Foliar spray of salicylic acid and ascorbic acid ameliorates the biochemical compounds in hybrid chillies

2.1 Plant material

The study was conducted at Vegetable Research Institute (VRI), Ayub Agricultural Research Institute Faisalabad under field conditions in Pakistan in 2020–2021. Vigorous and uniform seedlings were transplanted into the open field under tunnel with a plastic sheet. We maintained the distance of 75 cm between one row to the other and plant to plant distance is 45 cm, 40 days after sowing (DAS). The experiment was carried out without the application of insecticides and fungicides. Weeds were kept under control by hand removing them. All the plots received regular and even cultural practices. There were 3 replicates per treatment and 10 plants per replicates. Foliar application of salicylic acid, ascorbic acid and their combination were applied 25 days after transplanting with 10 day intervals at the stages of planting, initiation of flowering and fruiting. The different levels of salicylic acid and ascorbic acid such as T1 (Control) T2 (SA 1 mM), T3 (SA 2 mM), T4 (AA 1 mM), T5 (AA 2 mM), T6 (SA 1 mM + AA 1 mM), T7 (SA 1 mM + AA 2 mM), T8 (SA 2 mM + AA 1 mM) and T9 (SA 2 mM + AA 2 mM) used in four hybrid varieties V1 = SV8233-HD (V2), SV5232-HY (V3), SV8883-HA (V4) and Golden Heart.

2.2 Vegetative growth parameter

Vegetative growth parameters i.e., Plant height (cm), Fruit weight (g), yield (g) root weight (g) shoot weight (g), Fruit width (mm) fruit length (mm), and pericarp thickness (mm) were estimated from five guarded plants in every treatment and their means was calculated.

2.3 Estimation of CAT activity

A reaction mixture containing 15 mM phosphate buffer (pH 7.0) and 15 mM H₂O₂ was used to estimate Catalase Enzyme Activity (CAT) using procedure described by Dhindsa et al. (1981). The absorbance fluctuations were taken at 240 nm after 40 s. The amount of enzyme activity equivalent to amount of enzyme required to reduce absorption from reaction mixture at 240 nm within one minute was equivalent to one unit of CAT.

2.4 Estimation of SOD activity

1000 ll of enzymatic extracts with 2.50 ml of 55.0 M methionine, 100 mM of phosphate buffer (pH 7.8), 300 ml nitrobluetetrasolium 0.75 mM and 60 ll of 0.1 mM riboflavin were kept in a test tube and incubated for 10 min in fluorescence light (40 lmol m-2 s-1) using method performed by the Xu et al. (2008). The absorbing solution read to 560 nm by a UV/visible spectrometer. One unit of SOD enzyme was determined as the amount of enzyme that inhibits 50% of photo-reduction NBT.

2.5 Estimation of POD activity

POD activity was calculated from 50 ml of enzyme extract placed into reaction mixture, 0.4% H2O2, potassium phosphate buffer to pH 6.1 and 1% guaiacol using by the Zhou and Leul (1998) method with absorbance at 470 nm.

2.6 Estimation of proline amount

A step wise procedure developed by Bates et al. (1973) was adopted to find out the amount of proline. Firstly, a homogenized leave sample of 0.2 g obtained through centrifugation at 18,000 g for 15 min was obtained. 2 ml of leave sample taken in a test tube was mixed with 2 ml glacial acetic acid and freshly prepared acid nin-hydrin solution (1.25 g nin-hydrin dissolved in 20 ml 6 m orthophosphoric acid and 30 ml glacial acetic acid). Heating of test tube solution at 100 °C for 1 h and cooled at 25 °C. Then, 4 ml of toluene were poured into the contents of the test tube in phase separation stage placing test tubes at vertical position for 10 min. The absorbance reading was observed at 520 nm and content of proline was amounted in lg g^-1on weight basis.

2.7 Estimation of proximate

Protein, carbohydrates, fat, crude fiber and ash content were measured by AOAC (2002).

2.8 Statistical analysis

The data recorded was subjected to analysis of variance following through R Studio (4.2.2). All graphical presentation by R Language (ggplot2) Scheme.

Result and Discussion.

3.1 Plant height (cm)

Analysis of variance showed significant differences between four varieties of chillies subjected to various levels of salicylic acid and ascorbic acid and their combination. However significant differences were observed in V4 plant height growth in response to different applications of salicylic acid and ascorbic acid. Out of nine salicylic acids, ascorbic acid and their combination, maximum plant growth was given by T9 (80.3 cm) whereas minimum plant growth in 33 cm were observed in T9 and T1 in chilli varieties. In variety V3 at the treatment T9 (58 cm) was observed in chillies i.e. minimum plant height growth was observed in T1 (34 cm). Moreover, maximum plant height at T9 (69 cm) less growth of plant height was observed (45 cm) in variety V2. Whereas the highest plant height in T9 (51.6 cm) and lowest plant height (33 cm) in variety of V1. It is depicted that different levels of salicylic acid, ascorbic acid and their combination strains significantly affected the plant height in chillies (Table 1).

