The present study reports the variable antioxidant profile resulting from inducers/elicitors on mustard varieties against Alternaria brassicicola. An experiment was conducted with four inducers/elicitors viz., Benzothiadiazole (BTH), Hydrogen peroxide, Jasmonic Acid and Salicylic acid at three different concentrations to evaluate their effect against Alternaria leaf blight of mustard in four varieties viz., TBM-204, Bullet, B-54 and B-9. The pathogen A. brassicicola was inoculated at 15 DAS. An attempt was made to study the underline biochemical changes which may have their influence on induced resistance. The component of first line defense mechanism, catalase (CAT) and non-enzymatic antioxidants like ascorbic acid were found to be more prevalent in the plants treated with BTH followed by Jasmonic acid, Salicylic acid and Hydrogen peroxide. Among the varieties tested, these enzymes were more prevalent in TBM-204 and Bullet and less prevalent in B-54 and B-9.

Mustard, Alternaria, catalase, ascorbic acid.

Brassica juncea, commonly known as Indian mustard, is a major cultivated brassica crop in north-west India, followed by limited cultivation of B. napus and B. rapa for vegetable oil production. Among oilseed crops, rapeseed mustard (B. juncea) occupies an important position globally in terms of production and consumption. India is the fourth largest producer of oilseeds in the world and stands second in Asia. Whereas, West Bengal stands fifth position in which area and production is 0.6 M ha and 0.7M t, respectively, with an average productivity of 1212 kg ha-1 (Ministry of Agriculture and Farmers Welfare, 2019). It is generally used as a vegetable, and grown mostly for seeds, which yield essential oil and condiment. Although, India is one of the leading oilseed producing countries of the world, it still not able to meet the requirement for its vast population. To meet the growing demand and make India self-sufficient for edible oils, productivity of the oilseed crops should be increased since the possibility of increasing land under oilseed crops is very limited (Economic survey, 2020-21). The development of high yielding varieties along with the new improved production technology leads to increase in production and productivity of mustard but the gap between potential yield and actual yields are broaden due to the different biotic and abiotic factors. Among them, fungal diseases of oilseed   Brassica are prevalent in India. The severe attack of these diseases deteriorates the quality and quantity of the seed and oil content. Among the diseases, Alternaria blight caused by Alternaria brassicicola (Berk.) Sacc. is the major constraint in production and destructive lethal disease of rapeseed mustard, reported from all the continents of the world causing 47% yield losses (Kolte, 1986) that may range up to 15-71% in productivity and 14.6-36 % in oil content (Meena et al., 2010). Apart from indiscriminate use of the pesticides, there is a need to develop strategies providing durable resistance, giving protection for a long time over a broad geographical area. Among such strategies, systemic acquired resistance (SAR) is an example of a defense mechanism offering long lasting disease resistance against a broad spectrum of pathogens and is promising for sustainable crop production in the future (Song and Goodman 2001). Therefore, the following study was conducted against Alternaria blight of mustard by using inducers.

A. Isolation and purification of A. brassicicola

Different infected plant parts, viz., leaves, pods and stems of infected mustard plants were collected in paper bags and brought to the laboratory for isolation of pathogen. The diseased portion of infected plant parts along with healthy portion were cut into bits of 8–10 mm, and surface sterilized with 1% sodium hypochlorite (NaOCl) solution for 30 sec, washed thrice with sterilized distilled water and were blot dried. Thereafter three-four bits were placed in each petriplate containing Potato Dextrose Agar (PDA) medium. The inoculated plates were incubated in BOD incubator at 22 ± 2°C and monitored at regular intervals and initial growth of the pathogen was sub-cultured into agar slants.

Pure culture of Alternaria was obtained by single spore isolation method. The spore suspension was prepared by scraping the surface of sporulating cultures and was added to lukewarm molten water agar and dispensed into sterilized petri plates. The petri plates were gently swirled for even distribution of the spores and kept for incubation at 25 ± 2°C for 12 h. Individual germinated spore, spaced out clearly was located on inverted water agar plates and marked with a glass marking pencil on the outside of the bottom dish using a compound microscope. Each marked spore was aseptically transferred into separate PDA slants. The culture was maintained and sub-cultured for further studies.  

