Characterization and Copper Sensitivity Evaluation of Pseudomonas savastanoi pv. savastanoi Isolates from Woody Hosts in Crete, Greece

Olive knot disease, caused by Pseudomonas savastanoi pv. savastanoi, is a significant economic threat to olive cultivation in Mediterranean regions. This study focused on the polyphasic characterization of fifty-five P. savastanoi (Psav) isolates from various hosts (olive, oleander, myrtle, and jasmine) isolated in Crete, employing phenotypic, molecular, and copper resistance analyses. Biochemical profiling using LOPAT and API20 NE tests confirmed 100% similarity between olive, jasmine and oleander isolates and the type strain CFBP1670T. Molecular characterization (BOX- and iaaL-PCR) revealed limited genetic variation among most isolates, with minor variations in myrtle, oleander, and Spanish broom strains. Multilocus sequence analysis further supported the close genetic relatedness of the Cretan isolates, while revealing limited internal differentiation. Copper resistance assays showed that most isolates were moderately sensitive (0.4 mM), while one oleander and one jasmine isolate were resistant (≥0.8 mM). In vitro evaluation of copper formulations showed that copper hydroxide, Bordeaux mixture, and tribasic copper sulfate effectively inhibited Psav growth, while copper oxide and copper oxychloride were largely ineffective. Finally, the copper resistance genes copA, copB, copD, copR, copS, and cusA were identified in all tested isolates regardless of their individual copper sensitivity levels, whereas copC, cusB, and cusC were not detected. This suggests that phenotypic variations in copper sensitivity likely stem from differences in gene expression rather than the mere presence or absence of these specific genes. These findings enhance the understanding of Psav diversity and provide a robust framework for optimizing sustainable management strategies for olive knot disease in Crete.