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, 14 (9), e0223216
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Sodium Alginate Potentiates Antioxidant Defense and PR Proteins Against Early Blight Disease Caused by Alternaria Solani in Solanum Lycopersicum Linn

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Sodium Alginate Potentiates Antioxidant Defense and PR Proteins Against Early Blight Disease Caused by Alternaria Solani in Solanum Lycopersicum Linn

Priya Dey et al. PLoS One.

Abstract

The use of biopolymers as elicitors in controlling plant diseases is gaining momentum world-wide due to their eco-friendly and non-toxic nature. In the present study, we have used an algal biopolymer (sodium alginate) and tested its applicability as an elicitor in inducing resistance factors against Alternaria solani, which causes early blight disease in Solanum lycopersicum (tomato plant). We have pre-treated tomato plants with different concentrations of sodium alginate (0.2%, 0.4%, and 0.6%) before A. solani infection. We found that sodium alginate has effectively controlled the growth of A. solani. In addition, a significant increase in the expression levels of SOD was observed in response to pathogen infection. The increased protease inhibitors activity further suggest that sodium alginate restrict the development of A. solani infection symptoms in tomato leaves. This corroborates well with the cell death analysis wherein increased sodium alginate pre-treatment results in decreased cell death. Also, the expression profile analyses reveal the induction of genes only in sodium alginate-pretreated tomato leaves, which are implicated in plant defense mechanism. Taken together, our results suggest that sodium alginate can be used as an elicitor to induce resistance against A. solani in tomato plants.

Conflict of interest statement

The affiliation of Acme Progen Biotech (India) Pvt. Ltd does not alter our adherence to PLOS ONE policies on sharing data and materials.

Figures

Fig 1
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
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
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
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
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
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
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
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
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
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
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
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.

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Grant support

Acme Progen Biotech (India) Pvt. Ltd has provided support in the form of RT-PCR study design, data collection and analysis.
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