Begomoviruses infecting common beans (Phaseolus vulgaris L.) in production areas in Cuba

Viral diseases caused by begomoviruses are economically important for their depressing impact on common bean production in Cuba. Mayabeque is a Cuban province where this crop is significantly grown and affectations by Bean golden yellow mosaic virus (BGYMV) have been detected in the last 30 years. Integrated pest management (IPM) programs in this crop have allowed controlling the disease for a long time. However, in prospections of the last years, an increase of the incidence of various yellowing symptoms typical of begomoviruses has been observed in common bean production areas. DNA was extracted from leaf samples taken from symptomatic plants. Non-radioactive nucleic acid hybridization and a specific PCR assay were used to detect BGYMV, Common bean severe mosaic virus, Common bean mottle virus, and Tobacco leaf curl Cuba virus. Of the 218 bean plants sampled, 89.5 % was positive to BGYMV; the presence of the rest of the begomovirus species was between 3 and 4% (3.08% of CBMoV, 3.08% of TbLCCuV and 4.32% of CBSMV). The viral DNA from some samples was analyzed by rolling circle amplification (RCA), by restriction fragment length polymorphism analysis using restriction enzymes, and by cloning and sequencing of the viral components. The DNA sequences from BGYMV isolates showed 98% of identity with the isolates reported in Cuba in 2003. The infection by Tobacco leaf curl Cuba virus (TbLCCuV) was confirmed also in fields in the Cuban western region. This is the first work where the DNA-B of TbLCCuV is identified. These studies will help to strengthen phytosanitary surveillance and management programs implemented in the country to control the whitefly-begomovirus complex in this economically important crop. Additional keywords: geminivirus; rolling circle amplification; whitefly; identification. Abbreviations used: BGYMV (Bean golden yellow mosaic virus); CBMoV (Common bean mottle virus); CBSMV (Common bean severe mosaic virus); IPM (integrated pest management); ORFs (Open reading frames); RCA (rolling circle amplification); TbLCCuV (Tobacco leaf curl Cuba virus). Authors’ contributions: Study design, interpretation of data, critical revision of the manuscript for important intellectual content: LCS, HGA and YMZ. Performed the experiments: LCS and HGA. Wrote the paper: LCS and YMZ. Citation: Chang-Sidorchuk, L.; González-Alvarez, H.; Martínez-Zubiaur, Y. (2018). Begomoviruses infecting common beans (Phaseolus vulgaris L.) in production areas in Cuba. Spanish Journal of Agricultural Research, Volume 16, Issue 2, e1006. https://doi. org/10.5424/sjar/2018162-11735 Received: 18 May 2017. Accepted: 25 Jun 2018. Copyright © 2018 INIA. This is an open access article distributed under the terms of the Creative Commons Attribution 4.0 International (CC-by 4.0) License. Funding: The Ministry of Foreign Affairs of Spain; Spanish Agency of International Cooperation for Development (MAECAECID) financed the fellowship granted to LCS. Competing interests: The authors have declared that no competing interests exist. Correspondence should be addressed to Yamila Martínez-Zubiaur: yamila@censa.edu.cu


Introduction
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(Varsani et al., , 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 virionsense 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.

Material and methods
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).

Detection of specific begomoviruses previously identified in Cuban fields
Molecular diagnostic tools such as nucleic acid hybridization and PCR were used to determine the presence of specific begomoviruses detected in common beans in our country.

Specific detection of BGYMV
The samples were analyzed by nucleic acid hybridization with a specific probe to identify the presence of BGYMV and determine its distribution in the localities studied.
The probe was obtained from a BGY MV clone from the Cuban isolate (Eche men día et al., 2001).The pri mers called MA 2163 (5'-GTAAATATGCGAGTGTCTC-CG-3') and MA 21 62 (5'-CTGG ACTTAC ACGTGGA-ATGG-3') were de sig ned using the inter genic re gion (IR) of the viral sequence from the Cuban isolate (Acc.No. AJ544531) obtained from the Genbank.The labelling of the non-radioactive probe was carried out according to the instructions in the reagent kit of PCR DIG Probe Synthesis Kit (Roche).The PCR was performed using a program that consisted in denaturation for 2 min at 94°C, followed by 30 cycles of reaction (1 min at 94ºC, 1 min at 57ºC, 50 s at 72ºC) and 5 min at 72ºC.PCR amplified products were analyzed by electrophoresis on 0.8% agarose gel containing ethidium bromide in 1X TE (10 mM Tris-HCl, EDTA 1 mM, pH 8.0).The PCR fragments obtained were approx.356 bp.DNA from plants infected with BGYMV was used as positive controls.
All DNA extracts (0.5-1 µg) of each sample were applied in a Hybond N+ membrane and set with UV light (120 mJ/cm 2 ) using a UV-crosslinker (Biorad).Pre-hybridization was conducted with a solution containing 5X SSC, 0.1% laurilsarcosine, 0.02% SDS and 2% blocking agent.Hybridization was conducted for 18 hours at 65 o C.There were three washings of 15 min each with the washing solution SSC and 0.1% SDS.

