The introduction of TYLCV into CR in 2012 was the second invasion event: a highly invasive OW monopartite begomovirus that can cause devastating losses to tomato production worldwide . Indeed, TYLCV has added a new challenge for tomato production in CR, as growers have reported increased losses due to begomoviruses disease since 2012 . However, what was less clear was how TYLCV interacts with the already existing bipartite begomoviruses ToYMoV and ToLCSiV. In Florida, the introduction of TYLCV led to a reduced incidence of the indigenous bipartite begomovirus ToMoV . Therefore, this provides an opportunity to investigate the invasion biology of these very different begomoviruses with the long-term objective of making predictions and management suggestions. In the infectivity experiments, interactions were revealed based on symptom severity and viral DNA accumulation. A general synergistic interaction was observed in which mixed infections resulted in more severe symptoms, with plants infected by all three viruses showing the most severe symptoms. A similar situations has been observed for the interaction of three indigenous begomoviruses in Brazil, as measured based on symptom severity . Furthermore, the interaction among begomoviruses detected in the present study is a type of neutral synergism, which has also been previously described, including for begomoviruses . For example, N. benthamiana and tomato plants co-inoculated with TYLCV and tomato yellow leaf curl Sardinia virus developed more severe symptoms than plants inoculated with either virus alone, curing cannabis and accumulation of each virus in co-infected plants was similar to that in single infections . Interestingly, we did detect an initial antagonistic or negative interference effect on ToYMoV and ToLCSiV accumulation at 7 dpi in all mixed infections, indicating it was not virus specific.

These results are consistent with a previous study showing a transient negative effect on viral accumulation early in mixed infections of tomato rugose mosaic virus and tomato yellow spot virus in N. benthamiana and tomato . The overall mechanism of antagonism in mixed infections remains to be elucidated, but may involve competition for host factors or stimulation of a more efficient defense response of the host . For example, potato spindle tuber viroid can interfere with TYLCSV accumulation in tomato by activation of the host DNA methylation pathways . The fact that TYLCV accumulation was not affectedduring mixed infections with NW bipartite begomoviruses in the present study could be due to the combine activities of multiple viral suppressor of gene silencing, e.g., C2, C4 and V2 . Taken together, our results with these three begomoviruses revealed the existence of temporary antagonism followed by a more sustained neutral synergism. We further showed that in mixed infections with TYLCV, TYLCD became dominant at 14 dpi and beyond, even in the presence of the two co-infecting NW tomato bipartite begomoviruses. Indeed, this dominant TYLCD phenotype has been observed in the tomato fields in Costa Rica since the introduction of TYLCV . Therefore, in terms of the invasion biology of these viruses, it appears they can effectively co-exist in tomato plants, which leads to more severe disease and the persistence of all three viruses in tomato production in CR. Another observation in mixed infections with TYLCV was that, although TYLCD symptoms eventually became dominant, symptoms induced by co-infecting bipartite begomoviruses appeared earlier , with TYLCD symptoms appearing at ~10 dpi. This observation can be explained in terms of the tissue tropism of these viruses. The more rapid appearance of mosaic/mottle symptoms in leaves may reflect the capacity to infect cells outside of the phloem, and more rapidly colonize and accumulate in plants.

