Comparative Evaluation of Biochemical Changes in Tomato (Lycopersicon esculentum Mill.) Infected by Alternaria alternata and Its Toxic Metabolites (TeA, AOH, and AME)

Abstract

In the present study, we have evaluated the comparative biochemical defense response generated against Alternaria alternata and its purified toxins viz. alternariol (AOH), alternariol monomethyl ether (AME), and tenuazonic acid (TeA). The necrotic lesions developed due to treatment with toxins were almost similar as those produced by the pathogen, indicating the crucial role of these toxins in plant pathogenesis. An oxidative burst reaction characterized by the rapid and transient production of a large amount of reactive oxygen species (ROS) occurs following the pathogen infection/toxin exposure. The maximum concentration of hydrogen peroxide (H₂O₂) produced was reported in the pathogen infected samples (22.2-fold) at 24 h post inoculation followed by TeA (18.2-fold), AOH (15.9-fold), and AME (14.1-fold) in treated tissues. 3,3'- Diaminobenzidine staining predicted the possible sites of H₂O₂ accumulation while the extent of cell death was measured by Evans blue dye. The extent of lipid peroxidation and malondialdehyde (MDA) content was higher (15.8-fold) at 48 h in the sample of inoculated leaves of the pathogen when compared to control. The cellular damages were observed as increased MDA content and reduced chlorophyll. The activities of antioxidative defense enzymes increased in both the pathogen infected as well as toxin treated samples. Superoxide dismutase (SOD) activity was 5.9-fold higher at 24 h post inoculation in leaves followed by TeA (5.0-fold), AOH (4.1-fold) and AME (2.3-fold) treated leaves than control. Catalase (CAT) activity was found to be increased upto 48 h post inoculation and maximum in the pathogen challenged samples followed by other toxins. The native PAGE results showed the variations in the intensities of isozyme (SOD and CAT) bands in the pathogen infected and toxin treated samples. Ascorbate peroxidase (APx) and glutathione reductase (GR) activities followed the similar trend to scavenge the excess H₂O₂. The reduction in CAT activities after 48 h post inoculation demonstrate that the biochemical defense programming shown by the host against the pathogen is not well efficient resulting in the compatible host-pathogen interaction. The elicitor (toxins) induced biochemical changes depends on the potential toxic effects (extent of ROS accumulation, amount of H₂O₂ produced). Thus, a fine tuning occurs for the defense related antioxidative enzymes against detoxification of key ROS molecules and effectively regulated in tomato plant against the pathogen infected/toxin treated oxidative stress. The study well demonstrates the acute pathological effects of A. alternata in tomato over its phytotoxic metabolites.

Keywords: Alternaria alternata; Polymerase chain reaction (PCR); pathogen; phytotoxic metabolites; toxins.

FIGURE 1

Gel electrophoresis of PCR products with primers AAF2/AAR3 of DNA from fungal isolates. Lane 1, molecular weight markers (1 kb ladder); lanes 2-3, Alternaria alternata (341 bp).

FIGURE 2

(A) Isolation, purification, characterization, and infection of pathogen A. alternata on tomato plant. (B) Infection on detached leaves of tomato plant by the A. alternata and its toxins, showing the area of necrotic lesions and control leaves without necrotic lesions.

FIGURE 3

UV absorption spectra recorded for standard metabolites and those isolated from A. alternata using HPLC. (A) absorption peaks for standard TeA at 239.6 nm and 278.7 nm, (B) absorption peaks for standard AOH at 256.1, 288.2, and 337.0 nm, (C) absorption peaks for standard AME at 240.8, 283.4, and 327.5 nm, (D-F) absorbance peaks as evaluated from metabolites TeA, AOH, and AME extracted from fungal pathogen.

FIGURE 4

Effect on chlorophyll content in tomato plants treated with pathogen and their metabolites after 48 h. The results are expressed as the mean of three replicates and vertical bars indicate the ±SD of the mean.

FIGURE 5

(I) Hydrogen peroxide production in tomato leaves as visualized by DAB staining. (A) control leaf; (B) microscopic view; (C) AME treated leaf; (D) microscopic view; (E) AOH treated leaf; (F) microscopic view; (G) TeA treated leaf; (H) microscopic view; (I) pathogen infected leaf; (J) microscopic view. (II) Cell death assay by Evans blue staining. (A) control leaf; (B) microscopic view; (C) AME treated leaf; (D) microscopic view; (E) AOH treated leaf; (F) microscopic view; (G) TeA treated leaf; (H) microscopic view; (I) pathogen infected leaf; (J) microscopic view.

FIGURE 6

(A) Production of Hydrogen peroxide (H₂O₂)_, (B) Superoxide dismutase (SOD), and (C) Lipid peroxidation (MDA content) in tomato plant at different time intervals infected by A. alternata and treated with its toxins. The results are expressed as the mean of three replicates and vertical bars indicate the ±SD of the mean.

FIGURE 7

(A) Ascorbate peroxidase (APx), (B) Catalase (CAT), and (C) Glutathione reductase (GR) enzymatic activity in tomato plant at different time intervals infected by A. alternata and treated with its toxins. The results are expressed as the mean of three replicates and vertical bars indicate the ±SD of the mean.

FIGURE 8

(A) Protein pattern (SDS-PAGE) of soluble protein extracted from stressed tomato plants leaves infected by A. alternata and treated with its toxins as compared to the control. Arrows indicate the increase and decrease in intensity after infection. Isoenzyme profile of (B) Superoxide dismutase (SOD), and (C) Catalase (CAT) infected by A. alternata and treated with its toxins.