Background During the first stages of development human infants are either fed human milk or human milk substitutes (infant formulas). The composition of infant formulas and human milk differ drastically, including a difference in protein constituents and bacterial load. Due to the high global frequency of infant formula use, the humanization of infant formulas to better reflect the complex nature of human milk is warranted. To better understand the role of human milk components, the fate and function of a key bacterial sensor in human milk, soluble CD14, was determined. Additionally, the microbiome of human milk was analyzed from a metagenomic standpoint in an attempt to determine which types of bacteria are present in human milk and what their potential biological function might be. Results In rodent models, ingested sCD14 persisted in the gastrointestinal tract and was transferred intact into the blood stream. Once transferred to the blood, ingested sCD14 retained its ability to recognize lipopolysaccharide and initiate an immune response in pups. This transfer of sCD14 across the epithelial barrier was also observed in human cells in vitro, where it appears to be dependent on Toll-like receptor 4. Using Illumina sequencing and the MG-RAST pipeline, the human milk metagenome of ten mothers was sequenced. DNA from human milk aligned to over 360 prokaryotic genera, and contained 30,128 open reading frames assigned to various functional categories. The DNA from human milk was also found to harbor immune-modulatory DNA motifs that may play a significant role in immune development of the infant. Conclusions Given the complex nature of human milk in comparison to its bovine or plant based substitutes, the results presented in this thesis warrant future modification of infant formulas to include non-nutritive bioactive components. Current human milk components not yet present in infant formulas include the diverse microbiome of human milk, the immune-modulatory DNAs which those microbes harbor, and bioactive human proteins such as sCD14.

Orientadores: Valéria Maia Merzel, Suzan Pantaroto de Vasconcellos / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-26T20:16:20Z (GMT). No. of bitstreams: 1 Dellagnezze_BrunaMartins_D.pdf: 5318956 bytes, checksum: ea46ea243dab1c96e35480b724029b55 (MD5) Previous issue date: 2015 / Resumo: A poluição é um problema mundial amplamente discutido, incluindo os derramamentos de petróleo ocorridos através de acidentes ou por atividades humana, os quais acarretam grande impacto ambiental e econômico. O processo de biorremediação utiliza micro-organismos, associados ou não a outros compostos como biossurfactantes e até mesmo enzimas, com o objetivo de transformar compostos orgânicos em inorgânicos, levando à formação de compostos inertes ou não tóxicos. Deste modo, a biorremediação representa um modo efetivo e sustentável para se tratar áreas contaminadas. Neste trabalho foi possível avaliar o potencial de clones metagenômicos obtidos a partir da construção de uma biblioteca fosmidial e de linhagens de bactérias, todos provenientes de amostras de petróleo de reservatórios brasileiros em escala de microcosmos e mesocosmos, visando futura aplicação em processos de biorremediação. Em um primeiro ensaio os micro-organismos foram avaliados na forma livre, em 50 mL de água do mar artificial e petróleo bruto como única fonte de carbono, a cada sete dias durante 21 dias. Posteriormente, os micro-organismos com melhor potencial de biodegradação foram selecionados e aprisionados em esferas de quitosana e testados novamente em microcosmos, em diferentes escalas, durante 21 e 30 dias. Com base nos resultados observados nos ensaios de degradação em microcosmos, um último ensaio foi realizado empregando-se um consórcio contendo quatro clones metagenômicos e uma linhagem de Bacillus subtilis, o qual foi avaliado em ensaio de mesocosmos em 3000 litros de água do mar não-estéril. Nesta etapa, parâmetros como a contagem total dos micro-organismos (DAPI) e a demanda biológica de oxigênio (DBO) foram avaliados, e a cromatografia gasosa (CG) foi empregada para avaliar a degradação de hidrocarbonetos do petróleo. Os resultados demonstraram a capacidade desses micro-organismos em degradar compostos do petróleo