Insights into Emerging Begomovirus–Deltasatellite Complex Diversity: The First Deltasatellite Infecting Legumes

Simple Summary Legumes play an important nutritional role in the diets of millions of people, mainly in developing countries, but their productivity is seriously affected by a variety of pathogens including viruses. In the last few decades, a number of whitefly-transmitted viruses have emerged mainly in tropical and subtropical areas worldwide including the DNA-containing begomoviruses. A survey of leguminous plants for the presence of begomoviruses was conducted in Venezuela, an understudied country in this regard, even when a variety of symptoms resembling those caused by begomoviruses has been observed in leguminous crops for 20 years. As a result, begomoviruses belonging to four novel species have been discovered and molecularly characterized. In addition, a novel deltasatellite, a small DNA molecule associated with some begomoviruses, has been found to be associated with cabbage leaf curl virus. This is the first time that a deltasatellite has been found to infect legumes. Our results illustrate the increasing complexity faced by researchers and breeders looking to develop control strategies against these emerging pathogens. Abstract Begomoviruses and associated DNA satellites are involved in pathosystems that include many cultivated and wild dicot plants and the whitefly vector Bemisia tabaci. A survey of leguminous plants, both crops and wild species, was conducted in Venezuela, an understudied country, to determine the presence of begomoviruses. Molecular analysis identified the presence of bipartite begomoviruses in 37% of the collected plants. Four of the six begomoviruses identified constituted novel species, and two others had not been previously reported in Venezuela. In addition, a novel deltasatellite (cabbage leaf curl deltasatellite, CabLCD) was found to be associated with cabbage leaf curl virus (CabLCV) in several plant species. CabLCD was the first deltasatellite found to infect legumes and the first found in the New World to infect a crop plant. Agroinoculation experiments using Nicotiana benthamiana plants and infectious viral clones confirmed that CabLCV acts as a helper virus for CabLCD. The begomovirus–deltasatellite complex described here is also present in wild legume plants, suggesting the possible role of these plants in the emergence and establishment of begomoviral diseases in the main legume crops in the region. Pathological knowledge of these begomovirus–deltasatellite complexes is fundamental to develop control methods to protect leguminous crops from the diseases they cause.


Introduction
Legumes are grown globally for human consumption, playing an important nutritional role in the diets of millions of people, mainly in developing countries. Additionally, they infecting three wild malvaceous plant species in Cuba [35]; (iii) monopartite Old World begomoviruses infecting sweet potato (Ipomoea batatas) and I. indica (sweepoviruses) in Spain and Portugal [21]; and (iv) a sweepovirus infecting Merremia dissecta in Venezuela [21]. In some cases, deltasatellites reduce or increase the accumulation of the helper begomovirus, but rarely modify the symptoms caused by them [40,41].
The aim of the present study was to investigate the presence of begomoviruses and associated DNA satellites infecting cultivated and wild leguminous plants in Venezuela. As a result, the presence of bipartite begomoviruses was shown in 37% of the collected samples from both crops and wild plants, with four begomoviruses constituting novel species. Additionally, a deltasatellite, constituting a novel species, was found infecting 15% of the collected samples. This is the first deltasatellite found in legumes and in a crop in the New World, and its dependence on the begomovirus cabbage leaf curl virus (CabLCV) for replication was shown experimentally.
The aim of the present study was to investigate the presen associated DNA satellites infecting cultivated and wild legumin As a result, the presence of bipartite begomoviruses was show samples from both crops and wild plants, with four begomov species. Additionally, a deltasatellite, constituting a novel spec 15% of the collected samples. This is the first deltasatellite found in the New World, and its dependence on the begomovirus cab bLCV) for replication was shown experimentally.

Plant Sample Collection and Molecular Identification
A survey was conducted in important leguminous producti Venezuela (localities from the states of Zulia, Mérida, and Truji tober 2017 (Figure 1). Forty-six leguminous (family Fabaceae, s with virus-like symptoms such as yellow mosaic, leaf rugose, and Plants included the economically important crops cowpea (Vigna mon bean (Phaseolus vulgaris, n = 4), black gram (Vigna mungo, n radiata, n = 1), faba bean (Vicia faba, n = 1), and wild plants (Rhyn roptilium bracteatum, n = 3; Desmodium scorpiurus, n = 4) ( Table 1, identification of the wild plants was molecularly confirmed b chloroplast rbcL and matK genes [42]. Leaf samples were dried u tained at room temperature in the dark until analysis.