3.2 Root weight (g) and shoot weight (g)

The data regarding the chillies plant growth responses to ascorbic and salicylic acid presented in Table 1 revealed that foliar of AA and SA applications significantly improved plant dry weight (g) and shoot weight (g) compared to control plants. The maximum value of shoot weight observed in T9 (161 g) and the minimum value of (71 g) in variety V4. The highest shoot weight of T9 (123.3 g) and the lowest weight in T1 (60.33 g) in variety V3. The shoot weight greater in T9 (141.0 g) and lower in control treatment of V2 as followed by V1 variety of chillies. Whereas the highest value of root weight in treatment of T9 (47.16 g) as compared to T1 (20.10 g) variety of V4. In variety V3 treatment T9 showed root weight (32.43 g) and minimum value in T1 (18.03 g). The values of root weight T9 (39.10 g) and T1 (16.10 g) in V2 as the same the highest value T9 (36.33 g) and lowest value T1 (13.46). The highest values of growth parameters were recorded, ascorbic and salicylic acid applied together with all concentrations as foliar with respect either individual or control plants.

3.3 Fruit width and fruit length (mm)

Different concentrations of SA and AA on different chilli varieties resulted in significant differences on fruit length constituting range of fruit length from 96.6 mm to 81.0 mm.The maximum fruit length (96.6 mm) was measured in T9 treatment for variety V3 followed by, 99.0 mm for V2 and (95.0 mm) for V1 respectively as compared to control. Similarly, greater fruit width(30.13 mm) was observed in treatment T9 and lowest (17.10 mm) in T1 for variety V4 which was statistically similar with variety V3, V2 and V1 of chillies.

3.4 Peri-carp thickness (mm)

The maximum value of peri-carp thickness in T9 (1.99 mm) and minimum (1.62 mm) for variety V4 was calculated. Moreover, in variety V3 the value was measured in T9 (1.89 mm) and T1 (1.48 mm). The greater value of peri-carp thickness of V2, V1 was showed (1.85 mm) and (2.10 mm) respectively as compared to control.

3.5 Fruit weight (g)

Significant impacts of SA and AA was estimated for fruit weight and maximum magnitude of the trait under study was observed in T9 (10.2 g) for V4 followed by (8.4 g) in V3. However, the highest fruit weight (11.6 g), (16.6 g) was recorded in treatment T9 in V2 and V1 plants as compared to T1 treatment.

3.6 Fat (%)

Fat percentage was decreased significantly through foliar spray of SA and AA with the greatest value of T1 (2.7%) in variety of V4. The fat (2.5%) of T1 variety V3 was recorded in plants. However, the lowest fat concentration (1.3%) at T9 in V3 and V1 was recorded. The amount of fat percent present in treatment T9 (1.6%) in V2 as compared to T1 treatment.

3.7 Ash (%)

A significant mean differences for ash percentage was estimated for different concentrations of foliar spray of SA and AA. The greatest value of T9 (9.2%), (8.2%) in varieties of V4 and V3 was recorded in plants. However, the greatest value of ash content (7.8%), (7.6%) was recorded in treatment T9 in V2 and V1 plants as compared to T1 treatment.

3.8 Protein (%)

Highest crude protein was found in T9 (23.48%) of V1 followed by (8.8 %) in V4. Lowest protein was recorded in V3, V2 in treatments in T9 (8.4 %) and (7.9%).

3.9 Carbohydrate (%)

The present results revealed high carbohydrate levels. Highest carbohydrates were found in T9 (57.27 %), followed by (52.49%) in varieties of V4 and V3 as compared to control. In varieties V2 and V1 (51.26 %), (48.87%) was measured in treatment T9 respectively.

4.1 Proline (%)

The data regarding proline content at various SA and AA foliar application revealed the mean differences for treatments and chilli varieties. Maximum proline contents of 19.1% was found in V2 followed by V1 having value of 18.5%. Furthermore, in treatment T9 maximum value of proline (%) was observed in V3 (17.65%) and V4 (17.85%)respectively.