The sap transmission of ToYMoV is evidence this virus is not phloem limited. In summary, we used the tomato begomovirus situation in CR to examine the invasion biology of three viruses, two of which were introduced. We first confirmed ToYMoV caused ToYMoD in CR and is a locally evolved NW bipartite begomovirus. We then used infectious clones to investigate interactions in mixed infections. We found that these viruses exhibited a neutral synergism, in which the viruses co-exist and induced more severe symptoms. In mixed infections with TYLCV, TYLCD became predominant. These results indicate that all three viruses are likely persisting in CR and causing more severe symptoms and losses, particularly in the presence of TYLCV. Thus, an effective management of these complexes will require an integrate approach, including the identification of varieties with resistance to all three viruses.The genus Begomovirus is comprised of a large and diverse group of plant viruses that possess a circular, single-stranded DNA genome encapsidated into twin quasi-icosahedral virions . These viruses infect dicotyledonous plants and cause numerous economically important diseases of fiber, fruit, ornamental and vegetable crops, mostly in tropical and subtropical regions of the world . Begomoviruses are transmitted, plant-to-plant, by whiteflies of the Bemisia tabaci cryptic species complex . The genome of begomoviruses is composed of either a single genomic DNA of ~ 2.8 kb or two ~2.6 kb DNA components , designated as DNA-A and DNA-B . The genomic DNA of monopartite begomoviruses is homologous to the DNA-A component of bipartite begomoviruses, and both are organized with overlapping virion -sense and complementary -sense genes transcribed in a bidirectional manner from an intergenic region , which contains the cis-acting elements involved in replication and gene expression .

In bipartite begomoviruses, an ~200 nucleotide noncoding sequence is shared between cognate DNA-A and DNA-B components, and this common region maintains the specificity of replication for these components. Otherwise, the sequences of the DNA-A and DNA-B components are different, and bothcomponents are needed for induction of typical disease symptoms . In terms of begomovirus evolution, continental drift is believed to have separated ancestral monopartite and bipartite begomoviruses, resulting in the predominance of monopartite begomoviruses in the Old World and bipartite ones in the New World . The subsequent independent diversification and evolution of OW and NW begomoviruses involved different combinations of mutation, recombination and acquisition and modification of foreign DNAs . For OW monopartite begomoviruses, acquisition of satellite DNAs has played a major role in evolution, whereas acquisition and modification of the DNA-B component was essential for bipartite begomoviruses, and allowed for pseudorecombination to act as an additional mechanism of evolution . Furthermore, the emergence of new begomoviruses has been facilitated by the global spread of the highly polyphagous B. tabaci species MEAM1, which can introduce mixtures of viral components/genomic DNAs into a diversity of plant species . Finally, human activities have led to the long distance intercontinental movement of numerous begomoviruses, blurring the geographic separation of OW and NW begomoviruses . The remarkable diversification of begomoviruses has been reflected in the appearance of diseases of crop and non-cultivated plants in tropical and subtropical regions worldwide. In these agroecosystems, it is common to observe non-cultivated plants showing striking golden/yellow mosaic symptoms, which are commonly associated with begomovirus infection. Inthe Caribbean Basin and other parts of Latin America, non-cultivated plants with these symptoms have been reported from species in the families Asteraceae, Capparaceae, Convolvulaceae, Euphorbiaceae, Fabaceae, Malvaceae, Nyctaginaceae and Solanaceae . Importantly, characterization of begomoviruses associated with these diseases has revealed substantial genetic divergence from viruses that cause economically important crop diseases, although there are some exceptions such as the golden/yellow mosaic symptoms of Malachra alceifolia associated with tobacco leaf curl Cuba virus infection in Jamaica , and mosaic and crumpling symptoms of Nicandra physaloides infected with tomato severe rugose virus in Brazil . This suggests that begomoviruses infecting crops and weeds have co-evolved independently with their hosts, with the practical implication that most of these symptomatic weeds are not major sources of inoculum for crop-infecting begomoviruses. However, these begomovirus-infected weeds can serve as a mixing vessels for evolution of viruses with the potential to infect crops . The family Malvaceae, commonly referred to as mallows, is comprised of >4225 species of annual and perennial plants .