B. Preparation of inoculum spray

Four mustard varieties, viz., TBM-204, Bullet, B-54and B-9 were collected from university instructional farm. The plantsof these varieties were raised in plastic pots (13 cm × 13 cm) containing 3 kg soil (sandyloam soil: FYM 3:1 w/w) in the net house, Department of Plant Pathology. For inoculation, A. brassicicola conidial suspension was prepared from nine-day old cultures by flooding the surface of the Petriplates with sterile distilled water and scraping the surface gently with a glass rod. The suspension was filtered through two layers of cheese cloth to eliminate mycelial fragments. Inoculum consisted of a conidial suspension adjusted to 1 ×104 conidia ml-1 using a haemocytometer. The plants were sprayed with freshly prepared conidial suspension using an atomizer at 15 DAS (Vishunavat and Kolte 2008).

C. Standardization of inducer concentrations

Benzothiadiazole (BTH) [S-methylbenzo-1, 2, 3-thiadiazole-7-carbothiate], hydrogen peroxide (H2O2), Jasmonic Acid (JA) and Salicylic Acid (SA) were used for seed treatment as inducers. Concentrations of these inducers were categorized as three levels viz., low, medium and high concentrations and standardized as BTH @ 0.25 mM, 0.75 mM, 1.5 mM, H2O2@ 1%, 2%, 3%, JA @ 1 mM, 2.5 mM, 4 mM and SA @ 0.5 mM, 1 mM, 2 mM.

Seed treatment was given by the standardize inducers for 1 h before sowing. For control treatment, seeds were soaked in sterilized distilled water. Spore suspension of the isolated pathogen was artificially inoculated at 15 DAS in three replications. The plants were covered with moist chamber consisting of transparent polythene sheet so that plant could maintain photosynthesis ability. After five days of inoculation, Alternaria blight incidence was visualized on leaves. The leaf samples were collected for three times at 15 DAS and 20 DAS (both uninoculated and inoculated samples) for biochemical analysis and are stored at   -20°C.

Biochemical analysis. Leaf samples of different mustard genotypes were analyzed for enzymatic antioxidant (Catalase) and non-enzymatic antioxidant (Ascorbic acid). For assay of catalase activity, fresh leaf sample (0.3 g) was extracted with 0.1 M phosphate buffer (pH 7.5) containing polyvinylpolypyrrolidone (PVP) and Triton X. The homogenate was centrifuged at 10,000 rpm for 30 min and supernatant was collected for enzyme assay. Catalase activity was determined by monitoring the disappearance of H2O2 at 240 nm (ε = 40 Mm-1 cm-1) (Aebi, 1984). The assay was performed using 2.8 ml of 100 mM phosphate buffer, 0.1 ml of H2O2 (1%) and 0.1 ml of plant extract. The molar extinction coefficient of hydrogen peroxide at 240 nm was taken as 0.04 sq. cm/μ mole. Enzyme activity was expressed as μ moles of hydrogen peroxide degraded/min/mg of protein. 

Ascorbic acid content was measured by using modified method of Davies and Masten (1991). Leaf samples were extracted using 4% of oxalic acid, using chilled mortar and pestle. Then homogenate was centrifuged at 10,000 rpm at 4°C for 30 min. One ml of supernatant was added with 2 ml of 1.72 mM 2, 6-dichlorophenolindophenol (2, 6-DCPIP) dye indicator in 3 ml cuvette and was measured at 518 nm immediately after mixing. Content of ascorbic acid was expressed as the mg of ascorbic acid per 100 grams of fresh sample.

Statistical analysis of data was performed by Analysis of Variance (ANOVA) using OP STAT software.

In order to assess the effect of inducers on biochemical responses, four different mustard varieties (TBM-204, Bullet, B-54 and B-9) were investigated against Alternaria blight infection.

