The genus Begomovirus is the most important and largest in number of species of all genera of the family Geminiviridae ( Brown et al. , 2015 ). The family Geminiviridae includes nine genera established on the basis of the phylogeny, organization of the genome, vector and host range ( Varsani et al. , 2014 , 2017 ; Zerbini et al. , 2017 ). Begomoviruses have a circular single-stranded DNA genome encapsidated in twinned icosahedral particles and are transmitted by the whitefly Bemisia tabaci ( Navas-Castillo et al. , 2011 ). Their genomes can be either monopartite or bipartite. The two genomic components of bipartite begomoviruses are designated DNA-A and DNA-B. DNA-A has six open reading frames (ORFs), two in the virion sense (AV1 and AV2) and four in the complementary sense (AC1, AC2 and AC3 and AC4). DNA-B has two ORFs, the virion-sense BV1 and complementary-sense BC1. The AV2 ORF is found in Old World bipartite begomoviruses, but not in New World viruses ( Rojas et al. , 2005 ). The two components of bipartite genomes share a common region (CR) of approximately 200 nucleotides that includes the origin of replication ( Fondong, 2013 ). In recent years, begomoviruses have caused significant yield losses in many crops due to their high rates of incidence and the disease severity caused in cultivated plants in the tropical and subtropical areas in the world. In the Americas, a series of whitefly-transmitted begomoviruses have been reported affecting bean crop. Most of them have been associated with leaf mosaic symptoms and important yield reductions ( Morales, 2010 ).

At the global level, Bean golden yellow mosaic virus (BGYMV) is the infection agent of one of the most dangerous diseases affecting common beans in Central America and the Caribbean. It has also been found in the Dominican Republic, Guatemala, El Salvador, Honduras, Nicaragua, and in Florida, USA ( Blair et al. , 1995 ; Mather et al. , 2003 ; Morales, 2005 ).

Common bean ( Phaseolus vulgaris L.) has a great importance in Cuba because it constitutes one of the basic food for the population. Regarding this crop, diseases with symptoms of golden mosaic have been associated with begomoviruses since the 70s ( Blanco et al. , 1984 ). The presence of golden mosaic disease became the main problem of common beans in the country ( Blanco & Faure, 1994 ; Vázquez, 1999 ). The molecular characterization of the begomovirus responsible for the disease showed it was caused by BGYMV ( Echemendía et al. , 2001 ).

The introduction of cultivars with a better behavior against this disease and the implementation of preventive and control measures into an integrated management program allowed reducing productive losses and improving the sustainability of the crop ( Echemendía et al. , 2010 ).

Despite of the pest management measures used to decrese the infection rates of BGYMV, an increase of the incidence of various yellowing symptoms typical of the presence of begomoviruses has been observed in prospections carried out in the main bean producing areas in the last three years. Recently, three new species have been reported affecting common beans in different geographic regions in Cuba: Common bean severe mosaic virus (CBSMV), Common bean mottle virus (CBMoV) in the western region ( Chang-Sidorchuk et al. , 2017 ) and Tobacco leaf curl Cuba virus (TbLCCuV) in the central region ( Leyva et al. , 2016 ).

The objective of the present study was to know the distribution of bipartite begomoviruses species in the main beans production areas in the Cuban western region.

Bean leaves with foliar mottling and bright mosaic symptoms were randomly collected from 218 plants in commercial fields at different localities in south west of Mayabeque province, during the growing season 2013-2016. The localities were Güines, San José de las Lajas, Batabanó, Jaruco, Quivicán and Melena del Sur. Ten to twelve samples were collected in each locality per year. The total DNA was extracted from all samples by the method described by Permingeat et al. (1998) .

Of all the common bean plants collected in this study, 145 were positive to BGYMV when they were analyzed by hybridization. The fields from Batabanó and Quivicán localities showed the highest infection values, 100% and 91% respectively, whereas the occurrence in the fields of San José de las Lajas displayed the lowest percentage (8.8%). The negative signal in the samples analyzed suggests the presence of other plant pathogens, such as phytoplasmas, that cause symptoms of yellowing in common bean plants ( Zamora et al. , 2012 ).

The PCR showed that between 3% and 4% of the samples corresponded to new begomovirus species detected in Cuba ( Leyva et al. , 2016 ; Chang-Sidorchuk et al. , 2017 ). Five samples were positive to CBMoV in Güines, seven samples to CBSMV in Güines and Jaruco, and five samples to TbLCCuV in Güines and Quivicán, making evident the low distribution of the new begomovirus species. Of all the plants sampled, 162 (74.3%) showed infection by some of the analyzed begomoviruses. BGYMV was the predominating virus, being present in 89.5% of the samples.