bruto, tanto hidrocarbonetos alifáticos como aromáticos. Em microcosmos, na forma livre, as linhagens de Dietzia maris e Micrococcus sp. apresentaram o melhor desempenho, alcançando ao final de 21 dias 99% de degradação de hidrocarbonetos alifáticos e de 63-99% de degradação de aromáticos (fenantreno e metilfenantreno). Dentre os clones, o clone 2B apresentou o melhor desempenho para degradar tanto hidrocarbonetos alifáticos (47%) como aromáticos (94%). Na forma aprisionada, os micro-organismos também apresentaram capacidade para degradar petróleo bruto em mesocosmos, exibindo valores de degradação de 90 a 100 % para hidrocarbonetos saturados e 70 a 100% para aromáticos, ao final de 30 dias de avaliação. Os resultados indicam um resultado promissor e inédito, onde um consórcio combinado contendo clones metagenômicos e Bacillus subtillis pode ser futuramente utilizado em estratégias de bioaumento, em sistemas de contenção, como ferramenta para biorremediação de ambientes contaminados com hidrocarbonetos / Abstract: Pollution is a global environmental problem widely discussed, including oil spills that occur accidentally or due to human activities, which cause huge environmental and economic impacts. Bioremediation process uses biological agents, associated or not to other compounds like biosurfactants or even their enzymes, to mineralize or complex organic and inorganic pollutant compounds, transforming them into inert or non-toxic compounds. Thus, bioremediation represents an ecofriendly and effective way to treat impacted areas. In this work, the biodegradation potential of clones obtained from metagenomic libraries and bacterial isolates, all originated from Brazilian petroleum reservoirs, was evaluated in microcosm and mesocosm scale aiming at a future application in bioremediation process. In the first assay, microorganisms were evaluated as free cells, in 50 mL-volume of artificial seawater and using crude oil as sole carbon source. The experiment was monitored each seven days during 21 days. Further, the best performing microorganisms were selected, immobilized in chitosan beads and evaluated in microcosm assays, at different scales, during 21 and 30 days. Finally, in the last experiment, one consortium containing four metagenomic clones and a Bacillus subtilis strain was evaluated in mesocosmos assay in 3000 L-volume of non-sterile seawater. Parameters such as total counting of microorganisms by DAPI and biological oxygen demand (BOD) were evaluated, and petroleum degradation was monitored by chromatographic analysis. Results demonstrated the ability of the microorganisms to degrade aliphatic and aromatic hydrocarbons. In microcosms, using free cells, the strains of Dietzia maris and Micrococcus sp. showed the best performance, reaching 99% of aliphatic hydrocarbon degradation and 63-99% of aromatic compound degradation in 21 days. Among metagenomic clones, clone 2B presented the best performance to degrade aliphatic (47%) and aromatic hydrocarbons (94%). In chitosan beads, the microorganisms were also able to degrade crude petroleum, showing percentages between 90 and 100% for aliphatic hydrocarbons and 70 and 100% to aromatic. The results gathered in this work demonstrate that a microbial consortium containing metagenomic clones and one bacterial strain is able to achieve high extents of hydrocarbon degradation, offering a promising tool to be further used in bioaugmentation approaches for treating contaminated environments / Doutorado / Genetica de Microorganismos / Doutora em Genética e Biologia Molecular

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This diploma thesis deals with taxonomy independent methods for classification of metagenomic signals, aquired by a MinION sequencer. It describes the formation and character of metagenomic data and already existing methods of metagenomic data classification and their development. This thesis also evaluates an impact of the third generation sequencing techniques in the world of metagenomics and further specialises on the function of the Oxford Nanopore MinION sequencing device. Lastly, a custom method for metagenomic data classification, based on data obtained from a MinION sequencer, is proposed and compared with an already existing method of classification.

