The partial host range of ToYMoV was next investigated by agroinoculation and particle bombardment inoculation of a range of plant species with the infectious ToYMoV clones . In addition to N. benthamiana and tomato, symptomless DNA-A infections were detected in ~38% of agroinoculated pumpkin plants, whereas ToYMoV did not infect the solanaceous species N. tabacum and D. stramonium; cucumber, cantaloupe and honeydew melons; and C. amaranticolor plants . PCR tests with component-specific primers confirmed that all symptomatic plants tested were infected with the ToYMoV DNA-A and DNA-B components and revealed the symptomless infections of pumpkin plants. Plants agroinoculated with the empty vector control or bombarded with gold particles alone did not develop symptoms and were negative in PCR tests for ToYMoV/ToLCSiV DNA-A and DNA-B components. The infectivity and pathogenicity of the full-length cloned DNA-A and DNA-B components of ToLCSiV-[CR:Lib:02] were also assessed by particle bombardment inoculation of N. benthamiana and tomato seedlings. All bombarded were stunted and newly emerged leaves developed epinasty, crumpling and mild yellow mosaic/mottle by 14 dpb . By 21 dpb, cannabis racks symptoms became progressively milder . These results were consistent with those reported by Idris et al. and established that the crumpling and yellow mottle/mosaic symptoms of tomato leaves observed in Liberia in 2002 was caused by ToLCSiV .
Sap transmission is a property of some bipartite begomoviruses and can reveal insight into tissue tropism, i.e., phloem-limitation . To determine if ToYMoV is mechanically transmissible, sap prepared from symptomatic N. benthamiana leaves from plants infected with ToYMoV via agroinoculation was rub-inoculated onto leaves of N. benthamiana and tomato seedlings. In these experiments, ToYMoV was sap-transmitted to ~56% of N. benthamiana plants, but not to tomato plants . Disease symptoms in N. benthamiana plants infected followed sap transmission were indistinguishable from those infected after agroinoculation . In contrast, a 100% of N. benthamiana plants rub-inoculated with the sap-transmissible BDMV developed stunting, distortion and severe leaf epinasty by 14 dpi, and 100% of tomato plants rub-inoculated with ToMoV developed stunting, epinasty and yellow mottling by 14 dpi. In all these experiments, the presence of the ToYMoV DNA-A and DNA-B components was confirmed in newly emerged leaves of representative symptomatic plants by PCR tests with component-specific primers . The expected-size PCR fragments were amplified from symptomatic N. benthamina and tomato plants infected with BDMV and ToMoV , respectively, whereas no DNA fragments were amplified from an extract prepared from plants inoculated with buffer alone .
To further investigate the relationship between ToYMoV and ToLCSiV, pseudorecombination experiments were conducted in which N. benthamiana and tomato plants were agroinoculated with the DNA-A and DNA-B components of the wild-type viruses and the pseudorecombinants . N. benthamiana plants agroinoculated with the ToYMoV DNA-A and ToLCSiV DNA-B PR did not developed symptoms, although ToYMoV DNA-A only infections were detected in all inoculated plants . Plants agroinoculated with the ToLCSiV DNA-A and ToYMoV DNA-B PR also did not develop symptoms, but in contrast to the TA + SB PR, ToLCSiV DNA-A only infections were not detected. Similar results were obtained in equivalent experiments conducted with tomato plants, although rates for ToYMoV DNA only infections were lower . Together, these results established that the DNA components of these viruses cannot form infectious PRs, which is consistent with ToYMoV and ToLCSiV belonging to different lineages and having different iterons and IRDs.To understand the potential impact of these two invasion events on begomovirus disease of tomato in CR, we next compared single and mixed infections of these viruses in terms of symptom development and severity and viral DNA accumulation. Consistent with agroinoculation results, ToYMoV and ToLCSiV each induced stunting and epinasty, crumpling and yellow mosaic/mottle symptoms in newly emerged leaves of all agroinoculated tomato plants by 21 dpi . Here it should be note that the ToYMoD symptoms induced in cv. Glamour by these two viruses were indistinguishable.
