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

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Sobral, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8407309/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version 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. Culture medium Antimicrobial susceptibility Minimum inhibitory concentration Plant pathogenic bacteria Quarantine pathogen Liquid medium Figures Figure 1 Figure 2 1. Introduction 1.1. Xylella fastidiosa as an Established Pathogen Xylella fastidiosa is a Gram-negative, xylem-limited bacterium responsible for a wide range of destructive plant diseases. Its capacity to form dense, biofilm-like structures within xylem vessels results in hydraulic dysfunction, leaf scorch, fruit withering, and, in severe cases, host death [1,2]. The bacterium causes Pierce’s disease in grapevine, citrus variegated chlorosis, almond leaf scorch, and olive quick decline syndrome [1,2]. Table 1 summarises the various plant species known to be susceptible to X. fastidiosa . [Table1] First described in the late 19 th century in Californian vineyards, X. fastidiosa was historically restricted to the Americas, but its detection in Europe in 2013 marked its global expansion [3, 4]. The emergence in Portugal in 2023 poses a serious threat to its agriculture sector, particularly olives, almonds and grapes, with significant economic, ecological and cultural impacts. 1.2. Insect Vectors and Disease Transmission The dissemination of X. fastidiosa is tightly linked to xylem-feeding insects [5]. Among these, sharpshooter leafhoppers ( Cicadellinae ) and spittlebugs ( Aphrophoridae ) are described as the most important vectors [1, 5]. The spittlebug, highly abundant in Mediterranean landscapes, has been identified as the primary vector in European outbreaks. These insects possess specialised mouthparts that penetrate deep into the xylem vessels, enabling both acquisition and transmission of the pathogen [5]. Unlike many plant pathogens, X. fastidiosa does not require a latent period in the insect [1, 5]. Once acquired, it colonises the foregut and can be transmitted immediately and persistently throughout the insect’s lifetime [5]. Its wide host range and capacity to thrive in agricultural and non-crop habitats facilitate epidemic spread [1, 6]. This ecological plasticity challenges containment strategies, making vector biology a central focus of epidemiological research and disease management. 1.3. Challenges in Cultivation A major obstacle in X. fastidiosa research is its slow and fastidious in vitro growth. Unlike most bacterial phytopathogens, colony development requires specialised nutrient formulations and can take between 7 to 30 days, often resulting in very small colonies that are difficult to detect [3]. Optimal growth conditions include incubation at 26–28 °C, pH control between 6.5–6.9, and careful plate sealing to avoid desiccation. Faster growth (1–2 days) usually indicates presence of contaminants rather than X. fastidiosa [3]. Numerous media have been formulated to support its growth, with varying degrees of success (Table 2). Early formulations such as Periwinkle Wilt (PW) and Pierce’s Disease medium (PD2) supported primary isolation but were limited by poor reproducibility and by a more complex sterilisation process due to the requirement of bovine serum albumin [7, 8]. More recently defined formulations (e.g. XDM2, XDM3, XFM) have enabled growth and metabolic studies but X. fastidiosa propagation remain arduous and the media are technically demanding [9, 10]. [TABLE 2] 1.4. Objectives The objectives of this study were to develop a novel culture medium supporting the rapid and reproducible growth of X. fastidiosa under laboratory conditions. We evaluate its performance in both liquid and solid formulations, focusing on growth and colony formation time. Additionally, and as proof of concept the study aimed to assess the compatibility of the new medium with standard antibiotic susceptibility testing protocols and to determine the minimum inhibitory concentrations (MICs) of selected antibiotics, comparing the results with those previously reported in the literature. Overall, the work sought to provide a reliable and accelerated cultivation system that facilitates physiological and antimicrobial studies. 2. Materials and Methods This study was conducted at the Portuguese Yeast Culture Collection (PYCC) facility, which holds the necessary authorisations from the Portuguese Directorate-General for Food and Veterinary Affairs (DGAV) for handling X. fastidiosa . All experimental procedures were carried out in facilities complying with the appropriate biosafety level requirements to ensure safe and regulated manipulation of this pathogenand was handled under authorised quarantine-level biosafety facilities in accordance with EU phytosanitary regulations (ISPM 27 DP 25). 2.1 Bacterial strain It was obtained from the DSMZ culture collection 2683 PCE-RR (Braunschweig, Germany) and maintained at the Portuguese Yeast Culture Collection (PYCC, Caparica, Portugal). The X. fastidiosa strain used in this study was DSM 10026/PYCC 9740, originally isolated from grapevine ( Vitis vinifera ) in Florida, USA. 2.2 Culture media and growth conditions X. fastidiosa was cultured in five different media, namely Bosea (with and without activated charcoal), GYE, PD3 and XFG ( Xylella fastidiosa Growth), the latter being a laboratory-adapted formulation developed during this study. The Bosea medium was prepared with ACES (Biosynth, 7365-82-4) 1.0%, yeast extract (Biokar Diagnostics, A1202) 1.0%, activated charcoal (Thermo Fisher Scientific, 7440-44-0) 0.2% and agar (LabChem, 9002-18-0) 1.0% [14]. The GYE medium consisted of HEPES (Sigma-Aldrich, 7365-45-9) 1.0%, yeast extract (Biokar Diagnostics, A1202) 1.0%, L-glutamic acid (Sigma-Aldrich, 56-86-0) 0.11% and agar (LabChem, 9002-18-0) 1.0% [12]. The PD3 medium was prepared with tryptone (ThermoFisher Scientific, 211921) 0.4%, soytone (ThermoFisher Scientific, 212488) 0.2%, citric acid trisodium salt (Sigma-Aldrich, 68-04-2) 0.1%, disodium succinate (Sigma-Aldrich, 150-90-3) 0.1%, magnesium sulphate heptahydrate (AnalaR, 10034-99-8) 0.1%, dipotassium phosphate (Roth, 7758-11-4) 0.15%, potassium dihydrogen phosphate (Roth, 7778-77-0) 0.1%, soluble potato starch (Sigma-Aldrich, 9005-25-8) 0.2% and agar (LabChem, 9002-18-0) 1.8%. The medium was supplemented with 1% (v/v) of a sterile-filtered hemin chloride stock solution (0.1% bovine hemin chloride, Sigma-Aldrich, 16009-13-5, in 0.05 N NaOH, Sigma-Aldrich, 1310-73-2) [13]. The XFG medium was prepared with the same base formulation of PD3 but with the following modifications: sodium citrate dihydrate (Sigma-Aldrich, 6132-04-3) 0.1%, succinic acid (Sigma-Aldrich, 110-15-6) 0.1% instead of disodium succinate, agar (LabChem, 9002-18-0) reduced to 1.2%, and porcine hemin (≥97% purity, ThermoFisher Scientific, 16009-13-5) in substitution of bovine hemin. For XFG, the hemin solution was prepared fresh by dissolving 0.1% porcine hemin in 0, 25% NaOH (Sigma-Aldrich, 1310-73-2) solution. For the Bosea media (with and without activated charcoal) and the GYE medium, the gelling agent Gelrite (Sigma-Aldrich, 71010-52-1) was also used at the same concentration as agar. For each formulation, in addition to the solid media, liquid media were also prepared by omitting the gelling agent. For all media, the pH was adjusted to 6.9 using 5 M KOH (Sigma-Aldrich, 1310-58-3) prior to autoclaving at 121 °C for 15 minutes, an adaptation made due to the concentration of the gelling agent. For solid media, seven plates of each medium were poured. All plates were incubated at 25 °C for up to four weeks. The purity of the culture was confirmed by microscopy (Olympus Model BX50f-3) and sequencing. 2.3 Growth curves Growth dynamics were assessed in PD3 and XFG liquid cultures at 25 °C with agitation at 100rpm. To perform biological replicates four independent flasks, each containing 50mL of medium, were inoculated from four independent pre-inocula X. fastidiosa at an initial optical density at 600 nm (OD₆₀₀) of 0.005 (≈1 × 10⁶ cfu). Optical density was monitored using a TURNER SP-850 spectrophotometer over a period of 15 days. At the end of the 15-day period, a sample from each flask was plated onto the corresponding solid medium. The purity of the culture was subsequently confirmed by microscopy (Olympus Model BX50f-3) and sequencing. 2.4 Molecular validation by PCR The taxonomic identity of the bacteria was confirmed by 16S rRNA gene sequencing. DNA was extracted from a single colony resuspended in 0.5 mL of sterile water, boiled at 100 °C for 2 min, and centrifuged at 11,000 × g for 2 min. The resulting supernatant was used as the template for PCR. Amplifications were performed using Thermo Scientific DreamTaq DNA polymerase (EP0701) with the universal primers 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-ACGGCTACCTTGTTACGACTT-3′). PCR products were resolved by 1% agarose gel electrophoresis in TAE buffer (NZYtech agarose 9012-36-6) at 100 mV. The DNA ladder used was Thermo Scientific GeneRuler DNA Ladder Mix (SM0323) and Bio-Rad UView 6× Loading Dye (166-0531). Gels were visualised using a Bio-Rad Gel Doc EZ Imager and the bands of 1000 bp. PCR products were sequenced by STAB VIDA (Portugal), and sequences were analysed by Standard Nucleotide BLAST against the NCBI database (NCBI Genome, accessed on 11 November 2025). 2.5 Serial dilution assay for antimicrobial testing Three-day pre-inocula incubated at 25 °C in XFG were diluted to OD₆₀₀ of 0.005 (≈ 1 × 10⁶ cfu) and dispensed into sterile 96-well microplates (Labbox, MICP-00F-100). Two-fold (1:2) serial dilutions were prepared in the first ten wells of a 96-well plate. Medium-only and medium with bacteria (without antimicrobial) were used as negative and positive controls, respectively. The antimicrobial was tested at concentrations ranging from 250 to 0.50 µg/mL. Microplates were incubated at 25 °C for 72 h and subsequently examined for visible bacterial growth. The antimicrobials tested were chloramphenicol (Sigma-Aldrich, CAS 56-75-7), kanamycin (Sigma-Aldrich, CAS 25389-94-0), ampicillin (Sigma-Aldrich, CAS 69-52-3), rifampicin (Sigma-Aldrich, CAS 13292-46-1) and tetracycline (Sigma-Aldrich, CAS 64-75-5). 