Ces travaux de thèse reposent sur le double objectif de proposer des approches innovantes pour l’étude des relations entre un hôte et son microbiote, et de les appliquer à la description fine de l’holobionte du puceron du pois par des données métagénomiques. Les relations symbiotiques façonnent le fonctionnement et l’évolution de tous les organismes, mais restent décrites de manière imparfaite, notamment à cause de la difficulté à caractériser la diversité génomique des partenaires microbiens constitutifs des holobiontes. L’essor des technologies de séquençage métagénomique révolutionne l’étude de ces systèmes, mais pose également des problèmes méthodologiques pour analyser les jeux de données métagénomiques. La métagénomique est ici appliquée au puceron du pois, un modèle d’étude des relations symbiotiques qui abrite une communauté bactérienne d’une complexité modérée, idéale pour développer de nouvelles approches de caractérisation de la diversité microbienne. Cette thèse vise à mieux décrire la communauté de symbiotes qu’abrite cet holobionte, notamment en distinguant les différents niveaux de variabilité génomique en son sein. Nous présentons une démarche pour l’analyse métagénomique d’holobiontes, qui repose d’abord sur l’assignation taxonomiques des lectures par alignement à des génomes de référence ou préalablement assemblés, puis sur la recherche de variants génomiques. L’étude de génotypes complets de symbiotes permet de retracer l’histoire évolutive des relations hôte-microbiote avec une résolution élevée. Chez le puceron du pois, nous mettons en évidence des niveaux et structures de diversité génomique différents selon les symbiotes, que nous proposons d’expliquer par les modalités de transmission ou l’histoire évolutive propre à chacun des partenaires microbiens. Cette approche repose sur la disponibilité d’un génome de référence adapté, qui est souvent difficile à obtenir en métagénomique. Dans un second temps, nous présentons donc une méthode d’assemblage guidé par référence en contexte métagénomique. Cette méthode se déroule en deux temps : le recrutement et l’assemblage de lectures par alignement sur un génome de référence distant, puis l’assemblage de novo ciblé des régions manquantes, permis par des développements complémentaires apportés au logiciel MindTheGap. Comparativement à un assembleur métagénomique, cette méthode permet l’assemblage d’un seul génome en un temps réduit, et permet de détecter d’éventuels variants structuraux sur le génome ciblé. Appliqué au puceron du pois, MindTheGap a réalisé l’assemblage du symbiote obligatoire Buchnera en un seul contig, et a permis d’identifier différents variants structuraux du bactériophage APSE. Ces travaux ouvrent la voie à la fois à une caractérisation plus précise des relations hôte-microbiote chez le puceron du pois par des approches fonctionnelles et métaboliques, ainsi qu’à l’application des outils présentés à des systèmes plus complexes. / The aim of this PhD thesis is to develop innovative approaches to characterize host-microbiota relationships, and to apply them to finely explore the pea aphid microbiota using metagenomic data. Symbiotic relationships play a major role in the life and evolution of all organisms, but are imperfectly described, essentially because of the difficult characterization of the genomic diversity of the microbial partners. The rise of high throughput metagenomic sequencing is a game changer for the study of those systems, but also raises methodological issues to analyze large metagenomic datasets. Metagenomic is here applied to the pea aphid holobiont, a model system for the study of symbiotic relationships, sheltering a moderately complex microbial community. This level of complexity seems to be ideal to develop new approaches for the strain-resolved characterization of host-microbiota relationships. This thesis aims at a better description of this symbiotic community by distinguishing several scales of metagenomic diversity. In a first part, we present a framework for the metagenomic analysis of holobionts, relying first on the taxonomic assignation of reads by alignment to reference or newly assembled genomes, and then on the detection of genomic variants. Whole genome variant profiles make possible to track the evolutionary history of host-microbiota associations with a high resolution. In the case of the pea aphid, we highlight different scales and structures for the metagenomic diversity of the different symbionts, accounting for different transmission modes or evolutionary histories specific to each microbial partner. This framework is based on the availability of a suitable reference genome, that may be hard to obtain in a metagenomic context. In a second part, we therefore present a novel method for reference guided genome assembly from metagenomic data. This method is based on two steps. First, the recruitment and assembly of reads by mapping metagenomic reads on a distant reference genome, and second, the de novo assembly of the missing regions, allowed by the development of an improved version of the software MindTheGap. Compared to a standard metagenomic assembler, this methods makes possible to assemble a single genome in a reasonable time, and allows to detect eventual structural variations within the targeted genome. When applied to the pea aphid holobiont, MindTheGap yielded single contig assemblie of the obligatory symbiont Buchnera aphidicola, and helped to identify different structural variants of the bacteriophage APSE. This works paves the way to a finer characterization of host-microbiota interactions, and to the application of the presented approaches to more complex systems.

Cyanobateria are promising cell factories due to their minimal nutrient requirements and utilization of asmospheric carbon di-oxide as its sole carbon source. In particular, polyhydroxybutyrate (PHB) is an industrially useful bio plastic that is produced naturally by some cyanobacteria. Furthermore, PHB biosynthetic pathway is a starting point for production of the biofuel, 1-butanol. There has been much genetic engineering effort toward increasing the production of PHB from cyanobacteria. These have been focused on increasing the pool of acetyl-CoA precursor, or increasing the amount of the reductant NADPH. The upstream process for increasing these reactants is complex and involves many genes. In this contect, cyanobacteria libraries will contribute to reveal genes or gene fragments that are responsible for production of PHB, alkanes and other high value compounds. In pursuit of finding these novel genes or genefragments, a transcription factor library is created in this study with 50 transcription factors. Furthermore, the process is optimized towards the creation of genomic fragment library and metagenomic fragment library with 26 diverse strains. Membersof the transcription factor library are over-expressed by a PHB - producing host Synechocystis PCC 6803 and the process towards creation of genomic and metagenomic libraries is optimized. The members of the metagenomic library can be screened for increased PHB, alkanes, lactate and other high value products and the potential members can be isolated and characterized.