Specific detection of CBSMV, CBMoV and TbLCCuV
Species-specific set of primers were designed by using DNA-A sequences (Table 1) of the following begomovirus: CBSMV (KX011476), CBMoV (KX011473) and TbLCCuV (AM050143).Primer sequences were chosen by multiple alignment analysis of the respective DNA-A sequences using MEGA 6.0 (Tamura et al., 2013).The primer sequences were evaluated for their ability to form homo-and heterodimers as well as hairpins by using Oligo 7 (Rychlik, 2007).
For PCR amplification, mixtures (total volume 25 μl) containing GoTaq Green Master Mix (Promega, Madison, WI, USA) with a final concentration of 1.5 mM MgCl 2 , 100 μM each dNTPs and forward and reverse primers (Table 1) at 10 pmol each were prepared.Full-length clones of three begomoviruses (Morán et al., 2006;Chang-Sidorchuk et al., 2017) served as positive controls for evaluation of the PCR.
PCR was run in a thermocycler (Touch T960) using the following program for primers CBSFw1/CBSRev2 and CBMoFw1/CBMoRev2: an initial denaturation step at 94ºC for 5 min followed by 30 cycles at 94ºC for 30 s, 55ºC for 1 min, 72ºC for 30 s, and a final extension of 72ºC for 7 min.For primers TbLFw1/TbLRev2 the cycling conditions were: 1 cycle at 94ºC for 5 min; 35 cycles at 94ºC for 1 min, 53ºC for 1 min and 72ºC for 1min, followed by a final extension cycle at 72ºC for 10 min.Ten microliters of the amplified products were electrophoresed on a 1% agarose gel containing (10 mg/mL) ethidium bromide for 1 h and visualized under an UV transiluminator.

Genome amplification
The rolling-circle amplification (RCA) on the extracted DNA was carried out using φ29 DNA polymerase (TempliPhi kit, GE Healthcare).Three DNA positive for specific diagnostic (samples 18, 127, 148) were selected from localities with positive samples to new species (Güines, Jaruco, Quivicán) for comparing the molecular characteristic with BGYMV isolated in 2001 (Echemendía et al., 2001).As TbLCCuV had only been reported in the bean crop in the central region of the country (Leyva et al., 2016), a positive sample (sample 8), obtained for specific PCR, was analyzed to confirm its presence in the west of the country and to complete the characterization.
RCA products were digested with HpaII, BamHI, EcoRI, HindIII, NcoI, NheI, SacI and PstI.The restriction fragments of approximately 2.6 kbp, obtained with the single cutting enzymes PstI, NcoI, EcoRI and HindIII were selected.These fragments were cloned using pGem-T-Easy and pBluescrip SK II (+) previously digested with the same restriction enzyme.The plasmids with the cloned viral fragments were purified according to Serghini et al. (1989) and the putative viral sequences present in the clones selected were completely sequenced (Macrogen Inc., Seoul, South Korea).

Sequence analysis
Sequences obtained were initially assembled with the program SeqMan (DNAStar, Inc., Madison, WI, USA) and analyzed using the Blastn algorithm for preliminary species assignment according to the recommendations of the Geminiviridae Study Group of the International Committee on Taxonomy of Viruses.Percentage identities with the most closely related begomoviruses were calculated with the program SDT (Species Demarcation Tool) version 1.0 (Muhire et al., 2014).Genomic sequences were aligned with the MUSCLE module implemented in MEGA 6.0 (Tamura et al., 2013).Phylogenetic trees were inferred by the neighbour-joining and maximum likelihood methods.A phylogeny test was conducted by the bootstrap method (1000 replicates).

Results and discussion
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).(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 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 HpaII 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 HpaII 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.  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. ,

Figure 1 .
Figure 1.Phylogenetic trees based on an alignment of the complete nucleotide sequences of DNA-A of ToLCCuV and BGYMV (A) and DNA-B of ToLCCuV (B) isolate obtained in this work (highlight) with the genomes of a selected begomovirus obtained from the GenBank.The trees were generated using the maximum likelihood algorithm in MEGA 6.The numbers at nodes represent percentage bootstrap values (1000 replicates).ACMV was used as outgroup.

Table 1 .
List of primers used for amplification of begomoviruses in PCR.