Exploring virus ecology and diversity in cultivated and wild Poaceae communities in Belgium

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