Redefining Xylella fastidiosa Cultivation: A Growth System for Faster Physiological and Antimicrobial Studies

Abstract Background : Xylella fastidiosa is a xylem-limited, Gram-negative phytopathogenic bacterium responsible for severe diseases affecting a wide range of economically important crops worldwide. Its recent expansion into Europe has reinforced its status as a major quarantine pathogen. However, progress in understanding its physiology, pathogenicity, and antimicrobial susceptibility its slow and fastidious growth under laboratory conditions, which typically requires 7-30 days for detectable development. These limitations reduce experimental throughput, reproducibility, and the feasibility of quantitative assays. The present study aimed to develop and evaluate a novel culture medium capable of supporting rapid and reproducible growth of X. fastidiosa, while remaining compatible with standard physiological and antimicrobial testing protocols. Results : We report the development of a new laboratory-adapted culture medium that consistently supports accelerated growth of X. fastidiosa strain DSM 10026/PYCC 9740 in both liquid and solid formulations. In liquid culture, bacterial populations reached the stationary phase within three days, while visible and isolated colonies formed on solid medium within the same time frame. Growth kinetics were confirmed by optical density measurements, demonstrating a shortened lag phase and reproducible exponential growth. Importantly, the medium was fully compatible with broth microdilution assays. Proof-of-concept antimicrobial susceptibility testing revealed high sensitivity to rifampicin and tetracycline (MIC = 0.25 µg/mL), and to chloramphenicol and ampicillin (MIC = 0.99 µg/mL). These MIC values are consistent with, and in some cases lower than, those previously reported using conventional media, supporting the robustness and applicability of the proposed system. Conclusions : This study presents a rapid, reproducible, and versatile cultivation system for X. fastidiosa that overcomes key limitations of existing media. The new medium enables reliable antimicrobial testing and quantitative growth analyses, thereby facilitating physiological, metabolic, and control-related studies. Its implementation is expected to significantly advance research on this high-risk quarantine pathogen and support the development of effective disease management strategies.