DNA Extraction and Full-Length Begomovirus and Deltasatellite Genome Cloning fr Field Samples
Total DNA was extracted from dried leaf tissue using a modified CTAB meth and was used as a template for rolling-circle amplification (RCA) using φ29 DNA merase (TempliPhi kit, GE Healthcare, Buckinghamshire, UK). Amplified RCA pr were digested with a set of restriction enzymes (HpaII, which recognizes a 4-nt si BamHI, EcoRI, HindIII, NcoI, NheI, and SalI, which recognize 6-nt sites). Restriction ucts were analyzed in 1% agarose electrophoresis gels in Tris-acetate-EDTA buff were stained with ethidium bromide and visualized under UV light. The selected di RCA products (~2.7 and ~0.7 kbp) corresponding to putative full-length begomovi nome components and deltasatellite genomes, respectively, were cloned into pBlu II SK (+) (Stratagene, La Jolla, CA, USA). Recombinant plasmid DNA was transf into Escherichia coli DH5α by electroporation, and selected clones were sequenced b rogen Inc. (Seoul, South Korea).

DNA Extraction and Full-Length Begomovirus and Deltasatellite Genome Cloning from Field Samples
Total DNA was extracted from dried leaf tissue using a modified CTAB method [43] and was used as a template for rolling-circle amplification (RCA) using ϕ29 DNA polymerase (TempliPhi kit, GE Healthcare, Buckinghamshire, UK). Amplified RCA products were digested with a set of restriction enzymes (HpaII, which recognizes a 4-nt site, and BamHI, EcoRI, HindIII, NcoI, NheI, and SalI, which recognize 6-nt sites). Restriction products were analyzed in 1% agarose electrophoresis gels in Tris-acetate-EDTA buffer that were stained with ethidium bromide and visualized under UV light. The selected digested RCA products (~2.7 and~0.7 kbp) corresponding to putative full-length begomoviral genome components and deltasatellite genomes, respectively, were cloned into pBlueScript II SK (+) (Stratagene, La Jolla, CA, USA). Recombinant plasmid DNA was transformed into Escherichia coli DH5α by electroporation, and selected clones were sequenced by Macrogen Inc. (Seoul, South Korea).

Sequence Analyses
The comparison of initial sequence similarity was performed using the BLAST program (https://blast.ncbi.nlm.nih.gov/Blast.cgi, accessed on 1 September 2021). Sequences were aligned with MUSCLE [44], and pairwise identity scores were calculated using SDT (sequence demarcation tool) [45]. DNA secondary structure prediction for the putative secondary stem-loop structure present in deltasatellites was performed by free energy minimization using the UNAfold web server (http://www.unafold.org/mfold/applications/ dna-folding-form.php, accessed on 1 September 2021) [46,47]. MEGA 7 [48] was used for phylogenetic analysis using the neighbor-joining method [49] and the evolutionary distances were computed using the p-distance method [50].

Construction of Infectious Clones and Plant Agroinoculation
Begomovirus (DNA-A and DNA-B) and deltasatellite infectious clones were constructed from monomeric genomic components cloned from sample V9. Inserts from monomeric clones were released from the plasmids, re-ligated, and subjected to RCA. Each RCA product was partially digested to produce dimeric molecules that were cloned in a plasmid vector. The inserts of the dimeric clones were excised and subcloned in a binary vector. Ligation reactions were transformed in Escherichia coli DH5α by electroporation (25 µF, 200 Ω, 2500 V) in a Gene Pulser Xcell Electroporation System (Bio-Rad, Hercules, CA, USA). Clones were verified by digestion, and those with inserts of the expected size were sequenced at Macrogen Inc. (Seoul, South Korea). Head-to-tail dimeric constructs were transferred to Agrobacterium tumefaciens strain C58C1 by electroporation using the conditions described above. Details on restriction enzymes and vectors used for cloning are shown in Table S1.
For agroinoculation assays, A. tumefaciens cultures harboring each dimeric construct were added (1:1000) to YEP liquid media containing kanamycin (50 µg mL −1 ) and rifampicin (50 µg mL −1 ) and grown at 28 • C for two days. Cultures were centrifuged at 3100× g for 20 min at 4 • C. The cultures were decanted from the media, and the pellets were resuspended in Agrobacterium inoculation buffer (10 mM MES, 10 mM MgCl 2 , and 150 µM acetosyringone, pH 5.6). The optical density was adjusted to 1 at 600 nm. Nicotiana benthamiana plants at the four-leaf stage were inoculated with A. tumefaciens cultures by stem puncture inoculation. Plants were maintained in an insect-free growth chamber (25 • C during the day and 18 • C at night, 70% relative humidity, with a 16-h photoperiod at 250 µmol s −1 m −2 of photosynthetically active radiation) for further analysis.