Catalase: Study on catalase (CAT), indicated that the activity was significantly decreased in all the varieties with regard to inducers and biotic stress in comparison to control. All the treatments in all the four varieties tested were depicting decreasing trend to overcome the infection of the pathogen. 

Inducers tested at all concentrations significantly increased the amount of catalase compared to control in all the varieties. Significantly high catalase activity was found in BTH followed by JA, SA and least activity was found in H2O2. With the increase in concentration the catalase activity and the percent increase over control was also increased (Tables 1-4).

Interaction between inducers and varieties revealed that Catalase activity was observed to be lesser in infected leaves as compared to the healthy one and the varieties B-54 and B-9 expressed less catalase activity than the varieties TBM-204 and Bullet which can consider having low resistance against Alternaria blight (Table 5, Fig. 1).

Above findings are strongly supported by the report of Subhani et al. (2018). The decline in catalase activity is regarded as a general response to many stresses (Jung, 2004; Pan  et al., 2006; Liu et al., 2008). The reduction of CAT activity is due to the inhibition of enzyme synthesis or change in the assembly of enzyme subunits under stress conditions. Decrease in catalase activity was also an indication of scavenging the hydrogen peroxide activity after infection.

Result indicate that the high levels of catalase played an important role in reducing damage caused by pathogen by dismutating O2• − and catalyzing H2O2 in TBM-204 and Bullet. However, in B-54 and B-9 varieties an uncontrollable production of reactive oxygen species may couple with lesser activity of catalase created the oxidative stress, and led to the membrane damage and finally appeared as a symptom in plant. 

Ascorbic acid: Study on non-enzymatic antioxidant revealed that the level of ascorbate was gradually increased significantly over their respective untreated controls with the increasing stress period in all the varieties tested.

In variety TBM-204, ascorbic acid content at 15 DAS was ranged from 34.95 – 83.25 mg/100 g FW while it was 35.54 - 119.61 and 39.85 - 125.54mg/100 g FW at 20 DAS and 5 DAI respectively (Table 6). In variety Bullet, ascorbic acid content at 15 DAS was ranged from 30.40 - 42.22mg/100 g FW while it was 31.31 - 63.84 and 33.84 - 94.95mg/100 g FW at 20 DAS and 5 DAI respectively (Table 7). In variety B-54, ascorbic acid content was ranged from 27.83 - 39.08, 30.40 - 43.84 and 31.23 - 84.67mg/100 g FW at 15 DAS, 20 DAS and 5 DAI respectively (Table 8). And it was 16.40 - 22.19, 22.70 – 35.54 and 24.67 – 72.10 at 15 DAS, 20 DAS and 5 DAI respectively (Table 9) in variety B-9. In the present study, varieties TBM-204 and Bullet in which maximum ascorbic acid (125.54 and 94.95 mg/100 g FW) under Alternaria blight infection were expected to have highest resistance, while varieties B-54 and B-9which possessed comparatively low ascorbic acid (84.67 and 72.10 mg/100 g FW) were considered to have low resistance against Alternaria blight (Table 10, Fig. 2).

Among the four inducers tested, BTH at high concentration showed high amount of ascorbic acid followed by medium and low. Later jasmonic acid showed significant high levels of ascorbate followed by salicylic acid and H2O2 in all the days of sampling.

Table 1: Effect of inducers at different concentrations on the Catalase activity (μmol of H2O2/min/mg of protein) in the mustard (TBM-204) against A. brassicicola.

DAS: Days after sowing; DAI: Days after pathogen inoculation; L: Low; M: Medium; H: High

Table 2: Effect of inducers at different concentrations on the Catalase activity (μmol of H2O2/min/mg of protein) in the mustard (Bullet) against A. brassicicola.

DAS: Days after sowing; DAI: Days after pathogen inoculation; L: Low; M: Medium; H: High

Table 3: Effect of inducers at different concentrations on the Catalase activity (μmol of H2O2/min/mg of protein) in the mustard (B-54) against A. brassicicola.