Members of this family are distributed worldwide, and occur in temperate, tropical and subtropical regions . Some species are important crops, such as cotton and okra ; others are grown as ornamentals or for medicinal purposes; and others are considered invasive weeds, e.g., Abutilon spp., Sida spp. and Malachra spp. . Moreover, weed dryer these malvaceous weeds are commonly infected by begomoviruses and develop striking golden/yellow mosaic symptoms . As part of a long-term study to characterize begomoviruses causing golden/yellow mosaic symptoms in weeds and assesses the potential of these viruses to cause diseases of crop plants in the Dominican Republic , we describe here the molecular and biological properties of two bipartite begomoviruses associated with these symptoms in Malachra sp. and Abutilon sp. plants on Hispaniola. Sequence and phylogenetic analyses together with infectivity studies with infectious clones were used to establish that the symptoms in Malachra sp. were caused by the crop-infecting bipartite begomovirus TbLCuCV, whereas those in Abutilon sp. were caused by a new species of weed-infecting begomovirus for which the name Abutilon golden yellow mosaic virus is proposed. Host range experiments showed that TbLCuCV also induced moderate to severe disease symptoms in Nicotiana benthamiana, tobacco and common bean plants plants. In contrast, AbGYMV induced mild or no symptoms in these plants, indicating a high degree of adaptation to Abutilon sp. from the DO and low potential to cause crop diseases. TbLCuCV and AbGYMV are closely related species in the Abutilon mosaic virus lineage of NW begomoviruses and we present evidence that recombination and pseudore combination play a role in the evolution of these viruses.To detect begomovirus DNA-A and DNA-B components, PCR tests were performed with the degenerate primer pairs PAL1v1978/PAR1c496 and PCRc1/PLB1v2040, respectively . PCR-amplified fragments were purified with the QIAquick gel extraction kit and directly sequenced with the PAL1v1978/PAR1c496 and PCRc1/PLB1v2040 primers. To estimate the number and genetic diversity of begomovirus DNAs present in the samples and to identify single-cutting restriction enzymes for obtaining full-length clones, restriction fragment length polymorphism analyses of circular DNAs generated by rolling circle amplification with Φ-29 DNA polymerase were performed . The RCA products were first digested with the fourbase-cutting enzyme MspI to generate RFLPs for estimating the number of begomovirus DNA components infecting the samples. Next, RCA products were digested with selected six-basecutting enzymes to identify sites in each DNA component for obtaining full-length clones. The linearized DNA components were ligated into pGEM11Z  or pSL1180 digested with the appropriate enzyme. Recombinant plasmids having the full-length DNA-A and DNA-B components were identified by restriction enzyme digestion and DNA sequence analyses. Based upon sequencing and RCA results, the begomovirus isolates from the M1 and M4 samples were selected for further studies. Thus, full-length DNA-A and DNA-B clones were obtained from sample M1 , sample M2 , sample M3 and sample M4 .The complete sequences of the cloned full-length DNA-A and DNA-B components of the bipartite begomoviruses from samples M1-M4 were determined and analyzed with Vector NTI advance software . Pairwise nt sequence alignments were performed with MUSCLE within the Species Demarcation Tool v.1.2, and with full-length DNA-A and DNA-B sequences of the ten begomoviruses with the highest identities revealed by the BLASTn search . The Vector NTI advance software was used to make more extensive comparisons, including individual open reading frames and non-translated regions from both components. The cis-acting elements involved in begomovirus replication were identified as described in Argüello-Astorga and Ruiz-Medrano .For the phylogenetic analyses, we used the complete nt sequences of the DNA-A and DNAB components of: the bipartite begomoviruses from the M1-M4 samples; TbLCuCV isolates from CU ; the ten most identical viruses revealed by the BLASTn search; and selected viruses representing the AbMV, Brazil, squash leaf curl virus , bean golden yellow mosaic virus and Boerhavia golden mosaic virus lineages of NW begomoviruses. Multiple sequence alignments for the DNA-A and DNA-B component sequences were generated with the MAFFT algorithm implemented in the Guidance2 Server . The alignment quality was analyzed, and unreliable regions were removed with the GUIDANCE algorithm . The resulting alignments were then exported as Nexus files. Phylogenetic trees were constructed with a Bayesian inference and Markov chain Monte Carlo simulation implemented in MrBayes V3.2 . The best-fit model of nt substitution for each data set was determined with the program MrModeltest V2.2 . The analyses were carried out by running 2,000,000 generations and sampling at every 100 generations, resulting in 20,000 trees. The first 10% of samples were discarded as a burn-in. Trees were visualized with Archaeopteryx tree viewer and exported in Newick format .