Few genomic and metagenomic studies have focused on freshwater systems in the last years. Most of the studies carried out on these particular environments so far rely on microscopy, physiology, phenotypic observations, individual genes and 16S rRNA sequencing. Here, we shed light on microbial communities from oligotrophic and mesotrophic freshwater systems using high-throughput deep sequencing metagenomics and genome-resolved metagenomics. We have focused on the study of ubiquitous and cosmopolitan microbial groups from two temperate Spanish reservoirs (Tous, Amadorio). Among these, we studied freshwater picocyanobacteria from Synechococcus and Cyanobium genera, which so far have not been well characterized at the genomic level, compared to the marine representatives. In particular, we were able to isolate two of the most abundant picocyanobacteria from Tous reservoir, which were previously studied via metagenomics. These picocyanobacteria are not only abundant in this reservoir but are widely distributed in different freshwater and brackish systems. In this work we also shed light on some of the first freshwater representatives of the phylum Verrucomicrobia, that are ecologically uncharacterized in freshwater systems about which relatively little is known. We discovered a wide range of metabolisms in these microbes, ranging from nitrogen fixation and photoheterotrophy via rhodopsin pumps to important contributions in the degradation of recalcitrant matter and polysaccharides. We also include the first metagenomic study of the microbial communities under the ice waters of the largest (by volume) ultraoligotrophic lake in the world, Lake Baikal. This study has provided a first glimpse and a particular microbial composition on the sub-ice, having found an unusual fraction of Verrucomicrobia and new microbial lineages from many typical freshwater phyla, including the first freshwater representative of the groups I/II of SAR11 lineage and novel genomes of Proteobacteria, Thaumarchaeaota, Gemmatimonadetes, Cyanobacteria, Planctomycetes, Bacteroidetes, Acidobacteria, Nitrospirae, Verrucomicrobia or Actinobacteria.

Tetrodotoxin (TTX) is a lethal neurotoxin isolated mainly from the organs of wild puffer fishes. Although the neurotoxicity mechanisms of TTX are well known, the TTX origin and the biosynthetic mechanisms inside its hosts remain unresolved. In recent decades, the numerous reports of TTX-producing bacteria strongly suggested its bacterial origin. However, this origin is currently being challenged by the low and inconsistent TTX productions in vitro by the previously reported TTX-producing bacteria. Culturable TTX-producing bacteria were frequently isolated and reported from the guts of TTX-bearing animals including puffer fishes, however, these bacteria were estimated to account for 0.1% of the total gut bacteria. Moreover, the identification and functions of the non-culturable gut bacteria participating in TTX biosynthesis have never been reported. I hypothesize that the puffer fish gut bacteria and the entire gut environment serve as a functional integrality responsible for TTX biosynthesis. In this study, 16S rRNA amplicon metagenomics pipeline was established to profile the entire gut bacterial structures of both toxic and non-toxic puffer fishes respectively. UniFrac based principal coordinate analysis showed that bacterial diversities were significantly different (P-value < 0.001) between the gut environments of toxic puffer fishes and the non-toxics. Vibrio and Cyanobacteria were identified as centralities of gut bacteria co-occurrence network in toxic puffer fishes, implying their key roles in TTX biosynthesis. The results of metagenome prediction and gene set enrichment indicated that arginine biosynthesis was significant enriched (P-value < 0.05) in the toxic group. To further investigate the roles of key bacteria and arginine biosynthesis in producing TTX, metabolomics pipeline was established along with 16S rRNA amplicon metagenomics to monitor the dynamics of metabolites and bacterial compositions in guts of toxic puffer fishes during their detoxification process. The average TTX concentrations in the liver after a 60-day culture (6.41 ± 3.00 µg/g) was found significantly lower (P-value < 0.01) than that of the same species from the wild (31.86 ± 22.20 µg/g). The relative abundance of Vibrio was found positively correlated with the liver TTX concentrations. With the increase of culture periods, the relative abundance of Vibrio and Cyanobacteria decreased. In addition, both the metabolites and functional genes in arginine biosynthesis metabolic pathway were found significantly down-regulated (P-value < 0.05). These results indicated that both Vibrio and Cyanobacteria bacterial symbionts participated in TTX biosynthesis using arginine as a potential precursor in the gut environment of toxic puffer fishes.

