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Insecticides --- formulations --- Surfactants --- Pesticide persistence --- residues --- toxicity --- Biotechnology

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Ecology of plant viruses examines complex interactions among plant-associated viruses, their hosts and their vectors, and the environment. Starting at the end of the 19th century, virus ecology followed the development of new technologies for virus detection and characterization, from host symptomatology to biochemistry, microscopy, serological and molecular techniques. Most recently, high throughput sequencing (HTS) technologies allowed for the first time, to characterize all or nearly all viruses in a sample without a priori information about which viruses might be present, that is the study of the viral metagenome or virome. This thesis reviewed the major advances in virus ecology, in relation to the development of virus detection technologies. It focused on the viral metagenomics and detailed the opportunities and challenges associated to each step of a virome-based study using HTS technologies (field sampling, laboratory work and bioinformatics analyses).The research conducted in the framework of this thesis explored the diversity and ecology of viruses infecting a plant family of economical and ecological importance: the Poaceae plants or grasses. The study was performed in the region of the Belgian National Park "Burdinale-Mehaigne". Combining untargeted HTS-based analysis and targeted virus detection by RT-PCR in thousands of grass samples, virome characterization was unrolled down to the species taxonomical rank to support an analysis at three different levels: in global plant communities, in plant populations from same species, and in individual plants. A global view of the Poaceae virome and its ecology could be obtained through various ecological and epidemiological analyses: examination of virus richness, prevalence and co-infection, network and clustering analyses, Akaike information corrected criterion (AICc) calculation, or Spatial Analysis by Distance IndicEs (SADIE).First, we investigated the virome in three different Poaceae communities presenting a contrasted biodiversity (in terms of grass species richness) and a gradient of human management (i.e. cereal crops, grazed pastures and mowed grasslands). A diversified and largely unknown virome was identified in cultivated and non-cultivated Poaceae, with at least seventy virus species, among which fifty previously unknown, from to eighteen virus families and twenty-nine genera. Viruses with persistent lifestyles belonging to Alphachrysovirus, Partitivirus and Totivirus genera represented a large part of this virome, reaching 60% of the viruses detected and mostly novel virus species. A positive correlation was found between virus species and grass species richness in the plant communities, with very few or no virus species detected in cereal crops while a diversified virome was observed in wilder communities with up 26 virus species in grasslands. It illustrated the influence of plant diversity and land use on the distribution of viral communities. In addition, virome comparison over years revealed complex virus-plant relationships within the Poaceae community, separating plant virus and mycovirus models.Second, virome was characterized and compared among Poaceae species, investigating the influence of plant traits (i.e. lifespan, height, occurrence) on virus richness observed. Significant higher virus richness was determined in perennial grasses compared to annuals, and low occurrence species presented specific virus species that were not observed in dominant species. Virome network and clustering analysis revealed the presence of both ubiquitous (or generalist) virus species and specialist viruses limited to one or a few host species, with specialist viruses dominating the Poaceae virome. Among all plant species examined, the perennial ryegrass (Lolium perenne) was demonstrated to represent an important virus reservoir and will thus constitute an interesting model for future studies in virus ecology. Analysis in individual plants showed contrasted prevalences, co-infections and spatial distributions among plant communities, plant species and virus species. Interactions between viruses were also explored and revealed positive and negative viral associations depending on the grass species.Third, a more-in-depth analysis was performed for two novel virus species belonging to Secoviridae family that were identified in abundance in Poaceae communities and species: Poaceae Liege nepovirus A (PoLNVA) and Poaceae Liege virus 1 (PoLV1). The analysis focused on rough bluegrass (Poa trivialis L.) for which almost complete virus genomes from both viruses could be obtained. Sequence and phylogenetic analyses placed PoLNVA in the genus Nepovirus, while low levels of amino acid identity for both Pro-Pol and CP regions could define PoLV1 as an unclassified secovirid, between the genera Waikavirus and Sequivirus. PoLNVA and PoLV1 were detected in eleven wild Poaceae species, sometimes with high prevalence (e.g. 86% of L. perenne and 74% of P. trivialis samples infected by PoLNVA), highlighting their significant presence and large host range within Poaceae. Virus transmission was also investigated and PoLNVA was found to be seed-transmitted.In summary, this thesis revealed the complex structure of viral communities in nature (in terms of richness, prevalence, co-infection, host range, spatial distribution and genetic structure) and improved our understanding of virus diversity ecology in agro-ecological landscapes, illustrated here with the Poaceae family. A couple of virus species were further characterized but dozens of other novel virus species remains to be examined and will likely provide new insights about the ecology and phylogeny of Poaceae viruses. Other fields of investigation concern the diversity of virus transmission agents and other plant pathogens (i.e. bacteria, fungi), in order to obtain a holistic view of the Poaceae phytobiome. A particular attention should be paid to fungi due to the abundance of so-called mycoviruses in Poaceae. Much remains to be done to unravel the secrets of the virus ecology in wild and cultivated Poaceae, a domain still in its infancy.