Equivalent tomato plants agroinoculated with the infectious clone of TYLCV were stunted and developed the characteristic symptoms of tomato yellow leaf curl disease , including upward leaf curling, crumpling and interveinal and marginal yellowing in newly emerged leaves by 21 dpi . Tomato plants co-inoculated with ToYMoV and ToLCSiV were not more stunted than plants infected with each virus alone, but co-infected plants developed more severe epinasty, crumpling and yellow mosaic/mottle by 21 dpi . By 21 dpi, tomato plants co-infected with ToYMoV or ToLCSiV and TYLCV were more stunted and showed mostly TYLCD symptoms . The symptoms of ToLCSiV and ToYMoV appeared earlier that those of TYLCV . However, by 21 dpi, TYLCD symptoms had become dominant, regardless of the combination . Furthermore, plants having mixed infections with TYLCV showed a marked symptoms transition in leaves, in which the crumpling and yellow mosaic/mottle associated with bipartite begomoviruses initially appeared in the lower leaves, whereas those of TYLCV appeared later and became dominant by 21 dpi . Finally, plants infected with all three viruses were severely stunted and foliar symptoms appeared at ~7 dpi, eventually developed unusually severe TYLCD symptoms . These results revealed either a synergistic interaction or an additive effect in plants co-inoculated with these viruses. In order to measure virus accumulation over time, samples of newly emerged leaves were collected from tomato plants agroinoculated with each virus alone and in all combinations at 7, 14 and 21 dpi, and viral DNA accumulation was quantified by qPCR. In single infections, all viruses were detected in newly emerged leaves and had accumulated to similar levels in newly emerged leaves by 7 dpi, even though symptoms had not appeared in plants infected with TYLCV . Levels of TYLCV and ToLCSiV increased by 14 and 21 dpi, whereas those of ToYMoV remained the same or decreased slightly . In mixed infections, ToYMoV DNA levels were significantly reduced at 7 dpi, and similar trend was observed for ToLCSiV; however, levels of both viruses increased by 14 and 21 dpi and were not significantly different from those of plants infected with these viruses alone . TYLCV DNA levels were unaffected by the co-infecting bipartite begomoviruses. Thus,other than this transient negative effect, there was no evidence of any virus having levels consistently reduced in the presence of a co-infecting virus. Variability was high or increased for the bipartite in mixed infections, especially with TYLCV . In contrast, the viral DNA levels of TYLCV were not affected in mixed infection with either or both of the bipartite begomoviruses . Levels of variability for TYLCV were higher at 7 dpi for single and mixed infections, but then became much lower at 14 and 21 dpi . Interestingly, overall TYLCV variability was greater in single infected compared with plants having mixed infections. Together, our results suggest that these viruses can co-infect tomato plants without negatively impacting replication of co-infecting viruses.In the present study, we further analyzed the three emergence/introduction events for tomato-infecting begomoviruses in CR to investigate the invasion biology of these viruses, i.e., indoor grow rack the investigation of interactions among these viruses and the susceptible host and how this will impact begomovirus disease of the tomato crop in CR.
