Fig 1.
Manifestation of disease symptoms on tomato leaves on day 8 after A. solani inoculation.
A: Untreated control leaves; B: Untreated S. lycopersicum leaves inoculated with A. solani; C: S. lycopersicum leaves pretreated with 0.2% sodium alginate; D: S. lycopersicum leaves pretreated with 0.2% sodium alginate and inoculated with A. solani; E: S. lycopersicum leaves pretreated with 0.4% sodium alginate; F: S. lycopersicum leaves pretreated with 0.4% sodium alginate and inoculated with A. solani; G: S. lycopersicum leaves pretreated with 0.6% sodium alginate; H: S. lycopersicum leaves pretreated with 0.6% sodium alginate and inoculated with A. solani. ds: disease symptom (early blight).
Fig 2.
Fluorescence microscopy analysis of A. solani colonization within tomato leaves on day 8.
A: Untreated control leaves; B: Untreated S. lycopersicum leaves inoculated with A. solani; C: S. lycopersicum leaves pretreated with 0.2% sodium alginate; D: S. lycopersicum leaves pretreated with 0.2% sodium alginate and inoculated with A. solani; E: S. lycopersicum leaves pretreated with 0.4% sodium alginate; F: S. lycopersicum leaves pretreated with 0.4% sodium alginate and inoculated with A. solani; G: S. lycopersicum leaves pretreated with 0.6% sodium alginate; H: S. lycopersicum leaves pretreated with 0.6% sodium alginate and inoculated with A. solani. cd: Cell death.
Fig 3.
SEM analysis of A. solani infection within tomato leaves on day 8 (500× magnification).
A: Untreated control leaves; B: Untreated S. lycopersicum leaves inoculated with A. solani; C: S. lycopersicum leaves pretreated with 0.2% sodium alginate; D: S. lycopersicum leaves pretreated with 0.2% sodium alginate and inoculated with A. solani; E: S. lycopersicum leaves pretreated with 0.4% sodium alginate; F: S. lycopersicum leaves pretreated with 0.4% sodium alginate and inoculated with A. solani; G: S. lycopersicum leaves pretreated with 0.6% sodium alginate; H: S. lycopersicum leaves pretreated with 0.6% sodium alginate and inoculated with A. solani. cd: Cell death.
Fig 4.
Histochemical analysis of O2− production.
The O2− production around the site of cell death in the tomato leaves was detected by NBT staining. Compared with the control leaves (water control), sodium alginate-pretreated leaves infected with A. solani showed a time-dependent increase in the dark blue precipitation due to the reaction of NBT with O2−. A1–A5: Untreated control leaves; B1–B5: Untreated S. lycopersicum leaves inoculated with A. solani; C1–C5: S. lycopersicum leaves pretreated with 0.2% sodium alginate; D1–D5: S. lycopersicum leaves pretreated with 0.2% sodium alginate and inoculated with A. solani; E1–E5: S. lycopersicum leaves pretreated with 0.4% sodium alginate; F1–F5: S. lycopersicum leaves pretreated with 0.4% sodium alginate and inoculated with A. solani; G1–G5: S. lycopersicum leaves pretreated with 0.6% sodium alginate; H1–H5: S. lycopersicum leaves pretreated with 0.6% sodium alginate and inoculated with A. solani.
Fig 5.
Histochemical analysis of H2O2 production.
H2O2 production around the site of cell death in tomato leaves was assessed using DAB staining. Compared with the untreated control leaves, sodium alginate-pretreated leaves infected with A. solani showed a time-dependent increase in H2O2 accumulation, evident as a reddish-brown stain formed by the reaction of DAB with H2O2. A1–A5: Untreated control leaves; B1–B5: Untreated S. lycopersicum leaves inoculated with A. solani; C1–C5: S. lycopersicum leaves pretreated with 0.2% sodium alginate; D1–D5: S. lycopersicum leaves pretreated with 0.2% sodium alginate and inoculated with A. solani; E1–E5: S. lycopersicum leaves pretreated with 0.4% sodium alginate; F1–F5: S. lycopersicum leaves pretreated with 0.4% sodium alginate and inoculated with A. solani; G1–G5: S. lycopersicum leaves pretreated with 0.6% sodium alginate; H1–H5: S. lycopersicum leaves pretreated with 0.6% sodium alginate and inoculated with A. solani.
Fig 6.
Time course study of the levels of (A) H2O2 and (B) lipid peroxidation. Time course study of the levels of (A) H2O2 and (B) lipid peroxidation in tomato leaves pretreated with water or sodium alginate followed by A. solani infection. The levels were measured in the leaves after the following treatments: Control, A. solani inoculation; 0.2% sodium alginate pretreatment, 0.2% sodium alginate pretreatment followed by A. solani infection; 0.4% sodium alginate pretreatment, 0.4% sodium alginate pretreatment followed by A. solani infection; 0.6% sodium alginate pretreatment, 0.6% sodium alginate pretreatment followed by A. solani infection. The values represent the mean ± standard error of the mean (n = 3). The asterisks (*, **, ***) indicate that the mean values are significantly different from those of the control at the same time point; ***P < 0.001, **P < 0.01 and *P < 0.05, ns: not significant based on Student’s t-test.
