Impact assessment of different agricultural practices in grapevine soil and must microbiota

Viticulture, an agricultural activity of great economic, social and cultural relevance, currently faces challenges that compromise its viability, such as climate change, soil degradation, biodiversity loss and the environmental impact of conventional practices. In this context, the microbiota within the viticultural ecosystem plays a critical role in crop health and wine quality. Thanks to next-generation sequencing (NGS) technologies, it is now possible to characterize the composition and functionality of these microbial communities. However, most studies on emerging practices in viticulture have not incorporated a microbiological perspective, limiting our understanding of their real impact on the agroecosystem. In this thesis, we hypothesized that agricultural practices significantly influence the structure and functionality of vineyard microbial communities. The main objective of the thesis was to assess the impact of emerging vineyard management strategies on microbial ecosystems. Specifically, the study aimed to: i) evaluate the effect of organic mulches on the fungal and bacterial communities of soil and must, ii) characterize the impact of five rootstocks under irrigation and drought conditions on the rhizosphere bacterial community and iii) determine how irrigation with ozonated water influences soil bacterial and fungal populations. Three field studies were conducted between 2019 and 2024 in vineyards within the Rioja Qualified Designation of Origin (DOCa) in three representative locations — Logroño, Aldeanueva de Ebro, and Arenzana de Abajo — selected for their edaphoclimatic conditions and vineyard management practices. The three management practices included: i) application of organic mulches (straw, grapevine pruning debris and spent mushroom post), compared with conventional treatments (herbicide and tillage); ii) use of five rootstocks under irrigation and water stress conditions; and iii) irrigation with ozonized water. A total of 246 samples from soil, rhizosphere and must were collected. Total DNA was extracted and analysed using NGS technologies: metabarcoding (16S rRNA and ITS2) for taxonomic profiling, and shotgun metagenomics for functional analysis. The results showed that vintage and location were the primary factors influencing both bacterial and fungal microbiota in soil and must. The evaluated agricultural practices had a smaller, though detectable, impact —primarily in soil and rhizosphere, rather than in must— and their effects varied depending on the treatment type and environmental context. In the organic mulch experiment, significant changes in bacterial beta diversity and fungal alpha diversity were observed in the third year, indicating delayed but notable long-term effects of these practices. However, no relevant changes in the abundance of pathogenic or beneficial fungal taxa were detected. Additionally, there was limited overlap between soil and must microbiota, except for some fermentative fungal taxa, which challenge the hypothesis of microbial migration from soil to must. In the rootstock trial, irrigation emerged as the main modulating factor on rhizosphere bacterial communities. However, under drought conditions, genotype-specific differences emerged, highlighting the role of rootstock in stress scenarios. Key microbial taxa and functions associated with drought were also identified, which may be of great interest in future research. Finally, irrigation with ozonated water did not affect soil bacterial and fungal microbiota, including beneficial bacterial and fungal groups (PGPB) and pathogens (GTD), which raises questions about the potential of ozone as a sustainable irrigation strategy. In conclusion, the impact of agricultural practices on vineyard microbiota is variable and context-dependent, often requiring multiple years to become evident. The effects observed also differ between microbial groups, with bacteria and fungi responding differently. This thesis underscores the importance of evaluating emerging practices over multiple years and under specific conditions to better understand and optimize viticultural management through a microbiological lens.