Begomovirus and Deltasatellite Detection
Begomovirus genome components (DNA-A and DNA-B) and deltasatellite genomes were detected in agroinoculated plants by molecular hybridization. At 38 days postinoculation (dpi), the apical leaves were used for tissue blots of petiole cross-sections (tissue printing) performed on positively charged nylon membranes. Hybridization of the membranes was carried out using specific digoxigenin-labelled DNA probes. Probes were synthesized by PCR according to the DIG-labelling detection kit (Roche Diagnostics, Mannheim, Germany) with primers and amplification programs shown in Table S2. Hybridization was carried out under high stringency conditions [washing steps at 65 • C in 0.19× SSC (15 mM NaCl and 1.5 mM sodium citrate) and 0.1% sodium dodecyl sulfate] following standard procedures. Membranes were treated with CDP-Star (Roche Diagnostic, Mannheim, Germany), and hybridization signals were detected on X-ray film (Kodak, Rochester, NY, USA) following a conventional photographic process. Cabbage leaf curl virus detection in field-infected plants was also carried out by PCR using specific primers designed to synthesize the above-mentioned probes.

Known and Novel New World Begomoviruses Infect Leguminous Plants in Venezuela
Analysis of leaf samples by digestion of RCA products with several restriction enzymes and PCR revealed that 37% (17 of 46) of the plants sampled were infected by begomoviruses (samples V1-V4, V6-V13, V20-V23, and V35) ( These samples were collected in the states of Zulia (three municipalities) and Mérida (one municipality) ( Table 1). GenBank accession numbers of full-length begomovirus genome components (DNA-A and DNA-B) isolated in this work are listed at the end of the manuscript and included in Table S3.
Eleven samples (three cowpea [V1 and V2], two black gram [V12 and V13], one D. scorpiurus [V8], and five R. minima [V9, V10, V20, V22, and V23]) were infected by CabLCV, a typical New World begomovirus ( Table 1). The complete genome sequences of DNA-A and DNA-B of CabLCV from eight infected plants from different hosts were sequenced after RCA and cloning (samples V2 and V35 from cowpea, V13 from black gram and V9, V10, V20, V22, and V23 from R. minima). CabLCV isolates showed a high sequence identity (96.5-100% for DNA-A and 93.6-100% for DNA-B). These isolates were also closely related (96.6-97.6%) to the isolate of CabLCV infecting common bean (MH359390) and R. minima (MH359394) in Ecuador [12]. In samples V1 (cowpea), V8 (D. scorpiurus), and V12 (black gram), CabLCV was detected by PCR with specific primers (Table S2) for the DNA-A and DNA-B components. Although CabLCV was first found to infect cabbage in the United States [51], the virus was found later infecting wild plants of the family Fabaceae in Mexico (Desmodium sp. and Rhynchosia sp.) [52], Jamaica (Rhynchosia sp.) (KP641347-KP641350), and Ecuador (Mucuna pruriens) [12]. Recently, CabLCV has also been found to infect leguminous crops including soybean in Cuba and common bean, cowpea, and pigeon pea in Ecuador [12]. These findings, along with those reported here, highlight the importance of the presence of CabLCV in legumes. Begomoviruses isolated from the other infected plants were different from CabLCV, although they also showed the typical genome organization of bipartite New World begomoviruses.
DNA-A isolated from R. minima samples V11 and V21 were 99% identical and showed the highest sequence identity (87.2-87.5%) with an isolate of Rhynchosia golden mosaic virus (RhGMV, EU339938) infecting R. minima in Mexico. DNA-B from the same samples showed an identity of 98.5%, and the highest sequence identity (80.2%) was with a RhGMV isolate (DQ356429) infecting soybean in Mexico. Interestingly, the two plants infected by this virus were sampled in different municipalities of the state of Zulia, Maracaibo, and Sucre, separated by about 180 km by Lake Maracaibo. This suggests that this virus is probably spread, at least in other municipalities of the state of Zulia, that were not surveyed.