Tetrodotoxin (TTX) is a lethal neurotoxin isolated mainly from the organs of wild puffer fishes. Although the neurotoxicity mechanisms of TTX are well known, the TTX origin and the biosynthetic mechanisms inside its hosts remain unresolved. In recent decades, the numerous reports of TTX-producing bacteria strongly suggested its bacterial origin. However, this origin is currently being challenged by the low and inconsistent TTX productions in vitro by the previously reported TTX-producing bacteria. Culturable TTX-producing bacteria were frequently isolated and reported from the guts of TTX-bearing animals including puffer fishes, however, these bacteria were estimated to account for 0.1% of the total gut bacteria. Moreover, the identification and functions of the non-culturable gut bacteria participating in TTX biosynthesis have never been reported. I hypothesize that the puffer fish gut bacteria and the entire gut environment serve as a functional integrality responsible for TTX biosynthesis. In this study, 16S rRNA amplicon metagenomics pipeline was established to profile the entire gut bacterial structures of both toxic and non-toxic puffer fishes respectively. UniFrac based principal coordinate analysis showed that bacterial diversities were significantly different (P-value < 0.001) between the gut environments of toxic puffer fishes and the non-toxics. Vibrio and Cyanobacteria were identified as centralities of gut bacteria co-occurrence network in toxic puffer fishes, implying their key roles in TTX biosynthesis. The results of metagenome prediction and gene set enrichment indicated that arginine biosynthesis was significant enriched (P-value < 0.05) in the toxic group. To further investigate the roles of key bacteria and arginine biosynthesis in producing TTX, metabolomics pipeline was established along with 16S rRNA amplicon metagenomics to monitor the dynamics of metabolites and bacterial compositions in guts of toxic puffer fishes during their detoxification process. The average TTX concentrations in the liver after a 60-day culture (6.41 ± 3.00 µg/g) was found significantly lower (P-value < 0.01) than that of the same species from the wild (31.86 ± 22.20 µg/g). The relative abundance of Vibrio was found positively correlated with the liver TTX concentrations. With the increase of culture periods, the relative abundance of Vibrio and Cyanobacteria decreased. In addition, both the metabolites and functional genes in arginine biosynthesis metabolic pathway were found significantly down-regulated (P-value < 0.05). These results indicated that both Vibrio and Cyanobacteria bacterial symbionts participated in TTX biosynthesis using arginine as a potential precursor in the gut environment of toxic puffer fishes.

Les approches de métagénomique basées sur l’utilisation des techniques de séquençage haut débit ont ouvert une nouvelle ère pour la découverte non biaisée et la caractérisation génomique des virus. Comme pour les autres virus, de telles études montrent que la diversité des virus phytopathogènes a jusqu’à tout récemment été fortement sous-estimée. Ces virus constituant une composante potentiellement importante des écosystèmes naturels ou des agrosystèmes anthropisés, il est important d’explorer la diversité des virus associés aux populations végétales et de comprendre les forces structurant cette diversité dans le temps et dans l’espace. Dans le même temps, le développement de telles études reste confronté à des questions d’ordre méthodologique concernant, par exemple, le choix des populations d’acides nucléiques à séquencer, la reproductibilité des analyses ou la disponibilité d’une stratégie permettant de comparer de façon fiable la richesse virale dans différents environnements. Dans le présent travail, le virome associé à des populations végétales échantillonnées dans différents écosystèmes, avec un focus sur les adventices et les plantes sauvages, a été caractérisé par des approches de métagénomique par séquençage haut débit. Dans ces travaux, l’analyse bioinformatique de la richesse du virome a été conduite par deux approches, l’une classique basée sur l’annotation Blast pour l’identification des familles virales présentes dans un échantillon, et l’autre, décrite et validée ici, qui permet de classifier les séquences virales métagénomiques en unités taxonomiques opérationnelles (operational taxonomic units, OTUs) utilisées comme proxy des espèces virales. Toujours dans une perspective méthodologique, les résultats obtenus avec des pools complexes de plantes représentatifs de la diversité végétale au site d’échantillonnage (approche « tondeuse à gazon ») ont permis de comparer les performances des deux techniques actuellement utilisées pour enrichir les séquences virales, la purification d’ARN bicaténaires (double-stranded RNA, dsRNA) ou d’acides nucléiques associés aux virions (virion-associated