Calcium modulation of bacterial wilt disease on potato

Abstract

Ralstonia solanacearum species complex (RSSC) is a phytopathogenic bacterial group that causes bacterial wilt in several crops, being potato (Solanum tuberosum) one of the most important hosts. The relationship between the potato plant ionome (mineral and trace elements composition) and the resistance levels to this pathogen has not been addressed until now. Mineral content of xylem sap, roots, stems and leaves of potato genotypes with different levels of resistance to bacterial wilt was assessed in this work, revealing a positive correlation between divalent calcium (Ca) cation concentrations and genotype resistance. The aim of this study was to investigate the effect of Ca on bacterial wilt resistance, and on the growth and virulence of RSSC. Ca supplementation significantly decreased the growth rate of Ralstonia pseudosolanacearum GMI1000 in minimal medium and affected several virulence traits such as biofilm formation and twitching motility. We also incorporate for the first time the use of microfluidic chambers to follow the pathogen growth and biofilm formation in conditions mimicking the plant vascular system. By using this approach, a reduction in biofilm formation was observed when both, rich and minimal media, were supplemented with Ca. Assessment of the effect of Ca amendments on bacterial wilt progress in potato genotypes revealed a significant delay in disease progress, or a complete absence of wilting symptoms in the case of partially resistant genotypes. This work contributes to the understanding of Ca effect on virulence of this important pathogen and provides new strategies for an integrated control of bacterial wilt on potato.

Importance: Ralstonia solanacearum species complex (RSSC) includes a diverse group of bacterial strains that cause bacterial wilt. This disease is difficult to control due to pathogen aggressiveness, persistence, wide range of hosts, and wide geographic distribution in tropical, subtropical, and temperate regions. RSSC causes considerable losses depending on the pathogen strain, host, soil type, environmental conditions, and cultural practices. In potato, losses of $19 billion per year have been estimated for this pathogen worldwide. In this study, we report for the first time the mineral composition found in xylem sap and plant tissues of potato germplasm with different levels of resistance to bacterial wilt. This study underscores the crucial role of calcium (Ca) concentration in the xylem sap and stem in relation to the resistance of different genotypes. Our in vitro experiments provide evidence of Ca's inhibitory effect on the growth, biofilm formation, and twitching movement of the model RSSC strain R. pseudosolanacearum GMI1000. This study introduces a novel element, the Ca concentration, which should be included into the integrated disease control management strategies for bacterial wilt in potatoes.

Keywords: bacterial wilt; calcium; disease resistance; plant ionome.

FIG 1

Calcium quantification in leaves, stems, and roots of potato plants. Interspecific potato breeding lines with different levels of bacterial wilt resistance including 13001.79 S and 09509.6 R genotypes and susceptible control (cv. Chieftain) were analyzed by ICP-OES. Each column represents calcium content (n = 3). * indicates significantly different concentrations according to Tukey’s multiple comparison test (P < 0.001). Vertical bars represent standard errors of the means.

FIG 2

Growth curves of R. pseudosolanacearum in rich medium (CPG) (A) and MM (B) alone or supplemented with CaCl₂ 1.0 mM (CPG+Ca and MM+Ca, respectively). Growth curves were carried out in 96-well plates with shaking and bacterial concentration was estimated by measuring OD600 nm. Vertical bars represent standard errors of the means (n = 16).

FIG 3

Calcium effect on biofilm formation of R. pseudosolanacearum in rich medium (CPG). (A) Biofilm formation assessed in 96-well plates with CPG and CPG supplemented with CaCl₂ 1.0 mM (CPG+Ca). ** represents significantly different according to ANOVA and Tukey’s multiple comparison test (P = 0.003). Vertical bars represent standard errors of the means (n = 6). (B) Biofilm biomass quantification by confocal laser scanning microcopy (CLSM) three days after inoculation in CPG and CPG supplemented with CaCl₂ 1.0 mM (CPG+Ca). *** represents significant differences in biofilm biomass according to Student t-test comparison (P = 0.0001). Vertical bars represent standard errors of the means (n = 9). Two-dimensional (C and D) and three-dimensional (E and F) images of biofilm formed in CPG (C and E) or CPG supplemented with Ca (D and F).

FIG 4

Calcium effect on R. pseudosolanacearum cell twitching motility. (A) Colony fringe width of R. pseudosolanacearum cultured on CPG and CPG supplemented with CaCl₂ 1.0 mM (CPG+Ca). *** represents significant differences in colony fringe width according to Student t-test comparison (P < 0.0001). Vertical bars represent standard errors of the means (n = 16). (B) Representative micrographs of colony fringes of R. pseudosolanacearum cultured on agar plates with treatments mentioned in A.

FIG 5

R. pseudosolanacearum growth in CPG and MM under flow conditions in MCs. Image was captured 8 and 48 h after inoculation. Scale bar is shown at the bottom left of the figure.

FIG 6

Calcium effect on R. pseudosolanacearum growth under flow conditions in MCs. (A) R. pseudosolanacearum growth in CPG and CPG supplemented with CaCl₂ 1.0 mM (CPG+Ca). Images were captured 19, 29, and 34 h after inoculation. Dark arrow shows biofilm formation. (B) R. pseudosolanacearum growth in MM and MM supplemented with CaCl₂ 1.0 mM (MM+Ca). Images were captured 12, 30, and 72 h after inoculation.

FIG 7

Bacterial wilt progress curves after soil inoculation with R. pseudosolanacearum in plants treated with SS and SS supplemented with CaCl₂ 20 mM (SS+Ca). Vertical bars represent standard errors of the means (n = 6). Each data point represents the average wilting rating using a scale ranging from 0 (asymptomatic plant) to 4 (all leaves wilted). Values of area under the disease progress curve (AUDPC) for the average wilting rating followed by the same letter were non-significantly different. Significant effects involving genotypes and treatments were found using ANOVA and Tukey’s multiple comparison test (P = 0.0003).

FIG 8

Relative changes of calcium concentration in leaves of 09509.6 R and susceptible control (cv. Chieftain) genotypes. Plants were treated with SS and SS supplemented with CaCl₂ 20 mM (SS+Ca). After 13 days of treatment, plants were inoculated with R. pseudosolanacearum. Calcium concentration was measured in leaves sampled one day before and 12 days after inoculation by ICP-OES. (A) Percentage changes of Ca concentrations of infected plants relative to healthy plants. * represents significant differences in Ca concentration according to Student t-test comparison (P < 0.05). (B) Percentage changes of Ca concentrations of resistant genotype relative to susceptible genotype. * represents significant differences in Ca concentration according to Student t-test comparison (P < 0.05). Each column represents the average of percentage of change of calcium concentration and vertical bars represent standard errors of the means (n = 6).