CHARACTERIZATION OF THE BIODIVERSITY OF SICILIAN OLIVE TREE MICROBIOTA AND EVALUATION OF ENDOPHYTES AS PLANT GROWTH PROMOTING AND POTENTIAL BIOCONTROL AGENTS

Plants are the primary producers in terrestrial ecosystems and coexist with a wide community of microorganisms that inhabit almost all plant organs, both above and below ground, both inside and outside their tissues. These microorganisms are involved in intricate networks of interactions that influence plant health and have a significant impact on ecosystem functions. While our understanding of plant microbiota continues to grow, we still lack the ability to fully control these communities, determine their complete ecological roles, or predict their responses to environmental changes. Despite the important cultural and economic role of the olive tree, the bacterial and fungal communities associated with olive trees have received less attention compared to the microbiota of other crops, especially in Sicily, a region renowned for its biodiversity and natural vulnerability to climate change. This thesis, conducted as part of a three-year PhD program, explores the endophytic communities hosted in Sicilian olive trees, emphasizing the crucial role of plant-associated microorganisms in both plant relationships and in maintaining olive tree health and disease control through a holistic and sustainable approach.The experimental studies are organised into chapters as follows.Chapter 1 highlights the significance of olive cultivation in Sicily, focusing on the adaptive strategies that olive trees have developed to cope with various abiotic stressors. Particular attention is given to the resilience of three traditional Sicilian cultivars—'Nocellara del Belice’, ‘Nocellara Etnea’, and ‘Nocellara Messinese’—with an emphasis on microbiota mediation and its mechanisms for enhancing plant growth and health. The first chapter also introduces the various pests and diseases that threaten olive trees, with a focus on pathogens affecting the vascular system, their impact on olive production, and potential integrated control methods.Chapter 2 presents a culture-independent metabarcoding method for studying bacterial and fungal endophytes, offering an effective protocol for DNA extraction and purification—typically a limiting factor in studying wood-associated microbiota. Metabarcoding of plant endophytic communities is often hampered by the co-amplification of host DNA during polymerase chain reaction (PCR) of phylogenetic marker genes. This chapter introduces an innovative method involving the use of peptide nucleic acid clamps to prevent host DNA amplification, facilitating the efficient metabarcoding of endophytic bacterial communities in olive trees. The chapter also investigates phyllosphere microbial communities to provide a comprehensive picture of Sicilian olive microbiota biodiversity and offers insights into endophyte assemblages based on statistical analyses of various factors influencing microbiota diversity and microbe-microbe interactions.Olive microbiota hosted by twigs of the three different Sicilian olive cultivars and wild olive accessions were affected by phenological phases and farming systems both in diversity and composition. Bacterial and fungal communities showed positive correlations more frequently than negative ones. The strongest positive correlations were observed between fungus Neosetophoma rosarum, and bacteria Pseudomonas sp. and Kineococcus mangrovi. While Sphingomonas sp. Was involved in negative correlations with two fungi Pseudocercospora and Biscogniauxia rosacearum. Fungal communities were mainly categorized as saprotroph represented by wood rotting fungi, more abundantly found in wild accessions, during the winter and under organic management.Chapter 3 outlines the sampling, isolation, and identification methods used to assess the endophytic microbiota of Sicilian olive cultivars and wild accessions from different phyllosphere tissues, employing a culturomic approach. This approach was used to identify the optimal conditions for establishing a functional microbial collection from leaves and twigs of three Nocellara cultivars and a wild olive population across four phenological stages (winter dormancy, flowering, fruit set, and fruit maturation) and two farming systems (organic and conventional). Additionally, alpha and beta diversity measures were inferred using statistical analyses to describe the structure, composition, and potential ecological roles of endophytic communities. The results revealed that a more extensive fungal consortium, compared to bacterial communities, characterizes the olive microbiota, with twigs hosting a greater diversity of microorganisms than leaves. Furthermore, during winter dormancy, a higher diversity in taxa and microbial composition was observed. Wild olives displayed greater species richness and distinct endophytic compositions. Differences in endophytic community structure between the two agricultural management systems were less pronounced. Culturable bacterial endophytes were characterized to identify strains with plant growth-promoting traits. This phase of the research was conducted at the Department of Microbiology and Parasitology at the University of Seville, under the supervision of Prof. Salvadora Navarro de la Torre. Functional characterization revealed that most of bacterial isolates produced siderophores, followed by nitrogen-fixing abilities, and indole-3-acetic acid (IAA) synthesis. Bacillus spp. dominated the enzymatic activities, including amylase, protease, and lipase production. Additionally, top-performing endophytes along with a soil-borne fungus (Trichoderma harzianum), were tested in vitro for antifungal activity against major olive vascular pathogens, including Verticillium dahliae, Neofusicoccum parvum, and Neofusicoccum vitifusiforme. Bacillus licheniformis displayed a relevant antifungal activity against the olive fungal pathogen Neofusicoccum vitifusiforme.Chapter 4 consolidates current insights into the diversity, composition, and ecological determinants of phyllosphere-associated endophytes in olives, with particular attention to bacterial and fungal taxa. Emphasis was placed on host-related factors—such as genotype, organ specificity, developmental stage, and plant age—that modulate microbiota composition as well as geographic location, environmental conditions, and seasonal dynamics that exert significant influence on endophyte diversity and distribution. A further overview was given on the influence of agricultural management practices, as well as biotic and abiotic stressors, on the stability and functionality of the endophytic microbiota. Of particular interest were several cultivable microbial taxa—including Bacillus, Paenibacillus, Pantoea, Aureobasidium, and Penicillium—which showed antagonistic properties against key olive pathogens, highlighting their promise as biological control agents.Finally, concluding reflections and future perspectives were provided, acknowledging that the findings of this thesis contribute to a better understanding of olive microbiota biodiversity, distribution, and function in relation to crop health. This research paves the way for developing targeted integrated approaches that, when combined with other sustainable cultivation systems, can be transferred to Sicilian olive farming.