Figure 1.
Ionome of X. fastidiosa synthetic medium (PD2) versus field-collected grape xylem sap.
PD2 batches (n = 4) and xylem sap from different grape varieties (n = 6) were analyzed via ICP-OES. Values are presented as molar concentrations and represent average and standard errors of the mean (SEM).
Figure 2.
Box-plot representation of mineral element concentrations in X. fastidiosa biofilm or planktonic cells.
The box represents the interquartile range for the samples and the whiskers the total range (n = 40) A) Cu B) Zn C) Mn, D) Ca, E) K, F) Na, G) Fe, and H) Mg. Significant differences are observed in Cu, Zn, Mn, Ca and K (t-test p<0.05, see Table 1).
Table 1.
Mineral element concentration in Xylella fastidiosa in the planktonic and biofilm physiological states.
Figure 3.
Quantification of attachment using crystal violet detection assay with variable Cu concentrations.
Biofilm is assessed using absorbance at 600 nm of crystal violet staining after washing of the microtiter plate. Biofilm fraction is compared to the average biofilm content in PD2 media. A) Cu added up to 200 µM resulted in increased surface attachment, while attachment decreased when 200–600 µM Cu was added. Planktonic growth was unaffected by copper addition across the complete range 0–600 µM. The experiment was repeated three times, and each experiment contained three replicates. Asterisks indicate significant difference between copper supplemented and non-supplemented PD2 according to t-test (p<0.05). B) Copper chelation by the extracellular only chelator, bathocuproine sulfate, BCS, inhibits attachment at concentrations greater than ∼70 µM. The experiment was repeated three times, and one representative experiment is shown. In both panels solid lines represent biofilm quantification, while dotted line planktonic cells quantification. Copper or BCS concentrations are in µM.
Figure 4.
Evaluation of cell-to-cell aggregation of X. fastidiosa at different Cu concentrations.
Time-lapse micrographs showing cell attachment and the formation of cell aggregates inside microfluidic chambers. Images were captured at 1, 2, 3, and 5 days post inoculation. PD2 or PD2 supplemented with 50 µM or 400 µM Cu was used as growth media.
Figure 5.
Cu addition to PD2 affects biofilm formation and cell-to-cell interactions.
Cells grown in PD2 (n = 3) were compared to cells grown in 50 µM CuSO4 (n = 3) or 400 µM CuSO4 (n = 3). The planktonic cells (A, C, E) were separated from biofilm (B, D, F) and measured by ICP-OES for copper content (log 10 scale)(A, B), total optical density as a measure of cells in different states (C, D) and settling rate as a measure of cell-to-cell aggregation (E, F). N.D. not detectable due to low cell numbers. Data presented as mean and standard error of the mean.
Figure 6.
Zn addition to PD2 affects biofilm formation and cell-to-cell interactions.
Cells grown in PD2 (n = 3) were compared to cells grown in 0.25 mM ZnCl (n = 3) or 2.5 mM ZnCl (n = 3). The planktonic cells (A, C, E) were separated from biofilm (B, D, F) and measured by ICP-OES for zinc content (log 10 scale)(A, B), total optical density as a measure of cells in different states (C, D) and settling rate as a measure of cell-to-cell aggregation (E, F). N.D. not detectable due to low cell numbers. Data presented as mean and standard error of the mean.
Figure 7.
Mn addition to PD2 affects biofilm formation and cell-to-cell interactions.
Cells grown in PD2 (n = 3) were compared to cells grown in 0.25 mM MnCl2 (n = 3) or 2.5 mM MnCl2 (n = 3). The planktonic cells (A, C, E) were separated from biofilm (B, D, F) and measured by ICP-OES for manganese content (log 10 scale)(A, B), total optical density as a measure of cells in different states (C, D) and settling rate as a measure of cell-to-cell aggregation (E, F). Data presented as mean and standard error of the mean.