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

Bacterial canker, caused by Pseudomonas syringae pv. actinidiae (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 Actinidia chinensis var. deliciosa 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 in vitro 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 Erwinia iniecta (MIC 2.8 mM). A subset of 25 isolates combined all four PGP traits, and several also exhibited antagonism against Psa in vitro, among them Bacillus pumilus 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 in planta validation and the development of sustainable, microbiome-informed approaches for Psa control.