3. Results and Discussion 3.1 Obtaining isolated colonies in solid media Solid medium assays were conducted to identify an appropriate medium to support the growth of X. fastidiosa , thereby providing the basis for subsequent experiments in liquid culture (Figure 1). Marked differences were observed among the tested formulations in terms of the time required for colony formation and the total number of colonies recovered. Various contaminations events occurred throughout this process, more frequently in media that required longer incubation periods for visible growth. [Figure 1] Growth on Bosea medium was first detected after approximately 14 days of incubation, and the number of colonies increased slowly thereafter. For Bosea medium lacking activated charcoal, no growth was observed. This finding underlines the critical role of charcoal in this formulation. Activated charcoal likely supports bacterial survival by adsorbing inhibitory compounds and toxic metabolites produced endogenously by X. fastidiosa during growth [1, 2, 15]. It is well established that this bacterium produces phytotoxins and other secondary metabolites that can accumulate in closed systems, ultimately compromising its own viability [1, 2, 16]. The presence of charcoal therefore functions as a detoxifying element, maintaining a microenvironment more conducive to sustained growth. In contrast, GYE medium supported visible growth only after 34 days, with a low number of colonies. PD3 medium supported faster and more abundant growth than the traditional Bosea medium. First signs of growth were obtained at day 8, with well-defined isolated colonies visible from day 9. However, in the XFG medium colonies became visible as early as day 3 and isolated colonies were consistently observed by day 4, indicating a significantly accelerated growth rate and reproducibility (n=7). These observations clearly demonstrate the improved performance of the XFG medium for the isolation of X. fastidiosa . The presence of X. fastidiosa was confirmed by PCR analysis and further validated by microscopic examination (Supplementary Material S1 and S2). A major distinction in growth PD3 media and XFG media with Bosea media is the use of hemin and lack of charcoal. Both function as growth-enhancing supplements but through different mechanisms. Charcoal acts non-specifically, adsorbing toxic an inhibitors compounds, thus improving the physicochemical environment for bacterial proliferation [15]. In contrast, hemin acts as a defined biochemical cofactor, providing iron in a biologically available form and supporting essential enzymatic processes involved in respiration and oxidative stress defence [17]. While both can be considered growth-promoting factors, only hemin is indispensable as a nutrient, whereas charcoal is an environmental modulator [15, 17]. Our data support the use of XFG for the cultivation of X. fastidiosa on solid media. In addition, PD3 and XFG allow efficient bacterial growth in liquid culture while allows turbidity measurements, making them suitable for downstream quantitative assays. In substitution for the agar, the gelling agent Gelrite as also tested. However, growth was found to be inconsistent under those conditions. 3.2 Liquid Medium and Growth curve Based on their superior growth rate, high colony yield, and compatibility with both solid and liquid formats, PD3 and XFG were selected as the media of choice for subsequent experiments. The growth curves of X. fastidiosa in liquid culture in the PD3 and XFG media at 25°C are presented in Figure 2. The growth curve in XFG medium demonstrates that the bacterium entered a steep exponential phase shortly after inoculation (48 hours), with optical density (OD₆₀₀) increasing steadily until approximately 96 hours (0.05h -1 ). At this point, the culture approached the onset of the stationary phase, indicating that the maximum cell density was reached within four days. This rapid progression was particularly noteworthy when compared with previous reports, in which X. fastidiosa required much longer periods [18–20]. The reduced lag phase observed here further shows that cells readily adapt to the XFG formulation, suggesting that the medium provides an adequate nutritional environment. [Figure 2] In contrast, growth in PD3 was markedly slower, with an extended lag phase and a delayed exponential phase that only became evident after approximately 200 hours of incubation. The final OD₆₀₀ achieved in PD3 remained comparable to that of XFG, but the prolonged adaptation period highlights the lower efficiency of this formulation in supporting initial bacterial proliferation (0.07h -1 ). These findings further underscore the superior performance and reproducibility (n=4) of XFG in promoting X. fastidiosa growth under controlled liquid culture conditions. As before, the presence of X. fastidiosa in all tested media was confirmed by sequencing the 16S rRNA gene. The maximum OD attained in XFG (~0.25–0.27) is consistent with the modest biomass yield expected for a fastidious bacterium, yet the speed with which this level was reached clearly demonstrates the efficiency of the medium [18-20]. 3.2.1 Performance of XFG relative to existing culture media Among the various media historically developed for the cultivation of X. fastidiosa , PD3 has been regarded as a robust solid medium, largely owing to the replacement of bovine serum albumin (BSA) in PD2 by soluble potato starch, which improved reproducibility and eliminated the need for post-sterilisation supplementation [7, 8, 12]. Building on this formulation, the newly developed XFG medium incorporates targeted modifications, that mechanistically can account for its enhanced performance. The replacement of disodium succinate with succinic acid facilitates direct assimilation through the tricarboxylic acid cycle, increasing the availability of metabolically accessible carbon and thereby shortening the lag phase [21]. Similarly, sodium citrate dihydrate provides a more balanced ionic environment than trisodium citrate, reducing the alkaline load while preserving citrate’s role as both a carbon source and metal chelator [22]. This adjustment improves the bioavailability of essential divalent cations such as magnesium, which supports enzymatic activity and nucleic acid stability [22]. The substitution of bovine-derived hemin chloride with highly purified porcine hemin ensures a more consistent and bioavailable source of iron–protoporphyrin IX, thereby improving respiratory efficiency and reducing batch-to-batch variability [17, 23]. Finally, and for the solid formulation, the reduction in agar concentration enhances nutrient and oxygen diffusion within the solid medium, enabling earlier colony visualisation without compromising the gel consistency [10]. As a result of these modifications, XFG supported detectable growth from day 3, whereas PD3 required at least 9 days for colony isolation. These differences highlight the benefits of optimising the carbon source, ionic composition, agar content and hemin purity. Additionally, growth in liquid medium is faster and easier to handle. Although this procedure has been briefly described in previous studies, it typically involves initial inoculation (twice) on media containing charcoal (BCYE or Bosea), followed by transfer to a suspension in succinate-citrate-phosphate buffer, and finally into liquid PD3 medium [24, 25]. 3.3 Antimicrobial assay The absence of activated charcoal does not impair growth in liquid culture. Instead, XFG supports rapid biomass accumulation while retaining optical clarity, which is indispensable for quantitative assays. XFG appears to mitigate inhibitory effects during the exponential phase, supporting rapid and reproducible population expansion. The clarity of the medium and the reliable kinetics observed make XFG particularly suitable for downstream applications, including antimicrobial testing by serial dilution assays, which would be precluded in charcoal-supplemented formulations. To evaluate the suitability of XFG for quantitative antimicrobial testing, the standard broth microdilution (serial dilution) approach was employed. This widely used technique provides a direct measurement of the minimal inhibitory concentration (MIC) of a compound against a given organism [24]. The MIC is defined as the lowest concentration that prevents visible growth [24]. This method is particularly advantageous because it is economical, scalable to multiple agents and concentrations, and readily yields quantitative endpoints that can be compared across experiments and replicates [24, 25]. Five antibiotics, representing distinct mechanistic classes (β-lactam, phenicol, aminoglycoside, ansamycin, tetracycline) were selected to test the microdilution approach under standardised conditions in XFG (Table 2). [Table 3] Several in vitro surveys and targeted susceptibility studies have previously identified tetracycline, ampicillin, kanamycin and rifampicin among the more active antibiotics against X. fastidiosa isolates, supporting our observation of low MICs for rifampicin and tetracycline and intermediate activity for kanamycin [26-27]. Comparative interpretation of the MIC profile obtained here indicates that rifampicin and tetracycline exhibited the greatest inhibitory potency against the tested isolates in XFG (MIC = 0.25 µg/mL), followed by ampicillin and chloramphenicol (MIC ≈ 0.99 µg/mL), while kanamycin showed the highest MIC (3.90 µg/mL) among the five antibiotics tested. This pattern broadly mirrors the susceptibility spectrum reported in the literature [26-28]. The observed variation in MIC values among antibiotics likely reflects differences in their mechanisms of action and cellular uptake, as well as the intrinsic permeability barriers characteristic of X. fastidiosa . Furthermore, differences between our results and those reported in the literature may be related to the use of different media, as well as variations in antimicrobial activity protocols [26–28]. 3.4 Future applications The development of XFG as a liquid medium supporting reproducible and relatively rapid growth of X. fastidiosa has significant implications for future research. It may enables the study of biofilm formation, which in planta occurs within xylem vessels and is central to pathogenicity and vascular occlusion [1,2,20,29]. XFG will allow quantitative biofilm assays, including crystal violet staining, confocal microscopy, and flow-cell studies [20,29,30], providing insights into biofilm structure, regulation and environmental triggers. In addition, XFG facilitates systematic antimicrobial testing under controlled conditions, enabling standardised MIC determinations, time-kill assays, and biofilm-associated tolerance studies. This supports screening of conventional antibiotics, novel small molecules, plant-derived compounds, and bacteriophage enzymes [26–28]. Beyond antimicrobial evaluation, XFG allows detailed investigation of X. fastidiosa physiology and metabolism. Its optical clarity permits kinetic analyses of nutrient uptake, carbon utilisation, and metabolic fluxes using isotopic labelling and metabolomics [31]. Transcriptomic and proteomic studies in liquid culture further enable dynamic profiling of gene expression in response to environmental or chemical stimuli, providing new insights into pathogenicity and stress responses [32]. 