Bootstrap values above 50% are shown (1000 replicates)

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Thirty-seven leaf samples from affected fields in the departments of Valle del Cauca (municipalities of Rozo, Palmira and Pradera) and Quindío (municipality of Quimbaya) were collected, and DNA was extracted from 20 mg of silica-dried leaves or 100 mg of fresh leaves using cetyltrimethylammonium bromide (CTAB). Samples from fields surveyed between 2008 and 2014 in the same region were also included in this study. PCR detection of begomovirus was carried out using generic primers targeting the DNA-A and the DNA-B components [5]. Twenty-six samples tested positive with at least one of these primer sets, and 22 PCR fragments were sent for sequencing (Macrogen). A copy of the complete genome of BLCrV was obtained by rolling-circle amplification (RCA) using phi29 polymerase (New England Biolabs, USA) as reported previously [6]. RCA fragments of ~2.5-2.9 kb obtained by digestion with EcoRI were cloned and sequenced (Macrogen, Korea). The genome organization obtained was that of a typical bipartite begomovirus genome, including a common region (CR) of 195 nt (93.7% identity) containing the characteristic iteron, TATA box and invariant nonanucleotide sequences (Fig. 1B). DNA-A (GenBank accession number KX857725, 2598 nt) has one virus-sense open reading frame (ORF) encoding the capsid protein (AV1) and four ORFs on the complementary-sense strand, encoding the replication-associated protein (Rep/AC1), a transcriptional activator protein (TrAP/AC2), a replication enhancer protein (REn/AC3) and the AC4 protein (AC4). DNA-B (GenBank accession KX857726, 2538 nt) has one virus-sense ORF encoding the nuclear shuttle protein (BV1) and a complementary-sense ORF (BC1). Sequence analysis using the MUSCLE option in SDT v1.2, following current recommendations for begomovirus classification [7], indicated that BLCrV should be regarded as a member of a new species of the genus Begomovirus. DNA-A has the highest nt sequence identity (84.17%) to tomato mild yellow leaf curl Aragua virus (73.17% for DNA-B). Phylogenetic analysis using MEGA v5 [8] showed that BLCrV is more closely related to begomoviruses associated with leaf curling symptoms than to leaf-mosaic-associated begomoviruses (Fig. 1C). Sequence comparisons of a portion of the AC1 gene with the corresponding region from viruses obtained from samples collected in previous years confirmed the presence of BLCrV in Colombia since at least 2008 in all symptomatic plants (nt sequence identity >98%). Interestingly, leaf mosaic symptoms were observed in some affected plants that were positive for BLCrV but not for other begomoviruses. (Table 1).

Table 1 Samples evaluated in this study. DNA-A and DNA-B PCR products were obtained using generic primers [5], and sequences were confirmed for the samples indicated by an asterisk (*). Samples from symptomless plants are shown in bold. Samples 4 (F199) and 13 (HA) were selected for RCA. Only sample 13 (HA) was fully sequenced. ND, not determined
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In the bean-breeding program at CIAT, plants have been bred successfully for virus resistance for decades, mainly focusing on BGMV and BGYMV in Mesoamerican bean gene pools [9]. On the other hand, in the Andean bean-breeding program, due to its product focus, plants are not bred for geminivirus resistance. Our observations on recent local outbreaks indicate that breeding lines of the Mesoamerican gene pool show some resistance to BLCrV, while genotypes of the Andean gene pool (representing most of the varieties grown in Colombia) are significantly more susceptible (not shown). Climate change is expected to aggravate this as whitefly populations colonize higher-altitude areas [1, 2]. These results underscore the need for continued virus diagnostics and surveillance in order to formulate proper management strategies. Introgression of begomovirus resistance sources into Andean bean breeding lines is under way.