DAS: Days after sowing; DAI: Days after pathogen inoculation; L: Low; M: Medium; H: High

Table 4: Effect of inducers at different concentrations on the Catalase activity (μmol of H2O2/min/mg of protein) in the mustard (B-9) against A. brassicicola.

DAS: Days after sowing; DAI: Days after pathogen inoculation; L: Low; M: Medium; H: High

Table 5: Effect of inducers at different concentrations on the Catalase activity (μmol of H2O2/min/mg of protein) in the mustard varieties against A. brassicicola

Fig. 1. Effect of inducers at different concentrations on the Catalase activity in the mustard varieties against A. brassicicola.

Table 6: Effect of inducers at different concentrations on the ascorbic acid content (mg/100 g FW) in the mustard (TBM-204) against A. brassicicola

    DAS: Days after sowing; DAI: Days after pathogen inoculation; L: Low; M: Medium; H: High

Table 7: Effect of inducers at different concentrations on the ascorbic acid content (mg/100 g FW) in the mustard (Bullet) against A. brassicicola.

    DAS: Days after sowing; DAI: Days after pathogen inoculation; L: Low; M: Medium; H: High

Table 8: Effect of inducers at different concentrations on the ascorbic acid content (mg/100 g FW) in the mustard (B-54) against A. brassicicola.

DAS: Days after sowing; DAI: Days after pathogen inoculation; L: Low; M: Medium; H: High

Table 9: Effect of inducers at different concentrations on the ascorbic acid contentmg/100 g FW) in the mustard (B-9) against A. brassicicola.

(DAS: Days after sowing; DAI: Days after pathogen inoculation; L: Low; M: Medium; H: High

Table 10: Effect of inducers at different concentrations on the ascorbic acid content (mg/100 g FW) in the mustard varieties against A. brassicicola.

Fig. 2. Effect of inducers at different concentrations on the ascorbic acid content in the mustard varieties against A. brassicicola.

Above findings are strongly supported by the report of Mallick et al. (2017). Ascorbic acid, acts as powerful antioxidant in tissues and an enhanced level has also been observed in stressed plants as a resistance index against the pathogen (Gupta et al., 2012). When the ROS level increases in plants that are exposed to stress, enhanced production of non enzymatic antioxidants in plant cells like ascorbic acid will play a crucial role in minimizing ROS induced oxidative stress (Gill and Tuteja 2010). In this study it is indicated that the less or reduced accumulation of ascorbic acid in B-54 and B-9 varieties favours the oxidative stress and invasion of pathogen deep into the host and finally cause the injury or symptoms in the plant. Necrotrophs appear to stimulate ROS production in the infected tissue to induce cell death that facilitates subsequent infection (Govrin and Levine 2000). This might suggest that the redox state of acorbate could be a defensive response in resistant varieties TBM-204 and Bullet against Alternaria. Moreover, the changes in total concentration and redox state of ascorbic acid can regulate the expression of pathogenesis-related (PR) proteins (Foyer and Noctor 2005), induce the accumulation of phytoalexins (De Gara et al., 2003).

The activation of antioxidant mechanism of the plant, where both, enzymatic antioxidant like catalase and non-enzymatic antioxidant like ascorbic acid were comparatively found to be more in TBM-204 and Bullet through which we can consider them as resistant varieties. The high levels of catalase played an important role in reducing the damage caused by the pathogen by dismutating O2•− and catalyzing H2O2 in resistant in coordination with the non-enzymatic antioxidants. However, it can be assumed that in B-54 and B-9 varieties an uncontrollable production of reactive oxygen species may be coupled with the less potentiality of antioxidant system created the oxidative stress.

M. Divya, Srikanta Das  and Sunita Mahapatra  (2024). Evaluation of Antioxidants in Different Mustard Cultivars Against Alternaria Blight. Biological Forum – An International Journal, 16(6): 135-142.