4. Conclusion The XFG medium represents a substantial methodological advance for the cultivation of Xylella fastidiosa . By improving nutrient accessibility, diffusion, and iron availability, XFG supports faster, more consistent growth in both solid and liquid formats, overcoming major limitations of previous media. Its compatibility with quantitative and antimicrobial assays enables robust determination of MICs and expands the experimental toolkit available for X. fastidiosa research. XFG is thus expected to facilitate both fundamental physiological studies and the development of effective management strategies for this quarantine pathogen. Declarations Acknowledgments and Funding This work is financed by national funds from FCT - Fundação para a Ciência e a Tecnologia, I.P., in the scope of the Research Unit on Applied Molecular Biosciences - UCIBIO (UID/04378) and the project LA/P/0140/2020 (DOI: 10.54499/LA/P/0140/2020) of the Associate Laboratory Institute for Health and Bioeconomy - i4HB. Author Contributions Conceptualization, E.C.-C. and C.P.; investigation, E.C.-C., T.M and C.C.; writing—original draft preparation, E.C.-C.; writing—review and editing, all authors; funding acquisition, R.G.S. and C.P. All authors have read and agreed to the published version of the manuscript. 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In vitro activity of antimicrobial compounds against Xylella fastidiosa , the causal agent of the olive quick decline syndrome in Apulia (Italy). FEMS Microbiol Lett. 2018;365(5):fnx281. https://doi.org/10.1093/femsle/fnx281 Del Grosso C, Grandi L, Lombardi T, D’Attoma G, Schmitt N, De Michele VR, et al. In vitro high-throughput screening of the antimicrobial activity of different compounds against Xylella fastidiosa subsp. pauca . Chem Biol Technol Agric. 2025;12(1):15. https://doi.org/10.1186/s40538-025-00734-w Marques LLR, Ceri H, Manfio GP, Reid DM, Olson ME. Characterization of biofilm formation by Xylella fastidiosa in vitro . Plant Dis. 2002;86(6):633–638. https://doi.org/10.1094/PDIS.2002.86.6.633 Gouran H, Gillespie H, Nascimento R, Chakraborty S, Zaini PA, Jacobson A, et al. The secreted protease PrtA controls cell growth, biofilm formation and pathogenicity in Xylella fastidiosa . Sci Rep. 2016;6:31098. https://doi.org/10.1038/srep31098 Balan AS, Tranchina G, Bonanno F, Caruso T, Marra FP, Di Vaio C, et al. A systems biology framework integrating cross-species transcriptomics and PPI networks for Xylella fastidiosa resistance gene identification. BMC Plant Biol. 2025;25(1):1062. https://doi.org/10.1186/s12870-025-07102-8 Dourado MN, Pierry PM, Feitosa-Junior OR, Uceda-Campos G, Barbosa D, Zaini PA, et al. Transcriptome and secretome analyses of endophyte Methylobacterium mesophilicum and pathogen Xylella fastidiosa interacting show nutrient competition. Microorganisms. 2023;11(11):2755. https://doi.org/10.3390/microorganisms11112755 Tables Table 1: Major plant species affected by Xylella fastidiosa , associated diseases, year and country of first report. [1-6] Plant species (scientific name – common name) Associated disease First report (year) Country Prunus persica – peach Phony peach disease 1885 United States of America Vitis spp. – grapevines Pierce’s disease 1892 United States of America Medicago sativa – alfalfa Alfalfa dwarf 1970 United States of America Prunus dulcis – almond Almond leaf scorch 1974 United States of America Prunus domestica – plum Plum leaf scald 1982 Brazil Citrus spp. – sweet orange Citrus variegated chlorosis 1987 Brazil Prunus avium – sweet cherry Cherry leaf scorch / decline 1987 United States of America Platanus spp. – sycamore Sycamore leaf scorch 1988 United States of America Quercus spp. – oak Bacterial leaf scorch of oaks 1991 United States of America Coffea arabica – coffee Coffee leaf scorch 1995 Brazil Acer saccharum – sugar maple Bacterial leaf scorch 1996 United States of America Ficus carica – fig Fig scorch 1997 United States of America Nerium oleander – oleander Oleander leaf scorch 1998 United States of America Ulmus americana – American elm Bacterial leaf scorch 1999 United States of America Pyrus spp. – pear Pear leaf scorch 2001 United States of America Vaccinium spp. – blueberry Blueberry bacterial leaf scorch 2009 United States of America Olea europaea – olive Olive leaf scorch 2013 Italy Carya illinoinensis – pecan Pecan bacterial leaf scorch 2017 United States of America Table 2: Summary of culture media reported for X. fastidiosa . The table lists the principal formulations used in laboratory research, their key components, first description and typical growth times under standard incubation conditions (26–28 °C, pH 6.5–7.0). Medium Key composition First description Growth time (days) Liquid growth Notes / Limitations BCYE ACES buffer; yeast extract; activated charcoal; L-cysteine; ferric pyrophosphate; agar. [11] 10-20 No Originally developed for Legionella , this medium was later applied to Xylella , supporting the growth of certain strains. PW Peptone; tryptone; MgSO₄; phosphates; hemin; BSA; agar/gelrite. [7] 10-30 No Historically useful for primary isolation. Requires serum proteins added after sterilisation. PD2 Soytone; tryptone; trisodium citrate; disodium succinate; KH₂PO₄; K₂HPO₄; BSA (post-sterilisation); hemin; agar. [8] 10-30 No Classical isolation medium. Poor reproducibility GYE / PYE Yeast extract enriched with glutamate (GYE) or phosphate (PYE). [12] 10-30 No Poor reproducibility. PD3 PD2 variant with potato starch replacing BSA. [13] 7-10 Yes Reproducible. XDM2 / XDM3 Salts; glucose; vitamins; amino acids: serine, methionine, glutamine, asparagine (XDM2), aspartic acid (XDM3); gelrite. [9] 10-20 No Slow growth. XFM Citrate; succinate; glutamine; asparagine; cysteine; hemin; salts; gelrite. [10] 15-30 No Slow growth. Bosea ACES; yeast extract; activated charcoal; agar/gelrite. [14] 7-14 No Charcoal-based medium akin to BCYE but without cysteine/iron supplement. Supports several X. fastidiosa strains. XFG Sodium citrate dihydrate; succinic acid; tryptone; soytone; MgSO₄·7H₂O; KH₂PO₄; K₂HPO₄; soluble potato starch; porcine hemin; agar. This study 3-5 Yes Laboratory-adapted formulation derived from PD3. Supports rapid and reproducible growth in both solid and liquid formats Table 3: Minimal inhibitory concentrations (MICs) of five antibiotics against X. fastidiosa grown in XFG medium were determined. The antibiotics belong to different mechanistic classes. MIC values were measured by broth microdilution and expressed in µg/mL, with each antibiotic tested in triplicate. In the literature, agar dilution and disk diffusion methods have been used with PD3 and BCYE media. Antibiotic Antibiotic class Main mechanism of action Minimal inhibitory concentration (µg/mL) Minimal inhibitory concentration in literature (µg/mL) [26-28] Ampicillin β-lactam Inhibits cell wall synthesis by blocking transpeptidase activity. 0.99 2-8 Chloramphenicol Phenicol Inhibits protein synthesis by binding to the 50S ribosomal subunit (peptidyl transferase inhibition) 0.99 1-4 Kanamycin Aminoglycoside Inhibits protein synthesis by binding to the 30S ribosomal subunit. 3.90 4-8 Rifampicin Ansamycin Inhibits bacterial RNA polymerase, blocking transcription. 0.25 0.5-1 Tetracycline Tetracycline class Inhibits protein synthesis by binding to the 30S ribosomal subunit. 0.25 1-4 Additional Declarations No competing interests reported. Supplementary Files S1.jpg S1: Molecular identification of Xylella fastidiosa . (A) Agarose gel electrophoresis of the 16S rRNA gene amplified from X. fastidiosa colonies grown on different media. Well 1: DNA ladder; 2: Bosea; 3: GYE; 4–5: PD3; 6–7: XFG. A clear ~1,000 bp band corresponds to the expected 16S fragment. (B) BLASTn results showing ≥99% identity of the amplified sequence with X. fastidiosa 16S rRNA reference (AF192343), confirming species identity. S2.jpg S2: Light micrograph of X. fastidiosa cells stained using the Gram staining technique. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8407309","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Method Article","associatedPublications":[],"authors":[{"id":584310273,"identity":"500b546a-1c20-4938-af30-c50a10538493","order_by":0,"name":"Eduardo Costa-Camilo","email":"","orcid":"","institution":"UCIBIO Applied Molecular Biosciences Unit, Department of Life Sciences, NOVA School of Science and Technology, Universidade NOVA de Lisboa, 2829-516","correspondingAuthor":false,"prefix":"","firstName":"Eduardo","middleName":"","lastName":"Costa-Camilo","suffix":""},{"id":584310274,"identity":"d085eec8-6a8b-4163-a4da-05fca6bd2ac9","order_by":1,"name":"Tomás Martins","email":"","orcid":"","institution":"Universidade Nova de Lisboa","correspondingAuthor":false,"prefix":"","firstName":"Tomás","middleName":"","lastName":"Martins","suffix":""},{"id":584310275,"identity":"5034db42-97f6-4c87-9f77-35ce07d3bb58","order_by":2,"name":"Cláudia Carvalho","email":"","orcid":"","institution":"PYCC – Portuguese Yeast Culture Collection, Departamento de Ciências da Vida, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, Caparica, Portugal","correspondingAuthor":false,"prefix":"","firstName":"Cláudia","middleName":"","lastName":"Carvalho","suffix":""},{"id":584310276,"identity":"f2968c74-722b-4c16-9984-497dcbf245c5","order_by":3,"name":"Rita G. Sobral","email":"","orcid":"","institution":"UCIBIO Applied Molecular Biosciences Unit, Department of Life Sciences, NOVA School of Science and Technology, Universidade NOVA de Lisboa, 2829-516","correspondingAuthor":false,"prefix":"","firstName":"Rita","middleName":"G.","lastName":"Sobral","suffix":""},{"id":584310277,"identity":"dd831ac2-eb8b-4a84-a447-48302ac90734","order_by":4,"name":"Carla Pinheiro","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA4klEQVRIie3RsQrCMBCA4SuBuIR2jQj1FSKCkw/TUOisWwfBuMTFWQq+iGNKoFMfoNAOiuCsm4KDqZtItW6C+YeDO/jIEACb7VcLQAEGcHbxY0WiNUEsB2ZWR9SnT6l6YNqKeGuFd/tt5bs9ncVqdgNWLgSaXpoJLYMO4/lxiN0oKlTGgFWpQMmbV1gvwJRLzSUho/IsDCm4QKQFmddkkn5DAmwItCK05EtmyEASHFKVDUnXEJ1EzcTbhNn+KnW/v0LpSc183y3C9DAZNxPzC/JpJfXQ74AJv57QB2Kz2Wz/1R32Uk/Sc7cg3AAAAABJRU5ErkJggg==","orcid":"","institution":"UCIBIO Applied Molecular Biosciences Unit, Department of Life Sciences, NOVA School of Science and Technology, Universidade NOVA de Lisboa, 2829-516","correspondingAuthor":true,"prefix":"","firstName":"Carla","middleName":"","lastName":"Pinheiro","suffix":""}],"badges":[],"createdAt":"2025-12-19 17:53:43","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8407309/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8407309/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":101792753,"identity":"8599dc84-a740-4426-8e4f-a20b899d4191","added_by":"auto","created_at":"2026-02-03 16:14:47","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":111997,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend.\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8407309/v1/703876e8ca28863d22df2e04.jpg"},{"id":101792755,"identity":"b3dccb93-e91d-415c-b213-e751b79bfe11","added_by":"auto","created_at":"2026-02-03 16:14:47","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":89830,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend.