Functional Profiling of Kiwifruit Phyllosphere Bacteria: Copper Resistance and Biocontrol Potential as a Foundation for Microbiome-Informed Strategies

Bacterial canker, caused by <i>Pseudomonas syringae</i> pv. <i>actinidiae</i> (Psa) is a major threat to global kiwifruit production. Copper-based bactericides remain widely used, but increasing resistance highlights the urgency of developing sustainable alternatives. Understanding the functional capabilities of phyllosphere bacteria under copper pressure is critical for designing microbiome-informed management strategies. This study provides a culture-based functional inventory of bacteria associated with <i>Actinidia chinensis</i> var. <i>deliciosa</i> leaves from Portuguese orchards under long-term copper management, aiming to identify native taxa with traits relevant to plant health and resilience. A total of 1058 isolates were recovered and grouped into 261 Random Amplification of Polymorphic DNA (RAPD) clusters, representing 58 species across 29 genera. Representative strains were screened for Plant Growth-Promoting (PGP) traits (Indole-3-acetic acid (IAA), siderophore production, phosphate solubilization, ammonia production), copper tolerance, and <i>in vitro</i> antagonism against Psa. Copper resistance was widespread (53.3% of isolates with MIC ≥ 0.8 mM), including the first evidence of a highly copper-resistant PSA strain in Portuguese kiwifruit orchards and an exceptionally resistant non-pathogenic strain closely related to <i>Erwinia iniecta</i> (MIC 2.8 mM). A subset of 25 isolates combined all four PGP traits, and several also exhibited antagonism against Psa <i>in vitro</i>, among them <i>Bacillus pumilus</i> consistently supressed pathogen growth. Notably, antagonistic and multifunctional traits co-occurred in some isolates, highlighting promising candidates for integrated biocontrol strategies. Overall, the findings reveal a functionally diverse and copper-resilient collection of cultured bacteria, offering both challenges and opportunities for microbiome-based disease management. This work establishes a robust functional basis for subsequent <i>in planta</i> validation and the development of sustainable, microbiome-informed approaches for Psa control.