DNA-A from an M. bracteatum sample (V3) showed the highest identity (81.6%) with an isolate of CabLCV (KP641349) infecting R. minima in Jamaica. DNA-B from the same sample showed the highest identity (74.3%) with an isolate of bean golden mosaic virus (BGYMV, L01636) infecting common bean in the Dominican Republic [14].
DNA-A from sample V6 of D. scorpiurus showed the highest identity (82.3%) with an isolate of tomato yellow mottle virus (KC176780) from Costa Rica. DNA-B from the same sample was most closely related (73.2%) to bean latent virus (BLV, MN158326) from Mexico [16].
DNA-A and DNA-B from sample V7 of D. scorpiurus showed the highest identity (82 and 73%, respectively) with two isolates of CabLCV (KT192632 and KT381194) infecting soybean in Cuba.
The current begomovirus species demarcation criteria established that a new isolate should be considered to belong to a novel species if the highest percentage of pairwise identity (full-length genome for monopartite begomoviruses or DNA-A component for bipartite begomoviruses) with known begomoviruses is <91%. Thus, begomoviruses isolated from samples V11 and V21, V3, V6, and V7 would constitute novel species and the following names are proposed: Rhynchosia mottle virus (RhMoV, isolates VE-Rh V11-17 and VE-Rh V21-17 from samples V11 and V21, respectively), Macroptilium mottle virus The analysis of phylogenetic relationships of the DNA-A and DNA-B genomes described in this work with isolates of other begomoviruses native to the New World infecting leguminous plants showed that the novel begomoviruses belong to different lineages. Ca-bLCV isolates are included in a major clade with high bootstrap support (100%). Within this clade, a minor clade includes isolates from Venezuela (described in this work) and Ecuador [12], all of which were found to infect plants of the family Fabaceae (Figure 3). Deltasatellite is 91% nucleotide sequence identity when considering full-length sequences. Thus, the deltasatellite described here would constitute a novel species. Considering the association of this deltasatellite with CabLCV, the name Cabbage leaf curl deltasatellite is proposed for this species.   DNA-Bs of Macroptilium mottle virus and bean leaf crumple virus were grouped in sister branches, as occurs with DNA-Bs of DesMV and DesYSV. However, their DNA-As were not grouped together. This represents an additional example of the dissimilar evolutionary history undergone by both begomovirus genome components [53]. The same happens, for example, with Rhynchosia rugose golden mosaic virus (RhRGMV); DNA-A is phylogenetically related to viral isolates infecting bean, soybean, and Wissadula amplissima ( Figure 3A), while DNA-B is included in the clade that contains all the CabLCV isolates ( Figure 3B).
Of all 46 symptomatic plants, only 17 were shown to be infected by begomoviruses. This could be explained because unspecific symptoms could be caused by RNA viruses (not analyzed here), other pathogens, or even nutritional deficiencies. The identification of novel begomovirus species in legumes stressed the importance of these viruses as a limiting factor in the production of these crops in Central and South America and the Caribbean. Additionally, the presence of begomoviruses in cultivated and wild legumes suggests a reservoir role for the non-cultivated plants in the emergence of viruses that could be a threat to nearby crops. The role of wild plants in begomovirus emergence has been proposed in the case of numerous begomoviruses infecting, for example, tomato crops [54][55][56][57][58][59].