\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8407309/v1/9b7c934b19c23c5e6d9cd7ef.jpg"},{"id":106959594,"identity":"f13adea2-2121-4bae-8155-f2acabc5750a","added_by":"auto","created_at":"2026-04-15 09:12:00","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1135189,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8407309/v1/8a0beba2-96c6-4377-bd7b-a280e97fa849.pdf"},{"id":101792752,"identity":"a7134606-76a1-4b4c-aee6-f18ab820faf9","added_by":"auto","created_at":"2026-02-03 16:14:47","extension":"jpg","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":144175,"visible":true,"origin":"","legend":"\u003cp\u003eS1: Molecular identification of \u003cem\u003eXylella fastidiosa\u003c/em\u003e. (A) Agarose gel electrophoresis of the 16S rRNA gene amplified from \u003cem\u003eX. fastidiosa\u003c/em\u003ecolonies grown on different media. Well 1: DNA ladder; 2: Bosea; 3: GYE; 4–5: PD3; 6–7: XFG. A clear ~1,000 bp band corresponds to the expected 16S fragment.\u003cbr\u003e\n(B) BLASTn results showing ≥99% identity of the amplified sequence with \u003cem\u003eX. fastidiosa\u003c/em\u003e 16S rRNA reference (AF192343), confirming species identity.\u003c/p\u003e","description":"","filename":"S1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8407309/v1/bca40af07a43431382c73ca3.jpg"},{"id":101792751,"identity":"828ecf22-3dda-441b-8cb3-1ed856d5521c","added_by":"auto","created_at":"2026-02-03 16:14:47","extension":"jpg","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":82595,"visible":true,"origin":"","legend":"\u003cp\u003eS2: Light micrograph of \u003cem\u003eX. fastidiosa\u003c/em\u003e cells stained using the Gram staining technique.\u003c/p\u003e","description":"","filename":"S2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8407309/v1/521add2be2c67038d92319a3.jpg"}],"financialInterests":"No competing interests reported.","formattedTitle":"Redefining Xylella fastidiosa Cultivation: A Growth System for Faster Physiological and Antimicrobial Studies","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003e\u003cstrong\u003e1.1. \u003cem\u003eXylella fastidiosa\u003c/em\u003e as an Established Pathogen\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eXylella fastidiosa\u0026nbsp;\u003c/em\u003eis a Gram-negative, xylem-limited bacterium responsible for a wide range of destructive plant diseases. Its capacity to form dense, biofilm-like structures within xylem vessels results in hydraulic dysfunction, leaf scorch, fruit withering, and, in severe cases, host death [1,2]. The bacterium causes Pierce’s disease in grapevine, citrus variegated chlorosis, almond leaf scorch, and olive quick decline syndrome [1,2]. Table 1 summarises the various plant species known to be susceptible to \u003cem\u003eX. fastidiosa\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003e[Table1]\u003c/p\u003e\n\u003cp\u003eFirst described in the late 19\u003csup\u003eth\u003c/sup\u003e century in Californian vineyards, \u003cem\u003eX. fastidiosa\u003c/em\u003e was historically restricted to the Americas, but its detection in Europe in 2013 marked its global expansion [3, 4]. The emergence in Portugal in 2023 poses a serious threat to its agriculture sector, particularly olives, almonds and grapes, with significant economic, ecological and cultural impacts.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e1.2. Insect Vectors and Disease Transmission\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe dissemination of \u003cem\u003eX. fastidiosa\u003c/em\u003e is tightly linked to xylem-feeding insects [5]. Among these, sharpshooter leafhoppers (\u003cem\u003eCicadellinae\u003c/em\u003e) and spittlebugs (\u003cem\u003eAphrophoridae\u003c/em\u003e) are described as the most important vectors [1, 5]. The spittlebug, highly abundant in Mediterranean landscapes, has been identified as the primary vector in European outbreaks. These insects possess specialised mouthparts that penetrate deep into the xylem vessels, enabling both acquisition and transmission of the pathogen [5]. Unlike many plant pathogens, \u003cem\u003eX. fastidiosa\u003c/em\u003e does not require a latent period in the insect [1, 5]. Once acquired, it colonises the foregut and can be transmitted immediately and persistently throughout the insect’s lifetime [5].\u003c/p\u003e\n\u003cp\u003eIts wide host range and capacity to thrive in agricultural and non-crop habitats facilitate epidemic spread [1, 6]. This ecological plasticity challenges containment strategies, making vector biology a central focus of epidemiological research and disease management.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e1.3. Challenges in Cultivation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA major obstacle in \u003cem\u003eX. fastidiosa\u003c/em\u003e research is its slow and fastidious \u003cem\u003ein vitro\u003c/em\u003e growth. Unlike most bacterial phytopathogens, colony development requires specialised nutrient formulations and can take between 7 to 30 days, often resulting in very small colonies that are difficult to detect [3]. Optimal growth conditions include incubation at 26–28 °C, pH control between 6.5–6.9, and careful plate sealing to avoid desiccation. Faster growth (1–2 days) usually indicates presence of contaminants rather than \u003cem\u003eX. fastidiosa\u0026nbsp;\u003c/em\u003e[3].\u003c/p\u003e\n\u003cp\u003eNumerous media have been formulated to support its growth, with varying degrees of success (Table 2). Early formulations such as Periwinkle Wilt (PW) and Pierce’s Disease medium (PD2) supported primary isolation but were limited by poor reproducibility and by a more complex sterilisation process due to the requirement of bovine serum albumin [7, 8]. More recently defined formulations (e.g. XDM2, XDM3, XFM) have enabled growth and metabolic studies but \u003cem\u003eX. fastidiosa\u003c/em\u003e propagation remain arduous and the media are technically demanding [9, 10].\u003c/p\u003e\n\u003cp\u003e[TABLE 2]\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e1.4. Objectives\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe objectives of this study were to develop a novel culture medium supporting the rapid and reproducible growth of \u003cem\u003eX. fastidiosa\u003c/em\u003e under laboratory conditions. We evaluate its performance in both liquid and solid formulations, focusing on growth and colony formation time. Additionally, and as proof of concept the study aimed to assess the compatibility of the new medium with standard antibiotic susceptibility testing protocols and to determine the minimum inhibitory concentrations (MICs) of selected antibiotics, comparing the results with those previously reported in the literature. Overall, the work sought to provide a reliable and accelerated cultivation system that facilitates physiological and antimicrobial studies.\u0026nbsp;\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cp\u003eThis study was conducted at the Portuguese Yeast Culture Collection (PYCC) facility, which holds the necessary authorisations from the Portuguese Directorate-General for Food and Veterinary Affairs (DGAV) for handling \u003cem\u003eX. fastidiosa\u003c/em\u003e. All experimental procedures were carried out in facilities complying with the appropriate biosafety level requirements to ensure safe and regulated manipulation of this pathogenand was handled under authorised quarantine-level biosafety facilities in accordance with EU phytosanitary regulations (ISPM 27 DP 25).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.1 Bacterial strain\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIt was obtained from the DSMZ culture collection 2683 PCE-RR (Braunschweig, Germany) and maintained at the Portuguese Yeast Culture Collection (PYCC, Caparica, Portugal). The \u003cem\u003eX. fastidiosa\u003c/em\u003e strain used in this study was DSM 10026/PYCC 9740, originally isolated from grapevine (\u003cem\u003eVitis vinifera\u003c/em\u003e) in Florida, USA.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.2 Culture media and growth conditions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eX. fastidiosa\u003c/em\u003e was cultured in five different media, namely Bosea (with and without activated charcoal), GYE, PD3 and XFG (\u003cem\u003eXylella fastidiosa\u003c/em\u003e Growth), the latter being a laboratory-adapted formulation developed during this study.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe Bosea medium was prepared with ACES (Biosynth, 7365-82-4) 1.0%, yeast extract (Biokar Diagnostics, A1202) 1.0%, activated charcoal (Thermo Fisher Scientific, 7440-44-0) 0.2% and agar (LabChem, 9002-18-0) 1.0% [14].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe GYE medium consisted of HEPES (Sigma-Aldrich, 7365-45-9) 1.0%, yeast extract (Biokar Diagnostics, A1202) 1.0%, L-glutamic acid (Sigma-Aldrich,\u0026nbsp;56-86-0) 0.11% and agar (LabChem, 9002-18-0) 1.0% [12].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe PD3 medium was prepared with tryptone (ThermoFisher Scientific, 211921) 0.4%, soytone (ThermoFisher Scientific, 212488) 0.2%, citric acid trisodium salt (Sigma-Aldrich, 68-04-2) 0.1%, disodium succinate (Sigma-Aldrich, 150-90-3) 0.1%, magnesium sulphate heptahydrate (AnalaR, 10034-99-8) 0.1%, dipotassium phosphate (Roth, 7758-11-4) 0.15%, potassium dihydrogen phosphate (Roth, 7778-77-0) 0.1%, soluble potato starch (Sigma-Aldrich, 9005-25-8) 0.2% and agar (LabChem, 9002-18-0) 1.8%. The medium was supplemented with 1% (v/v) of a sterile-filtered hemin chloride stock solution (0.1% bovine hemin chloride, Sigma-Aldrich,\u0026nbsp;16009-13-5, in 0.05 N NaOH, Sigma-Aldrich, 1310-73-2) [13].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe XFG medium was prepared with the same base formulation of PD3 but with the following modifications: sodium citrate dihydrate (Sigma-Aldrich, 6132-04-3) 0.1%, succinic acid (Sigma-Aldrich, 110-15-6) 0.1% instead of disodium succinate, agar (LabChem, 9002-18-0) reduced to 1.2%, and porcine hemin (\u0026ge;97% purity, ThermoFisher Scientific, 16009-13-5) in substitution of bovine hemin. For XFG, the hemin solution was prepared fresh by dissolving 0.1% porcine hemin in 0, 25% NaOH (Sigma-Aldrich, 1310-73-2) solution.\u003c/p\u003e\n\u003cp\u003eFor the Bosea media (with and without activated charcoal) and the GYE medium, the gelling agent Gelrite (Sigma-Aldrich, 71010-52-1) was also used at the same concentration as agar. For each formulation, in addition to the solid media, liquid media were also prepared by omitting the gelling agent.\u003c/p\u003e\n\u003cp\u003eFor all media, the pH was adjusted to 6.9 using 5 M KOH (Sigma-Aldrich, 1310-58-3) prior to autoclaving at 121 \u0026deg;C for 15 minutes, an adaptation made due to the concentration of the gelling agent. For solid media, seven plates of each medium were poured. All plates were incubated at 25 \u0026deg;C for up to four weeks. The purity of the culture was confirmed by microscopy (Olympus Model BX50f-3) and sequencing.