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Importance of associated weeds and cropping systems in shaping the viromes of horticultural crops. Ayoub Maachi PhD Thesis

Metagenomics based on high throughput sequencing (HTS) has opened a new era of unbiased discovery and genomic characterization of viruses. As for other viruses from other kingdoms, metagenomic studies indicate that the diversity of plant viruses was until recently far underestimated. As potentially important components of unmanaged and cultivated ecosystems, there is a need to explore the diversity of the viruses associated with plant populations and to understand the drivers shaping their diversity in space and time.

At the same time, the development of such studies is still challenged by methodological questions concerning, for example, the choice of target nucleic acid populations, or the reproducibility of the analyses. HTS approaches coupled with more traditional methods can be used to study interactions up to the ecosystem scale. These tools allow to analyze viromes in different ecosystems, uncovering plant virus diversity and helping us to better understand plant virus epidemiology, ecology, and evolution. Thus, the overall objective of this thesis was to use HTS to study the importance of associated weeds and cropping systems in shaping the viromes of horticultural crops. a first stage, as described in the first chapter of this thesis, we used two RNA input methods and developed a bioinformatics pipeline for the analysis of HTS data. Then, we applied them for the detection of viruses in a collection of tomato samples showing virus-like symptoms. The results of the virus identification were further confirmed by conventional methods and the pipeline was the basis for the further studies of the thesis. We found that the dsRNA input provided better coverage in comparison to the total RNA input for most viruses. However, the dsRNA method generated many troubles during the sequencing process, failing often to meet the requirements for library preparation calling for the need of a pre-amplification step. In addition, it was time consuming, making it unsuitable for handling large number of samples. Using our pipeline, we successfully reported three viruses previously undescribed infecting tomato crops in Spain: tomato fruit blotch virus, lettuce ring necrosis virus and olive latent virus. In a second stage, as described in the second chapter of this thesis, we analyzed and compared the viromes of melon and three common weeds found in the edges of the crops. We determined the incidence of the viruses infecting melon, and we uncovered the role(s) that these weeds may play in virus ecology and evolution by conducting population genetics, phylogenetic and migration studies on the viruses overlapping between the melon and the weeds. We found that cucurbit aphid borne yellows virus (CABYV) and tomato leaf curl New Delhi virus (ToLCNDV) were infecting both melon and Ecballium elaterium. The CABYV population was diverse and structured by the host. The ToLCNDV population was much less diverse. The analysis of potential virus migration fluxes between the two hosts showed no evidence of migration between CABYV populations of the two hosts. For ToLCNDV, evidence of migration from melon to E. elaterium but not the other way around was found. In a third stage, described in chapter three of this thesis, we characterized and described in detail the genomes of two novel viruses which may constitute new putative species, found in the surrounding weeds of melon crops. The incidence of these two viruses in weeds was also determined. Lastly, as described in chapter four, we studied the influence of different agro-ecosystems on viromes’ structure and diversity by analyzing the viromes in lettuce farms conducted under ecological or conventional practices. The viromes of weeds associated with this crop were also characterized in both systems. We found a limited overlap of the lettuce viromes between both cropping systems, indicating the importance of the cropping system in determining the viruses affecting the crop, and no virus overlap was found between l

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