The three emergence/introduction events were: local evolution of ToYMoV, introduction of an invasive NW bipartite begomovirus from elsewhere in the region and introduction of the OW monopartite TYLCV. However, because our approach required infectious clones of each virus, we first generate infectious clones and agroinoculation systems for isolates of ToYMoV and ToLCSiV from CR. In the case of ToYMoV, this allowed us to complete the characterization of the virus, including fulfilling Koch’s postulates for the ToYMoD. Host range studies performed via agroinoculation showed that ToYMoV has a relatively narrow host range, infecting and causing symptoms in tomato and N. benthamiana, but not in all solanaceous species . This is consistent with previous reports in which ToYMoV was never found in association with begomovirus diseases on pepper in CR . Cucurbits do not appear to be a host of ToYMoV, with the exception of a small number of symptomless DNA-A only infections of pumpkin. The sap transmissibility of ToYMoV, albeit only to N. benthamiana plants, indicates some capacity for cell-to-cell movement and egress from the phloem. Taken together with previous results, ToYMoV is a locally evolved virus that has been associated with ToYMoD in CR for over a 30 yrs period, with relatively little divergence over ~20 years . Thus, these results suggests that ToYMoV has reached stabilizing selection and that it is well-adapted to tomato. Furthermore, reports of ToYMoV from NI and Panama indicated capacity for regional spread in Central America. The placement of ToYMoV in the SLCuV lineage was expected, based on a previous study , but our phylogenetic analyses further indicated that the four ToYMoV isolates comprise a strongly supported clade that is distinct from the rest of the viruses in the SLCuV lineage. Thus, this may represent a new lineage that is closely related to the SLCuV lineage. This hypothesis is supported by several lines of evidence. First, although the closest relatives of ToYMoV are members of the SLCuV lineage, the relatively low identities , indicate a long period of diversification via random mutation . Second, the ToYMoV common region are different from those of members of the SLCuV lineage, such that they would be predicted to not form infectious PRs. Third, the ToYMoV AC4 differs from those of members of the SLCuV lineage in size and in possessing an N-terminal myristoylation motif. Finally, there has been substantial host adaptation of ToYMoV to infect tomato, whereas most of the members of the SLCuV lineage are adapted to and cause disease in cucurbits. These two lineages probably arose from an ancestral begomovirus infecting a non-cultivated plants species in the region, and then underwent local evolution to adapt to infect tomato or cucurbits.The long branch that separates ToYMoV and the other member of the SLCuV lineage also was indicative of recombination playing a role in viral evolution . Indeed, a recombination event was detected in the well-known hot-spot region of the begomovirus genomic DNA/DNA-A component, and includes the Rep binding site, thereby changing replication specificity . The overall biological significance of recombination in this region is still not clear, but it has been suggested to be involved in host adaptation, including a role for AC4 . Thus, it is notable that acquire a very different AC4 via the recombination even. Thus, ToYMoV is an indigenous virus that locally evolved to infect tomato and that this was likely mediated by indigenous whiteflies . The detection of ToLCSiV, first described in MX in 1989 , in Costa Rica in 1998 was the first invasion event: the introduction of an invasive NW bipartite begomovirus from the region. Furthermore, the detection of ToYMoV and ToLCSiV in samples with stunting and yellow mosaic/mottle of leaves collected from Liberia in 2002 and from Grecia in 2012 , is consistent with both viruses causing ToYMoD and with ToLCSiV having spread and become established in CR. Mixed infections of ToYMoV and ToLCSiV will allow for genetic interactions between these viruses. However, the finding that ToYMoV and ToLCSiV did not form infectious PRs is consistent with these viruses coming from distinct lineages, and having divergent regulatory sequences. Thus, a replication incompatibility must likely underlie the lack of infectivity of the PRs. Finally, although these results seem to rule out this genetic mechanism as a means ofvariability, the viruses can clearly co-infect tomato plants and exist in mixed infections, thereby allow for recombination to occur. An interesting observation from the PRs experiments was the different capacities of the DNA-A components of ToYMoV and ToLCSiV to systemically infect N. benthamiana and tomato plants. DNA-A alone infections have been previously reported for NW and OW bipartite begomoviruses in agroinoculated plants . As in these previous reports, the ToYMoV DNA-A infections were symptomless, consistent with the need of the DNA-B component for symptom development of bipartite begomoviruses . The mechanism underlying the virusspecific DNA-A alone infections is not known, but it may be involved the AC4 . Furthermore, the of N. benthamiana and tomato by the ToYMoV DNA-A reflects the susceptibility of these species to begomovirus infection, and maybe reveal how NW monopartite begomoviruses may have emerged .