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.3 Growth curves\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGrowth dynamics were assessed in PD3 and XFG liquid cultures at 25 \u0026deg;C with agitation at 100rpm. To perform biological replicates four independent flasks, each containing 50mL of medium, were inoculated from four independent pre-inocula \u003cem\u003eX. fastidiosa\u003c/em\u003e at an initial optical density at 600 nm (OD₆₀₀) of 0.005 (\u0026asymp;1 \u0026times; 10⁶ cfu). Optical density was monitored using a TURNER SP-850 spectrophotometer over a period of 15 days. At the end of the 15-day period, a sample from each flask was plated onto the corresponding solid medium. The purity of the culture was subsequently confirmed by microscopy (Olympus Model BX50f-3) and sequencing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.4 Molecular validation by PCR\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe taxonomic identity of the bacteria was confirmed by 16S rRNA gene sequencing. DNA was extracted from a single colony resuspended in 0.5 mL of sterile water, boiled at 100 \u0026deg;C for 2 min, and centrifuged at 11,000 \u0026times; g for 2 min. The resulting supernatant was used as the template for PCR.\u003c/p\u003e\n\u003cp\u003eAmplifications were performed using Thermo Scientific DreamTaq DNA polymerase (EP0701) with the universal primers 27F (5\u0026prime;-AGAGTTTGATCCTGGCTCAG-3\u0026prime;) and 1492R (5\u0026prime;-ACGGCTACCTTGTTACGACTT-3\u0026prime;). PCR products were resolved by 1% agarose gel electrophoresis in TAE buffer (NZYtech agarose 9012-36-6) at 100 mV. The DNA ladder used was Thermo Scientific GeneRuler DNA Ladder Mix (SM0323) and Bio-Rad UView 6\u0026times; Loading Dye (166-0531).\u003c/p\u003e\n\u003cp\u003eGels were visualised using a Bio-Rad Gel Doc EZ Imager and the bands of 1000 bp. PCR products were sequenced by STAB VIDA (Portugal), and sequences were analysed by Standard Nucleotide BLAST against the NCBI database (NCBI Genome, accessed on 11 November 2025).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.5 Serial dilution assay for antimicrobial testing\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThree-day pre-inocula incubated at 25 \u0026deg;C in XFG were diluted to OD₆₀₀ of 0.005 (\u0026asymp; 1 \u0026times; 10⁶ cfu) and dispensed into sterile 96-well microplates (Labbox, MICP-00F-100). Two-fold (1:2) serial dilutions were prepared in the first ten wells of a 96-well plate. Medium-only and medium with bacteria (without antimicrobial) were used as negative and positive controls, respectively. The antimicrobial was tested at concentrations ranging from 250 to 0.50 \u0026micro;g/mL. Microplates were incubated at 25 \u0026deg;C for 72 h and subsequently examined for visible bacterial growth. The antimicrobials tested were chloramphenicol (Sigma-Aldrich, CAS 56-75-7), kanamycin (Sigma-Aldrich, CAS 25389-94-0), ampicillin (Sigma-Aldrich, CAS 69-52-3), rifampicin (Sigma-Aldrich, CAS 13292-46-1) and tetracycline (Sigma-Aldrich, CAS 64-75-5).\u003c/p\u003e"},{"header":"3. Results and Discussion","content":"\u003cp\u003e\u003cstrong\u003e3.1 Obtaining isolated colonies in solid media\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSolid medium assays were conducted to identify an appropriate medium to support the growth of \u003cem\u003eX. fastidiosa\u003c/em\u003e, thereby providing the basis for subsequent experiments in liquid culture (Figure 1). Marked differences were observed among the tested formulations in terms of the time required for colony formation and the total number of colonies recovered. Various contaminations events occurred throughout this process, more frequently in media that required longer incubation periods for visible growth.\u003c/p\u003e\n\u003cp\u003e[Figure 1]\u003c/p\u003e\n\u003cp\u003eGrowth on Bosea medium was first detected after approximately 14 days of incubation, and the number of colonies increased slowly thereafter. For Bosea medium lacking activated charcoal, no growth was observed. This finding underlines the critical role of charcoal in this formulation. Activated charcoal likely supports bacterial survival by adsorbing inhibitory compounds and toxic metabolites produced endogenously by \u003cem\u003eX. fastidiosa\u003c/em\u003e during growth [1, 2, 15]. It is well established that this bacterium produces phytotoxins and other secondary metabolites that can accumulate in closed systems, ultimately compromising its own viability [1, 2, 16]. The presence of charcoal therefore functions as a detoxifying element, maintaining a microenvironment more conducive to sustained growth. In contrast, GYE medium supported visible growth only after 34 days, with a low number of colonies.\u003c/p\u003e\n\u003cp\u003ePD3 medium supported faster and more abundant growth than the traditional Bosea medium. First signs of growth were obtained at day 8, with well-defined isolated colonies visible from day 9.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eHowever, in the XFG medium colonies became visible as early as day 3 and isolated colonies were consistently observed by day 4, indicating a significantly accelerated growth rate and reproducibility (n=7). These observations clearly demonstrate the improved performance of the XFG medium for the isolation of \u003cem\u003eX. fastidiosa\u003c/em\u003e. The presence of X. fastidiosa was confirmed by PCR analysis and further validated by microscopic examination (Supplementary Material S1 and S2).\u003c/p\u003e\n\u003cp\u003eA major distinction in growth PD3 media and XFG media with Bosea media is the use of hemin and lack of charcoal. Both function as growth-enhancing supplements but through different mechanisms. Charcoal acts non-specifically, adsorbing toxic an inhibitors compounds, thus improving the physicochemical environment for bacterial proliferation [15]. In contrast, hemin acts as a defined biochemical cofactor, providing iron in a biologically available form and supporting essential enzymatic processes involved in respiration and oxidative stress defence [17]. While both can be considered growth-promoting factors, only hemin is indispensable as a nutrient, whereas charcoal is an environmental modulator [15, 17]. Our data support the use of XFG for the cultivation of \u003cem\u003eX. fastidiosa\u003c/em\u003e on solid media. In addition, PD3 and XFG allow efficient bacterial growth in liquid culture while allows turbidity measurements, making them suitable for downstream quantitative assays.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn substitution for the agar, the gelling agent Gelrite as also tested. However, growth was found to be inconsistent under those conditions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.2 Liquid Medium and Growth curve\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBased on their superior growth rate, high colony yield, and compatibility with both solid and liquid formats, PD3 and XFG were selected as the media of choice for subsequent experiments. The growth curves of \u003cem\u003eX. fastidiosa\u003c/em\u003e in liquid culture in the PD3 and XFG media at 25\u0026deg;C are presented in Figure 2. The growth curve in XFG medium demonstrates that the bacterium entered a steep exponential phase shortly after inoculation (48 hours), with optical density (OD₆₀₀) increasing steadily until approximately 96 hours (0.05h\u003csup\u003e-1\u003c/sup\u003e). At this point, the culture approached the onset of the stationary phase, indicating that the maximum cell density was reached within four days. This rapid progression was particularly noteworthy when compared with previous reports, in which \u003cem\u003eX. fastidiosa\u003c/em\u003e required much longer periods [18\u0026ndash;20]. The reduced lag phase observed here further shows that cells readily adapt to the XFG formulation, suggesting that the medium provides an adequate nutritional environment.\u003c/p\u003e\n\u003cp\u003e[Figure 2]\u003c/p\u003e\n\u003cp\u003eIn contrast, growth in PD3 was markedly slower, with an extended lag phase and a delayed exponential phase that only became evident after approximately 200 hours of incubation. The final OD₆₀₀\u0026nbsp;achieved in PD3 remained comparable to that of XFG, but the prolonged adaptation period highlights the lower efficiency of this formulation in supporting initial bacterial proliferation (0.07h\u003csup\u003e-1\u003c/sup\u003e). These findings further underscore the superior performance and reproducibility (n=4) of XFG in promoting \u003cem\u003eX. fastidiosa\u003c/em\u003e growth under controlled liquid culture conditions. As before, the presence of \u003cem\u003eX. fastidiosa\u003c/em\u003e in all tested media was confirmed by sequencing the 16S rRNA gene.\u003c/p\u003e\n\u003cp\u003eThe maximum OD attained in XFG (~0.25\u0026ndash;0.27) is consistent with the modest biomass yield expected for a fastidious bacterium, yet the speed with which this level was reached clearly demonstrates the efficiency of the medium [18-20].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.2.1 Performance of XFG relative to existing culture media\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAmong the various media historically developed for the cultivation of \u003cem\u003eX. fastidiosa\u003c/em\u003e, PD3 has been regarded as a robust solid medium, largely owing to the replacement of bovine serum albumin (BSA) in PD2 by soluble potato starch, which improved reproducibility and eliminated the need for post-sterilisation supplementation [7, 8, 12]. Building on this formulation, the newly developed XFG medium incorporates targeted modifications, that mechanistically can account for its enhanced performance. The replacement of disodium succinate with succinic acid facilitates direct assimilation through the tricarboxylic acid cycle, increasing the availability of metabolically accessible carbon and thereby shortening the lag phase [21]. Similarly, sodium citrate dihydrate provides a more balanced ionic environment than trisodium citrate, reducing the alkaline load while preserving citrate\u0026rsquo;s role as both a carbon source and metal chelator [22]. This adjustment improves the bioavailability of essential divalent cations such as magnesium, which supports enzymatic activity and nucleic acid stability [22]. The substitution of bovine-derived hemin chloride with highly purified porcine hemin ensures a more consistent and bioavailable source of iron\u0026ndash;protoporphyrin IX, thereby improving respiratory efficiency and reducing batch-to-batch variability [17, 23]. Finally, and for the solid formulation, the reduction in agar concentration enhances nutrient and oxygen diffusion within the solid medium, enabling earlier colony visualisation without compromising the gel consistency [10].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAs a result of these modifications, XFG supported detectable growth from day 3, whereas PD3 required at least 9 days for colony isolation. These differences highlight the benefits of optimising the carbon source, ionic composition, agar content and hemin purity. Additionally, growth in liquid medium is faster and easier to handle. Although this procedure has been briefly described in previous studies, it typically involves initial inoculation (twice) on media containing charcoal (BCYE or Bosea), followed by transfer to a suspension in succinate-citrate-phosphate buffer, and finally into liquid PD3 medium [24, 25].