Fig 7.
Time course study of the levels of (A) CAT, (B) GPX, and (C) SOD. Time course study of the levels of (A) CAT, (B) GPX, and (C) SOD in tomato leaves pretreated with sodium alginate followed by A. solani infection. The levels were measured in tomato leaves after the following treatments: Control, A. solani inoculation; 0.2% sodium alginate pretreatment, 0.2% sodium alginate pretreatment followed by A. solani infection; 0.4% sodium alginate pretreatment, 0.4% sodium alginate pretreatment followed by A. solani infection; 0.6% sodium alginate pretreatment, 0.6% sodium alginate pretreatment followed by A. solani infection The values represent the mean ± standard error of the mean (n = 3). The asterisks (*, **, ***) indicate that the mean values are significantly different from those of the control at the same time point; ***P < 0.001, **P < 0.01, and *P < 0.05, ns: not significant based on Student’s t-test.
Fig 8.
GPX activity revealed by guaiacol and H2O2 staining in sodium alginate-pretreated tomato leaves infected with A. solani.
Each lane contains 40 μg of enzyme sample extracted from tomato leaves harvested at different time points after the following treatments: Lane 1: Control; Lane 2: A. solani inoculation; Lane 3: 0.4% sodium alginate pretreatment; Lane 4: 0.4% sodium alginate pretreatment followed by A. solani inoculation; Lane 5: 0.6% sodium alginate pretreatment; Lane 6: 0.6% sodium alginate pretreatment followed by A. solani infection. A: 12 h; B: 24 h; C: 36 h; D: 48 h. The (-) and (+) signs indicate the direction of migration of enzyme samples.
Fig 9.
SOD activity revealed by NBT and riboflavin staining in sodium alginate-pretreated tomato leaves infected with A. solani.
Each lane contains 40 μg of enzyme sample extracted from tomato leaves harvested at different time points after the following treatments: Lane 1: Control; Lane 2: A. solani inoculation; Lane 3: 0.2% sodium alginate pretreatment; Lane 4: 0.2% sodium alginate pretreatment followed by A. solani inoculation; Lane 5: 0.4% sodium alginate pretreatment; Lane 6: 0.4% sodium alginate pretreatment followed by A. solani infection; Lane 7: 0.6% sodium alginate pretreatment; Lane 8: 0.6% sodium alginate pretreatment followed by A. solani infection. A: 12 h; B: 24 h; C: 36 h; D: 48 h. The (-) and (+) signs indicate the direction of migration of enzyme samples.
Fig 10.
Changes in A) trypsin and B) chymotrypsin inhibitory activities. Changes in A) trypsin and B) chymotrypsin inhibitor activities in tomato leaves harvested at different time points after the following treatments: Control, A. solani inoculation, 0.2% sodium alginate pretreatment, 0.2% sodium alginate pretreatment followed by A. solani infection; 0.4% sodium alginate pretreatment, 0.4% sodium alginate pretreatment followed by A. solani infection; 0.6% sodium alginate pretreatment, 0.6% sodium alginate pretreatment followed by A. solani infection The values represent the mean ± standard error of the mean (n = 3). The asterisks (*, **, ***) indicate that the mean values are significantly different from those of the control at the same time point; ***P < 0.001, **P < 0.01, and *P < 0.05, ns: not significant based on Student’s t-test.
Fig 11.
Analysis of cell death in tomato leaves.
The tomato leaves for cell death analysis were harvested on day 8 after the following treatments: Control, A. solani inoculation; 0.2% sodium alginate pretreatment, 0.2% sodium alginate pretreatment followed by A. solani infection; 0.4% sodium alginate pretreatment, 0.4% sodium alginate pretreatment followed by A. solani infection; 0.6% sodium alginate pretreatment, 0.6% sodium alginate pretreatment followed by A. solani infection. The values represent the mean ± standard error of the mean (n = 3). The asterisks (*, **, ***) indicate that the mean values are significantly different from those of the control at the same time point; ***P < 0.001, **P < 0.01, and *P < 0.05, ns: not significant based on Student’s t-test.
Fig 12.
Relative expression of A) SA-dependent, B) JA-dependent, and C) ET-dependent defense genes. Relative expression of A) SA-dependent, B) JA-dependent, and C) ET-dependent defense genes in tomato leaves harvested on day 8 after the following treatments: Control, A. solani inoculation; 0.4% sodium alginate pretreatment, 0.4% sodium alginate pretreatment followed by A. solani infection; 0.6% sodium alginate pretreatment, 0.6% sodium alginate pretreatment followed by A. solani inoculation. The relative gene expressions of SA-dependent NPR1, PR2, and PR4 transcripts; JA-dependent LoxD transcript; and ET-dependent ACO1 transcript were calculated using the comparative Ct method. One-way ANOVA was used to determine whether the sample means differed significantly at ***P < 0.001, **P < 0.01, or *P < 0.05. The error bars represent the 95% confidence interval calculated from ANOVA. The values represent means (of three replicates) ± standard error of the mean.