Evaluation of Italian flora biodiversity as a source of natural compounds to develop new sustainable strategies for the control of Pseudomonas syringae pv. actinidiae

Pseudomonas syringae pv. actinidiae (Psa), the causal agent of kiwifruit bacterial canker, represents one of the most severe threats to kiwifruit production worldwide. Since its first report in Japan in 1989, the disease has spread to all major kiwifruit-producing countries, including Italy, New Zealand, and Chile, where epidemic outbreaks associated with highly virulent biovars have caused extensive economic losses. Current disease management in Europe relies largely on copper-based compounds, whose limited efficacy, phytotoxicity, and contribution to the emergence of copper-resistant strains highlight the urgent need for alternative and environmentally sustainable control strategies. This thesis explores natural products as alternative tools for the management of Psa infections, with particular emphasis on strategies that reduce bacterial virulence rather than bacterial growth. Although initially motivated by the control of Psa, the experimental framework was designed to investigate virulence modulation across the broader Pseudomonas syringae species complex, exploiting conserved regulatory features of the Type III Secretion System (T3SS). In parallel, bio-based formulations derived from agro-industrial by-products were investigated for their ability to stimulate early plant defence responses and enhance host resilience to infection. A multi-level experimental approach, combining large-scale biological screening, reporter-based assays, molecular and phenotypic analyses, transcriptomic profiling, chemical characterisation, and in planta experiments, was employed. The screening of extracts from the Mediterranean flora revealed that they represent a valuable reservoir of compounds with antimicrobial and/or antivirulence activities. Three plant extracts, namely Ailanthus altissima, Euphorbia pithyusa and Metrosideros excelsa, displayed antibacterial activity against Psa, with bactericidal or bacteriostatic effects. On the other hand, three extracts from Ceratonia siliqua, Empetrum hermaphroditum, and Philadelphus coronarius emerged as the most effective in interfering with T3SS induction, highlighting an alternative and complementary antivirulence strategy for pathogen control. Among these, Ceratonia siliqua displayed the most robust and consistent antivirulence activity, leading to a coordinated transcriptional and phenotypic reprogramming characterised by the repression of the hrp/hrc gene cluster, the inhibition of motility- and chemotaxis-associated functions and the attenuation of T3SS-dependent host responses. In contrast, Empetrum hermaphroditum and Philadelphus coronarius exhibited strong effects at specific experimental levels (transcriptomic or reporter-based), which were not consistently maintained across downstream phenotypic or in planta assays, underscoring the necessity of multi-level validation strategies. Further metabolomic analysis and bio-guided fractionation of the C. siliqua extract revealed that galloylated polyphenols emerged as relevant contributors to T3SS inhibition, and gallic acid was identified as a representative compound capable of reproducing key antivirulence effects without detectable impact on bacterial growth. In addition to pathogen-targeting strategies, the analysis of agro-industrial by-product hydrolysates showed that, while most formulations lacked direct activity against Psa, specific coffee-derived oligosaccharide hydrolysates were able to trigger early plant immune responses, highlighting a complementary host-directed strategy. Overall, this work identifies the Ceratonia siliqua extract and galloylated polyphenols, including gallic acid, as promising candidates for future antivirulence strategies targeting conserved T3SS regulation, while underscoring the potential of integrating pathogen- and host-directed approaches within the development of sustainable plant disease management frameworks.