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.3 Antimicrobial assay\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe absence of activated charcoal does not impair growth in liquid culture. Instead, XFG supports rapid biomass accumulation while retaining optical clarity, which is indispensable for quantitative assays. XFG appears to mitigate inhibitory effects during the exponential phase, supporting rapid and reproducible population expansion. The clarity of the medium and the reliable kinetics observed make XFG particularly suitable for downstream applications, including antimicrobial testing by serial dilution assays, which would be precluded in charcoal-supplemented formulations.\u003c/p\u003e\n\u003cp\u003eTo evaluate the suitability of XFG for quantitative antimicrobial testing, the standard broth microdilution (serial dilution) approach was employed. This widely used technique provides a direct measurement of the minimal inhibitory concentration (MIC) of a compound against a given organism [24]. The MIC is defined as the lowest concentration that prevents visible growth [24]. This method is particularly advantageous because it is economical, scalable to multiple agents and concentrations, and readily yields quantitative endpoints that can be compared across experiments and replicates [24, 25].\u003c/p\u003e\n\u003cp\u003eFive antibiotics, representing distinct mechanistic classes (\u0026beta;-lactam, phenicol, aminoglycoside, ansamycin, tetracycline) were selected to test the microdilution approach under standardised conditions in XFG (Table 2).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e[Table 3]\u003c/p\u003e\n\u003cp\u003eSeveral\u003cem\u003e\u0026nbsp;in vitro\u003c/em\u003e surveys and targeted susceptibility studies have previously identified tetracycline, ampicillin, kanamycin and rifampicin among the more active antibiotics against \u003cem\u003eX. fastidiosa\u003c/em\u003e isolates, supporting our observation of low MICs for rifampicin and tetracycline and intermediate activity for kanamycin [26-27]. Comparative interpretation of the MIC profile obtained here indicates that rifampicin and tetracycline exhibited the greatest inhibitory potency against the tested isolates in XFG (MIC = 0.25 \u0026micro;g/mL), followed by ampicillin and chloramphenicol (MIC \u0026asymp; 0.99 \u0026micro;g/mL), while kanamycin showed the highest MIC (3.90 \u0026micro;g/mL) among the five antibiotics tested. This pattern broadly mirrors the susceptibility spectrum reported in the literature [26-28]. The observed variation in MIC values among antibiotics likely reflects differences in their mechanisms of action and cellular uptake, as well as the intrinsic permeability barriers characteristic of \u003cem\u003eX. fastidiosa\u003c/em\u003e. Furthermore, differences between our results and those reported in the literature may be related to the use of different media, as well as variations in antimicrobial activity protocols [26\u0026ndash;28].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.4 Future applications\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe development of XFG as a liquid medium supporting reproducible and relatively rapid growth of \u003cem\u003eX. fastidiosa\u003c/em\u003e has significant implications for future research. It may enables the study of biofilm formation, which in planta occurs within xylem vessels and is central to pathogenicity and vascular occlusion [1,2,20,29]. XFG will allow quantitative biofilm assays, including crystal violet staining, confocal microscopy, and flow-cell studies [20,29,30], providing insights into biofilm structure, regulation and environmental triggers.\u003c/p\u003e\n\u003cp\u003eIn addition, XFG facilitates systematic antimicrobial testing under controlled conditions, enabling standardised MIC determinations, time-kill assays, and biofilm-associated tolerance studies. This supports screening of conventional antibiotics, novel small molecules, plant-derived compounds, and bacteriophage enzymes [26\u0026ndash;28].\u003c/p\u003e\n\u003cp\u003eBeyond antimicrobial evaluation, XFG allows detailed investigation of \u003cem\u003eX. fastidiosa\u003c/em\u003e physiology and metabolism. Its optical clarity permits kinetic analyses of nutrient uptake, carbon utilisation, and metabolic fluxes using isotopic labelling and metabolomics [31]. Transcriptomic and proteomic studies in liquid culture further enable dynamic profiling of gene expression in response to environmental or chemical stimuli, providing new insights into pathogenicity and stress responses [32].\u003c/p\u003e"},{"header":"4. Conclusion","content":"\u003cp\u003eThe XFG medium represents a substantial methodological advance for the cultivation of \u003cem\u003eXylella fastidiosa\u003c/em\u003e. By improving nutrient accessibility, diffusion, and iron availability, XFG supports faster, more consistent growth in both solid and liquid formats, overcoming major limitations of previous media. Its compatibility with quantitative and antimicrobial assays enables robust determination of MICs and expands the experimental toolkit available for \u003cem\u003eX. fastidiosa\u003c/em\u003e research. XFG is thus expected to facilitate both fundamental physiological studies and the development of effective management strategies for this quarantine pathogen.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments and Funding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work is financed by national funds from FCT - Funda\u0026ccedil;\u0026atilde;o para a Ci\u0026ecirc;ncia e a Tecnologia, I.P., in the scope of the Research Unit on Applied Molecular Biosciences - UCIBIO (UID/04378) and the project LA/P/0140/2020 (DOI: 10.54499/LA/P/0140/2020) of the Associate Laboratory Institute for Health and Bioeconomy - i4HB.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization, E.C.-C. and C.P.; investigation, E.C.-C., T.M and C.C.; writing\u0026mdash;original draft preparation, E.C.-C.; writing\u0026mdash;review and editing, all authors; funding acquisition, R.G.S. and C.P. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that this research was conducted in the absence of any commercial or financial relationships that could be construed as potential conflicts of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eSicard A, Zeilinger AR, Vanhove M, Schartel TE, Beal DJ, Daugherty MP, et al. \u003cem\u003eXylella fastidiosa\u003c/em\u003e: insights into an emerging plant pathogen. Annu Rev Phytopathol. 2018;56:181\u0026ndash;202. https://doi.org/10.1146/annurev-phyto-080417-045849\u003c/li\u003e\n\u003cli\u003ePurcell AH, Hopkins DL. Fastidious xylem-limited bacterial plant pathogens. Annu Rev Phytopathol. 1996;34:131\u0026ndash;151. https://doi.org/10.1146/annurev.phyto.34.1.131\u003c/li\u003e\n\u003cli\u003eRapicavoli J, Ingel B, Blanco-Ulate B, Cantu D, Roper C. \u003cem\u003eXylella fastidiosa\u003c/em\u003e: an examination of a re-emerging plant pathogen. Mol Plant Pathol. 2018;19(4):786\u0026ndash;800. https://doi.org/10.1111/mpp.12585\u003c/li\u003e\n\u003cli\u003eLoureiro T, Serra L, Martins \u0026Acirc;, Cortez I, Poeta P. \u003cem\u003eXylella fastidiosa\u003c/em\u003e dispersion on vegetal hosts in demarcated zones in the North Region of Portugal. Microbiol Res. 2024;15(3):1050\u0026ndash;1072. https://doi.org/10.3390/microbiolres15030069\u003c/li\u003e\n\u003cli\u003eRanieri E, Zitti G, Riolo P, Isidoro N, Ruschioni S, Brocchini M, et al. Fluid dynamics in the functional foregut of xylem-sap feeding insects: a comparative study of two \u003cem\u003eXylella fastidiosa\u003c/em\u003e vectors. J Insect Physiol. 2020;120:103995. https://doi.org/10.1016/j.jinsphys.2019.103995\u003c/li\u003e\n\u003cli\u003eCastro C, DiSalvo B, Roper MC. \u003cem\u003eXylella fastidiosa\u003c/em\u003e: a reemerging plant pathogen that threatens crops globally. PLoS Pathog. 2021;17(9):e1009813. https://doi.org/10.1371/journal.ppat.1009813\u003c/li\u003e\n\u003cli\u003eDavis MJ, Purcell AH, Thomson SV. Isolation medium for the Pierce\u0026rsquo;s disease bacterium. Phytopathology. 1980;70:425\u0026ndash;429.\u003c/li\u003e\n\u003cli\u003eDavis MJ, French WJ, Schaad NW. Axenic culture of the bacteria associated with phony disease of peach and plum leaf scald. Curr Microbiol. 1981;6:309\u0026ndash;314.\u003c/li\u003e\n\u003cli\u003ede Macedo Lemos EG, Alves LMC, Campanharo JC. Genomics-based design of defined growth media for the plant pathogen \u003cem\u003eXylella fastidiosa\u003c/em\u003e. FEMS Microbiol Lett. 2003;219(1):39\u0026ndash;45. https://doi.org/10.1016/S0378-1097(02)01189-8\u003c/li\u003e\n\u003cli\u003eAlmeida RP, Mann R, Purcell AH. \u003cem\u003eXylella fastidiosa\u003c/em\u003e cultivation on a minimal solid defined medium. Curr Microbiol. 2004;48(5):368\u0026ndash;372. https://doi.org/10.1007/s00284-003-4219-x\u003c/li\u003e\n\u003cli\u003eFeeley JC, Gibson RJ, Gorman GW, Langford NC, Rasheed JK, Mackel DC, et al. Charcoal-yeast extract agar: primary isolation medium for \u003cem\u003eLegionella pneumophila\u003c/em\u003e. J Clin Microbiol. 1979;10(4):437\u0026ndash;441. https://doi.org/10.1128/jcm.10.4.437-441.1979\u003c/li\u003e\n\u003cli\u003eCampanharo JC, Lemos MVF, Lemos EGD. Growth optimization procedures for the phytopathogen \u003cem\u003eXylella fastidiosa\u003c/em\u003e. Curr Microbiol. 2003;46(2):99\u0026ndash;102. https://doi.org/10.1007/s00284-002-3829-z\u003c/li\u003e\n\u003cli\u003eWells JM, Raju BC, Nyland G. 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Insight into biological strategies and main challenges to control the phytopathogenic bacterium \u003cem\u003eXylella fastidiosa\u003c/em\u003e. Front Plant Sci. 2025;16:1608687. https://doi.org/10.3389/fpls.2025.1608687\u003c/li\u003e\n\u003cli\u003eSil R, Chakraborti AS. Major heme proteins hemoglobin and myoglobin with respect to their roles in oxidative stress\u0026ndash;a brief review. Front Chem. 2025;13:1543455. https://doi.org/10.3389/fchem.2025.1543455\u003c/li\u003e\n\u003cli\u003eFeil H, Purcell AH. Temperature-dependent growth and survival of \u003cem\u003eXylella fastidiosa in vitro\u003c/em\u003e and in potted grapevines. Plant Dis. 2001;85(12):1230\u0026ndash;1234. https://doi.org/10.1094/PDIS.2001.85.12.1230\u003c/li\u003e\n\u003cli\u003eKung SH, Almeida RP. Natural competence and recombination in the plant pathogen \u003cem\u003eXylella fastidiosa\u003c/em\u003e. Appl Environ Microbiol. 2011;77(15):5278\u0026ndash;5284. https://doi.org/10.1128/AEM.0073\u003c/li\u003e\n\u003cli\u003eMoll L, Badosa E, Planas M, Feliu L, Montesinos E, Bonaterra A. Antimicrobial peptides with antibiofilm activity against \u003cem\u003eXylella fastidiosa\u003c/em\u003e. Front Microbiol. 2021;12:753874. https://doi.org/10.3389/fmicb.2021.753874 \u003c/li\u003e\n\u003cli\u003eNghiem NP, Kleff S, Schwegmann S. Succinic acid: technology development and commercialization. Fermentation. 2017;3(2):26. https://doi.org/10.3390/fermentation3020026\u003c/li\u003e\n\u003cli\u003e[22 Phillips CA. The effect of citric acid, lactic acid, sodium citrate and sodium lactate, alone and in combination with nisin, on the growth of \u003cem\u003eArcobacter butzleri\u003c/em\u003e. Lett Appl Microbiol. 