Benzoic Acid and Its Hydroxylated Derivatives Suppress Early Blight of Tomato (Alternaria solani) via the Induction of Salicylic Acid Biosynthesis and Enzymatic and Nonenzymatic Antioxidant Defense Machinery

Abstract

Tomato early blight, caused by Alternaria solani, is a destructive foliar fungal disease. Herein, the potential defensive roles of benzoic acid (BA) and two of its hydroxylated derivatives, ρ-hydroxybenzoic acid (HBA), and protocatechuic acid (PCA) against A. solani were investigated. All tested compounds showed strong dose-dependent fungistatic activity against A. solani and significantly reduced the disease development. Benzoic acid, and its hydroxylated derivatives, enhanced vegetative growth and yield traits. Moreover, BA and its derivatives induce the activation of enzymatic (POX, PPO, CAT, SlAPXs, and SlSODs) and non-enzymatic (phenolics, flavonoids, and carotenoids) antioxidant defense machinery to maintain reactive oxygen species (ROS) homeostasis within infected leaves. Additionally, BA and its hydroxylated derivatives induce the accumulation of salicylic acid (SA) and its biosynthetic genes including isochorismate synthase (SlICS), aldehyde oxidases (SlAO1 and SlAO2), and phenylalanine ammonia-lyases (SlPAL1, SlPAL2, SlPAL3, SlPAL5, and SlPAL6). Higher SA levels were associated with upregulation of pathogenesis-related proteins (SlPR-1, SlPR1a2, SlPRB1-2, SlPR4, SlPR5, SlPR6), nonexpressor of pathogenesis-related protein 1 (SlNPR1), and salicylic acid-binding protein (SlSABP2). These findings outline the potential application of BA and its hydroxylated derivatives as a sustainable alternative control strategy for early blight disease and also deciphering the physiological and biochemical mechanisms behind their protective role.

Keywords: Alternaria; antioxidant; benzoic acid; early blight; protocatechuic acid; reactive oxygen species (ROS); salicylic acid; tomato; ρ-hydroxybenzoic acid.

Figure 1

Chemicals and experimental design used in this study. (A–C) Chemical structure of benzoic acid (BA), ρ-hydroxybenzoic acid (HBA), and protocatechuic acid (PCA), respectively. Molecular weight/molar mass (g·mol⁻¹) is mentioned between parentheses under the chemical formula of each compound. (D) Experimental design, application times, and sampling points used in this study. Growth development stages are corresponding to the phenological growth stages on BBCH-scale [32,33].

Figure 2

In vitro antifungal activity of benzoic acid (BA) and its hydroxylated derivatives (ρ-hydroxybenzoic acid and protocatechuic acid) against Alternaria solani. (A) Antifungal activity of BAand its hydroxylated derivatives A. solani. (B) Mycelial radial growth inhibition (%) of A. solani after the treatment with BA and its hydroxylated derivatives. Values represent the means ± standard deviation (means ± SD) of six biological replicates (n = 6). Different letters indicate statistically significant differences between treatments (p < 0.05).

Figure 3

Probit regression analysis of the inhibition effects of benzoic acid (BA) and its hydroxylated derivatives (ρ-hydroxybenzoic acid and protocatechuic acid) against Alternaria solani. (A) The positive control (difenoconazole fungicide), (B) benzoic acid, (C) ρ-hydroxybenzoic acid, and (D) protocatechuic acid. Blue dots present the mean of six replicates (n = 6). The dose-response regression lines are presented as black solid lines. The 95% CI (confidence intervals) for the estimated regression are light-blue-shaded and edged with dotted lines. Regression equations, Cox, and Snell R2, Nagelkerke R2, and p-value based on the F test (p < 0.05) were also obtained and presented within the graphs. The experiment was repeated twice with similar results.

