During the last two decades, the spread of the highly polyphageous biotype B of B. tabaci has greatly contributed to the worldwide emergence of begomoviruses. These devastating viruses are one of the most important threats for tomato production in tropical and subtropical environments. Within the European Union, begomoviruses are listed on the EPPO A2 alert list and diagnostics tools are essential to prevent dispersions and new introductions. Several PCR-based methods [28, 29] and real-time PCR [15, 16] have been reported recently for the detection and the differentiation of strains and species of begomoviruses. We present here five real-time PCR assays including an internal report for the relative quantification of different begomoviruses in tomato plants. Those duplex real-time PCR assays are associated with a novel strategy for a unique quantification standard consisting in the cloning of both viral and internal report targets in the same plasmid. We developed and successfully applied these real-time PCR assays for the specific detection and quantification of a wide range of begomoviruses including the two emerging strains of TYLCV (IL and Mld strains), the PYMV and the ToLCKMV-like viruses.
As described previously in others studies [30, 31], we used an internal report to validate and normalize the entire experiment including the processes of sampling, DNA extraction and DNA amplification. Following Mason et al. , we selected the Solanum lycopersicum 25S ribosomal RNA gene as internal report and we optimized the real-time PCR to amplify, in the same reaction, both viral and host DNA targets. The original feature of our assay was the design of a unique standard quantification comprising both the viral and the internal report targets. Recently, Lay et al  described a similar approach with the cloning of two Epstein-Barr virus targets in a single quantification standard. Here, we have conceived a quantification standard comprising not only the five viral targeted amplicons but also the internal report target. This approach is very useful to reduce the laborious stages of preparation of quantification standards containing known amounts of each target to a single step, and thus reduces the time and the cost of the whole assay.
The real-time PCR assays developed in this study were optimized to detect and quantify both the viral and host DNA in multiplex reactions respecting the MIQE guidelines . PCR amplifications of the internal report cover six (TYLCV-IL, PYMV-A and ToLCKMV-like viruses) to seven (TYLCV-Mld and PYMV-B) orders of magnitude. Viral detection and quantification are possible in the range of 2 × 109 to 103 viral DNA copies/μL except for PYMV-A and ToLCKMV-like (2 × 108 to 103 viral DNA copies/μL). Those real-time PCR assays provide an accurate detection and quantification of the targeted viruses, with a higher detection limit than the ones previously described by others studies on RNA viruses [34, 35] or DNA viruses  albeit of only a 10-fold. Advantageously, our real-time PCR assays are able to quantify both the host and viral DNA in a single run, making a direct normalisation of the quantification possible.
We successfully used the real-time PCR developed with experimentally inoculated plants to compare viral accumulation at 10, 20 and 30 dpi. These three successive viral quantifications, although unable to reflect the entire kinetics of viral accumulation, were sufficient to observe different patterns of viral accumulation between the bipartite and the monopartite begomoviruses and between the different strains and species of monopartite begomoviruses analysed.
The comparison of relative loads of viral DNA demonstrated that the bipartite PYMV accumulated more viral DNA than the two strains of TYLCV and ToLCKMV in tomato plants at each date considered. To our knowledge, it is the first demonstration of the higher viral load of a bipartite begomovirus than monopartite begomoviruses. The DNA-B component of begomoviruses encodes two viral proteins with essential functions in intra- and inter-cellular efficient movement  and can contribute to symptom production . Although the origin of the DNA-B remains unclear , this component must provide selective advantages with enhanced viral fitness . A contrario to TYLCV, the capacity of bipartite begomoviruses to escape from the phloem cells and infect the surrounding tissues could be a key element in this difference of the viral accumulation observed [7, 37]. This wider tissue tropism gives the opportunity to infect more plant cells and may be the major determinant in our observed difference in viral accumulation. Our data revealed strong differences in the viral load between the bipartite and the monopartite begomoviruses at the leaf-level but the question of viral accumulation in a single infected cell remains open.
In the case of PYMV, interestingly, no difference was observed between the two genomic components accumulation at 10, 20 and 30 dpi with the higher viral loads reached at 10 dpi for both components. DNA-B of bipartite begomoviruses is necessary for viral infection, and so the two components must be co-transmitted to spread and induce systemic symptomatic infections  (for exception see ). Our data provide new insights into the replication of bipartite begomoviruses and suggest that both PYMV molecules accumulate at the same level from the early to the late phase of the infection, ensuring a further efficient transmission although we cannot exclude differential time to reach this peak viral load during the first ten days post-infection.
We also revealed differences in the patterns of viral accumulation between the two strains of TYLCV (Mld and IL strains). Experimental work using TYLCV, the monopartite ToLCV form Australia and TYLCCNV showed that the ORF C4 is implicated in viral movement , symptom development  and bypass defence mechanisms of the host [41, 42]. Considering the recombinant nature of TYLCV-IL, which shares a common origin for a portion of its genome comprising the C4 ORF with ToLCV-Asian-like ancestors , we can hypothesize that the C4 protein of TYLCV-IL is in part responsible for the higher fitness observed compared to the Mld strain.
Finally, we revealed strong differences between the two strains of TYLCV and the ToLCKMV. The accumulation kinetics of these two species differed both in the quantity of viral DNA produced, and in the time to reach this peak viral load. Such differences could have strong epidemiological consequences increasing the probability for an insect vector to acquire a virus from a plant with higher viral load during a longer timeframe and thus contributing to their preferential dispersions. In this study, we compared two species of monopartite begomoviruses with very different impacts and areas of distribution. While the TYLCV is considered as one of the most emergent plant viruses and has succeeded in spreading worldwide , the ToLCKMV-like viruses are for now confined to the Comoros archipelago with a minor impact on local production . Those two contrasted epidemiological profiles coincide with the strong differences in biological properties revealed by our study, such as the higher fitness. Our study thus suggests possible reasons for the successful spread of the emergent TYLCV, as compared to the indigenous and area-restricted ToLCKMV-like viruses.