1999;29(6):424\u0026ndash;428. https://doi.org/10.1046/j.1472-765X.1999.00668.x\u003c/li\u003e\n\u003cli\u003eSander A, Kretzer S, Bredt W, Oberle K, Bereswill S. Hemin-dependent growth and hemin binding of \u003cem\u003eBartonella henselae\u003c/em\u003e. FEMS Microbiol Lett. 2000;189(1):55\u0026ndash;59. https://doi.org/10.1111/j.1574-6968.2000.tb09205.x\u003c/li\u003e\n\u003cli\u003eHossain TJ. Methods for screening and evaluation of antimicrobial activity: a review of protocols, advantages, and limitations. Eur J Microbiol Immunol. 2024;14(2):97\u0026ndash;115. https://doi.org/10.1556/1886.2024.00035\u003c/li\u003e\n\u003cli\u003eMaugeri G, Lychko I, Sobral R, Roque AC. Identification and antibiotic-susceptibility profiling of infectious bacterial agents: a review of current and future trends. Biotechnol J. 2019;14(1):1700750. https://doi.org/10.1002/biot.201700750\u003c/li\u003e\n\u003cli\u003eKuzina LV, Miller TA, Cooksey DA. In vitro activities of antibiotics and antimicrobial peptides against the plant pathogenic bacterium \u003cem\u003eXylella fastidiosa\u003c/em\u003e. Lett Appl Microbiol. 2006;42(5):514\u0026ndash;520. https://doi.org/10.1111/j.1472-765X.2006.01898.x\u003c/li\u003e\n\u003cli\u003eBleve G, Gallo A, Altomare C, Vurro M, Maiorano G, Cardinali A, et al. In vitro activity of antimicrobial compounds against \u003cem\u003eXylella fastidiosa\u003c/em\u003e, the causal agent of the olive quick decline syndrome in Apulia (Italy). FEMS Microbiol Lett. 2018;365(5):fnx281. https://doi.org/10.1093/femsle/fnx281\u003c/li\u003e\n\u003cli\u003eDel Grosso C, Grandi L, Lombardi T, D\u0026rsquo;Attoma G, Schmitt N, De Michele VR, et al. \u003cem\u003eIn vitro\u003c/em\u003e high-throughput screening of the antimicrobial activity of different compounds against \u003cem\u003eXylella fastidiosa subsp. pauca\u003c/em\u003e. Chem Biol Technol Agric. 2025;12(1):15. https://doi.org/10.1186/s40538-025-00734-w\u003c/li\u003e\n\u003cli\u003eMarques LLR, Ceri H, Manfio GP, Reid DM, Olson ME. Characterization of biofilm formation by \u003cem\u003eXylella fastidiosa in vitro\u003c/em\u003e. Plant Dis. 2002;86(6):633\u0026ndash;638. https://doi.org/10.1094/PDIS.2002.86.6.633\u003c/li\u003e\n\u003cli\u003eGouran H, Gillespie H, Nascimento R, Chakraborty S, Zaini PA, Jacobson A, et al. The secreted protease PrtA controls cell growth, biofilm formation and pathogenicity in \u003cem\u003eXylella fastidiosa\u003c/em\u003e. Sci Rep. 2016;6:31098. https://doi.org/10.1038/srep31098\u003c/li\u003e\n\u003cli\u003eBalan AS, Tranchina G, Bonanno F, Caruso T, Marra FP, Di Vaio C, et al. A systems biology framework integrating cross-species transcriptomics and PPI networks for \u003cem\u003eXylella fastidiosa\u003c/em\u003e resistance gene identification. BMC Plant Biol. 2025;25(1):1062. https://doi.org/10.1186/s12870-025-07102-8\u003c/li\u003e\n\u003cli\u003eDourado MN, Pierry PM, Feitosa-Junior OR, Uceda-Campos G, Barbosa D, Zaini PA, et al. Transcriptome and secretome analyses of endophyte \u003cem\u003eMethylobacterium mesophilicum\u003c/em\u003e and pathogen \u003cem\u003eXylella fastidiosa\u003c/em\u003e interacting show nutrient competition. Microorganisms. 2023;11(11):2755. https://doi.org/10.3390/microorganisms11112755\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTable 1: Major plant species affected by \u003cem\u003eXylella fastidiosa\u003c/em\u003e, associated diseases, year and country of first report. [1-6]\u003c/p\u003e\n\u003cdiv\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"919\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 33.9499%;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePlant species (scientific name \u0026ndash; common name)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 34.6028%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eAssociated disease\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11.6431%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eFirst report (year)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 19.8041%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCountry\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 33.9499%;\"\u003e\n \u003cp\u003e\u003cem\u003ePrunus persica\u003c/em\u003e \u0026ndash; peach\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 34.6028%;\"\u003e\n \u003cp\u003ePhony peach disease\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11.6431%;\"\u003e\n \u003cp\u003e1885\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 19.8041%;\"\u003e\n \u003cp\u003eUnited States of America\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 33.9499%;\"\u003e\n \u003cp\u003e\u003cem\u003eVitis spp.\u003c/em\u003e \u0026ndash; grapevines\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 34.6028%;\"\u003e\n \u003cp\u003ePierce\u0026rsquo;s disease\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11.6431%;\"\u003e\n \u003cp\u003e1892\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 19.8041%;\"\u003e\n \u003cp\u003eUnited States of America\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 33.9499%;\"\u003e\n \u003cp\u003e\u003cem\u003eMedicago sativa\u003c/em\u003e \u0026ndash; alfalfa\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 34.6028%;\"\u003e\n \u003cp\u003eAlfalfa dwarf\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11.6431%;\"\u003e\n \u003cp\u003e1970\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 19.8041%;\"\u003e\n \u003cp\u003eUnited States of America\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 33.9499%;\"\u003e\n \u003cp\u003e\u003cem\u003ePrunus dulcis\u003c/em\u003e \u0026ndash; almond\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 34.6028%;\"\u003e\n \u003cp\u003eAlmond leaf scorch\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11.6431%;\"\u003e\n \u003cp\u003e1974\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 19.8041%;\"\u003e\n \u003cp\u003eUnited States of America\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 33.9499%;\"\u003e\n \u003cp\u003e\u003cem\u003ePrunus domestica\u003c/em\u003e \u0026ndash; plum\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 34.6028%;\"\u003e\n \u003cp\u003ePlum leaf scald\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11.6431%;\"\u003e\n \u003cp\u003e1982\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 19.8041%;\"\u003e\n \u003cp\u003eBrazil\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 33.9499%;\"\u003e\n \u003cp\u003e\u003cem\u003eCitrus spp.\u003c/em\u003e \u0026ndash; sweet orange\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 34.6028%;\"\u003e\n \u003cp\u003eCitrus variegated chlorosis\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11.6431%;\"\u003e\n \u003cp\u003e1987\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 19.8041%;\"\u003e\n \u003cp\u003eBrazil\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 33.9499%;\"\u003e\n \u003cp\u003e\u003cem\u003ePrunus avium\u003c/em\u003e \u0026ndash; sweet cherry\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 34.6028%;\"\u003e\n \u003cp\u003eCherry leaf scorch / decline\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11.6431%;\"\u003e\n \u003cp\u003e1987\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 19.8041%;\"\u003e\n \u003cp\u003eUnited States of America\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 33.9499%;\"\u003e\n \u003cp\u003e\u003cem\u003ePlatanus spp.\u003c/em\u003e \u0026ndash; sycamore\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 34.6028%;\"\u003e\n \u003cp\u003eSycamore leaf scorch\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11.6431%;\"\u003e\n \u003cp\u003e1988\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 19.8041%;\"\u003e\n \u003cp\u003eUnited States of America\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 33.9499%;\"\u003e\n \u003cp\u003e\u003cem\u003eQuercus spp.\u003c/em\u003e \u0026ndash; oak\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 34.6028%;\"\u003e\n \u003cp\u003eBacterial leaf scorch of oaks\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11.6431%;\"\u003e\n \u003cp\u003e1991\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 19.8041%;\"\u003e\n \u003cp\u003eUnited States of America\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 33.9499%;\"\u003e\n \u003cp\u003e\u003cem\u003eCoffea arabica\u003c/em\u003e \u0026ndash; coffee\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 34.6028%;\"\u003e\n \u003cp\u003eCoffee leaf scorch\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11.6431%;\"\u003e\n \u003cp\u003e1995\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 19.8041%;\"\u003e\n \u003cp\u003eBrazil\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 33.9499%;\"\u003e\n \u003cp\u003e\u003cem\u003eAcer saccharum\u003c/em\u003e \u0026ndash; sugar maple\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 34.6028%;\"\u003e\n \u003cp\u003eBacterial leaf scorch\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11.6431%;\"\u003e\n \u003cp\u003e1996\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 19.8041%;\"\u003e\n \u003cp\u003eUnited States of America\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 33.9499%;\"\u003e\n \u003cp\u003e\u003cem\u003eFicus carica\u003c/em\u003e \u0026ndash; fig\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 34.6028%;\"\u003e\n \u003cp\u003eFig scorch\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11.6431%;\"\u003e\n \u003cp\u003e1997\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 19.8041%;\"\u003e\n \u003cp\u003eUnited States of America\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 33.9499%;\"\u003e\n \u003cp\u003e\u003cem\u003eNerium oleander\u003c/em\u003e \u0026ndash; oleander\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 34.6028%;\"\u003e\n \u003cp\u003eOleander leaf scorch\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11.6431%;\"\u003e\n \u003cp\u003e1998\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 19.8041%;\"\u003e\n \u003cp\u003eUnited States of America\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 33.9499%;\"\u003e\n \u003cp\u003e\u003cem\u003eUlmus americana\u003c/em\u003e \u0026ndash; American elm\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 34.6028%;\"\u003e\n \u003cp\u003eBacterial leaf scorch\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11.6431%;\"\u003e\n \u003cp\u003e1999\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 19.8041%;\"\u003e\n \u003cp\u003eUnited States of America\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 33.9499%;\"\u003e\n \u003cp\u003e\u003cem\u003ePyrus spp.\u003c/em\u003e \u0026ndash; pear\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 34.6028%;\"\u003e\n \u003cp\u003ePear leaf scorch\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11.6431%;\"\u003e\n \u003cp\u003e2001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 19.8041%;\"\u003e\n \u003cp\u003eUnited States of America\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 33.9499%;\"\u003e\n \u003cp\u003e\u003cem\u003eVaccinium spp.