Figure 4

Benzoic acid and its hydroxylated derivatives, ρ-hydroxybenzoic acid (HBA) and protocatechuic acid (PCA), decrease the disease evaluation of early blight caused on tomatoes by Alternaria solani under greenhouse conditions. (A) Typical symptoms of early blight disease on tomato leaves at 7 days post-treatment (dpt) with 100 ppm of BA or one of its hydroxylated derivatives. (B) Disease progress curves of early blight disease on tomato leaves after the treatment with BA or one of its hydroxylated derivatives. (C) The area under disease progress curve (AUDPC) of early blight disease on tomato leaves after the treatment with BA or one of its hydroxylated derivatives. Values represent the means ± standard deviation (means ± SD) of six biological replicates (n = 6). Different letters indicate statistically significant differences between treatments (p < 0.05).

Figure 5

Benzoic acid (BA) and its hydroxylated derivatives, ρ-hydroxybenzoic acid (HBA) and protocatechuic acid (PCA), improve the growth variables and enhance fruit yield and its components of Alternaria solani-infected tomato plants under greenhouse conditions. (A) Plant height (cm), (B) number of leaves plant⁻¹, (C) total area per leaf (cm²), (D) shoot fresh weight (g plant⁻¹), (E) shoot dry weight (g plant⁻¹), (F) number of flowers plant ⁻¹, (G) number of fruits plant ⁻¹, (H) fruit yield (Kg plant ⁻¹), and (I) fruit yield increase over control (%). Values represent the means ± standard deviation (means ± SD) of six biological replicates (n = 6). Different letters indicate statistically significant differences between treatments (p < 0.05). Mock: 0.2% dimethyl sulfoxide (DMSO)-treated (mock control), FC: difenoconazole-treated (fungicide), BA: benzoic acid-treated, HBA: ρ-hydroxybenzoic acid-treated, and PCA: protocatechuic acid-treated.

Figure 6

Benzoic acid (BA) and its hydroxylated derivatives, ρ-hydroxybenzoic acid (HBA) and protocatechuic acid (PCA), enrich the biochemical profile of Alternaria solani-infected tomato plants under greenhouse conditions. (A) Chlorophyll a content (mg g⁻¹ FW), (B) chlorophyll b content (mg g⁻¹ FW), (C) total carotenoids content (mg g⁻¹ FW), (D) total soluble phenolics (mg GAE g⁻¹ FW), and (E) total soluble flavonoids (mg RE g⁻¹ FW). Values represent the means ± standard deviation (means ± SD) of six biological replicates (n = 6). Different letters indicate statistically significant differences between treatments (p < 0.05).

Figure 7

Benzoic acid (BA) and its hydroxylated derivatives, ρ-hydroxybenzoic acid (HBA) and protocatechuic acid (PCA), alleviate oxidative stress and enhance the antioxidant defense-related enzymes in Alternaria solani-infected leaves under greenhouse conditions. (A) In situ histochemical localization of hydrogen peroxide (H₂O₂) using DAB-based staining at 72 hpt with BA or one of its hydroxylated derivatives, (B) H₂O₂ content (nmol g⁻¹ FW) after the treatment with BA or one of its hydroxylated derivatives, (C) in situ histochemical visualization of superoxide anion (O₂^•−) using NBT-based staining at 72 hpt with BA or one of its hydroxylated derivatives, (D) O₂^•− content (nmol g⁻¹ FW) after the treatment with BA or one of its hydroxylated derivatives, (E) peroxidase activity (μM Tetraguaiacol g⁻¹ FW min⁻¹), (F) polyphenol oxidase activity (Arbitrary units), and (G) catalase activity (μM H₂O₂ g⁻¹ FW min⁻¹). Values represent the means of six biological replicates (n = 6), while whiskers reflect the standard deviation (means ± SD). (H–K) Relative gene expression of cytosolic ascorbate peroxidase 1 (SlAPX1), cytosolic ascorbate peroxidase 2 (SlAPX2), superoxide dismutase [Cu-Zn] 1 (SlCuSOD1), and iron superoxide dismutase (SlFeSOD), respectively. Values represent the means ± standard deviation (means ± SD) of five biological replicates (n = 5). Different letters indicate statistically significant differences between treatments (p < 0.05). Mock: 0.2% dimethyl sulfoxide (DMSO)-treated (mock control), FC: difenoconazole-treated (fungicide), BA: benzoic acid-treated, HBA: ρ-hydroxybenzoic acid-treated, and PCA: protocatechuic acid-treated.