4.2 Antioxidant enzymatic activity. SOD (ug-1Fw), POD (ug-1Fw), CAT (ug-1Fw).

The maximum value of SOD influenced by the SA and AA application. The results showed that maximum SOD content present in treatment (T9) (1.40 ug-1Fw) V4 (1.39 ug-1Fw) V3. The highest value of SOD in T9 (1.28 ug-1Fw), (1.12 ug-1Fw) V2 and V1 respectively. The maximum POD contents were recorded in treatments T9 (17.9 ug-1Fw), (17.75 ug-1Fw), (17.65 ug-1Fw) and (17.30 ug-1Fw) of varieties V4, V2, V3 and V1 respectively. The highest value of CAT contents in the treatment of T9 (6.15 ug-1Fw) variety of V4. Least value of CAT contents in pepper plant was seen in (5.70 ug-1Fw), (5.53 ug-1Fw) and (5.19 ug-1Fw) varieties of V1, V3, V4 respectively.

4.3 Yield (g)

The yield of pepper varieties was estimated by the foliar application of SA, AA and their combination. The largest yield of T9 (217.66 g) in variety V3 as compared to T1 treatment. The yield was measured (215.33 g) in V4 of T9. The highest value of yield estimated in (208.6 g) and (205.3 g) V1 and V2 as compared to control. It cleared that ascorbic acid and salicylic acid at their different levels of applications significantly increased all varieties yield compared to control.

5.1 Plant height (cm)

The foliar application of SA mitigated the growth reducing effects constituted due to various abiotic stresses thus, improved plant height, number of leaves plant per, chlorophyll content, fruit quality and yield as compared to control (Table 1). Such inferences were in accordance with the result findings of Zaghlool et al. (2006) who concluded additive association with plant growth and fruit yield parameters with the application of SA. Moreover, in addition to increased plant growth parameters like plant height, stem diameter, number of branches, number of leaves, root length as well fresh and dry weight the nitrogen, phosphorus, potassium uptake percentages were also increased with increasing concentrations of ascorbic acid as foliar application (Mazhar et al., 2011).

5.2 Yield

The impact of different formulation of SA significantly improved plant growth and becomes cause of increase in yield of the plant. Results showed that foliar spraying of ascorbic acid at 100 mg/l increased yield and improved plant characteristics such as, number of total and folded leaves, fresh weight of total and folded leaves, leaf area per plant, plant fresh and dry weight and leaves dry matter and total soluble solid (Jerry et al., 2011; Nafeesa et al., 2019).

5.3 Fruit width (mm) and fruit length (mm)

The foliar applications of SA indicated positive influence on some fruit characteristics, earliness, yield, chlorophyll contents in leaves and plant growth. However, SA treatments had no effect on pH, AA and TA of tomato (Yildrim and Dursun, 2009).

Biochemical attributes

5.4 Carbohydrate

Many previous observations, while for different crops, demonstrated that the application of SA altered the growth, photosynthesis and carbohydrates metabolism of maize and sugar beet. (Khaled and colleagues, 2020). Carbohydrates contents have positive relation with the foliar application of ascorbic acid due to reducing effects of plant stress on plant growth. The massive increase in total contents of carbohydrates due to foliar application of ascorbic acid in our case was resembled with Mazhar et al. (2011).

5.5 Proline

Foliar application of salicylic acid helpful to increase the proline contents in linseed (Bakry et al., 2012). The increase in proline content to combat the reducing effects of abiotic plant stresses matched the results with Nazi et al., 2016 who conducted experiment on cucumber in arid conditions and found Ascorbic acid stimulation in the chlorophyll a, proline, relative water content and Ci concentration).

5.6 Fat

Salicylic acid increased the fatty acid content under the drought stress in purslane plant. However, our outcomes contrast to the Saheri et al (2020). According to Aml et al. (2011) exogenous treatment of ascorbic acid boosted all fatty acids in the sunflower crop.

5.7 Fiber and ash

Out results correlates with the Meena et al. (2012) by the foliar spray of ascorbic acid and salicylic acid increase the fiber and ash contents in Aswagandha (Withania somnifera L.Dunal).

5.8 Protein

Highest values of protein content were noted with the treatment application of Acetyl Salicylic Acid at concentrations 0.5 mM in grain. Our results correlate with the Matysiak et al. (2020). Stating that spray of ascorbic acid increases the protein in maize at the concentration of 8 g /l. Our results of protein are similar to Lamiaa et al. (2020).

5.9 Enzymatic Activities:

In broad beans, foliar application of SA enhanced the activities of antioxidants enzymes and all growth parameters (Azooz et al., 2011). The use of AA along with 0.03% Zn combination enhanced photosynthetic pigmentation, vegetative growth, ion uptake, antioxidant activity in lettuce (Noreen et al., 2021).