\u003c/em\u003e \u0026ndash; blueberry\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 34.6028%;\"\u003e\n \u003cp\u003eBlueberry bacterial leaf scorch\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11.6431%;\"\u003e\n \u003cp\u003e2009\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 19.8041%;\"\u003e\n \u003cp\u003eUnited States of America\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 33.9499%;\"\u003e\n \u003cp\u003e\u003cem\u003eOlea europaea\u003c/em\u003e \u0026ndash; olive\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 34.6028%;\"\u003e\n \u003cp\u003eOlive leaf scorch\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11.6431%;\"\u003e\n \u003cp\u003e2013\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 19.8041%;\"\u003e\n \u003cp\u003eItaly\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 33.9499%;\"\u003e\n \u003cp\u003e\u003cem\u003eCarya illinoinensis\u003c/em\u003e \u0026ndash; pecan\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 34.6028%;\"\u003e\n \u003cp\u003ePecan bacterial leaf scorch\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11.6431%;\"\u003e\n \u003cp\u003e2017\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 19.8041%;\"\u003e\n \u003cp\u003eUnited States of America\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003eTable 2: Summary of culture media reported for \u003cem\u003eX. fastidiosa\u003c/em\u003e. The table lists the principal formulations used in laboratory research, their key components, first description and typical growth times under standard incubation conditions (26\u0026ndash;28 \u0026deg;C, pH 6.5\u0026ndash;7.0).\u0026nbsp;\u003c/p\u003e\n\u003cdiv\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" class=\"fr-table-selection-hover\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eMedium\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eKey composition\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eFirst description\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eGrowth time (days)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eLiquid growth\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eNotes / Limitations\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eBCYE\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eACES buffer; yeast extract; activated charcoal; L-cysteine; ferric pyrophosphate; agar.\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e[11]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e10-20\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eNo\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eOriginally developed for \u003cem\u003eLegionella\u003c/em\u003e, this medium was later applied to \u003cem\u003eXylella\u003c/em\u003e, supporting the growth of certain strains.\u003c/p\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003ePW\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003ePeptone; tryptone; MgSO₄; phosphates; hemin; BSA; agar/gelrite.\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e[7]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e10-30\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eNo\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eHistorically useful for primary isolation. Requires serum proteins added after sterilisation.\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003ePD2\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eSoytone; tryptone; trisodium citrate; disodium succinate; KH₂PO₄; K₂HPO₄; BSA (post-sterilisation); hemin; agar.\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e[8]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e10-30\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eNo\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eClassical isolation medium. Poor reproducibility\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eGYE / PYE\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eYeast extract enriched with glutamate (GYE) or phosphate (PYE).\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e[12]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e10-30\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eNo\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003ePoor reproducibility.\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003ePD3\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003ePD2 variant with potato starch replacing BSA.\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e[13]\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e7-10\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eYes\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eReproducible.\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eXDM2 / XDM3\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eSalts; glucose; vitamins; amino acids: serine, methionine, glutamine, asparagine (XDM2), aspartic acid (XDM3); gelrite.\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e[9]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e10-20\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eNo\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eSlow growth.\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eXFM\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eCitrate; succinate; glutamine; asparagine; cysteine; hemin; salts; gelrite.\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e[10]\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e15-30\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eNo\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eSlow growth.\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eBosea\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eACES; yeast extract; activated charcoal; agar/gelrite.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e[14]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e7-14\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eNo\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eCharcoal-based medium akin to BCYE but without cysteine/iron supplement. Supports several \u003cem\u003eX. fastidiosa\u003c/em\u003e strains.\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eXFG\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eSodium citrate dihydrate; succinic acid; tryptone; soytone; MgSO₄\u0026middot;7H₂O; KH₂PO₄; K₂HPO₄; soluble potato starch; porcine hemin; agar.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e3-5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eYes\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eLaboratory-adapted formulation derived from PD3. Supports rapid and reproducible growth in both solid and liquid formats\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003c/br\u003e\n\u003cp\u003eTable 3: Minimal inhibitory concentrations (MICs) of five antibiotics against \u003cem\u003eX. fastidiosa\u003c/em\u003e grown in XFG medium were determined. The antibiotics belong to different mechanistic classes. MIC values were measured by broth microdilution and expressed in \u0026micro;g/mL, with each antibiotic tested in triplicate. In the literature, agar dilution and disk diffusion methods have been used with PD3 and BCYE media.\u003c/p\u003e\n\u003cdiv\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" class=\"fr-table-selection-hover\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eAntibiotic\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eAntibiotic class\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eMain mechanism of action\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eMinimal inhibitory concentration (\u0026micro;g/mL)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eMinimal inhibitory concentration in literature (\u0026micro;g/mL) [26-28]\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eAmpicillin\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026beta;-lactam\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eInhibits cell wall synthesis by blocking transpeptidase activity.\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e0.99\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e2-8\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eChloramphenicol\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003ePhenicol\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eInhibits protein synthesis by binding to the 50S ribosomal subunit (peptidyl transferase inhibition)\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0.99\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e1-4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eKanamycin\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eAminoglycoside\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eInhibits protein synthesis by binding to the 30S ribosomal subunit.\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e3.90\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e4-8\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eRifampicin\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eAnsamycin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eInhibits bacterial RNA polymerase, blocking transcription.\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0.25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0.5-1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eTetracycline\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eTetracycline class\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eInhibits protein synthesis by binding to the 30S ribosomal subunit.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0.25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e1-4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Culture medium, Antimicrobial susceptibility, Minimum inhibitory concentration, Plant pathogenic bacteria, Quarantine pathogen, Liquid medium","lastPublishedDoi":"10.21203/rs.3.rs-8407309/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8407309/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground\u003c/strong\u003e: 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.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e: 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.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions\u003c/strong\u003e: 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.\u003c/p\u003e","manuscriptTitle":"Redefining Xylella fastidiosa Cultivation: A Growth System for Faster Physiological and Antimicrobial Studies","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-03 16:14:42","doi":"10.21203/rs.3.rs-8407309/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"d88cf271-a898-4586-91e0-f29fe9f0d1ec","owner":[],"postedDate":"February 3rd, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-02-03T16:14:42+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-03 16:14:42","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8407309","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8407309","identity":"rs-8407309","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}