The restriction analysis with Hpa II enzyme of the RCA products of the four samples showed distinct restriction patterns. But, based on the sequencing, it was established that samples 18, 127 and 148 were infected with BGYMV despite the differences found in the restriction patterns. Differences in the restriction patterns of these samples could be due to nucleotide changes in the sequences of the viral components A and B of this species. The enzyme Hpa II recognizes a cut sequence of four nucleotides (GGCC) and mutating a nucleotide in any of the isolates affects the restriction patterns. However, as it was then demonstrated with the complete sequencing, these changes were not superior to the species demarcation criteria established for begomoviruses ( Brown et al. , 2015 ).

The sequenced fragment of the sample 18 (1490 bp) (KY886918) included a partial fragment of the genes BV1 and BC1 belonging to the component DNA-B of BGYMV, with an identity of 99% with the Cuban isolate KU145406.

The complete nucleotide sequence of the viral DNA components of the samples 127 and 148 were obtained, which had a respective length of 2644 (KX185517) and 2645 (KX185518) nucleotides. Full-length DNA-A comparison using SDT program showed that both isolates had 98% similarity between them and with the BGYMV (AJ544531) previously described in Cuba ( Echemendía et al. , 2001 ). Using both methods, similar results were obtained after the two different phylogenetic analyses. These isolates were grouped into the same clade with the rest of the BGYMV isolates described in Florida, Puerto Rico, Mexico, and Guatemala (Fig. 1).

The analysis with Blastn of the viral sequences obtained from the two clones derived from the sample 8 showed that these sequences corresponded to the DNA-A (2609 nt) and DNA-B (2567 nt) genomic components of a bipartite begomovirus. BLASTn search and analysis with the SDT program showed that DNA-A (KX011471) from the sample 8 exhibited its highest nucleotide sequence identity (97.8%) with the DNA-A of TbLCCuV (AM050143), a begomovirus described in Cuba infecting tobacco ( Morán et al. , 2006 ) and recently found infecting common bean in Sancti Spiritus province ( Leyva et al. , 2016 ).

BLASTn search and analysis with the SDT program showed that DNA-B (GQ355487) from the sample 8 exhibited its highest nucleotide sequence identity (82%) with the DNA-B of Wissadula golden mosaic virus (GQ355487), which corresponded to the phylogenetic relationships observed in Figure 1. Both components had a common viral region of 203 bp with a 98.5% identity and four copies of identical iterons (two inverted), indicating that both viral components found in the sample 8 were a cognate pair. This is the first work where the DNA-B of tobacco leaf curl Cuba virus (TbLCCuV) is identified; it had not been previously reported in this species.

The presence of TbLCCuV in common beans confirms the adaptive capacity of this species to infect more than one host. This may be an epidemiological threat to other crops of economic importance in the country and suggests the need for further epidemiological studies to ascertain the impact on the production of the crops.

These results show that the dramatic increase of incidence and severity of begomoviruses symptoms on common beans is mostly associated with the presence of BGYMV with high molecular stability respect to the isolate reported in 2001. But in addition, other factors influence, such as the begomovirus species TbLCCuV, CBSMV, CBMoV, and the presence of B. tabaci MEAM1 ( Muniz et al. , 2011 ). The wide distribution of begomoviruses in Mayabeque province is largely due to the presence of B. tabaci (Middle East Asia Minor 1), which feeds on a wide range of plants, making it more likely to acquire and transmit a high number of begomoviruses to new potential hosts ( Muniz et al. , 2011 ). These results emphasize the need to continue the viral diagnosis and surveillance in order to design proper management strategies.

The incidence of BGYMV decreased during the end of 90´s and first years of the 2000's due to the introduction of resistant cultivars accompanied by management and prevention programs ( Echemendía et al. , 2007 ). However, symptoms similar to those caused by begomoviruses have recently been observed to increase in common beans production areas, possibly influenced by the limited access to new cultivars and increases in seed diversity introduced by farmers, given by the need to increase food, ignoring the plant breeding programs for resistance to begomoviruses ( de la Fé-Montenegro, 2016 ).

This study indicates that the infection with begomoviruses is a problem in beans production and suggests the occurrence of multiplex begomovirus species, making possible viral recombination, which needs to be taken seriously in crop management strategies and guide the participatory breeding programs implemented in the country to return to the introduction of sources of resistance to begomoviruses and in particular to BGYMV.

Thanks are due to Dr. Eduardo Sistachs and MSc. Leyanis Aguilar for the critical review and translation into English of the manuscript.