Figure 1.
High zinc concentrations suppress disease symptoms in Thlaspi caerulescens.
A. T. caerulescens plants growing on 10 µM zinc during an outbreak of mildew (Erysiphe sp.) in the glasshouse. B. T. caerulescens plants growing on 300 µM zinc during the same outbreak of mildew in the glasshouse. C. T. caerulescens plants were grown for 10 weeks on nutrient solution containing 0.04, 10, 30, or 300 µM ZnSO4. Leaves were infiltrated with P. syringae pv. maculicola M4 suspended in 10 mM MgCl2 at 108 cfu/ml and photographed 96 hours after inoculation. Scale bars represent 10 mm.
Figure 2.
T3SS mutants of Pseudomonas syringae pv. maculicola are unable to colonise Thlaspi caerulescens.
P. syringae pv. maculicola M4 (Psm M4), P. syringae pv. maculicola ES4326 (Psm 4326) and two T3SS mutants of Psm (hrpS− and hrcN−) were inoculated into fully expanded leaves of T. caerulescens at 106 cfu/ml. Three samples were taken for each strain, zinc treatment and time point, each consisting of three leaf discs pooled together. Values are means±SE (n = 3). The mean growth of the four strains at each zinc treatment over 5 days post-inoculation was compared in ANOVAs; growth of the four strains was found to differ significantly in all treatments except in the 300 µM zinc treatment, where no strain was able to grow (P<0.0005 for 0.04 and 10 µM Zn; P = 0.001 for 30 µM Zn; P = 0.169 for 300 µM Zn). Within each zinc treatment, Bonferroni simultaneous comparisons were used to determine which means differed significantly at 5 days post-inoculation, and these are marked with different letters. The inset shows symptoms observed in leaves from T. caerulescens plants grown on 0.04 µM zinc 72 hours after inoculation with wild-type Psm ES4326 and the Psm hrcN− mutant at 106 cfu/ml compared with uninoculated control leaves.
Figure 3.
High metal concentrations inhibit bacterial growth in Thlaspi caerulescens.
T. caerulescens plants were treated with a range of zinc (A), nickel (B), or cadmium (C) concentrations. Nine leaves of each of six plants were infiltrated with P. syringae pv. maculicola M4 suspended in 10 mM MgCl2 at 106 cfu/ml and leaves sampled at 0, 2 and 5 days after inoculation. Six samples were taken per time point and treatment, each sample consisting of three leaves pooled from one plant. Plant zinc, nickel and cadmium treatments were significant predictors of Psm growth at both day 2 and day 5 (ANOVAs; P<0.0005). Bonferroni simultaneous comparisons were carried out; means that were not significantly different are marked with the same letter. Values are means ± SE (n = 6). The experiment was repeated twice with similar results.
Figure 4.
Apoplast extracts from Thlaspi caerulescens plants grown in high metal concentrations inhibit bacterial growth.
Apoplast extracts obtained from T. caerulescens plants treated with the same zinc (A), nickel (B), or cadmium (C) concentrations used in the bacterial colonization assays shown in Figure 3 were used as a growth medium for P. syringae pv. maculicola M4. Six samples of 100 µl of apoplast were used for each treatment. Values are means ± SE (n = 6). The experiment was performed three times with similar results, and the results shown are from one experiment representative of the three. Concentrations of all three metals were significant predictors of growth at 18 and 24 hours (ANOVAs: P<0.0005 in all cases except P = 0.015 for cadmium at 24 h). Bonferroni simultaneous comparisons (α = 1%) show that all zinc and nickel treatments resulted in significantly less growth than the control at 18 h and 24 h, as did all cadmium treatments up to 18 h.
Figure 5.
Metal concentrations in zinc-, nickel- and cadmium-treated plants.
Data represent average values from three independent sets of plants. Metal content was determined by atomic absorption spectrophotometry. A, C, E: Apoplastic concentrations of zinc, nickel and cadmium, respectively. B, D, F: Bulk-leaf concentrations of zinc, nickel and cadmium, respectively, expressed both as molar concentration calculated on the basis of leaf fresh biomass, and as mass concentration relative to leaf dry biomass. Values are means ± SE (n = 6). Horizontal bars in A, C and F indicate metal concentrations representing the respective IC50 values for P. syringae pv. maculicola M4 determined experimentally in extracted apoplast (i.e. Zn = 0.12 mM; Ni = 0.025 mM; Cd = 0.01 mM); IC50 values measured in LB were higher (Zn = 0.63 mM; Ni = 0.78 mM; Cd = 0.22 mM).
Table 1.
Summary of bacterial growth across treatments in relation to metal concentration and metal-dependent growth inhibition of P. syringae pv. maculicola in vitro.
Figure 6.
Zinc tolerance of P. syringae pv. maculicola M4 and four mutants.
Five µl of bacterial suspension at an OD600 of 0.2 were inoculated into 200 µl of KB broth supplemented with zinc at 0 to 20 mM. The graph shows the percentage increase in OD600 48 hours after inoculation, relative to the increase in OD600 over 48 hours observed for the same strain in the absence of zinc. At least four samples were analysed for each treatment. Values are means ± SE (n = 8).
Table 2.
P. syringae pv. maculicola M4 mutants with altered zinc tolerance.
Figure 7.
P. syringae pv. maculicola M4 mutants with altered zinc tolerance show differential growth in Thlaspi caerulescens.
T. caerulescens plants were treated with 0.04, 10, 30, or 300 µM zinc. Nine leaves of three plants were infiltrated with either P. syringae pv. maculicola M4 or one of four zinc-tolerance mutants (9A6, 9A3, 7C11, or 10C1) suspended in 10 mM MgCl2 at 106 cfu/ml. Leaves were sampled for bacterial counts at 0 and 5 days after inoculation. Each replicate consisted of three leaves from one plant, giving a total of three replicates per time point and treatment. Values are means ± SE (n = 3). The experiment was performed three times with similar results, and the results shown are from one experiment representative of the three. ANOVAs were used to test for a significant effect of plant zinc treatment on the growth of each bacterial strain. No effect was detected for the high tolerance mutant 9A3 (P = 0.13). For the other strains, growth was found to be dependent on zinc (9A6: P = 0.01; wild-type, 7C11 and 10C1: P<0.0005 in each case). Where a significant effect was found, Bonferroni simultaneous comparisons (α = 5%) were carried out. Within each strain, means marked with the same letter were not significantly different.
Figure 8.
Bacterial endophytes isolated from a natural population of Thlaspi caerulescens exhibit high zinc tolerance.
To determine IC50 values for zinc, 5 µl of bacterial suspension at an OD600 of 0.2 were inoculated into 200 µl of KB broth supplemented with zinc at 0 to 20 mM. OD600 was measured after incubation, with continuous shaking, at 28°C for 48 hours, and IC50 values were calculated from the resulting dose–response curves. Values are means ± SE (n = 3). Diamonds = Hafna mine endophytes; triangles = plant pathogenic bacteria isolated from non-metal-accumulating crop plants; squares = mutants of P. syringae pv. maculicola generated in the present work. Abbreviations: DC3000, Pseudomonas syringae pv. tomato DC3000; X.c.c., Xanthomonas campestris pv. campestris 8004; P. cic 3109, Pseudomonas cichorii NCPPB3109; P. cic 907, P. cichorii NCPPB907; P. cic 943, P. cichorii NCPPB943; Ea286, Erwinia amylovora Ea286; B728a, P. syringae pv. syringae B728a.
Table 3.
Primers used in qRT-PCR.