Figure 8

Benzoic acid (BA) and its hydroxylated derivatives, ρ-hydroxybenzoic acid (HBA) and protocatechuic acid (PCA), induce the salicylic acid (SA) biosynthesis pathway in Alternaria solani-infected leaves under greenhouse conditions. (A) Schematic representation of the SA biosynthesis pathway. (B–E) Endogenous content (ng·g⁻¹ FW) of _L-phenylalanine, t-cinnamic acid (tCA), BA, and SA, respectively, as detected in tomato leaves after the treatment with BA or one of its hydroxylated derivatives using GC-MS. (F) Relative gene expression of isochorismate synthase (SlICS), (G,H) relative gene expression of two aldehyde oxidase (SlAO1 and SlAO2, respectively), and (I–M) relative gene expression of five phenylalanine ammonia-lyases (SlPAL1, SlPAL2, SlPAL3, SlPAL5, and SlPAL6, respectively). Values represent the means ± standard deviation (means ± SD) of five biological replicates (n = 5). Different letters indicate statistically significant differences between treatments (p < 0.05). Mock: 0.2% dimethyl sulfoxide (DMSO)-treated (mock control), FC: difenoconazole-treated (fungicide), BA: benzoic acid-treated, HBA: ρ-hydroxybenzoic acid-treated, and PCA: protocatechuic acid-treated.

Figure 9

Benzoic acid (BA) and its hydroxylated derivatives, ρ-hydroxybenzoic acid (HBA) and protocatechuic acid (PCA), induce the expression of pathogenesis-related proteins in A. solani-infected leaves under greenhouse conditions. (A–F) Relative gene expression of six pathogenesis-related proteins (PR) included SlPR-1, SlPR1a2, SlPRB1-2, SlPR4, SlPR5, and SlPR6, respectively. (G) Relative gene expression of nonexpressor of pathogenesis-related protein 1 (SlNPR1). (H) Relative gene expression of the salicylic acid-binding protein (SlSABP2). Values represent the means ± standard deviation (means ± SD) of five biological replicates (n = 5). Different letters indicate statistically significant differences between treatments (p < 0.05). Mock: 0.2% dimethyl sulfoxide (DMSO)-treated (mock control), FC: difenoconazole-treated (fungicide), BA: benzoic acid-treated, HBA: ρ-hydroxybenzoic acid-treated, and PCA: protocatechuic acid-treated.

Figure 10

Schematic representation of a hypothetical model for the potential defensive roles of benzoic acid (BA) and its hydroxylated derivatives, ρ-hydroxybenzoic acid (HBA) and protocatechuic acid (PCA), in tomato against A. solani. Briefly, BA and its hydroxylated derivatives attenuate the harmful effect of A. solani on tomato plants via a complex multilayered defense system that includes: (i) BA and its hydroxylated derivatives (HBA and PCA) might have potent antifungal activity against A. solani. (ii) BA and its derivatives neutralize and maintain ROS homeostasis within infected leaves via the induction of multilayered antioxidative machinery that includes enzymatic (include POX, PPO, CAT, APXs, and SODs) and non-enzymatic (phenolics, flavonoids, and carotenoids) antioxidant defenses. (iii) BA and its hydroxylated derivatives induce the SA-mediated pathway, which is implicated in ROS homeostasis and associated with defense response for fungal phytopathogens to reduce disease development (disease severity and AUDPC). The SA-mediated defense relies on two major components. The first component includes SA and its biosynthetic genes (PALs, AOs, and ICS), whereas the second component includes pathogenesis-related proteins (SlPR-1, SlPR1a2, SlPRB1-2, SlPR4, SlPR5, SlPR6), nonexpressor of pathogenesis-related protein 1 (SlNPR1), and salicylic acid-binding protein (SlSABP2). Solid lines with arrows represent positive reactions, whereas blunt-ended dotted lines indicate negative regulation. For more details and the full names and abbreviations, see the main text.