Biotechnological strategies for the adaptation of olive trees (Olea europaea L.) to climate change

The focus of this doctoral thesis is on olive trees (Olea europaea L. subsp. europaea), the dominant tree crop over large areas of the Mediterranean Basin. The drought resistance and nutritional efficiency of the olive tree allow it to survive in arid and semi-arid areas where other crops fail (Martins et al., 2024), making it a key species for the economic livelihood of local communities. From an ecological point of view, it provides habitat and food for a variety of species (such as birds, insects and mammals), and its extensive root system stabilises the soil, preventing erosion (Grego, 2022). Like most crops, the olive tree is facing important problems and challenges related to adverse environmental conditions, either biotic or abiotic ones, a situation that can be worsened in a climate change scenario. In this regard, the most threatened olive crops will be those with sensitive cultivars and management methods that do not maximise their resilience. Terrestrial agroecosystems and their management can play a key role in mitigating climate change: maintaining the carbon sink role of ecosystems and productive low-emission agriculture is an ambitious challenge given the continuous increase in resource use, on which a constantly growing population depends. The use of sustainable management systems such as organic fertilisation, pruning residue recycling, livestock integration and controlled deficit irrigation has been encouraged in olive agroecosystems to improve ecosystem services and ensure the multifunctionality of olive groves (Palese et al., 2013). Agronomic management can also shape the microbiome associated with olive trees, which in turn influences the dynamics that enable the plant to cope with environmental stress. The olive tree microbiome, i.e. the heterogeneous population of bacteria, fungi and archaea, that live associated with the host plant, essential for environmental sustainability, is valuable for the growth, health and adaptation of the olive tree to environmental stresses (Avramidou et al., 2024). In fact, some plant growth-promoting microorganisms (PGPMs) can exert direct effects through nitrogen fixation or phosphorus solubilisation or even by modulating hormones. Some microbial groups can act indirectly on the host through mechanisms that lead to induced systemic resistance, osmotic adjustment, siderophore production, antibiotic production, and cell wall-degrading enzymes (Dias et al., 2024). Olive trees have the capacity to undergo “stress imprinting” due to previous exposure to abiotic stress. The responses resulting from this mechanism depend not only on the plant's intrinsic ability to cope with a specific stress, but also on habitat conditions and the availability of symbionts. Specific plant growth-promoting rhizobacteria (PGPR) in the soil can induce a preconditioned state of high alert, enabling rapid and robust defence responses in the event of stress (Singh et al., 2024). The literature shows that beneficial microorganisms significantly affect both soil health and crop productivity, by solving several problems related to soil stress, soil fertility, soil degradation and plant growth. The interactions between genotype and the microbiome have only recently been analyzed, and few studies have focused on the particular priming capacity of the olive holobiont, which has nevertheless proven to be an excellent ally in coping with drought stress. Furthermore, especially in contexts characterised by arid environments, there are few studies that have analysed the contribution of genotype, climatic conditions and management practices at the same time, making it difficult to find a universal model that explains how all these variables influence the composition of the microbiome associated with olive trees. Therefore, to test the hypothesis that sustainable management systems reshape the taxonomic profiles of the olive tree microbiome and that inoculated microbial consortia (PGPR) induce priming for drought tolerance, this thesis has been organised into six chapters in order to clarify the interactions between the olive microbiome, agronomic practices and resilience/priming capacity in response to water stress. The first chapter (INTRODUCTION) provides a general overview of olive groves in the Mediterranean, starting with their botanical characteristics, through their history and their current economic and ecological role. Attention is paid to the problem of drought in agriculture and the concept of holobionts is integrated and priming and stress memory strategies in the plant world are defined. The second chapter (STATE OF THE ART) summarises current knowledge, starting with the definition of bacterial and fungal communities in the above- and below-ground parts of the olive tree, and then illustrates how genotype, soil and environmental pressures guide microbial assembly. It then analyses the main olive grove management techniques and their specific impacts on the microbiome, culminating in an examination of the physiological, biochemical and molecular responses activated by the olive tree under water stress, including priming mechanisms against drought. Follow the third chapter (OBJECTIVES) that defines the hypotheses, general and specific objectives of the thesis. Chapter 4 (RESULTS) contains two research articles. In particular, section 4.1 refers to the paper “Management affects the diversity and functions of root- and leaf-associated microbiomes: implications for olive resilience”, published in the journal Frontiers in Plant Science. This study reports on the differences in composition and function of the microbial communities of the Olea europaea L. subsp. europaea 'Ortice' when subjected to different agronomic management practices. While section 4.2 reports the article “Priming and memory in ancient olive cultivars: Enhancing drought resilience through rhizosphere microbial consortia under repeated stress cycles” published in the journal Environmental and Experimental Botany. This study examined the physiological, biochemical and molecular responses of three olive cultivars (Olea europaea L. subsp. europaea 'Ortice', Olea europaea L. subsp. europaea 'Ortolana', Olea europaea L. subsp. europaea 'Racioppella') when subjected to repeated water deficits. In addition, the resilience effects of a PGPR consortium (Bacillus subtilis, Pseudomonas fluorescens) were evaluated. The fifth chapter (INTEGRATED DISCUSSION), aims to highlight how agricultural management practices directly influence the assembly of olive tree microbial communities, their ability to cope with environmental stresses (in terms of stress memory) and, the limitations of the studies and possible solutions. The sixth final chapter (CONCLUSION) concludes the manuscript by summarising the main results obtained, and finally the implications of a biotechnological solution for sustainable agriculture. In this thesis, we observed how conservation-focused management (organic and traditional) increases the microbiome associated with the rhizosphere (enrichment of fungal biomarkers) and improves its functionality (abundant presence of bacteria of the Actinomycetales order). The different resilience capacities of three olive tree genotypes under drought conditions were explored ('Ortolana' maintained a certain degree of water stability, 'Ortice' proved to be plastic and adaptive, while 'Racioppella' showed a certain sensitivity). The effect of the PGPR consortium increased the accumulation of osmolytes and antioxidants, and the gene expression of aquaporins was reduced during drought events. In summary, the results show that the plant microbiome is not just a passive recipient of environmental influences, but a dynamic actor that adapts and, in turn, influences the plant's ability to tolerate stress. This bidirectional interaction, in which microbial communities are shaped by both agronomic management and extreme environmental conditions, is key to understanding the resilience of the plant holobiont (Singh et al., 2024).