Deltasatellites Belonging to a Novel Species Are Associated with CabLCV Infecting Wild and Cultivated Leguminous Plants
Deltasatellites were found to infect seven of the 11 leguminous plants infected by CabLCV including cowpea (samples V1 and V2), black gram (V12 and V13), D. scorpiurus (V8), and R. minima (V9 and V10) ( Table 1). GenBank accession numbers of full-length deltasatellite genomes isolated in this work are listed at the end of the manuscript and included in Table S3. These plants were collected in two municipalities in the state of Zulia and one municipality in the state of Mérida. Deltasatellite isolates showed identities of 100% between them and the highest identity (73.9%) with a previously characterized deltasatellite, tomato yellow leaf distortion deltasatellite 2 (KU232893), associated with the bipartite begomovirus tomato yellow leaf distortion virus isolated from Sidastrum micranthum in Cuba [40] (Figure 4). The current threshold for species demarcation in the genus Deltasatellite is 91% nucleotide sequence identity when considering full-length sequences. Thus, the deltasatellite described here would constitute a novel species. Considering the association of this deltasatellite with CabLCV, the name Cabbage leaf curl deltasatellite is proposed for this species.
The cloned and sequenced deltasatellites, one per sample, were 666 nt long and contained typical features found in these subviral agents (i.e., a stem-loop containing the conserved nonanucleotide TAATATTAC, a short region with sequence identity with the betasatellite-conserved region, and an A-rich region) [35]. The secondary stem-loop located close to begomovirus iteron-like sequences, which are also typical of deltasatellites, was not evident. However, a more detailed analysis localized the iteron-like sequences of the deltasatellites, which were characterized here after successful alignment with the corresponding region of other New World deltasatellites ( Figure 5A). These sequences were associated with the secondary stem-loop in previously characterized deltasatellites [35,39]. Figure 5B shows the predicted secondary structures for the genomic region where the iteron-like sequences are located, both in the deltasatellites characterized in this work and the other New World deltasatellites analyzed in Figure 5A. These analyses showed a gradation among New World deltasatellites, both for the length of the iteron-like sequences and the Gibbs free energy (dG) of the predicted secondary stem-loops, with a high negative correlation between both parameters (R 2 = 0.7108) ( Figure S1). This suggests that the predicted secondary stem-loop, although it seems to be a universal feature in deltasatellites, presents a significant variation in the level of structuration, which could be related to the presence/absence of a specific function.
virus; PepLRV, pepper leafroll virus; RhGMHaV, Rhynchosia golden mosaic Havana virus; RhGMV, Rhyn-n mosaic virus; RhMMV, Rhynchosia mild mosaic virus; RhRGMV, Rhynchosia rugose golden mosaic virus; ean blistering mosaic virus; SiMMV, Sida micrantha mosaic virus; ToYMoV, tomato yellow mottle virus. * orresponds to DNA-B of RhRGMV. The OW begomovirus East African cassava mosaic virus (EACMV) was utgroup. Bootstrap values are shown for supported branches (>50%). The bars below the trees indicate the cleotide substitutions per site. The cloned and sequenced deltasatellites, one per sample, were 666 nt long and contained typical features found in these subviral agents (i.e., a stem-loop containing the conserved nonanucleotide TAATATTAC, a short region with sequence identity with the betasatellite-conserved region, and an A-rich region) [35]. The secondary stem-loop located close to begomovirus iteron-like sequences, which are also typical of deltasatellites, was not evident. However, a more detailed analysis localized the iteron-like sequences of the deltasatellites, which were characterized here after successful alignment with the corresponding region of other New World deltasatellites ( Figure 5A). These sequences were associated with the secondary stem-loop in previously characterized deltasatellites [35,39]. Figure 5B shows the predicted secondary structures for the genomic region where the iteron-like sequences are located, both in the deltasatellites characterized in this work and the other New World deltasatellites analyzed in Figure 5A. These analyses showed a gradation among New World deltasatellites, both for the length of the iteron-like sequences and the Gibbs free energy (dG) of the predicted secondary stem-loops, with a Phylogenetic analysis including one isolate of each deltasatellite species recognized to date placed cabbage leaf curl deltasatellite (CabLCD) within the cluster that includes New World deltasatellites ( Figure 6). CabLCD is the first deltasatellite found to infect legumes and was the first found in the New World to infect a crop plant. Prior to this work, deltasatellites had been found in the New World in wild plants associated with the New World begomoviruses Sida golden yellow vein virus, tomato yellow leaf distortion virus, and Desmodium leaf distortion virus infecting malvaceous plants (Sida micrantha, Malvastrum coromandelianum, and Corchorus siliquosus) in Cuba [35] and the sweepovirus sweet potato leaf curl virus infecting the convolvulaceous Merremia dissecta in Venezuela [21]. Deltasatellites have also been detected in the Americas in whiteflies using vector-enabled metagenomic approaches in Puerto Rico [60] and Florida (United States) [61]. However, in the Old World, some deltasatellites have been found in economically important crops: ToLCD in tomato in Australia [39] and SPLCD1 in sweet potato in Spain [21].
The presence of a deltasatellite infecting plants of the family Fabaceae suggests that these subviral agents are probably capable of infecting members of additional plant families and are associated in nature with a wider range of helper begomoviruses than previously assumed. high negative correlation between both parameters (R 2 = 0.7108) ( Figure S1). This suggest that the predicted secondary stem-loop, although it seems to be a universal feature in de tasatellites, presents a significant variation in the level of structuration, which could b related to the presence/absence of a specific function.  portant crops: ToLCD in tomato in Australia [39] and SPLCD1 in sweet potato in Spain [21].
The presence of a deltasatellite infecting plants of the family Fabaceae suggests that these subviral agents are probably capable of infecting members of additional plant families and are associated in nature with a wider range of helper begomoviruses than previously assumed.