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Japanese persimmon (Diospyros kaki T) crop has increased dramatically during the last decade in Spain, particularly in the region of Valencia. This region (Eastern of Spain) accounts for 90% of the total area of Japanese persimmon cultivated in Spain. Furthermore, 90% of the crop is represented by almost only one cultivar: 'Rojo Brillante'. The Persimmon cryptic virus (PeCV) was discovered for the first time in Italy, in 2014. Afterward, it was detected in spring 2015, in the region of Valencia in some Japanese persimmons showing symptoms of leaf chlorosis. It is the first virus, detected in Spain, infecting Japanese persimmon. It is composed of two double stranded RNA coding a RNA dependent RNA polymerasegene (dsRNA-1; 1,577 bp) and a coat protein gene (dsRNA-2; 1,491 bp) encapsidated separately in isometric particles. This virus belongs to the genus Deltapartitivirus in the family Partitiviridae. Viruses of this family are not transmitted horizontally and symptoms induced on hosts are unclear. Deep-sequencing (Illumina Miseq) targeting small interfering RNA (siRNA) in synergy with conventional cloning followed by Sanger sequencing were carried out to recover the complete genome of PeCV. In addition, a survey and a RT-PCR test were performed for 35 leaf samples in 6 parcels to determine the dispersion of the virus in the Valencia area. Finally, a study of variability was realized on fragments of about 600 bp on both RdRp and CP genes to evaluate the genetic diversity. Illumina deep-sequencing allowed to recover a near complete genome for both segments (dsRNA-1 and dsRNA-2). The cloning into plasmid vector followed by Sanger sequencing permitted to recover the near complete genome of dsRNA-1 segment whereas only 24.5% of genome of dsRNA-2 segment was recovered. This incomplete recovery of the dsRNA-2 segment is due to a lack of specificity of the reverse primer used to amplify this segment. The comparison between Illumina sequence and Sanger sequence revealed very high similitude for both segments (more than 99% of similitude) although some base dissimilarities remain unclear. Besides, sequences recovered by deep-sequencing and Sanger sequencing showed very high similitude with the Italian strain of PeCV (99.8% for dsRNA-1 and 99.7% for dsRNA-2). In addition, analysis by deep-sequencing showed that the siRNA of 21 and 22 nucleotides in length were the most produced during viral infection demonstrating that Dicer types DCL2 and DCL4 are the most prevalent in the antiviral RNA silencing mechanisms against PeCV. Furthermore, deep-sequencing of siRNA appears to be efficient in the diagnostic of viral infection by detecting 2,568 reads (0.037% of total reads) belonging to the PeCV in a sample of 6,774,813 reads ranging from 1 to 50 nucleotides. On the other hand, the survey and the RT-PCR test revealed a high presence of PeCV (31 samples infected among 35 samples collected) in every parcel surveyed. Persimmon cryptic virus was detected in both asymptomatic and symptomatic trees. The study of variability did not show any variability among the samples tested. As Japanese persimmons are clonally propagated by grafting in the region of Valencia, we suggest that the wide presence of PeCV is due to an unique source of some infected parent plants clonally propagated for years and it is strengthened by the use of almost only one cultivar. Furthermore, symptoms induced by PeCV remain unclear but it does not cause serious economic damages on persimmon crops.