nucleic acids, VANA). Les résultats obtenus par les deux approches ont mis en évidence des viromes riches mais montrent que l’approche dsRNA devrait être préférée pour l’analyse de tels pools complexes car elle permet de façon reproductible une description plus complète du phytovirome, à l’exception des virus ADN. Les viromes caractérisés montrent, pour les populations végétales de milieux cultivés ou non gérés tempérés échantillonnées, une forte incidence virale (jusqu’à 86.5% dans 126 pools monospécifiques collectés sur une période de deux ans) et confirment la prédominance des virus dsRNA qui représentent plus de 70% des OTU identifiés. Alors qu’une proportion significative des virus ssRNA détectés sont déjà connus, plus de 90% des virus dsRNA détectés sont nouveaux et n’avaient pas été caractérisés auparavant. Un effort important en culturomique visant à comparer le phytovirome avec le mycovirome de cultures fongiques obtenues à partir des mêmes échantillons végétaux a révélé un nombre remarquablement faible d’OTUs partagés, renforçant le questionnement sur la nature, phytovirus ou mycovirus, des virus dsRNA identifiés dans les viromes des plantes. La composition en OTU des viromes analysés s’est révélée variable entre sites d’échantillonnage mais aussi très dynamique dans le temps, avec seulement une très faible fraction des OTUs ré-échantillonnés de façon stable dans la même population végétale sur une période de deux ans. Pris dans leur ensemble, ces travaux exploratoires permettent de mieux raisonner les choix méthodologiques pour l’étude des viromes associés aux plantes et étendent notre connaissance de la diversité des phytovirus, en particulier dans des espèces végétales sauvages largement négligées, apportant des points de référence importants pour de nouveaux travaux en écologie et en évolution virale. / Metagenomics based on high throughput sequencing (HTS) has opened a new era of unbiased discovery and genomic characterization of viruses. As for other viruses, such 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 faced by methodological questions concerning, for example, the choice of target nucleic acids populations, the reproducibility of the analyses or the implementation of a strategy to accurately compare virus richness in different environments. In the present thesis the phytovirome associated with plant populations sampled in various ecosystems, with an emphasis on wild plant or weed species was characterized using HTS-based metagenomics. In these experiments, the bioinformatic analysis of the virome complexity was performed using two strategies, a classical one based on Blast-based contigs annotation for the identification of the viral families present in a sample and a novel one, described and validated here, and which allows to classify the metagenomic viral sequences into operational taxonomic units (OTUs) as a proxy to viral species. Also from the methodological perspective, the results obtained using complex plant pools such as those used in the “lawn-mower” sampling strategy allowed to compare the performance of the two currently used viral enrichment methods, double-stranded RNA (dsRNA) or Virion-associated nucleic acids (VANA) purification. The results indicate both of approaches uncovered rich viromes and suggest that the dsRNA approach should be preferred when analyzing complex plant pools since it consistently provided a more comprehensive description of the analysed phytoviromes, with the exception of the DNA viruses. The virome characterization results obtained showed, for the temperate plant populations from unmanaged and cultivated sampling sites, a high virus incidence (up to 86.5% in 126 single species pools collected over a two-year period) and confirmed the predominance of dsRNA viruses with greater than 70% of the phytovirome OTUs. While a significant proportion of detected single-stranded RNA (ssRNA) viruses are already known agents, more than 90% of the dsRNA viruses are novel and had not previously been characterized. A large scale culturomics effort to contrast the phytovirome with the mycovirome of fungal cultures obtained from the same plant samples revealed an extremely low number of shared OTUs, further deepening the debate about the phytovirus or mycovirus nature of the dsRNA viruses identified in plant viromes. The OTU composition of the analyzed phytoviromes varied significantly between sampling sites but was also shown to be highly dynamic over time, with a very low proportion of OTUs consistently re-sampled in the same plant population over a 2 years period. Taken together, these exploratory studies allow a more reasoned choice of methodology for the study of plant-associated viromes and expand our knowledge of plant virus diversity especially in neglected wild plant populations, providing important references for the further viral ecology and evolution studies.