Cabbage Leaf Curl Virus Acts as a Helper Virus for CabLCD
Nicotiana benthamiana plants agroinoculated with CabLCV alone or in the presence of CabLCD were analyzed by tissue printing hybridization of apical leaves. In two independent experiments, all CabLCV and CabLCV/CabLCD inoculated plants became symptomatic, showing severe stunting, leaf deformation, and mild leaf yellowing ( Figure 7A). Tissue print hybridization with a probe specific for each genome component of CabLCV  (Table 2). No differences in symptomatology were observed in the CabLCV-infected plants in the presence or absence of CabLCD ( Figure 7B). The inability of deltasatellites to influence the symptomatology caused by the helper begomoviruses has been observed in most cases experimentally analyzed to date. Known examples are the New World Sida golden yellow vein virus (SiGYVV)/Sida golden yellow vein deltasatellite 1 (SiGYVD1) and tomato yellow leaf distortion virus/tomato yellow leaf distortion deltasatellite 2 (ToYLDD2) both in the natural malvaceous host plants and the experimental host N. benthamiana, and tomato leaf deformation virus (ToLDeV)/SiGYVD1 and ToLDeV/ToYLDD2 in N. benthamiana [40]. The same has been observed in coinfections of sweet potato leaf curl deltasatellite 1 (SPLCD1) and the bipartite Old World begomovirus tomato leaf curl New Delhi virus (ToLCNDV), the bipartite New World begomovirus SiGYVV, the monopartite New World begomovirus ToYLDeV or the curtovirus beet curly top virus in N. benthamiana; SPLCD1 and ToLCNDV in zucchini; and SPLCD1 and the sweepovirus sweet potato leaf curl virus (SPLCV) in most natural host plants [41]. Exceptions include the slight decrease in symptomatology of SPLCD1 and SPLCV observed in Ipomoea setosa and I. nil and SPLCD1 and tomato yellow leaf curl virus or tomato yellow leaf curl Sardinia virus in N. benthamiana and tomato.
The same has been observed in coinfections of sweet potato leaf curl deltasatellite 1 (SPLCD1) and the bipartite Old World begomovirus tomato leaf curl New Delhi virus (ToLCNDV), the bipartite New World begomovirus SiGYVV, the monopartite New World begomovirus ToYLDeV or the curtovirus beet curly top virus in N. benthamiana; SPLCD1 and ToLCNDV in zucchini; and SPLCD1 and the sweepovirus sweet potato leaf curl virus (SPLCV) in most natural host plants [41]. Exceptions include the slight decrease in symptomatology of SPLCD1 and SPLCV observed in Ipomoea setosa and I. nil and SPLCD1 and tomato yellow leaf curl virus or tomato yellow leaf curl Sardinia virus in N. benthamiana and tomato.

Conclusions
In this study, six bipartite begomoviruses were found to be associated with symptomatic leguminous plants, both cultivated and wild, in Venezuela. Interestingly, four of the begomoviruses reported here constitute novel species. CabLCV, a begomovirus previously described in other countries from the Americas, was the most widespread in the analyzed samples. This is the first report of CabLCV in Venezuela and the first time that this virus has been found infecting black gram and Desmodium scorpiurus. In addition, a novel deltasatellite species was described associated with CabLCV, Cabbage leaf curl deltasatellite. This is the first time that a deltasatellite has been found infecting a species of the Fabaceae family as well as economically important crops in the Americas. The results obtained in this study contribute to the explanation of the causes of symptoms that have been observed in leguminous plants in the last 20 years in Venezuela. Understanding the actual prevalence of begomovirus-deltasatellite complexes is fundamental to the choice and improvement of control methods to prevent or eliminate the viruses that cause them, mainly in those countries where they are understudied.

Supplementary Materials:
The following are available online at https://www.mdpi.com/article/ 10.3390/biology10111125/s1: Figure S1: Correlation analysis between the length of the iteron-like sequences and the Gibbs free energy (dG) of the predicted secondary stem-loops of New World deltasatellites; Table S1: Cloning strategy to obtain the infectious dimer plasmids of cabbage leaf curl virus (CabLCV) DNA-A and DNA-B and cabbage leaf curl deltasatellite (CabLCD); Table S2: Primers and PCR conditions employed to prepare the DNA probes used in this work; Table S3: GenBank accession numbers of full-length begomovirus (DNA-A and DNA-B components) and deltasatellite genomes characterized in this work.