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Le Physostegia chlorotic mottle alphanucleorhabdovirus (PhCMoV) a été détecté pour la première fois par séquençage à haut débit en 2014 en Autriche. Les multiples détections du virus à travers l'Europe dans les années qui ont suivies, ainsi que les graves symptômes associés et les pertes de rendement qui en résultent sur les cultures légumières, suggèrent que ce pathogène viral pourrait représenter une menace émergente. En raison de la découverte récente de ce virus, sa biologie est très peu connue. C'est pourquoi cette étude a tenté de mieux caractériser le PhCMoV, dans le but ultime d'évaluer le risque phytosanitaire qu'il pourrait représenter. Pour ce faire, des essais en serre ont été menés afin de définir l'impact du PhCMoV sur divers cultures hôtes de solanacées inoculées mécaniquement. Il a été constaté que le PhCMoV entraînait des pertes de 70% à 93% du rendement commercialisable de tomates inoculées au stade de 3.5 semaines, selon le cultivar testé. Les chutes de productions commercialisables étaient principalement attribuables à un déclassement des tomates, ainsi qu'à une à éventuelle réduction du rendement total. Alors que l'infection par le PhCMoV n'a pas réduit le nombre de fruits par plante, le poids moyen des tomates infectées était inférieur jusqu'à plus de 40% de celui des fruits exempts du virus. Les défauts de qualité des fruits invendables résultaient principalement d'un mûrissement inégal, de marbrures et de divers motifs chlorotiques. De manière globale, l'infection par PhCMoV était également associée à des anomalies de croissance, à un éclaircissement des nervures et à une déformation des feuilles, quelle que soit l'espèce hôte. Néanmoins, les plantes malades pouvaient rester asymptomatiques, parfois jusqu'à la fructification ou le développement de repousses. Par ailleurs, le stade de développement de la plante au moment de l'inoculation s'est révélé exercer une influence considérable sur l'impact du virus. Parallèlement à ces bio-essais, des séquences de plants de tomates provenant de divers sites ont été soumises à des analyses bio-informatiques et le génome entier du PhCMoV, lorsqu'il a été détecté, a été reconstitué. La pureté de l'isolat utilisé comme source d'inoculation a été assurée et des analyses phylogénétiques ont suggéré un faible taux de mutation du virus, comme observé pour les autres virus de ce genre dont le mode de transmission par vecteur est de type persistant multipliant. Bien que ce vecteur soit toujours inconnu, diverses espèces de cicadelles trouvées dans les champs infectés par le PhCMoV ont été identifiées par codage à barres de l'ADN, et certaines d'entre elles ont été soumises à un test de transmission. Les résultats encouragent les études futures à se concentrer sur Anaceratagallia lithuanica et Eupteryx atropunctata en tant que vecteurs potentiels. L'impact considérable de ce virus, démontré au cours de cet essai, souligne le besoin crucial de poursuivre les recherches sur ce virus, afin de pouvoir mettre en œuvre des stratégies de contrôle appropriées en cas d'épidémie.

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Plant virus ecology is mainly studied on cultivated plants, due to the significant impact of viruses on crops yield and production. However, it was demonstrated that viruses are commonly present in wild plants, where they can be detrimental, commensal or even mutualistic. In fact, wild plant populations and viruses have coexisted for a long time, thereby resulting in complex interactions. Moreover, wild plants are often infected by a combination of several viruses, which still increases the complexity of these relations. One aspect of the plant virus ecology is the study of the role of the viruses on the population dynamics of their wild host plants, and the eventual impact of a virus infection on the demography of a wild plant population. This master's thesis examined this aspect by characterizing wild populations of the model plant Arabidopsis thaliana (L.) Heynh. from six locations in Central Spain, presenting low anthropic pressure : Marjaliza, Ciruelos de Coca, Carbonero, Menasalbas, Polánand Las Rozas. Demographic studies were performed in these populations, and combined to the detection of five common viruses infecting Brassicaceae : Cucumber mosaic virus (CMV, including two strains: Fny (subgroup I) and LS (subgroup II) ), Cauliflower mosaic virus (CaMV), Turnip mosaic virus (TuMV), Turnip yellow mosaic virus (TYMV), and Turnip crinkle virus (TCV). Molecular hybridization and quantitative real-time reverse transcription polymerase chain reaction(qRT-PCR) were used to detect them in leaf samples collected in two populations of A. thaliana, everytwo weeks, as well as from transplanted plants from all populations. Statistical analyses (Generalized Linear Model) were performed on the basis of the demographical, virus detection and quantification data. They revealed that virus infection has an effect on plant fitness, by prolonging the survival (i.e.plant lifespan), reducing the progeny production but maintaining the seed weight. In addition, high variation of the virus incidence was found during the sampling campaign for each virus and population, as well as for the total virus incidence. Nevertheless, for all studied locations, two peaks of incidence were observed in mid-March and mid-April, followed by a drastic drop, connected to the plants' mortality at that moment. Thus, the results obtained in this study confirm the role of the viruses on the population dynamics of their wild host plants. However their fine interpretation is complex and has to take into account the fact that, in nature, the virus infection, as well as the host reaction, are modulated by many interacting factors, such as : the environmental conditions impacting the plant development and the transmission of the virus, the virus genotype and the association of infecting viruses, the host genotype, and finally the effect of the environment on the antagonism-mutualism continuum of plant-virus interactions.